Detection and Analysis of Genetic Alterations

Detection and Analysis ofGenetic Alterations in Normal

Skin and Skin Tumours

Åsa SivertssonMed.Lic.

Royal Institute of TechnologyDepartment of Biotechnology

Stockholm 2002

Åsa Sivertsson

Department of BiotechnologyRoyal Institute of TechnologyStockholm Center for Physics, Astronomy and BiotechnologySE-106 91 StockholmSweden

Printed at Universitetsservice US ABBox 700 14100 44 StockholmSweden

ISBN 91-7283-379-3

Åsa Sivertsson (2002): Detection and analysis of genetic alterations in normal skinand skin tumoursDepartment of Biotechnology, Royal Institute of TechnologyStockholm, SwedenISBN 91-7283-379-3

ABSTRACT

The investigation of genetic alterations in cancer-related genes is useful for research,prognostic and therapeutic purposes. However, the genetic heterogeneity that oftenoccurs during tumour progression can make correct analysis challenging. Theobjective of this work has been to develop, evaluate and apply techniques that aresufficiently sensitive and specific to detect and analyse genetic alterations in skintumours as well as in normal skin.

Initially, a method based on laser-assisted microdissection in combination withconventional dideoxy sequencing was developed and evaluated for the analysis of thep53 tumour suppressor gene in small tissue samples. This method was shown tofacilitate the analysis of single somatic cells from histologic tissue sections. In twosubsequent studies the method was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53 immunoreactive and non-immunoreactive cells from different layers of sunexposed skin, as well as skinprotected from exposure, were analysed for mutations in the p53 gene. The resultsrevealed the structure of a clandestine p53 clone and provided new insight into thepossible events involved in normal differentiation by suggesting a role for alleledropout. The mutational effect of physiological doses of ultraviolet light A (UVA) onnormal skin was then investigated by analysing the p53 gene status in singleimmunoreactive cells at different time-points. Strong indications were found thatUVA (even at low doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis.After slight modifications, the p53 mutation analysis strategy was then used tocomplement an x-chromosome inactivation assay for investigation of basal cell cancer(BCC) clonality. The conclusion was that although the majority of BCC’s are ofmonoclonal origin, an occasional tumour with apparently polyclonal origin exists.

Finally, a pyrosequencing-based mutation detection method was developed andevaluated for detection of hot-spot mutations in the N-ras gene of malignantmelanoma. More than 80 melanoma metastasis samples were analysed by thestandard approach of single strand conformation polymorphism analysis(SSCP)/DNA sequencing and by this pyrosequencing strategy. Pyrosequencing wasfound to be a good alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simple and rapid technique.

Keywords: single cell, DNA sequencing, p53, mutation, UV, BCC, pyrosequencing,malignant melanoma, N-ras

Åsa Sivertsson, 2002

There is much pleasure to be gained from useless knowledgeBBBBeeeerrrrttttrrrraaaannnndddd RRRRuuuusssssssseeeellllllll

ISBN 91-7283-379-3

This thesis is based on the following publications, which in the text will be referred to

by their Roman numerals:

LIST OF PUBLICATIONS

I. Persson Å, Ling G, Williams C, Bäckvall H, Ponten J, Ponten F, Lundeberg J.(2000) Analysis of p53 mutations in single cells obtained from histologicaltissue sections. Anal Biochem 287(1): 25-31.

II. Persson Å/Ling G, Berne B, Uhlén M, Lundeberg J, Ponten F. (2001)Persistent p53 mutations in single cells from normal human skin. Am J Pathol159:1247-1253.

III. Persson Å, Wiegleb Edström D, Bäckvall H, Lundeberg J, Pontén F, Ros A-M, Williams C. (2002) The Mutagenic Effect of Ultraviolet A1 in HumanSkin-demonstrated by sequencing the p53 gene in single keratinocytes.Photodermatology, Photoimmunology and Photomedicine. In press.

IV. Asplund A, Sivertsson Å, Lundeberg J, Pontén F (2002) Analysis of x-chromosome inactivation patterns in human basal cell carcinoma revealsdiverse clonal organization: evidence of multicellular origin. Manuscript.

V. Sivertsson Å, Platz A, Hansson J, Lundeberg J. (2002) Pyrosequencing as analternative to SSCP for detection of N-ras mutations in human melanomametastases. Clin Chem. In press.

INTRODUCTION___________________________________________________ 1

Mutation detection_______________________________________________________ 1

Scanning methods _______________________________________________________ 2DNA sequencing ______________________________________________________________ 2Sanger sequencing _____________________________________________________________ 2SSCP _______________________________________________________________________ 4Other conformation-based methods ________________________________________________ 6Cleavage –based techniques _____________________________________________________ 8

Specific methods ________________________________________________________11Hybridisation-based techniques__________________________________________________ 11Methods based on allele-specific amplification ______________________________________ 12Oligonucleotide ligation assays __________________________________________________ 12Techniques based on polymerase extension _________________________________________ 13Pyrosequencing ______________________________________________________________ 16

CARCINOGENESIS________________________________________________ 19

Cancer of the skin _______________________________________________________21Ultraviolet radiation __________________________________________________________ 21Normal skin _________________________________________________________________ 24The tumour suppressor p53 _____________________________________________________ 26The p53 patch _______________________________________________________________ 27Non-melanoma skin cancer _____________________________________________________ 29Malignant melanoma __________________________________________________________ 30

PRESENT INVESTIGATION________________________________________ 32

Genetic analysis of skin and skin tumour samples. _____________________________32Sample handling and preparation for analysis_______________________________________ 32

Sample preparation _________________________________________________________ 32Microdissection____________________________________________________________ 33Laser microdissection _______________________________________________________ 33

p53 gene analysis ________________________________________________________35Development of method for single cell genetic analysis (I). _____________________________ 35

Applications ____________________________________________________________38Effects of UV radiation on the p53 gene in normal human epidermis (II, III) _______________ 38Sun-exposure and persistent p53 mutations (II) _____________________________________ 38p53 mutations induced by UVA1 (III) _____________________________________________ 41The clonality of BCC (IV) ______________________________________________________ 43Detection of N-ras hot-spot mutations in melanoma using pyrosequencing (V) ______________ 45

Abbreviations______________________________________________________ 47

Acknowledgements _________________________________________________ 48

References ________________________________________________________ 50

Å Sivertsson

1

INTRODUCTION

The human body consists of approximately 2x101 2 cells, each containing the

hereditary information of the genome in the form of 3x109 basepairs of

deoxyribonucleic acid (DNA). This DNA is arranged into 46 chromosomes and the

composition of the DNA bases within these macromolecules determines the genotype

and contributes to the phenotype of an individual. Consequently, alterations in

chromosomes may lead to genetically related diseases. Cells are constantly exposed to

agents and events that can be harmful to the DNA. Depurination, deamination,

oxidation and methylation of bases are endogenous events that can change the base

composition of DNA, while exogenous agents such as UV light, ionising radiation

and genotoxic substances may damage DNA by creating pyrimidine dimers, double-

strand breaks and adducts. Several different gatekeeper and DNA repair systems exist

to maintain DNA integrity, but occasionally these backup systems fail and mutations

arise. Mutations can be divided into two classes; gross alterations and subtle

alterations. Gross alterations involve changes in chromosome number, partial

deletions of chromosomes, chromosomal translocations and gene duplications, while

subtle alterations comprise of single base substitutions, insertions and deletions.

Mutations are common events in cancer and the ability to monitor them is therefore of

great interest for diagnostic and prognostic purposes as well as in understanding gene

function.

Mutation detection

Gross chromosomal aberrations can usually be detected by cytogenetical methods or

by DNA fragment analysis, while methods that are more sensitive and usually more

expensive must be used for detection of subtle alterations. A wide range of methods

already exists for detection of single (or a few) basepair changes. Specificity,

sensitivity, technical complexity and cost vary between the different methods and the

multitude of different techniques present indicates that the perfect technique for

detection of subtle alterations has not yet been described. The mutation detection

strategies can be divided into two categories depending on the alteration they identify.

Scanning methods detect uncharacterised sequence variations, while specific methods

Detection and analysis of genetic alterations in normal skin and skin tumours

2

identify previously characterised sequence variations such as polymorphisms and

mutation hotspots. Some of the more widely used methods as well as some promising

upcoming techniques will be described briefly below.

Scanning methods

In order to perform a comprehensive mutation analysis of a stretch of DNA the use of

scanning methods or DNA sequencing is required. The principle of some scanning

methods described below is shown in Figure 1. DNA sequencing is considered the

golden standard for identification of mutations while any mutation found by a

scanning method is also usually confirmed by DNA sequencing.

DNA sequencing

In 1977, two different DNA sequencing techniques based on the generation of single-

strand DNA ladders were described. Maxam-Gilbert sequencing takes advantage of

chemicals that cleave DNA at specific bases to create fragments of different lengths

subsequently separated by electrophoresis (Maxam, 1977). The toxicity of the

chemicals used for cleavage has precluded widespread use of this method, but it

provides an approach for sequencing templates unavailable for enzymatic sequencing

such as adduct modified DNA (Wilkins, 1985; Mao, 1995; Mao, 1992). The Sanger

sequencing technique, which is more widely used, relies on enzymatic chain

termination to generate a sequencing ladder for size separation (Sanger, 1977).

Sanger sequencing

Although Sanger sequencing was introduced 25 years ago, to date it is still the only

method able to scrutinise each position in an unknown sample of up to 800 basepairs

in length. This feature has lead to the continuous development of the technique via

automation, nucleotide labelling and enzyme engineering, despite it being a laborious

and expensive method.

The method is based on primer extension in the presence of a dideoxynucleotide,

which causes chain termination. Four parallel reactions are performed in which a

primer is hybridised to the single-stranded DNA template and extended by a DNA

polymerase in the presence of a low concentration of a chain terminating

dideoxynucleotide (ddNTP) (one in each reaction) and all four deoxynucleotides

(dNTPs). The synthesis is terminated whenever a ddNTP is incorporated instead of

Å Sivertsson

3

the corresponding dNTP resulting in a ladder of fragments terminated at stepwise

intervals. The fragments are separated according to size by electrophoresis and the

sequence of the target DNA is determined by reading the band pattern.

Conventionally, radioactively labelled nucleotides or primers have been used to

facilitate detection of the band patterns, but these labels have now generally been

replaced by fluorescent-based labelling strategies. Fluorescent dyes can be introduced

by using primers with a 5´ end-label (dye-primers) (Smith, 1986; Ansorge, 1986) or

alternatively by incorporation of labelled ddNTPs (dye-terminators) (Prober, 1987) or

labelled dNTPs (internal labelling) (Ansorge, 1992) during extension. The dye-primer

and the dye-terminator set-ups are compatible with the two different formats available

for slab-gel electrophoresis i. e. the four lane-one dye and the one lane-four dye

strategies. The four lane-one dye format generates easily interpreted data, since a

single dye is used and the mobility is equal in all lanes. The throughput, however, is

slow but an automated set-up is available in the automated laser fluorescence

sequencer (ALF) from Amersham Pharmacia. In the one lane-four dye format, four

different fluorophores are used and all four sequencing ladders are separated

simultaneously in a single lane using the set-up described by Smith (Smith, 1986).

This approach has become more common due to its higher throughput and is used in

the automated sequencers commercially available from manufacturers such as

Applied Biosystems. The requirement for processing raw data using base calling

algorithms may however slightly hamper the sensitivity of mutation detection and

quantification.

The ever-increasing need for higher throughput and cost reduction in DNA

sequencing has lead to the gradual replacement of the slab gels by capillary gel

electrophoresis (CGE) (Smith, 1991). CGE requires smaller sample volumes allowing

for a reduction in the amount of template DNA and reagents, thereby resulting in a

reduction in overall costs. In addition, faster sequencing runs are facilitated, since the

capillaries can more effectively dissipate heat allowing the use of higher voltages

during electrophoresis. Also in terms of sample analysis capillaries have an advantage

over slab gels in that tracking of lanes no longer needs to be considered.

In addition to the development of fluorescent labelling and automation, a significant

advance in the technology has been the modification and engineering of the enzymes

used in the DNA sequencing reaction. The first enzyme used was the Klenow


4

fragment, which due to its sequence dependent discrimination between ddNTPs

generated variations in the raw data and thus uneven peaks (Klenow, 1970). Higher

quality and more easily interpreted data was generated using the T7 DNA polymerase,

which showed even incorporation of all four ddNTPs (Tabor, 1987; Tabor, 1989;

Kristensen, 1988). This enzyme still offers the most uniform sequence data but has

the disadvantage of being temperature sensitive and easily degraded. However, after

the introduction of the thermostable polymerase derived from Thermus aquaticus

(Taq DNA polymerase) (Chien, 1976) the use of a linear PCR amplification mode for

the dideoxy reactions has become very popular. In cycle sequencing, one primer is

used to generate the Sanger fragments in an amplification reaction, which gives the

advantages of lower template requirement, generation of single stranded DNA

without time-consuming denaturation of double-strands and ease of automation

(Innis, 1988; Murray, 1989). Manipulations of the Taq DNA polymerase solved

initial problems with discrimination of the enzyme for dNTPs over ddNTPs and a

uniform peak pattern comparable to that of T7 DNA polymerase can now be

generated (Reeve, 1995; Tabor, 1995).

SSCP

Scanning methods usually provide a simple and low-cost assay to simultaneously

compare larger regions of DNA between normal and unknown samples to rapidly

eliminate non-mutant samples. Since its introduction in 1989, single strand

conformation polymorphism (SSCP) analysis has become one of the most popular

scanning strategies, which is probably due to its technical simplicity in combination

with its low cost and relatively high sensitivity (Orita, 1989). The method is based on

the sequence dependent mobility of single stranded DNA during electrophoresis.

Amplified DNA fragments are first denatured using agents such as formamide,

sodium hydroxide, urea or methylmercuric hydroxide (Humphries, 1997; Xie, 1997).

Despite the superior performance of methylmercuric hydroxide in some applications

its toxicity precludes its widespread use, with the result that formamide is the most

commonly used reagent today (Weghorst, 1993; Hongyo, 1993). The single stranded

DNA is then subjected to electrophoresis through a non-denaturing gel with the

mobility of the DNA fragment dependent on its adopted conformation, which is

influenced by its sequence and size. Thus, a single base alteration can be detected by

SSCP if a conformer displaying different mobility through the gel is generated.

Å Sivertsson

5

However, due to the absence of theoretical models to predict either the three-

dimensional structure of single-stranded DNA under a given set of conditions or the

resulting mobility of any given conformer, the optimal conditions for discriminating

between any two fragments differing by a single base must be empirically determined.

In addition to the sequence context and size of the DNA fragment, several parameters

such as the gel matrix composition, temperature during electrophoresis and

concentration of DNA have been found to affect the sensitivity of SSCP analysis. The

pH, ionic strength and composition of the electrophoresis buffer can also affect the

mobility of DNA during SSCP. However, it has been postulated that virtually all

mutations can be detected by using a combination of the three following conditions;

electrophoresis at room temperature with or without 5%-10% glycerol and at 4°C

without glycerol (Hayashi, 1993; Hayashi, 1991; Sheffield, 1993). The composition

of the gel matrix is important and optimal sensitivity have been achieved using high

concentrations of acrylamide with low crosslinking or alternatively by using the

commercially available Mutation Detection Enhancement (MED) gel (Ravnik-Glavac,

1994; Glavac, 1993). The difference in migration between fragments longer than 300

basepairs can be further enhanced by addition of 10-15 % glycerol or sucrose, or 5 %

of a denaturing agent such as urea. Addition of glycerol decreases the pH and thereby

weakens the electrostatic repulsion between the negative charges in the nucleic acid

backbone, which in turn may allow for greater stabilisation of the tertiary structure

(Kukita, 1997). The same effect can also be achieved by lowering the temperature

during electrophoresis or by using buffers with a low pH or higher salt concentration

(Kukita, 1997). Addition of primers to the SSCP template has also been shown to

stabilise the single stranded conformation resulting in better separation of the DNA

fragments (Almeida, 1998). In relation to the DNA fragment length, Sheffield and co-

workers has reported an effective size limit of 150-200 basepairs in order to achieve a

maximum sensitivity of 97 % (Sheffield, 1993), while other studies have shown

sensitivity greater than 95 % for fragments ranging from 200 to 900 basepairs (Fan,

1993; Ravnik-Glavac, 1994; Ravnik-Glavac, 1994; Highsmith, 1999). These varying

results may in part depend on the sequence composition of the samples. The detection

sensitivity is greater in GC rich templates which is due to the higher proportion of

hydrogen bonds formed between G and C residues resulting in a more intricate, and

thus more easily influenced folding of the strands (Highsmith, 1999).


6

The detection of fragments after electrophoresis can be achieved by autoradiography,

silver staining or through the use of fluorescent dyes (Hoshino, 1992; Hiort, 1994;

Iacopetta, 1998; Law, 1996). Autoradiography is time-consuming and involves the

use of radioactively labelled primers and has therefore gradually been replaced by

other methods. Analysis on automated sequencers using fluorescent-labelled primers

is another commonly used alternative which offers high-throughput in addition to

high sensitivity. The use of fluorescent dyes further allows for the loading of low

concentrations of DNA which prevents disturbing reannealing (Makino, 1992;

Iwahana, 1996; Ellison, 1996; Ellison, 1993; Iwahana, 1994).

Depending on the sequence analysed and the conditions used it has been reported that

between 60 % to 100 % of mutations present can be detected by SSCP (Martincic,

1996; Vidal-Puig, 1994; Ellis, 2000; Moore, 2000; Ellison, 1993; Ravnik-Glavac,

1994). Usually a sensitivity of 90% or less is reported using a single condition for

electrophoresis while a sensitivity of greater than 95 % is reached when several

different conditions are applied.

Recently, high-throughput methods combining SSCP and heteroduplex analysis (HA)

have been described (Kozlowski, 2001; Kourkine, 2002). By thermally denaturing and

rapidly cooling a mixture of wild type and mutant double-stranded DNA, single

stranded conformers as well as homo -and heteroduplexes will be formed. The sample

is then subjected to simultaneous SSCP and HA using capillary electrophoresis or

capillary array electrophoresis and fluorescent detection. This tandem approach

results in 100 % sensitivity for mutation detection, compared to a sensitivity of 90-

93% for SSCP and 75-81 % for HA.

Other conformation-based methods

Denaturing gradient gel electrophoresis (DGGE), HA and denaturing high

performance liquid chromatography (DHPLC) belong to a group of scanning methods

which are based on mobility shifts created during electrophoresis between DNA

fragments of different sequences but of equal lengths. Denaturing gradient gel

electrophoresis (Myers, 1985)[(Fischer, 1980; Fisher, 1983) uses a linearly

increasing gradient of a denaturing agent to exploit differences in the melting

properties of DNA duplexes with different sequences. During migration in the gel the

duplexes will progressively dissociate at discrete domains that have lower melting

temperatures (dependent on the sequence composition) which will cause a marked

Å Sivertsson

7

retardation of the fragments. A single base change may result in a different migration

pattern, thus allowing mutations to be detected. If homo- and heteroduplexes of wild-

type and mutant sequences are formed via denaturation and reannealing of the

amplified fragments and a GC-clamp is introduced (Myers, 1985; Sheffield, 1989;

Myers, 1985) (to prevent complete denaturation of high melting domains), the

sensitivity can be further increased. Under these conditions, detection of almost 100

% of single nucleotide changes in fragments ≤ 500 basepairs has been shown (Myers,

1985). Several variants of DGGE have been developed such as genomic DGGE

(Borresen, 1988), temperature gradient gel electrophoresis (TGGE) (Wartell, 1990)

and constant denaturant gel electrophoresis (CDGE) (Hovig, 1991). Denaturing

gradient gel electrophoresis is a sensitive method and has an advantage over other

similar methods in that it is possible to optimise the method through computer

simulation. A limitation of DGGE is that establishing the method is labour intensive

and it can only analyse fragments of less than 600 basepairs. Heteroduplex analysis

(Nagamine, 1989; Keen, 1991) is based on the difference in mobility through a native

gel due to conformational differences between homoduplex and heteroduplex DNA.

The sensitivity is high for detection of insertions and deletions but lower for detection

of single base substitutions (Bhattacharyya, 1989; Bhattacharyya, 1989). By using a

special gene matrix, the sensitivity for single base changes can be improved reaching

an estimated mutation detection rate of 80% (Perry, 1992). Conformation-sensitive

gel electrophoresis (CSGE) is a variant of this method where conditions are created to

increase the local denaturation around a mismatch to enhance the retardation

(Ganguly, 1993; Ganguly, 2002). Denaturing high-performance liquid

chromatography is based on the same principle, but the separation is performed in a

special HPLC column and thus gel pouring is avoided (Liu, 1998). Under partly

denaturing conditions, the method detects single-base substitutions as well as small

deletions and insertions in fragments of 100 to 1,500 basepairs with high efficiency

and sensitivity (Wagner, 1999; Jones, 1999). Detection of mutations in fragments

smaller than 100 basepairs is also possible but completely denaturing conditions must

be used (Oefner, 2000). The method has recently been adapted so that it can be

performed in capillaries, which gives the same degree of separation but increases the

throughput and decreases the sample and solution volumes (Xiao, 2001; Huber,

2001). Dideoxy fingerprinting (ddF) combines a modified DNA sequencing reaction

with non-denaturing gel electrophoresis (Sarkar, 1992). In ddF, PCR products are


8

subjected to a sequencing reaction containing only one dideoxy terminator and the

analysis is based on migration differences or the loss or gain of fragments. Although a

detection sensitivity of up to 100 % for detection of p53 mutations has been

demonstrated, the establishment of the method is difficult and time-consuming

(Sarkar, 1992; Martincic, 1996; Blaszyk, 1995).

Cleavage –based techniques

Another group of scanning methods relies on chemical or enzymatic cleavage of

mismatches. Chemical cleavage of mismatch (CCM) was developed by Cotton in

1988 as a modification of the Maxam-Gilbert DNA-sequencing method (Cotton,

1988). Amplified DNA fragments are first denatured and reannealed (forming homo-

and heteroduplexes), followed by chemical modification of mismatched cytosines and

thymines by hydroxylamine and osmium tetroxide, respectively. The modified bases

can then be cleaved by hot piperidine treatment, which facilitates the localisation of

mismatches through detection of the cleavage products. The throughput of CCM has

been increased through the use of single tube reactions and by adapting the method to

a solid phase assay format (Hansen, 1996; Ramus, 1996). Fluorescence assisted

mutation assay (FAMA) is a modification of CCM that uses fluorescently labelled

primers in the PCR to achieve a higher sensitivity in visualisation of the cleaved

product (Verpy, 1994). Chemical cleavage of mismatch is the most sensitive and

specific cleavage method (Cotton, 1989) and these modifications has allowed CCM to

be performed in a semi-automated fashion. However, the biohazardous chemicals

involved make the method less attractive for routine use, although improvements have

been made by for example substituting osmium tetroxide for potassium permanganate

(Lambrinakos, 1999).

Methods based on enzymatic cleavage have an advantage over CCM in that they

avoid the use of hazardous chemicals. However, they display varying sensitivities and

most enzymes do not cleave all types of mismatches. The types of enzymes

commonly used in various cleavage assays include endonucleases, ribonucleases and

exonucleases. Several different methods take advantage of enzymatic cleavage by

endonucleases. Cleavage Fragment Length Polymorphism (CFLP) (Brow, 1996)

relies on the enzyme Cleavase which induces endonucleolytic cleavage at the base of

single stranded stem-loop structures formed after cooling of the DNA without

Å Sivertsson

9

reannealing. These stem-loop structures are dependent on the primary sequence and

thus changes in the sequence may result in alterations of the cleavage profile, which

subsequently can be detected by capillary or denaturing gel electrophoresis. In

contrast to CFLP, other endonuclease assays rely on cleavage at or near the site of

mismatches in heteroduplexes. The bacteriophage proteins T4 endonuclease VII

(T4E7) and T7E1, sometimes referred to as resolvases, can localise and cleave

mismatches in large DNA duplexes with a sensitivity approaching 100 % (Mashal,

1995; Youil, 1995; Youil, 1996). Ribonuclease A cleavage is another screening

method for large fragments (1Kb), which was first described by Myers using

DNA:RNA hybrids (Myers, 1985). Mismatches formed after hybridisation of labelled

RNA probes with amplified DNA are cleaved by RnaseA and visualised by

electrophoresis. The sensitivity of the original method is approximately 60 % but a

sensitivity of 88-90 % can be achieved using a modified method described by

Goldrick. In the Non-Isotopic RNase Cleavage Assay (NIRCATM) (Goldrick, 1996;

Goldrick, 2001), RNA:RNA heteroduplexes formed by in vitro transcription of PCR

products are cleaved by RnaseTI and Rnase1 which cleave a wider range of

mismatches than RnaseA. The cleaved products are then separated by non-denaturing

agarose gel electrophoresis and visualised by ethidium bromide.

Exonuclease protection assays take advantage of mismatch-binding proteins for

mutation detection. The E. coli mismatch recognition protein MutS detects and binds

preferentially to single base mismatches and the resulting protein /DNA complex can

be visualised by a gel mobility shift assay (Lishanski, 1994). However, MutS is

unable to detect C:C mismatches and the low stability of MutS/DNA complexes may

also result in a loss of signal (Jiricny, 1988). MutS can also be used in the MutEx

exonuclease protection assay to localise mutations (Ellis, 1994) and in an in vitro

constructed MutHLS system to detect mismatches in heteroduplexes after PCR

amplification (Smith, 1996). Another E. coli protein Mut Y, has also been used by

itself or in combination with thymine glycosylase for mismatch detection (Hsu, 1994;

Lu, 1992). The enzyme is highly sensitive but recognises only G:A mismatches and

therefore detection of G:G or C:C mismatches is not possible even when glycosylase

is used. In Base Excision Sequence Scanning (BESS) (Hawkins, 1997; Hawkins,

1999) a limiting amount of dUTP is present during a single PCR amplification. The

amplified product is then cleaved at sites of dUTP incorporation and at dGTP sites


10

using two different excision reactions involving uracil-N-glycosylase/E. coli

endonuclease IV and dGTP modifications respectively. The resulting sets of

fragments correspond to the positions of deoxyguanine and deoxythymidine in the

sequence and can be analysed using standard sequencing gels or on an automated

sequencer. All mutation types can be detected and in most cases, exact identification

of the mutation and determination of its position is possible.

Figure 1. Principles of scanning methods used for detection of mutations at non-defined positions.

S

Scanning methods

SSCP

DGGE

HA

CCM

CFLP

MutS

MutHLS

RNase

MutY

Normal Mutant

S

SHL

Y

R

Gelshift

Gelshift

Gelshift

Gelshift

Cleavage

Cleavage

Cleavage

Cleavage

Cleavage

MutEx Cleavage protection

Exo

Exo

Exo

Exo

BESS CleavageU GU

Å Sivertsson

11

Specific methods

In contrast to scanning technologies, specific methods identify alterations at pre-

defined sites. Two types of alterations are of particular interest; mutations and single

nucleotide polymorphisms. Mutations can be distributed across the genome, but at

some sites a clustering tendency is observed. These so-called “hotspot mutations”

have been identified in for example cancer-related genes such as ras and p53. Single

nucleotide polymorphisms (SNP) have been estimated to occur once in every

thousand bases in the human genome. They represent nucleotide variations at certain

positions in coding and non-coding regions of the genome. SNPs in coding regions

may account for differences in drug response between individuals and also may be a

contributing factor in disease susceptibility (Evans, 1999), (Davignon, 1988; Bertina,

1994). The possible effect of SNPs in non-coding regions is not yet determined but

they are extremely useful as markers in population and genetics studies (Jorde, 2000;

Hacia, 1999). Some methods that have been developed to analyse both mutations and

SNPs and are tailor-made for detection of variations at specific sites are described

below and shown in Figure 2.

Hybridisation-based techniques

The possibility of detecting a single-base mismatch using hybridisation with allele

specific oligonucleotides (ASO) was demonstrated as early as 1979 (Wallace, 1979).

Since the invention of PCR, the principle of ASO has become widely used in various

assays. The method relies on the differences in hybridisation efficiency to the target

DNA between a fully complementary ASO probe and a probe containing a single

mismatch. The early ASO assays were performed in a dot-blot or reverse dot-blot

format immobilising either the amplified target DNA or the oligonucleotide probe set

respectively on a membrane (Saiki, 1988; Saiki, 1989). The hybridisation product was

detected using autoradiography or enzyme conjugates. The assay was later adapted to

a high-density microarray format, but neither format allows for perfect allele

discrimination (Wang, 1998; Cho, 1999). Increased stringency can be achieved using

DASH (dynamic allele-specific hybridisation) where the hybridisation is monitored

over a temperature gradient (Ririe, 1997),(Prince, 2001) or by using higher affinity

LNA (locked nucleic acid) probes instead of DNA (Orum, 1999). In some recently

developed real-time PCR-based ASO assays, fluorescence is emitted as a result of a

change in the physical distance between a fluorophore and a quencher molecule. In


12

the Molecular Beacon assay (Giesendorf, 1998; Vet, 1999; Tyagi, 1996; Smit, 2001)

fluorescence is emitted when the stem-loop structure of the probe is opened upon

perfect hybridisation to the DNA target sequence during the primer-annealing phase.

In the TaqManTM assay release of the quencher after exonuclease degradation of

perfectly annealed probes by the polymerase allows the fluorophore to emit a

fluorescent signal (Livak, 1995; Livak, 1999). Both assays are compatible with 96-

well or 384-well microtiter plate formats and facilitate the use of multiple

fluorophores. However, the need for fluorescent and quencher moieties on the probes

make these assays rather expensive.

The concept of ASO was the first step towards today’s oligonucleotide arrays, which

are used in various applications ranging from mutation detection to gene expression

studies. The theoretical principle of sequencing by hybridisation (SBH) was

independently described by two groups in the late 1980’s and is based on arrays of all

possible combinations of short immobilised oligonucleotides (Drmanac, 1989) (Lysov

Iu, 1988). Labelled target DNA is then hybridised to the array and the hybridisation

pattern is used to in silico reconstruct the target sequence. This method proved less

suitable for de novo sequencing but has been successfully used for mutation detection

in the p53 gene (Drmanac, 1998).

Methods based on allele-specific amplification

Several allele-specific PCR-based methods have also been used for sequencing of

defined alterations. Allele specific PCR primers with the 3´ terminus annealing at the

variant position are used in the PCR reaction, which results in amplification with only

the perfectly matched primer. Assays based on this principle are for example ASA

(allele specific amplification) (Okayama, 1989), ASPCR (allele specific PCR) (Wu,

1989) and PASA (PCR amplification of specific alleles) (Sommer, 1992).

Oligonucleotide ligation assays

To increase the specificity of ASO hybridisation a number of assays based on ASO

ligation have been developed. In the oligonucleotide ligation assay (OLA)

(Landegren, 1988) a ligation probe and probes specific to wild type and mutant alleles

are hybridised adjacent to each other on the target sequence. Since ligases can

effectively discriminate against mismatches, only perfectly matched probes will be

Å Sivertsson

13

ligated. Depending on the label used, the detection of the ligated products can be

carried out in an ELISA format or by electrophoretic separation in a DNA sequencer

instrument (Nickerson, 1990; Samiotaki, 1994), (Grossman, 1994). The OLA assays

have also been used in different microarray formats (Broude, 2001; Gerry, 1999) but

the need for several differently modified probes increases the cost significantly. In

ligase chain reaction (LCR) two pairs of probes are used together with a thermostable

ligase in a cyclic ligation reaction resulting in amplification of the target sequence in

cases where the probes are perfectly matched (Barany, 1991). Padlock probes

(Nilsson, 1994) (Nilsson, 1997) are linear oligonucleotides that have complementary

target sequences at both ends separated by a random DNA sequence. Upon

hybridisation to a target, the probe ends are ligated to form a circularised probe if

there is complete homology to the target. Signal amplification can then be achieved

by using the circularised product of successful padlock probe ligation as a template

for rolling circle amplification (RCA) which creates a long single stranded DNA

composed of tandem-repeats complementary to the padlock probe (Fire, 1995; Baner,

1998; Lizardi, 1998). A single tube assay combining ligation and rolling circle

amplification has also been described which increases the throughput significantly

(Qi, 2001).

Techniques based on polymerase extension

Minisequencing, also denoted PEX for single nucleotide primer extension (Syvänen,

1990), is based on discrimination of variants by single nucleotide extension at the site

of a mutation. A primer is hybridised to the target sequence immediately adjacent to

the variable position and a DNA polymerase is then used to extend the 3´ end of the

primer with a labelled dideoxynucleotide complementary to the nucleotide at the

variable site. The method has been adapted to various formats and detection

strategies, resulting in ELISA, electrophoresis and microarray based formats

(Nikiforov, 1994; Pastinen, 1996; Tully, 1996; Pastinen, 1997; Lindroos, 2001). The

minisequencing concept is also used in the commercially available SNaPshotTM assay

(Applied Biosystems) and in the improved variant MAPA (multiplex automated

primer extension) described by Makridakis and Reichardt (Makridakis, 2001).

MALDI-TOF–MS (matrix-assisted laser desorption ionisation - time-of-flight - mass

spectrometry) emerged as a method for DNA sequencing in the late 1980’s. An

innovation in the field of ionisation of macromolecules made it possible to perform


14

analysis of DNA in a system which was earlier limited to peptide analysis (Karas,

1988). DNA samples are desorbed, ionised and subjected to an electric field where the

molecules are accelerated proportional to their mass/charge ratio. Detection is based

on the time required for the molecules to reach the detector. MALDI-TOF-MS is a

rapid sequence analysis technique that permits simultaneous analysis of many DNA

strands in a heterogenous mixture. However, the limited read-length of 100

nucleotides resulting from sequencing of dideoxy generated DNA ladders make it less

attractive for de novo sequencing (Wu, 1994; Taranenko, 1998; Monforte, 1997). The

method has been successfully used to sequence exon 5 to 8 of the p53 gene (Fu,

1998), but the true potential probably lies in typing single point mutations and SNPs

(Griffin, 2000; Griffin, 2000; Li, 1999; Tang, 1999; Griffin, 1999). Several different

approaches for MALDI-TOF-MS have been described. In the PINPOINT assay (Haff,

1997; Haff, 1997; Ross, 1998) the target is subjected to primer extension by a single

base in the presence of all four ddNTPs and the mass added onto the primer then

defines the variable base in the subsequent MALDI-TOF-MS analysis. In

MassEXTEND (Little, 1997; Little, 1997), (Braun, 1997; Braun, 1997) and VSET

(very short extension)(Sun, 2000) which are similar assays, dNTP mixes containing a

single ddNTP or ddNTP mixes containing a single dNTP respectively, are used in the

primer extension reaction. Apyrase-Mediated Allele-Specific Extension (AMASE) is

another single-step extension approach recently described (Ahmadian, 2001). This

assay relies on extension of paired allele-specific primers in the presence of the

nucleotide degrading enzyme apyrase. Only a perfectly matched primer will give rise

to extension, since the slower reaction kinetics of a mismatched primer-template

configuration will allow for the apyrase to degrade the nucleotides before extension

can occur. Thus the acknowledged difficulties that polymerases have in

discriminating between certain mismatches can be circumvented. The assay has

recently been adapted to a microarray format and successfully used for SNP typing

(O'Meara, 2002).

Å Sivertsson

15

Figure 2. Principles of methods used for detection of mutations/variations at defined positions.

ASO

ASA

OLA

PEX

LCR

No amplification

No duplex

No ligation

No ligation / amplification

No extension

Normal Mutant

Specific methods

Beacons

Padlocks

AMASE

AC

AT

Fluorescence

No ligation / amplification

No extension


16

Pyrosequencing

The principle of sequencing-by-synthesis was described in 1985 (Melamede, 1985)

and led to the development of two approaches for DNA sequencing; direct detection

of labelled nucleotides (Canard, 1994; Metzker, 1994) and indirect detection of

released pyrophosphate (Hyman, 1988; Nyren, 1987), respectively. In the direct

approach, the low efficiency of incorporation of labelled nucleotides results in

unsynchronised extension and limits the read length to a few bases (Metzker, 1994).

The indirect approach relies on the use of enzymes to convert inorganic phosphate,

released upon sequential addition and incorporation of nucleotides, into light. This

approach was further developed and in 1993 a real-time sequencing method was

described (Nyren, 1993). Two important modifications to this real-time sequencing

strategy were the substitution of dATPαS for dATP and the introduction of the

nucleotide degrading enzyme apyrase which resulted in an improved method denoted

Pyrosequencing (Ronaghi, 1996; Ronaghi, 1998; Ronaghi, 2001) (Figure 3). The use

of dATPαS solved the problem of non-specific signals generated by luciferase after

each addition of dATP, while addition of apyrase dispensed with the need for

extensive washing to remove the excess of nucleotides after each addition.

Pyrosequencing is based on polymerase-assisted primer extension using sequential

addition and incorporation of nucleotides in the presence of adenosine phosphosulfate

(APS), D-luciferin, ATP sulfurylase, luciferase and apyrase. The pyrophosphate (PPi)

released upon incorporation of a nucleotide is used as substrate for ATP sulfurylase

generating ATP. This ATP is then used in a reaction catalysed by luciferase resulting

in the release of detectable light. After each addition, the excess nucleotides are

degraded by apyrase before the next nucleotide is added. If the added nucleotide does

not form a basepair with the DNA template, it is not incorporated by the polymerase

and thus no light will be generated. The nucleotide is then rapidly degraded by

apyrase. Pyrosequencing may also be performed directly on double-stranded DNA

using enzymatic removal of amplification primers and nucleotides (from the PCR) as

described by Nordström et al (Nordstrom, 2000).

To obtain high-quality pyrosequencing data, efforts are required to optimise the

relative amounts of the different enzymes involved. The most critical reactions in

pyrosequencing are those that compete for nucleotides i. e. DNA polymerisation and

nucleotide removal. The DNA polymerase should be in excess to ensure that

polymerisation takes place immediately upon nucleotide addition. Furthermore, the

Å Sivertsson

17

nucleotide concentration must be above the KM of the DNA polymerase to obtain

rapid polymerisation, but it should not be so high that it affects the fidelity of the

polymerase (Eckert, 1990; Cline, 1996). Finally, the degradation of nucleotides by

apyrase must be slower than the incorporation by DNA polymerase.

An interesting feature of pyrosequencing is the possibility to predetermine the

dispensation order of the nucleotides. For analysis of an unknown sequence, cyclic

dispensation of AGTC might be the best choice, while programmed dispensation can

be more suitable for sequencing of known mutations or SNPs. A programmed

nucleotide delivery approach can be used to keep synchronised extension of different

alleles in (or out of) phase and thus improve the discrimination between different

alleles.

A fully automated microtiter plate-based instrument, simultaneously analysing 96

samples is commercially available (Pyrosequencing AB) allowing sequencing of

SNPs in less than 10 minutes and analysis of short stretches of DNA in less than an

hour. Due to product accumulation and decreased enzymatic activity over time, the

pyrosequencing read-length is limited and despite reports of read-lengths above 100

bases, the effective limit is around 40 bases (Garcia, 2000; Gharizadeh, 2002).

Pyrosequencing has thus so far mostly been used for SNP typing (Ahmadian, 2000;

Gustafsson, 2001; Gustafsson, 2001; Fakhrai-Rad, 2002; Wasson, 2002; Andreasson,

2002) but it has been used in the typing of bacteria (Monstein, 2001) and viruses

(Gharizadeh, 2001; O'Meara, 2001), mutation screening (Garcia, 2000) and tag

library sequencing (Agaton, 2002; Nordstrom, 2001).


18

Figure 3. Schematic diagram of pyrosequencing.

TAGAAGGACCGTTTAAGT

ATCTT

3'

Polymerase

PPi

ATP

dTTP

Sulfurylase

Luciferase

Real-time monitoring

A T C TTDNA sequence:

5'

Nucleotides added:

Primer

dTMP + 2Pi

Apyrase

5'

Light

Å Sivertsson

19

CARCINOGENESIS

The disease of cancer is described in the papyri from Egypt (written around 1600 BC)

which are the earliest medical records known and probably date from sources as early

as 2500 BC. The ancient Egyptians used not only surgery but also pharmacological

and magical treatments to fight the disease and were able to distinguish between

benign and malignant tumours. However, up until the 17th century, cancer was

considered an illness caused by an excess of black bile and curable only in its earliest

stages. Although enormous progress has been made in understanding cancer in the

last century, the disease remains elusive. Cancer is now defined as a cellular disease,

characterised by a transformed cell population that displays net cell growth and an

anti-social behaviour, which has originated from one single cell. Many of the events

involved in this multi-step process, both genetic and epigenetic, have been defined but

the complexity of the cell machinery makes it difficult to determine the definitive

outcome of any single event. In general terms, carcinogenesis can be divided into

three stages: initiation, promotion and progression. Initiation of a cell is the result of

events leading to permanent genetic alterations that will be transferred to the daughter

cells, such as mutations in one or more proto-oncogenes or tumour-suppressor genes.

During promotion, the initiated cell or cells go through selective clonal expansion due

to a growth advantage over normal cells, e.g. by evading apoptosis. The end result of

promotion is a benign tumour and the events that turn this tumour malignant are

denoted progression. The genetic events during these different stages have been

described in detail for the development of metastasising colon cancer via mild and

severe dysplasia and adenoma, leading to the postulation that between three and six

mutations are needed to complete the carcinogenic process (Vogelstein, 1993). In the

first successful attempt to transform normal human cells to cancer cells using only

defined genetic events, it was shown that a minimum of four pathways involving

telomerase, the two tumour-suppressors p53 and pRb and the oncogene ras had to be

interrupted (Weitzman, 1999). From a cell physiological perspective, Hanahan and

Weinberg have identified and described six traits that are probably common to all

cancers (Hanahan, 2000). These traits are defined as; self-sufficiency in growth

signals, insensitivity to anti-growth signals, evasion of apoptosis, a limitless

replicative potential, sustained angiogenesis and the capacity of tissue invasion and

metastasis. Self-sufficiency in growth signals can be achieved through alterations in


20

growth signalling, for example by receptor over-expression, autocrine stimulation or

activation of the ras oncogene or members of its pathway. Disruption of the pRb

pathway, for example by mutation of pRb or TGF-β or inactivation of the tumour

suppressor p53, renders cells insensitive to anti-growth signals and thus stimulates

proliferation. In addition, inactivation of p53 is one way of evading apoptosis, which

can also be accomplished by production of survival factors such as IGF-2. An

unlimited replicative potential is achieved by maintaining telomere length by

upregulation of the enzyme telomerase or by interchromosomal exchange of

sequences. Furthermore, loss of p53 or activation of ras can give rise to the capability

of inducing and sustaining angiogenesis through downregulation of the angiogenesis

inhibitor thrombospondin and release of the negative transcriptional control on the

proangiogenic factor vascular endothelial growth factor (VEGF) respectively. The

final essential characteristic of a cancer cell is the ability to invade and metastasise,

which is acquired by alterations in expression of cell-cell adhesion molecules such as

E-cadherin and integrins and by upregulation of proteases. According to this

information and from experimental evidence, it is apparent that multiple genetic hits

are necessary for tumour development. However, due to the meticulous maintenance

of genomic integrity by monitoring and repair enzymes, mutations are such rare

events that the probability of acquiring the required number of genetic alterations

during a lifetime would be diminutive unless there are some changes in genomic

stability. Genome instability can be achieved by altering the function of proteins

involved in the genomic caretaker systems, thus creating cells with higher mutability

and the so-called “mutator phenotype”. The most prominent and most studied

member of these caretaker systems is the tumour suppressor p53 (discussed below)

that plays an important role by inducing either growth arrest or apoptosis in response

to DNA damage. Thus alterations in p53 or members of its pathway are not only

involved in reaching several of the above mentioned biological endpoints necessary

for cancer development but are also a prerequisite in creating genomic instability.

Å Sivertsson

21

Cancer of the skin

Skin cancer, which is the generic term for basal cell cancer (BCC), squamous cell

cancer (SCC) and malignant melanoma, is the most common cancer in the western

world. In the U.S.A. the incidence of non-melanoma skin cancer (NMSC) i. e. BCC

and SCC is estimated to be about 700 cases per 100,000 individuals per year with a

ratio of BCC to SCC of roughly 4:1, while the incidence of melanoma is 50 cases per

100,000 per year (Miller, 1994). This reflects an increase in incidence of 3-8% per

year since the 1960´s (Green, 1992; Glass, 1989). In Sweden, the numbers are lower

with an incidence of NMSC estimated to be about 100 cases per 100,000 individuals

and of melanoma estimated to be approximately 12 cases per 100,000 per year (Nylén,

2002). The increase in incidence over the last ten years is similar to that in the U.S.A.

and is at about 3-4 % per year. A connection between skin cancer and weather-beaten

skin has been discussed since the end of the 19th century and the relation to chronic

sun-exposure was promptly suggested (Hyde, 1906). Today, ultraviolet radiation

(UVR) is well established as a carcinogen and knowledge of the underlying molecular

mechanisms is constantly and rapidly increasing.

Ultraviolet radiation

Ultraviolet radiation in the solar electromagnetic spectrum extends from the UVA

band (315-400 nm) through the UVB band (280-315 nm) down to the high-energy

UVC band (190-280 nm) and vacuum UV (below 190 nm). The most energetic and

harmful UV radiation (wavelengths below 240 nm) is absorbed by oxygen in the outer

reaches of the atmosphere, while the stratospheric ozone formed in the process

absorbs part of the UVB radiation. UVA, which comprise the major part of the total

spectral energy of solar UV, is not absorbed at all (Diffey, 1999). Ultraviolet radiation

is highly photochemically active and reacts, depending on the wavelength, directly or

indirectly with different biomolecules such as DNA and proteins. UVB is directly

absorbed by DNA and exerts a genotoxic effect mainly by dimerizing pyrimidines

giving rise to cyclobutan pyrimidine dimers (CPD) and 6-4 pyrimidine-pyrimidone

photoproducts (6-4 PP) (Tornaletti, 1996) (Figure 4). If these products are not

removed during DNA repair, these lesions will induce the UV signature mutations C-

T and CC-TT (Hauser, 1986; Brash, 1987; Drobetsky, 1987; Armstrong, 1992).

Recent experimental studies have also shown that UVB can cause deletions and that


22

UV (A/B) can cause chromosomal aberrations in mammalian cells (Horiguchi, 2001;

Emri, 2000). UVA is usually subdivided into UVA1 (340-400 nm) and UVA2 (315-

340 nm), since they differ in their effect from a biological point of view (Fitzpatrick,

1986). The shorter wavelength range of UVA2 has a similar effect as UVB, while

UVA1 is not absorbed by DNA but is oxidative in nature and damages DNA by

generating reactive oxygen species (ROS) (Young, 1998; Cadet, 1997). Endogenous

photosensitisers such as porphyrines and NADH become excited by absorbing UVA

radiation and can then react directly with DNA (type I reaction) or give rise to ROS

by reacting with oxygen (type II reaction) (De Gruijl, 2000; Kawanishi, 2001). The

DNA damage caused by both type I and type II reactions appears most frequently to

generate 8-oxo-7,8-dihydroxyguanine (8-oxo-dG), which is a miscoding lesion

inducing G-T transversions (Cheng, 1992) (Figure 4). However, type II reactions to a

lesser extent may also induce the formation of the 5-OH-dC adduct leading to C-T

mutations (Wang, 1998; Jenkins, 2001) (Figure 4). It is generally accepted that DNA

damage caused by UV radiation is responsible for the induction of skin cancer. Both

UVB and UVA radiation have been proven to be fully carcinogenic in animal studies

with UVB radiation giving rise to actinic keratosis, SCC and BCC (De Gruijl, 1983),

while UVA radiation mainly induces papillomas and SCC (Kelfkens, 1991). Induction

of malignant melanoma has not yet been correlated to any particular UVR wavelength

but studies on opossum as well as retrospective studies both on users of tanning

salons and users of sunscreen implicates UVA (Ley, 1997; Westerdahl, 1995; Wolf,

1998; Swerdlow, 1988; Westerdahl, 1994). UVA radiation is also the cause of

photoageing of the skin and affects all layers down to the dermis. UVB radiation, on

the other hand, is mainly absorbed by the epidermis with only 2-10 % reaching the

basal layer compared to 20 % for UVA (Tyrrell, 1996).

Å Sivertsson

23

Figure 4. Structure of UV-induced photoproducts (A) and the DNA lesions commonly formed after

UVA irradiation (B).

UV radiation

S

P

S

P

5-OH-dC 8-OXO-dG

A

B

Adjacent thymines

S

S

P

N

O

C N

C C

H

O

CH3H

C

N

OC N

C C

H

O

CH3H

C

6-4 photoproduct

S

S

P

N

O

C N

C C

H

O

CH3

H

C

N

OC N

C COH

CH3H

C

H

S

S

P

N

O

C N

C C

H

O

CH3H

C

N

O

C N

C C

H

OH

CH3H

C

TT-dimer


24

Normal skin

The skin including the underlying subcutis amounts to 15% of our bodyweight and is

thereby our largest organ and also the most important barrier to protect us from the

environment. Its major functions are to prevent dehydration, physical protection,

protection against chemical agents and micro-organisms, heat regulation and sensory

functions. The skin is a constantly renewing tissue that consists of an epidermis

overlying a dermal layer in conjunction with subcutaneous tissues mostly made up of

fat (Figure 5). The epidermis is subdivided into four layers, the stratum basale (basal

layer), the stratum spinosum, the stratum granulosum and the stratum corneum.

Keratinocytes are the principal cell type of the epidermis and undergo cell division,

differentiation and maturation while gradually migrating to the skin surface to be shed

resulting in a turnover time of 15-30 days for epidermis. These cells are proposed to

make up the fine mosaic of epidermal proliferative units (EPU) which consists of a

column of transit-amplifying and differentiated keratinocytes overlaying

approximately 10 basal cells, in which one epidermal stem cell is responsible for

maintenance (Clausen, 1990; Potten, 1981; Potten, 1988). An investigation of the

EPU size by looking at the x-chromosome inactivation pattern in skin revealed a

mosaic pattern with clones of 20-350 basal cells, suggesting an EPU size of less than

35 cells in diameter (Asplund, 2001). Epidermal stem cells are slow-cycling cells that

reside in the basal layer of epidermis as well as in the bulge area of the hair follicle

(Figure 5). The latter have been shown to be bipotent and are thus able to give rise not

only to the hair follicle but also to the epidermis and may therefore be the ultimate

stem cells of the epidermis/hair follicle system (Taylor, 2000; Oshima, 2001). The

progeny of basal layer stem cells is transit amplifying cells that undergo a finite

number of divisions to generate the terminally differentiating cells that move upwards

from the basal layer. In the case of hair follicular stem cells, the progeny undergoes

two independent pathways of migration and differentiation. Cells can move upwards

to be part of the maintenance of the epidermis or migrate downwards during the

growth phase of the hair follicle giving rise to the lower part of a new hair follicle

(Taylor, 2000). The basal layer also contains melanocytes producing melanin for

protection against UV radiation. Melanin is produced in melanocyte-specific

organelles (melanosomes), which are phagocytised by the keratinocytes and

accompanies them to the skin surface. Melanin exerts its protective effect by

absorbing, reflecting and scattering UVR and possibly also acts as a free radical

Å Sivertsson

25

quencher. The production of melanin is induced by UV-light and by melanocyte

stimulating hormone excreted from the pituitary gland and from keratinocytes. The

melanocyte stemcells in mouse have recently been described as being localised in the

lower permanent portion of the hair follicle (Nishimura, 2002).

Figure 5. Histological section of normal epidermis (A) and a schematic picture of the

different layers of the skin (B).

Cornified layer

Granular layer

Spinous layer

Basal layer

Epidermis

Dermis

A

Epidermis

Dermis

Subcutis

Fat

Hair follicleSebaceous gland

Sweat gland

Basal l.Spinous l.

Granular l.

Cornified layerB


26

The tumour suppressor p53

The tumour suppressor gene p53 is an 11 exon gene spanning over 20,000 basepairs

on the long arm of chromosome 17. P53 has been proposed as the guardian of the

genome and is the most frequently mutated gene in all human cancers (Lane, 1992).

P53 mutations fall into two classes, those that affect the DNA-binding domain (Class

I) and those that change the conformation of the protein (Class II) (Ory, 1994). The

latter are associated with a more severe phenotype in vitro and exert a dominant

negative effect over the wild-type protein (Ory, 1994). The gene encodes a 393 amino

acid nuclear phosphoprotein that functions as a stress and DNA damage-inducible

sequence specific transcription factor that selectively activates genes involved in

differentiation, cell cycle control, DNA repair, senescence, angiogenesis and

apoptosis (el-Deiry, 1992; Hupp, 1992; Hall, 1996; Levine, 1997; Ford, 1997). The

p53 protein is constitutively expressed in small amounts and is under normal

conditions present in a latent form with a half-life of 5-20 minutes due to rapid

degradation. In response to DNA damaging agents e.g. gamma radiation, UV

radiation, alkylating or oxidising agents (Huang, 1996; Nelson, 1994), (Siegel, 1995)

or cellular stress in the form of hypoxia, hyperthermia, nucleotide depletion, serum

starvation, massive DNA demethylation or activated oncogenes (Graeber, 1994;

Linke, 1996; Matsumoto, 1997; Canman, 1997; Jackson-Grusby, 2001) the protein is

rapidly activated. Upon activation, p53 is altered to allow for homotetramerisation

which makes the protein functionally active as a transcription factor. Depending on

the type and duration of damage or stress, genes that cause either growth arrest,

apoptosis, altered DNA repair or altered differentiation are selectively activated. In

response to low doses of DNA damage or milder forms of stress, genes involved in

growth arrest and DNA repair including the cyclin-dependent kinase inhibitor p21Waf-

1 (G1 arrest), 14-3-3 Sigma (G2 arrest), Gadd45 (repair) and PCNA (repair) are

activated to allow for repair of the damage (Kastan, 1991; McKay, 1999). Severe

DNA damage or extreme stress instead triggers genes involved in the apoptotic

pathway such as bax, PIDD, NOXA, PUMA and p53AIP (Miyashita, 1995; Polyak,

1997). Regulation of activity occurs through phosphorylation and through reduction

of cysteines in the DNA binding domain. Acetylation was earlier thought to be deeply

involved in activation, but recent data does not support this view (Espinosa, 2001;

Nakamura, 2000). Instead it is speculated that acetylation might mark a p53-activated

promoter for subsequent inactivation by recruiting deacetylases (Prives, 2001).

Å Sivertsson

27

Phosphorylation can occur on more than 18 phosphoacceptor sites and although some

are phosphorylated under normal growth conditions (Meek, 1990; Gatti, 2000) most

of them are modified in response to damage or stress (Canman, 1998; Fuchs, 1998;

Chernov, 1998; Hirao, 2000; Siliciano, 1997; Higashimoto, 2000; Chehab, 1999).

Multiple kinases, some of which are able to act on the same sites, are involved in the

phosphorylation and the type of stress factor involved determines which kinases are

active. In response to UV radiation, ATR, Chk1 and JNK are preferentially activated

(Unsal-Kacmaz, 2002; Liu, 2000; Buschmann, 2001). Phosphorylation is not only

involved in transcriptional activation but also in p53 stability. Serine 15 and serine 20,

which are phosphorylated by the kinases ATM/ATR and Chk2 respectively, are

involved in the association of p53 with one of its master regulators, mdm-2 (Shieh,

1997). P53 transcriptionally upregulates the proto-oncoprotein mdm-2 that in turn

negatively regulates p53 by targeting it for ubiquitin-mediated proteasomal

degradation, which thus results in the low amount and short half-life of the p53

protein under normal conditions. The damage-induced phosphorylation of multiple

sites, including those involved in mdm-2 binding, releases p53 from mdm-2 and

stabilises the protein (Fuchs, 1998; Shieh, 1997). Disruption of the association with

mdm-2 is further responsible for the stabilisation of the p53 protein commonly

associated with p53 mutation.

The p53 patch

Typical damage to normal skin that is chronically exposed to UVR includes erythema,

edema, hyperplasia, sunburn cells and photoageing. The acute response to UVR is a

transient accumulation of the tumour suppressor protein p53 in the skin, which can be

detected by immunostaining. The distribution of the protein is wavelength dependent

with UVA inducing dispersed p53 immunostaining limited to the basal layer, while

UVB induces dispersed immunostaining throughout the entire epidermis (Campbell,

1993; Ren, 1996) (Figure 6). This p53 accumulation, which is due to the prolonged

half-life of the wild-type conformation of the protein, can be detected after 4 hours

with levels returning to normal within 120 hours (Pontén, 1995). In addition to the

dispersed pattern of p53 immunoreactive cells seen after UVR exposure, a compact

pattern with strong immunoreactivity localised to all cell nuclei in a sharply

demarcated area is also frequently found in skin (Figure 6) (Urano, 1995; Ren, 1996;

Jonason, 1996). These so-called “p53 patches” comprise of 10-3000 cells and can


28

cover as much as 4 % (there are 3-40 patches /cm2 depending on exposure) of the

epidermis with their size and frequency increasing in a dose-dependent manner with

sun-exposure. Although, increasing age has been correlated with an increase in size of

p53 patches an age-related increase in frequency is somewhat disputed (Ren, 1996;

Jonason, 1996; Tabata, 1999). The patches consist of clones of morphologically

normal cells arising from the epidermal-dermal junction or from hair follicles with

their incidence and location suggesting a possible role in skin carcinogenesis. In

addition, 30 -70 % of these clones harbour a p53 mutation (Jonason, 1996; Pontén,

1997; Tabata, 1999). However, although they are frequently found adjacent to BCC,

CIS (carcinoma in situ) and SCC they have so-far not been found to share the same

mutation (Pontén, 1997; Ren, 1996). Despite this fact, the early onset of p53 patches

in normal mouse skin following chronic UV irradiation has been suggested as an

indicator of tumour risk (Berg, 1996; Rebel, 2001). In another recent animal study, it

was demonstrated that the clonal expansion of patches was continually driven by UV

radiation and that the clone size represented a murine EPU or a multiple thereof

(Zhang, 2001). Thus stem cell compartments seem to act as physical barriers to clonal

expansion, but colonisation can occur under the influence of constant UV irradiation.

Perhaps an explanation for this is that the p53 mutation allows the patches to escape

the UV induced apoptosis which affects cells in neighbouring compartments (Ziegler,

1994).

Figure 6. Immunoreactive keratinocytes showing the compact pattern typical of a p53

patch (A) and the dispersed pattern common in response to UV damage (B).

A

B

Å Sivertsson

29

Non-melanoma skin cancer

Basal cell cancer and SCC are keratinocyte-derived tumours that partly share

aetiology and have therefore commonly been grouped together as non-melanoma skin

cancer. However, some important differences exist. Cutaneous SCC is believed to

arise from interfollicular basal stem cells and is what can be called a “classical”

cancer that develops through precursor lesions (dysplasia also denoted actinic or solar

keratosis) with the potential to progress into cancer in situ and further into

metastasising disease. It can develop in different parts of the skin but its behaviour

may differ depending on its location. The tumour never spontaneously regresses once

developed, but in accordance with the characteristics of precursors, dysplasias

frequently do regress (Marks, 1986). Basal cell cancer on the other hand is a slowly

growing, locally invasive tumour that lacks known precursors and almost never

progresses to metastasising disease. This inability to metastasise has been explained

by the unconditional dependence of the tumour on the specific loose connective

stroma recruited by the surrounding fibroblasts (van Scott, 1961). Its cellular origin is

thought to be either interfollicular basal cells retaining basal morphology or

keratinocytes in hair follicles or sebaceous glands (Kruger, 1999; Miller, 1993). Basal

cell cancer develops only in hair-bearing skin and mostly in sun-exposed areas, with

85 % appearing in the head and neck region. The major risk factor for developing

BCC and SCC is exposure to sunlight and especially UVB radiation, but while SCC is

associated with cumulative exposure, BCC development has been linked to early-age

sunburn and intermittent, intense exposure (Armstrong, 2001; Diffey, 1979; Kricker,

1995; Franceschi, 1996; Rosso, 1996; Zanetti, 1996). The correlation with UVR is

not as strong for BCC as SCC suggesting that other factors might be involved

(Kricker, 1995; Kricker, 1995). Other agents associated with BCC and SCC include

exposure to ionising radiation, arsenicals, polyaromatic hydrocarbons and psoralen-

plus-UVA therapy (Gailani, 1997; Stern, 1996). The genetic background of BCC and

SCC is similar, with mutation of the p53 gene being an early event during tumour

development. Mutations are present in approximately 50% of BCCs and up to 90 % of

SCCs with the majority being missense mutations carrying a UV-signature (Soehnge,

1997; Brash, 1991; Brash, 1996). In typical cases of SCC, one p53 allele carries a

point mutation and the other is deleted, while point mutations commonly affect both

alleles in BCC. Except for p53, the oncogene ras and other tumour suppressors

including p14ARF, p16INK4a and the ptch gene are found mutated in SCC. The ptch


30

gene is located on chromosome 9q and is involved in the repression of genes that

drive cell growth and differentiation. The ptch protein forms part of a receptor

complex together with the product of the Smoothened gene. Upon binding of the

sonic hedgehog to this complex, ptch conformation is changed and Smoothened is

activated, resulting in upregulation of its target genes. Mutations that inactivate ptch

therefore lead to constitutive activation of Smoothened, which in turn leads to

overexpression of GLI1 and uncontrolled cell proliferation. Germline mutations in the

ptch gene are responsible for the autosomal dominant disorder denoted Gorlin

syndrome, which is characterised by developmental abnormalities and multiple early

onset BCCs (Gorlin, 1995). The ptch gene is also the most commonly altered gene in

sporadic BCC and is lost and /or mutated in at least two thirds of the tumours

(Gailani, 1996). However, the frequency of UV signature mutations in this gene is

much lower than in p53, which indicates that factors other than UV are important for

BCC development. In tumours with intact ptch, other genes involved in ptch

signalling, such as Smoothened are mutated, indicating that inactivation of this

pathway is a necessary step for tumour formation.

Malignant melanoma

Malignant melanoma arises from melanocytes and is the most fatal of skin cancers.

Tumours mostly occur on the face, ears, neck, shoulders and back and are rare on the

buttocks and the soles of the feet (Green, 1993). There is a clear correlation between

melanoma and sunlight, especially intermittent sun-exposure that often results in

sunburn. The early stages of melanoma development are thought to begin with a

lentigo (freckle) that turns into a common aquired melanocytic naevus (mole) with a

dermal component which then develops further into a naevus with an aberrant cell

pattern. A lentigo results from the accumulation of normal melanocytes at the dermal-

epidermal junction, while a mole consists of normally appearing melanocytes that

extends suprabasally and dermally. The number of moles is proportional to sun-

exposure during early childhood and a high number is associated with an elevated

frequency of dysplatic naevi. However, moles routinely regress after age 15 in males

and age 25 in females (Nicholls, 1973). The melanoma begins when the naevus

develops a subpopulation of cells with nuclear atypia, denoted a dysplastic naevus,

which is then followed by a radial growth phase, a vertical growth phase and

metastasis. The rate of progression from naevi to melanoma is rather low, although

Å Sivertsson

31

the risk of transformation of a dysplatic naevus is almost 100 times as high as for a

common naevi (Kraemer, 1983). Melanomas, especially of the superficial spreading

type, are one of few human tumours that may spontaneously regress. Total regression

occurs in about 5 % of the tumours, while partial regression is seen in 10-50 % of the

tumours depending on their thickness (Blessing, 1992). On the genetic level, it seems

that mutations in or loss of the CDKN2A (INK4a) locus and mutations in the

oncogenes H-ras and N-ras play the most important roles in the development and

progression of malignant melanoma (Herbst, 1997; Healy, 1996; Ortonne, 2002). The

INK4a locus encodes both the CDK4/6 inhibitor p16INK4a and the mdm-2 binding

protein p14ARF. Thus loss of this locus disrupts both the pRb and the p53 pathways.

This is common in melanoma cell lines but in primary tumours the rate of

homozygous deletion is only 10 % with a mutation rate of 0-25 % (Pollock, 1995;

Ruas, 1998). The ras family of proto-oncogenes contains three functional members

(N-ras, H-ras and K-ras) encoding GTP-binding G-proteins, which in their active

state stimulate extracellular growth and differentiation (Crespo, 2000). The signalling

stops when the intrinsic GTPase activity turns the protein into its inactive GDP-bound

state. Mutations that lock the protein in the active state will result in continuous

activation of downstream targets and thus continuous proliferation. Such activating

mutations preferentially occur in codons 12, 13, 61 and 63 (Barbacid, 1987) of ras

and exert their effect by reducing the intrinsic GTPase activity of the protein in

addition to making it insensitive to GTPase-activating proteins (GAPs) (Der, 1986;

Polakis, 1993; Sweet, 1984). N-ras is the ras gene most commonly mutated in

cutaneous melanoma with a reported incidence rate of between 5% and 70 %

(Barbacid, 1987; van 't Veer, 1989; Albino, 1989; Jiveskog, 1998; Ball, 1994;

Demunter, 2001). This variability in the frequency of ras mutations can be explained

by the fact that the mutation rate varies with sun-exposure as well as with the location

of the tumour (Jiveskog, 1998; van 't Veer, 1989). Recently another gene in the ras

pathway, B-raf, was reported to harbour missense mutations in 66 % of malignant

melanomas (Davies, 2002). B-raf is one of three raf genes that are just downstream of

ras and code for cytoplasmic serine/threonine kinases regulated by binding ras.


32

PRESENT INVESTIGATION

This thesis is based on five papers describing the development, evaluation and

application of techniques for detection and analysis of genetic alterations in normal

skin and in various skin tumours. Initially a method based on conventional dideoxy

sequencing was developed and evaluated for the analysis of the p53 gene in small

tissue samples down to the single cell level. The method was then used to investigate

the effects of ultraviolet light on normal skin providing new insight into the possible

events involved in normal differentiation and skin carcinogenesis. After slight

modifications of the technique it was then used to complement an x-chromosome

inactivation assay for investigation of BCC clonality. Finally a pyrosequencing-based

method was developed and evaluated for hot-spot mutation detection in the N-ras

gene of malignant melanoma.

Genetic analysis of skin and skin tumour samples.

The investigation of genetic alterations in cancer-related genes is useful for research,

prognostic and therapeutic purposes and in the future may become a routine

procedure in cancer therapy and diagnostics (Harvey, 1995; Lowe, 1994). Sensitive

and reliable detection methods are of major importance for obtaining relevant results,

but a critical issue is the quality of the starting material, which largely affects the

outcome of the analysis.

Sample handling and preparation for analysis

To obtain good quality genetic material a number of factors such as sample selection,

preservation, preparation and handling are important. The tissue must be correctly

treated to be useful for pathological and immunohistochemical analysis as well as to

prevent DNA damage or degradation.

Sample preparation

Skin tumours such as BCC and melanoma can be surgically removed whereupon the

tissue is fixed and paraffin embedded or snap-frozen. Formalin fixation followed by

paraffin embedding is the traditional method and has resulted in invaluable archival

stores of different tissue samples. This method preserves cell morphology excellently

Å Sivertsson

33

and makes pathological analysis possible decades after excision, but however does

not protect the genetic material in the tissue. Formalin fixation has long been known

to damage DNA by making protein-DNA cross-links (Chalkley, 1975) as well as

decreasing the PCR amplification efficiency (Ben-Ezra, 1991). In recent years

evidence has also arisen of artefactual mutations appearing during the amplification of

DNA originating from formalin fixed material (Williams, 1999). Alcohol-based

fixatives improve the integrity and recovery of nucleic acids, but preservation of cell

morphology is somewhat impaired (Alaibac, 1997; Giannella, 1997). When frozen

unfixed tissues are used the preservation of morphology is satisfactory for most

applications (although it is inferior to that achieved with formalin) with a high

recovery rate of nucleic acid. Therefore, in this work only freshly frozen material

from excised skin tumours and skin punch biopsies have been used.

Microdissection

The ability to select specific areas, cell populations or even individual cells from an

excised material is of great interest. During the investigation of tumour material,

measures must be taken to limit the number of normal cells present in the sample to

prevent the wild type DNA in these cells from masking possible genetic alterations in

the DNA of tumour cells. This can be achieved by cryosectioning of the biopsies

followed by microdissection of the material from the sections. The tissue sections are

histologically stained and microdissection is performed under a microscope using a

fine surgical scalpel. Using this technique it is possible to specifically dissect tumour

nests by removing the surrounding normal tissue and to remove larger strokes of

normal cells infiltrating tumour tissue. However, the dissection of more complex

tissues is beyond the capability of this technique.

Laser microdissection

In recent years, a new technique based on laser-assisted microdissection microscopy

has addressed the issues of exact and defined sampling and has made possible the

retrieval of cell populations and even the retrieval of single cells from heterogenous

material. There are two main principles for this type of microdissection, laser

microbeam microdissection/laser pressure catapulting (LMM/LPC) developed by

Schutze (Schutze, 1994; Schutze, 1998) and laser capture microdissection (LCM)

developed by Liotta and co-workers (Emmert-Buck, 1996). In LMM, a 337-nm pulsed


34

nitrogen laser is used to ablate undesired biological material surrounding the cell or

cells of interest. The laser beam allows for fine focused microdissection (focus point

< 2µm) without damage to surrounding cells. Tissue sections mounted on membranes

or ordinary glass slides can be used and the isolated cell (or cells) can be retrieved

using a needle, glass capillary or through laser catapulting into the cap of a microfuge

tube (LPC). LCM uses a heat-generating infrared laser to fuse a thin temperature-

sensitive transparent film with selected parts of the tissue on an underlying

histological section. The film is mounted on a rigid flat cap and upon removal the

selected parts of the tissue are detached. These techniques have provided new

possibilities for analysis of tissue components and have become standard methods for

retrieving defined cell populations, including single cells from heterogeneous

samples, e. g. complex tumor and normal tissues (Suarez-Quian, 1999).

Figure 7. A schematic view of LMM/LPC for dissection of a small tissue sample.

Laser pressure catapulting

Microfuge tube cap

Laser-assisted dissection

Microscope slide

Tissue sectionMembrane

Nailpolish

PALM laser

Å Sivertsson

35

p53 gene analysis

Development of a method for single cell genetic analysis (I).

The development of laser-assisted dissection techniques opened up the possibility of

performing genetic analysis on a single cell allowing light to be shed on a multitude

of biological issues. In a previous study, the heterogeneity of a BCC harbouring two

p53 mutations was studied by analysing the p53 sequence of the two affected exons in

single cells retrieved from tissue sections of the tumour (Pontén, 1997). However, the

amplification of DNA from these single cells was not satisfactory in terms of

amplification rate and exon and allele representation. Both alleles of the two exons

analysed were amplified in only a fraction of the cells (14%). There may be several

reasons for the apparently inefficient amplification of these single cells. A total lack

of amplification may reflect damage or loss of the cell during transfer from the

microscope slide to the PCR tube, while allelic dropout (ADO) may indicate chemical

or physical alterations of the template. These alterations could include DNA

degradation, loss of chromosomes during sectioning and microdissection or structural

changes in the template rendering it unavailable for amplification. An alternative

explanation may also be the preferential amplification of amplicons generated in the

first few cycles over the original template. In the first paper of this thesis (I) these

issues were addressed in the development and evaluation of a method for analysing

exons 4 - 9 of the p53 gene in single cells obtained by laser-assisted microdissection

of histological sections. The protocol is based on a previously described

multiplex/nested approach where an outer PCR simultaneously amplifying exons 4 -9

of the p53 gene is followed by an inner nested amplification specific for each exon

(Berg, 1995). The inner PCR product is then subjected to direct sequencing on

automated DNA sequencers such as ALF, ABI 377 or ABI 3700. The PCR protocol

was adjusted to increase the probability of primer annealing and extension in the first

critical amplification cycles by decreasing the reaction volume and increasing

annealing and extension times. To diminish the risk of misincorporation and

subsequent artefactual mutations Taq DNA Polymerase was replaced with Pfu DNA

polymerase, which has an error rate of 1 in 6.5 x107 base-pairs (Andre, 1997).

Evaluation of the optimised protocol using single cells picked from a fresh culture of

the HaCaT cell line showed a dramatic increase in amplification efficiency. To

determine the allele pick-up frequency, the amplified products of exons 5 and 8, each


36

containing an inherent p53 mutation affecting different alleles, were sequenced.

Sequencing was carried out using dye terminators on the automated ABI 377 DNA

sequencer. A mutation signal of 50% for both mutations proved that both alleles were

amplified, while a 100 % mutation signal for either of the mutations indicated

amplification dependent ADO. It was demonstrated that both alleles and all six exons

were amplified in 50 % of the cells, which is an increase in efficiency of more than

300%. The improvement was considered satisfactory and the protocol was applied to

single cells retrieved from immunohistochemically stained tissue sections. Again the

amplification efficiency decreased as all exons were amplified in only 1 of 16 cells (6

%). This indicated that it was the quality of the DNA and not the amplification

procedure that was the critical factor. Measures taken to diminish DNA damage by

eliminating the immunohistochemical staining procedure and the proteinase K lysis

did however not affect the amplification efficiency. The rate of successful

amplification of all exons could however be increased to 70 % when EDTA, which

has been shown to protect DNA from degradation (Yagi, 1996) was added during the

different steps of sectioning and microdissection. Finally, when EDTA was present

during the sample preparation, allele representation was determined to 50 % by

sequencing single cells retrieved from a BCC with a known heterozygous mutation.

The ADO rates reported in the literature range from 10-60% using fresh cells and are

expected to be higher using cells from tissue sections (Garvin, 1998; Findlay, 1998;

Rechitsky, 1998). Other parameters affecting ADO includes cell type, length of

amplified fragments and degree of multiplexing, with an increase in length and

number of amplified fragments resulting in an increased ADO rate. Thus we accepted

that a lower ADO rate might be difficult to obtain considering the conditions used.

With this improved method, single cells selected on the basis of

immunohistochemistry, morphology or other features that are in stages of normal

development or malignant disease can be isolated and subsequently analysed for p53

gene mutations (Figure 8).

Å Sivertsson

37

Figure 8. Schematic illustration of the different steps involved in genetic analysis of

single cells from tissue sections.

Mt Ex 4 Ex 6Ex 5 Ex 8 Ex 9Ex 7 Ex 10 Ex 11

Mt Ex 4 Ex 6Ex 5 Ex 8 Ex 9Ex 7 Ex 10 Ex 11

Mt 4 5 6 7 8 9 10 11

Single cell microdissection

Outer multiplex PCR

Inner exon specific PCR

DNA sequencing


38

Applications

Effects of UV radiation on the p53 gene in normal human epidermis (II, III)

Sun exposure has been implicated in all types of human skin cancer, as well as in

photoageing of normal skin. Ultraviolet B (UVB) radiation is a causative agent of

both BCC and SCC, while UVA radiation is implicated in malignant melanoma. In

chronically sun-exposed normal human skin, a multitude of p53 immunoreactive

clusters of morphologically normal epidermal keratinocytes can be found (Pontén,

1995; Jonason, 1996; Ren, 1996). These so called p53 clones (or p53 patches) have

been shown to originate from putative stem cell compartments and commonly have an

underlying p53 mutation, which may facilitate their expansion by allowing their

escape from UV-induced apoptosis (Brash, 1996). A single dose of UVA or UVB

radiation will further induce transient overexpression of p53 protein in normal skin

(Pontén, 2001). In addition, scattered p53 immunoreactive keratinocytes can be found

after the acute UV response should have disappeared and also in skin that has been

UV-protected. The genetic background of these cells is unclear but due to the

development of precise laser-assisted microdissection and protocols for single cell

amplification these cells can now be analysed. In the following two papers, the effect

of sun exposure and UVA1 radiation on normal epidermis was investigated.

Sun-exposure and persistent p53 mutations (II)

Natural sun-exposure leads to a transient increase in the number of p53

immunoreactive cells in normal skin, a response believed to result from

overexpression of normal p53 protein and cell-cycle arrest in response to DNA

damage. However, it has previously been shown that in skin protected from sun-

exposure for two months (thus allowing for a complete skin turn-over) scattered p53

immunoreactive keratinocytes can also be found (Berne, 1998). To further investigate

the nature of these persistent morphologically normal p53 immunoreactive

keratinocytes, we applied the p53 gene analysis strategy on single cells from skin

subjected to ordinary daily summer sun and from adjacent skin that had been totally

protected from solar radiation by blue denim fabric (sun protection factor 1700). The

skin originated from three volunteers of different age and gender and

immunohistochemistry revealed a dispersed pattern of scattered p53 immunoreactive

cells throughout the epidermis with fewer immunoreactive cells in shielded skin

Å Sivertsson

39

compared to sun-exposed skin. A total of 72 scattered single cells from the different

layers of epidermis were successfully amplified and sequenced, which revealed the

presence of one or more p53 mutations in eight keratinocytes. In cells from sun-

exposed skin, three out of four mutations were typical UV signature mutations (C-T at

a dipyrimidine site). In shielded skin, a typical UV signature was found in one out of

six mutations and the remaining were two C-A transversions, two G-A transitions and

one C-T transition at non-dipyrimidine sites. The C-T transitions and C-A

transversions represent mutation types indicative of oxidative damage and thus

possibly related to UVA exposure as discussed in the Introduction and paper III. The

major findings of this study were that p53 mutations are frequent in normal skin and

that these mutations, which were all missense or truncating mutations, were found in

both immunoreactive and non-immunoreactive keratinocytes. The mutations found in

the chronically exposed skin may represent events affecting terminally differentiated

or transit amplifying cells that are already destined to be shed as a result of the

constant epidermal turnover. In this case, possible resulting defects in cell cycle

control or in the apoptosis pathway will have no effect and the mutated cells will

disappear by the natural course of cell shedding. In the shielded skin, at least two skin

turnovers had occurred since the last exposure to UV and therefore the presumably

UV-induced mutations appeared to be persistent. This we interpret as being consistent

with the mutated cells being the offspring of originally mutated epidermal stem cells.

The consequence of these mutations (if the skin had been continuously exposed)

might be a selective growth advantage leading to the formation of small p53 clones

such as those described below.

In one of the biopsies from shielded skin, a small cluster of p53 immunoreactive cells,

apprehended as a p53 clone, was found in addition to the scattered cells. The cluster

covered an area of 0.05 mm2 involving all layers of the epidermis. From the cluster,

2 5 single cells displaying different p53 immunoreactivity and originating from

different epidermal layers were successfully sequenced. Two dominating mutations,

affecting all but one of the 18 keratinocytes harbouring mutations, were found among

the five different mutations detected. They were typical UV induced mutations

affecting the core DNA binding domain of p53 and were present in 12

immunoreactive and 5 non-immunoreactive keratinocytes from all layers of the

epidermis (Figure 9). An interesting observation was that cells in the basal layer

harboured both mutations to a greater extent compared to suprabasal and superficial


40

cells. This phenomenon is difficult to fully explain by amplification dependent ADO

and might indicate that ADO is a part of the terminal differentiation pathway. The

visual appearance of the patch in addition to the sharing of mutations indicated that

the p53 cluster had clonal origin as opposed to the scattered cells, which had different

mutations. These findings further supported the theory of a mutated stem cell

continuously giving rise to transit amplifying cells and terminally differentiated cells.

These will spread within an area corresponding to the size of an epidermal

proliferative unit (EPU) unless the mutation confers a growth advantage and /or gives

the cells the ability to overcome growth restrictions. In this case, the clone was

approximately the estimated size of an EPU, which could be interpreted as either that

the mutations were not detrimental to growth control or that the clone had regressed

after withdrawal of UV radiation (i. e. the potential selective force). In our opinion,

since both mutations were missense and affected an important region of the gene the

latter interpretation is more plausible. As mentioned earlier, p53 clones are commonly

found in chronically sun-exposed skin but the consequence of their presence is so far

unknown. Although p53 clones are frequently found adjacent to SCCs and BCCs a

genetic link has not been established. However in mice, early emergence of p53

clones has been shown following chronic UV-irradiation and recently their

appearance was shown to be an indicator of tumour risk (Berg, 1996; Rebel, 2001).

This study provides additional insights into the structure of a clandestine p53 clone

and proposes a model for allele dropout during the normal epidermal differentiation

process.

Å Sivertsson

41

Figure 9. The topography of the p53 clone found in sunshielded normal epidermis. Twomissense mutations were present in all three layers of epidermis and found in immunoreactiveas well as non-immunoreactive cells.

p53 mutations induced by UVA1 (III)

UVA irradiation has long been used in the treatment of skin diseases such as atopic

dermatitis and schleroderma, as well as for cosmetic tanning. UVA radiation contains

less energy than UVB, does not give rise to the typical UV mutations frequently

found in SCCs and BCCs and has been considered to have a minor role in

photocarcinogenesis. However, UVA has been shown to induce squamous cell cancer

in mice (Matsui, 1991), malignant melanoma in opossum (Ley, 1997) and has also

been implicated in the increased risk of malignant melanoma in tanning bed (Autier,

1991; Walter, 1990; Westerdahl, 1994) and sunscreen users (Autier, 1995;

Westerdahl, 1995; Wolf, 1998). Similar to UVB radiation, UVA exposure is known to

result in a transient increase in p53 in normal human epidermis (Burren, 1998).

Recently persistently p53 immunoreactive keratinocytes were also shown after

suberythemal doses of UVA1 (Edstrom, 2001; Seite, 2000).

p53 non-immunoreactivekeratinocytes

p53 immunoreactivekeratinocytes

241 241 wt

wt wt

281 299

Superficialcells

Suprabasalcells

241 wt

241+281281

Basalcells

wt241

241

3

241+281 241+281 241+281281

wt 281+38

wt 241+281+219

281

241+281

241+281

281


42

Using our single cell analysis strategy (extended to include exons 10 and 11) we

investigated the p53 gene status of the dispersed p53 immunoreactive keratinocytes

found in normal epidermis after physiological doses of UVA1. Three volunteers with

skin types II-III were during a period of two weeks exposed to six physiological doses

(40 J/cm2) of UVA1 on a 40 cm2 area of the buttock. Punch biopsies were taken

before and at three different time points after exposure. The first time-point (directly

after exposure) was selected in order to detect the transient p53 response, while the

second (five days after exposure) was selected in order to detect possible persistent

p53 immunoreactivity after disappearance of the transient response. The last time-

point (8 weeks after last exposure) was selected so that at least one skin turnover

would have occurred after exposure. Immunostained sections from the different

biopsies all revealed a dispersed pattern of p53 immonoreactive cells with the amount

of reactive cells significantly increased directly after exposure and five days following

exposure in comparison to the skin prior to the treatment. Eight weeks after exposure

when skin shedding had occurred, the amount of reactive cells had returned to

background levels. The increase in the number of p53 overexpressing cells directly

after exposure could be explained by the transient response, while the persistence

after five days was interpreted as a sign that damage may have occurred in p53 or

parts of its pathway. Single keratinocytes displaying p53 immunoreactivity were

microdissected from sections of unexposed skin as well as from sections taken

directly after exposure and five days following exposure. Among the exposed cells,

three mutations were found in 26,000 basepairs sequenced, while no mutations were

detected after sequencing 15,900 basepairs of cells from unexposed skin. All three

mutations were G-T transversions, a mutation type that has been related to oxidative

damage and implicated as a result of UVA exposure (see Introduction)(Cheng, 1992;

De Gruijl, 2000; Kvam, 1997; Kielbassa, 2000; Kawanishi, 2001). Two of the

mutations, one missense mutation and one affecting intron sequence were found in

cells taken five days after exposure, while another intron sequence mutation was

found among the cells taken directly after completed exposure. The finding of

mutations in intron sequence were in accordance with our view that these mutations

had not yet been selected for and thus represented random events. Affected cells

would presumably be shed at the next skin turnover, unless these cells had stem cell

properties, in which case the mutations would be transferred to daughter cells.

Å Sivertsson

43

Support for the former theory was suggested in sections from 8 weeks after exposure

where skin shedding had occurred and a reduction of immunoreactive cells were

observed. In addition, the mutation rate was calculated using data from the study,

which revealed the obtained value to be more than 10,000 times higher than the

estimated value for spontaneous mutations. This further suggested the involvement of

UVA radiation in the induction of these mutations. Furthermore, the number of

mutations per base-pair was found to be well in accordance with the number found for

oxidative damage type of mutations in paper II representing the UVA mutation load

from normal sun-exposure. We interpret the findings of this study to show that even

low-dose UVA gives rise to a multitude of mutations randomly spread across the

genome, some of which will probably affect genes and their function and therefore

the role of UVA should not be disregarded in malignant skin disease.

The clonality of BCC (IV)

In paper IV, p53 gene analysis was used to complement an X-chromosome

inactivation assay to investigate the clonal nature of BCC. BCC usually appears to

consist of multiple foci surrounded by a characteristic connective stroma, which its

growth is heavily dependent on. This multifocal appearance may indicate a polyclonal

nature but both genetic studies and the results of tumour reconstitution from serial

sections (Pontén, 1997; Imayama, 1987) has lead to the belief that BCC is of

monoclonal origin. However, the tumour’s heavy dependence on stroma suggested a

possible parallel to another tumour, phylloid tumour of the breast, which also has both

stromal and epithelial components. This tumour has been shown to consist of a

compartment of monoclonal stromal cells, which give rise to polyclonal proliferation

of epithelial cells (Noguchi, 1993). In paper IV the clonal arrangement in both

tumours and their cognate stroma was resolved by studying the x-chromosome

inactivation patterns using a microsatellite marker in the HUMARA locus of the

androgen receptor gene. The x-chromosome inactivation assay is based on the natural

process of lyonization, which leads to random inactivation of one of the x-

chromosomes in all cells of the female body. In the in vitro assay the active allele is

fragmented by enzymatic cleavage, from which the methylated inactive allele is

protected. Subsequent amplification of a polymorphic microsatellite yields product

only from the intact inactivated allele and the obtained fragment length distinguishes


44

the maternal from the paternal allele. In this study, manual or laser-assisted

microdissection was utilised to obtain pure samples from 13 tumours and their

cognate stroma. X-chromosome analysis showed that 12 of these 13 tumours were

monoclonal and that all samples from these tumours showed the same inactivation

pattern. One tumour however, displayed two distinct x-chromosome inactivation

patterns, indicating it was of polyclonal origin. Three samples from this tumour

showed non-random inactivation of one allele, while the other allele was inactivated

in a fourth sample. This case was further investigated by loss of heterozygosity

(LOH) analysis of the ptch and p53 loci and additional sequencing of the p53 gene in

microdisssected samples from serial sections (Figure 10). Loss of heterozygosity of

the p53 and ptch loci as well as mutation of the p53 gene are frequent events in skin

cancer and may thus be used as possible clonality markers. The results of these

analyses further supported the conclusions from the x-chromosome inactivation

analysis. The three samples sharing inactivation pattern displayed LOH of the same

allele and in both loci, while the sample with a deviating x-chromosome pattern was

proven unaffected by LOH. Two of the three samples affected by LOH were further

shown to harbour a truncating mutation in exon 6 of the p53 gene not detected in the

deviating sample.

Another intriguing finding in this study was an apparent correlation between x-

chromosome allelic size difference and which allele was inactivated. This correlation

was further investigated by analysing x-chromosome inactivation in eight additional

BCC cases. In total, 20 of 21 analysed BCCs showed inactivation of the longer or

shorter allele depending on the allelic size difference, while no such bias was seen in

an earlier study on normal skin. The implication of this finding is not clear.

In conclusion, our findings support the theory that BCC is a tumour of monoclonal

origin and indicates that the surrounding connective stroma has a polyclonal nature.

However, our data is inconclusive due to the finding of a single polyclonal tumour.

Å Sivertsson

45

Figure 10. A schematic picture of the tumour displaying two distinct patterns of inactivation.

Detection of N-ras hot-spot mutations in melanoma using pyrosequencing (V)

Analogous to p53 mutations which are common alterations found early in BCC,

mutations in the N-ras gene are frequently found in cutaneous malignant melanoma.

In contrast to the p53 gene where mutations are found scattered in the coding region,

mutations in N-ras in malignant melanoma have so far been identified at a few

prominent positions and most commonly at codons 12, 13 and 61 (Barbacid, 1987;

Tormanen, 1992). One of the most common screening assays for such pre-defined

positions is the sensitive, simple and low-cost SSCP assay. However, the reliability of

this technique is disputed and therefore confirmatory sequencing must be performed.

In the final paper of this thesis we investigated the feasability of using

pyrosequencing for one-step detection and identification of N-ras mutations in clinical

samples of cutaneous melanoma.

To ensure that the pyrosequencing strategy was reliable and simple several parameters

were initially optimised and evaluated. A PCR approach was developed in which

fragments containing the two mutated sites (codon 12 – 13 of exon 1 and codon 61 of

exon 2) were co-amplified in an outer multiplex PCR followed by subsequent

individual inner PCRs. This approach was used to evaluate two different methods for

XL

XS

XS

XS

: long and short allele of p53

: long and short allele of ptch, D9S280: long and short allele of ptch, D9S180

: inactivation of long or short allele of X-chrom.XL/XS

Dashed lines represent alleles affected by LOH: p53 mutation


46

obtaining template DNA from tumour samples. Direct proteinase K lysis of the

dissected samples is a rapid approach resulting in a crude DNA extract, while a pure

extract can be obtained using more expensive and time-consuming commercially

available DNA extraction kits. Equal amplification efficiencies and pyrosequencing

results were obtained irrespective of which extract was used as a template for the

amplification. For the pyrosequencing analysis, nucleotide dispensation orders were

created to be able to distinguish between the peak patterns of the six most common

mutations at codons 12 and 13, as well as between the four most common mutations

at codon 61. The detection limit of the optimised method was then determined by

analysis of dilution series of cell line samples containing any of two common codon

61 mutations and was found to be satisfactory.

Evaluation on clinical samples were made by analysis of N-ras mutations in DNA

extracts from 82 melanoma metastases using the pyrosequencing method and an

earlier developed SSCP/Sanger sequencing method. Twenty-two codon 61 mutations,

representing four different mutation types, were found among the 71 samples

successfully amplified for use in SSCP/Sanger sequencing analysis. These mutations

were also detected and correctly identified by pyrosequencing. The high sensitivity of

pyrosequencing became evident when the concentrations of some mutations were

estimated to be less than 25% according to the relative intensities of the shifted bands

in the SSCP analysis. Another four codon 61 mutations were found among the 11

samples not analysed by SSCP and the resulting mutation frequency (32 %) correlates

well with previous studies (Jiveskog, 1998; Omholt, 2002; Platz, 1994).

We conclude that pyrosequencing is a possible alternative to SSCP /Sanger

sequencing for detection of N-ras hot-spot mutations in terms of reproducibility,

sensitivity, simplicity and speed. However, a possible limitation of the method is that

only a certain number of positions can be analysed simultaneously, in contrast to

SSCP where the entire amplified fragment is scanned. Also some effort must be put

into creating nucleotide dispensation profiles to obtain maximal sensitivity and

specificity. In developing this method we have provided a simple and rapid tool for

detection and identification of N-ras hot-spot mutations that may prove useful for

large-scale screening of melanoma patients for prognostic and diagnostic purposes.

Å Sivertsson

47

Abbreviations

ADO allele dropoutAMASE apyrase-mediated allele-specific extensionASA allele specific amplificationASO allele specific oligonucleotidesBCC basal cell cancerBESS base excision sequence scanningCCM chemical cleavage of mismatchCDGE constant denaturant gel electrophoresisCFLP cleavage fragment length polymorphismCGE capillary gel electrophoresisCIS cancer in situCSGE conformation-sensitive gel electrophoresisDASH dynamic allele-specific hybridisationddF dideoxy fingerprintingddNTP dideoxynucleotidetriphosphatedNTP deoxynucleotidetriphosphateDGGE denaturing gradient gel electrophoresisDHPLC denaturing high-performance liquid chromatographyDNA deoxyribonucleic acidEPU epidermal proliferative unitFAMA fluorescence assisted mutation assayGAP GTPase activating proteinHA heteroduplex analysisLCM laser capture microdissectionLCR ligase chain reactionLMM/LPC laser microbeam microdissection/laser pressure

catapultingLOH loss of heterozygosityMALDI-TOF–MS matrix-assisted laser desorption ionisation - time-of-

flight - mass spectrometryNIRCA non-isotopic RNase cleavage assayNMSC non-melanoma skin cancerOLA oligonucleotide ligation assayPCR polymerase chain reactionPEX single nucleotide primer extensionPPi pyrophosphateSBH sequencing by hybridisationSCC squamous cell cancerSNP single nucleotide polymorphismSSCP single strand conformation polymorphism analysisTGGE temperature gradient gel electrophoresisUVR ultraviolet radiation


48

Acknowledgements

I would like to thank everyone who has contributed to this thesis and has made theselast couple of years so enjoyable.

I am especially grateful to:

Joakim Lundeberg, my supervisor, for excellent guidance during this scientificjourney, for always being so positive, supportive and enthusiastic and for taking sogood care of your students.

Mathias Uhlén for accepting me as a PhD student and for being a true visionary witha fantastic skill for attracting good people, good money and lots of attention to thisdepartment.

My collaborators, co-authors and friends in Uppsala:Figge, for inspiring and fruitful discussions on biology, skin cancer and life ingeneral, for being so enthusiastic and, last but not least, for all nice meetings andfantastic X-mas parties.

Anna A for interesting discussions on a variety of subjects (some of which involvedscience…) and for the very funny and very long e-mails with your thoughts on lifeand science. Thank you so much also for the enormous effort you have put in to getthe last paper ready.

Helena for being a great and enthusiastic collaborator, for sharing the “passion“ foranalysing sequences, life and relationships, and for fun nights at Karlsson´s. Thankyou also for letting me use your beautiful UVB damage picture!

The late Jan Pontén for being such a great and enthusiasming scientist and for makingthe solving of the mysteries of EPUs and skin cancer seem like true adventures to me.

My collaborators and co-authors in Stockholm:Desiree, for a fun and fruitful collaboration and for being very patient and optimisticduring the time our paper was just bouncing around.

Anton Platz and Johan Hansson, for a great collaboration and for sharing yourknowledge on N-ras and melanoma.

Deirdre, for kindly reading and giving valuable comments on this thesis. (Note that noblame should fall on her for errors in this section, which I wrote with no concernwhatsoever for spelling and grammar)

The “Cancer group”: Jocke, Figge, Afshin, Max, Cecilia L, Cissi, Lotta, Anna G,Helena, Anna, Cecilia for very stimulating and interesting meetings and very nicelunches.

My roommates:Maria and Jenny for excellent company and vivid scientific discussions on everythingfrom relationships and shopping to work and philosophy. I would also like to thankJenny for helping me out with pictures and formats and a whole lot of other thingsduring the writing of this thesis.

Å Sivertsson

49

Kristofer for bearing with us despite the “girl talk” and for letting us turn up theheating, making our room the only nice and warm place on this floor.

Maria, for being not only an excellent roommate, but also a very good and supportivefriend AND an extremely nice sister-in-law. Thank you also for your successful effortin the matchmaking business…

Anna G and Lotta for filling the corridors with laughter and gossip and for planningevents and activities that help to create a great atmosphere.

Tove for nice chats, mental support and for sharing the burden of general anxietywhich, I think, is a compulsory phase of thesis writing.

Niklas Malmqvist for introducing me to “magic wands” and other features ofIllustrator.

Bahram for great help with sequencing and for not complaining about having to runp53 under special conditions, changing matrices and stuff.

Karin and Jenny for help with PCR and sequencing and for adding some culture (notcell culture but REAL culture) to this place.

All collegues, former and present, at the Department of Biotechnology for being sonice and helpful and for contributing (everyone in his or her own special way) to thefantastic atmosphere of this place. I feel very privileged to have had the opportunity toget to know you and to work with you, and I would like to mention you all by namebut you are so many and it is very late….

My friends, in and out of science, for being such great persons and for all the goodtimes we have shared.

Cissi, my best friend, for recruiting me to this department and paving the road for thesingle cell research, but above all for being such a great friend and for all the goodtimes we have had.

My sister with family for always being there for me with good advice, home-madefudge or whatever will do the trick.

My parents for endless support and encouragement and for believing in me andwishing me happiness whatever I do.

Micke, the love of my life, my husband, for loving me and being all I ever wished for.

Finally, I would also like to thank Micke for his generous contribution of geneticmaterial to the most challenging project of our lives so far, which has absolutelynothing to do with any of the projects in this thesis.


50

References

Agaton C, Unneberg P, Sievertzon M, Holmberg A, Ehn M, Larsson M, Odeberg J, Uhlen M andLundeberg J. (2002) Gene expression analysis by signature pyrosequencing. Gene 289:31-39.

Ahmadian A, Gharizadeh B, Gustafsson AC, Sterky F, Nyren P, Uhlen M and Lundeberg J. (2000)Single-nucleotide polymorphism analysis by pyrosequencing. Anal Biochem 280:103-110.

Ahmadian A, Gharizadeh B, O'Meara D, Odeberg J and Lundeberg J. (2001) Genotyping by apyrase-mediated allele-specific extension. Nucleic Acids Res 29:E121.

Alaibac M, Filotico R, Giannella C, Paradiso A, Labriola A and Marzullo F. (1997) The effect offixation type on DNA extracted from paraffin-embedded tissue for PCR studies in dermatopathology.Dermatology 195:105-107.

Albino AP, Nanus DM, Mentle IR, Cordon-Cardo C, McNutt NS, Bressler J and Andreeff M. (1989)Analysis of ras oncogenes in malignant melanoma and precursor lesions: correlation of point mutationswith differentiation phenotype. Oncogene 4:1363-1374.

Almeida TA, Cabrera VM and Miranda JG. (1998) Improved detection and characterization ofmutations by primer addition in nonradioisotopic SSCP and direct PCR sequencing. Biotechniques24:220-221.

Andre P, Kim A, Khrapko K and Thilly WG. (1997) Fidelity and mutational spectrum of Pfu DNApolymerase on a human mitochondrial DNA sequence. Genome Res 7:843-852.

Andreasson H, Asp A, Alderborn A, Gyllensten U and Allen M. (2002) Mitochondrial sequenceanalysis for forensic identification using pyrosequencing technology. Biotechniques 32:124-126, 128,130-123.

Ansorge W, Sproat BS, Stegemann J and Schwager C. (1986) A non-radioactive automated method forDNA sequence determination. J Biochem Biophys Methods 13:315-323.

Ansorge W, Voss H, Wiemann S, Schwager C, Sproat B, Zimmermann J, Stegemann J, Erfle H, HewittN and Rupp T. (1992) High-throughput automated DNA sequencing facility with fluorescent labels atthe European Molecular Biology Laboratory. Electrophoresis 13:616-619.

Armstrong BK and Kricker A. (2001) The epidemiology of UV induced skin cancer. J PhotochemPhotobiol B 63:8-18.

Armstrong JD and Kunz BA. (1992) Photoreactivation implicates cyclobutane dimers as the majorpromutagenic UVB lesions in yeast. Mutat Res 268:83-94.

Asplund A, Guo Z, Hu X, Wassberg C and Ponten F. (2001) Mosaic pattern of maternal and paternalkeratinocyte clones in normal human epidermis revealed by analysis of X-chromosome inactivation. JInvest Dermatol 117:128-131.

Autier P, Dore JF, Schifflers E, Cesarini JP, Bollaerts A, Koelmel KF, Gefeller O, Liabeuf A, LejeuneF, Lienard D and et al. (1995) Melanoma and use of sunscreens: an Eortc case-control study inGermany, Belgium and France. The EORTC Melanoma Cooperative Group. Int J Cancer 61:749-755.

Autier P, Joarlette M, Lejeune F, Lienard D, Andre J and Achten G. (1991) Cutaneous malignantmelanoma and exposure to sunlamps and sunbeds: a descriptive study in Belgium. Melanoma Res1:69-74.

Ball NJ, Yohn JJ, Morelli JG, Norris DA, Golitz LE and Hoeffler JP. (1994) Ras mutations in humanmelanoma: a marker of malignant progression. J Invest Dermatol 102:285-290.

Baner J, Nilsson M, Mendel-Hartvig M and Landegren U. (1998) Signal amplification of padlockprobes by rolling circle replication. Nucleic Acids Res 26:5073-5078.

Å Sivertsson

51

Barany F. (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase.Proc Natl Acad Sci U S A 88:189-193.

Barbacid M. (1987) ras genes. Annu Rev Biochem 56:779-827.

Ben-Ezra J, Johnson DA, Rossi J, Cook N and Wu A. (1991) Effect of fixation on the amplification ofnucleic acids from paraffin- embedded material by the polymerase chain reaction. J HistochemCytochem 39:351-354.

Berg C, Hedrum A, Holmberg A, Pontén F, Uhlén M and Lundeberg J. (1995) Direct solid-phasesequence analysis of the human p53 gene by use of multiplex polymerase chain reaction and alpha-thiotriphosphate nucleotides. Clin Chem 41:1461-1466.

Berg RJ, van Kranen HJ, Rebel HG, de Vries A, van Vloten WA, Van Kreijl CF, van der Leun JC andde Gruijl FR. (1996) Early p53 alterations in mouse skin carcinogenesis by UVB radiation:immunohistochemical detection of mutant p53 protein in clusters of preneoplastic epidermal cells.Proc Natl Acad Sci U S A 93:274-278.

Berne B, Ponten J and Ponten F. (1998) Decreased p53 expression in chronically sun-exposed humanskin after topical photoprotection. Photodermatol Photoimmunol Photomed 14:148-153.

Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA andReitsma PH. (1994) Mutation in blood coagulation factor V associated with resistance to activatedprotein C. Nature 369:64-67.

Bhattacharyya A and Lilley DM. (1989) The contrasting structures of mismatched DNA sequencescontaining looped-out bases (bulges) and multiple mismatches (bubbles). Nucleic Acids Res 17:6821-6840.

Bhattacharyya A and Lilley DM. (1989) Single base mismatches in DNA. Long- and short-rangestructure probed by analysis of axis trajectory and local chemical reactivity. J Mol Biol 209:583-597.

Blaszyk H, Hartmann A, Schroeder JJ, McGovern RM, Sommer SS and Kovach JS. (1995) Rapid andefficient screening for p53 gene mutations by dideoxy fingerprinting. BioTechniques 18:256-260.

Blessing K and McLaren KM. (1992) Histological regression in primary cutaneous melanoma:recognition, prevalence and significance. Histopathology 20:315-322.

Borresen AL, Hovig E and Brogger A. (1988) Detection of base mutations in genomic DNA usingdenaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes. Mutat Res 202:77-83.

Brash DE, Rudolph JA, Simon JA, Lin A, McKenna GJ, Baden HP, Halperin AJ and Ponten J. (1991)A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc NatlAcad Sci U S A 88:10124-10128.

Brash DE, Seetharam S, Kraemer KH, Seidman MM and Bredberg A. (1987) Photoproduct frequencyis not the major determinant of UV base substitution hot spots or cold spots in human cells. Proc NatlAcad Sci U S A 84:3782-3786.

Brash DE, Ziegler A, Jonason AS, Simon JA, Kunala S and Leffell DJ. (1996) Sunlight and sunburn inhuman skin cancer: p53, apoptosis, and tumor promotion. J Investig Dermatol Symp Proc 1:136-142.

Braun A, Little DP and Koster H. (1997) Detecting CFTR gene mutations by using primer oligo baseextension and mass spectrometry. Clin Chem 43:1151-1158.

Braun A, Little DP, Reuter D, Muller-Mysok B and Koster H. (1997) Improved analysis ofmicrosatellites using mass spectrometry. Genomics 46:18-23.


52

Broude NE, Woodward K, Cavallo R, Cantor CR and Englert D. (2001) DNA microarrays with stem-loop DNA probes: preparation and applications. Nucleic Acids Res 29:E92.

Brow MA, Oldenburg MC, Lyamichev V, Heisler LM, Lyamicheva N, Hall JG, Eagan NJ, Olive DM,Smith LM, Fors L and Dahlberg JE. (1996) Differentiation of bacterial 16S rRNA genes and intergenicregions and Mycobacterium tuberculosis katG genes by structure-specific endonuclease cleavage. JClin Microbiol 34:3129-3137.

Burren R, Scaletta C, Frenk E, Panizzon RG and Applegate LA. (1998) Sunlight and carcinogenesis:expression of p53 and pyrimidine dimers in human skin following UVA I, UVA I + II and solarsimulating radiations. Int J Cancer 76:201-206.

Buschmann T, Potapova O, Bar-Shira A, Ivanov VN, Fuchs SY, Henderson S, Fried VA, Minamoto T,Alarcon-Vargas D, Pincus MR, Gaarde WA, Holbrook NJ, Shiloh Y and Ronai Z. (2001) Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptionalactivities in response to stress. Mol Cell Biol 21:2743-2754.

Cadet J, Berger M, Douki T, Morin B, Raoul S, Ravanat JL and Spinelli S. (1997) Effects of UV andvisible radiation on DNA-final base damage. Biol Chem 378:1275-1286.

Campbell C, Quinn AG, Angus B, Farr PM and Rees JL. (1993) Wavelength specific patterns of p53induction in human skin following exposure to UV radiation. Cancer Res 53:2697-2699.

Canard B and Sarfati RS. (1994) DNA polymerase fluorescent substrates with reversible 3'-tags. Gene148:1-6.

Canman CE and Kastan MB. (1997) Role of p53 in apoptosis. Adv Pharmacol 41:429-460.

Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K, Appella E, Kastan MB andSiliciano JD. (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation of p53.Science 281:1677-1679.

Chalkley R and Hunter C. (1975) Histone-histone propinquity by aldehyde fixation of chromatin. ProcNatl Acad Sci U S A 72:1304-1308.

Chehab NH, Malikzay A, Stavridi ES and Halazonetis TD. (1999) Phosphorylation of Ser-20 mediatesstabilization of human p53 in response to DNA damage. Proc Natl Acad Sci U S A 96:13777-13782.

Cheng KC, Cahill DS, Kasai H, Nishimura S and Loeb LA. (1992) 8-Hydroxyguanine, an abundantform of oxidative DNA damage, causes G----T and A----C substitutions. J Biol Chem 267:166-172.

Chernov MV, Ramana CV, Adler VV and Stark GR. (1998) Stabilization and activation of p53 areregulated independently by different phosphorylation events. Proc Natl Acad Sci U S A 95:2284-2289.

Chien A, Edgar DB and Trela JM. (1976) Deoxyribonucleic acid polymerase from the extremethermophile Thermus aquaticus. J Bacteriol 127:1550-1557.

Cho RJ, Mindrinos M, Richards DR, Sapolsky RJ, Anderson M, Drenkard E, Dewdney J, Reuber TL,Stammers M, Federspiel N, Theologis A, Yang WH, Hubbell E, Au M, Chung EY, Lashkari D,Lemieux B, Dean C, Lipshutz RJ, Ausubel FM, Davis RW and Oefner PJ. (1999) Genome-widemapping with biallelic markers in Arabidopsis thaliana. Nat Genet 23:203-207.

Clausen OP and Potten CS. (1990) Heterogeneity of keratinocytes in the epidermal basal cell layer. JCutan Pathol 17:129-143.

Cline J, Braman JC and Hogrefe HH. (1996) PCR fidelity of pfu DNA polymerase and otherthermostable DNA polymerases. Nucleic Acids Res 24:3546-3551.

Å Sivertsson

53

Cotton RG and Campbell RD. (1989) Chemical reactivity of matched cytosine and thymine bases nearmismatched and unmatched bases in a heteroduplex between DNA strands with multiple differences.Nucleic Acids Res 17:4223-4233.

Cotton RG, Rodrigues NR and Campbell RD. (1988) Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamine and osmium tetroxide and its application to the study ofmutations. Proc Natl Acad Sci U S A 85:4397-4401.

Crespo P and Leon J. (2000) Ras proteins in the control of the cell cycle and cell differentiation. CellMol Life Sci 57:1613-1636.

Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ,Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V,Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA,Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ,Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL,Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR and Futreal PA.(2002) Mutations of the BRAF gene in human cancer. Nature 417:949-954.

Davignon J, Gregg RE and Sing CF. (1988) Apolipoprotein E polymorphism and atherosclerosis.Arteriosclerosis 8:1-21.

De Gruijl F, , Methods in Enzymology, Academic Press 2000, p. 359-366.

De Gruijl FR, Van Der Meer JB and Van Der Leun JC. (1983) Dose-time dependency of tumorformation by chronic UV exposure. Photochem Photobiol 37:53-62.

Demunter A, Stas M, Degreef H, De Wolf-Peeters C and van den Oord JJ. (2001) Analysis of N- andK-ras mutations in the distinctive tumor progression phases of melanoma. J Invest Dermatol 117:1483-1489.

Der CJ, Finkel T and Cooper GM. (1986) Biological and biochemical properties of human rasH genesmutated at codon 61. Cell 44:167-176.

Diffey BJ. (1999) Photodermatology, Arnold, London.

Diffey BL, Tate TJ and Davis A. (1979) Solar dosimetry of the face: the relationship of naturalultraviolet radiation exposure to basal cell carcinoma localisation. Phys Med Biol 24:931-939.

Drmanac R, Labat I, Brukner I and Crkvenjakov R. (1989) Sequencing of megabase plus DNA byhybridization: theory of the method. Genomics 4:114-128.

Drmanac S, Kita D, Labat I, Hauser B, Schmidt C, Burczak JD and Drmanac R. (1998) Accuratesequencing by hybridization for DNA diagnostics and individual genomics. Nat Biotechnol 16:54-58.

Drobetsky EA, Grosovsky AJ and Glickman BW. (1987) The specificity of UV-induced mutations atan endogenous locus in mammalian cells. Proc Natl Acad Sci U S A 84:9103-9107.

Eckert KA and Kunkel TA. (1990) High fidelity DNA synthesis by the Thermus aquaticus DNApolymerase. Nucleic Acids Res 18:3739-3744.

Edstrom DW, Porwit A and Ros AM. (2001) Effects on human skin of repetitive ultraviolet-A1(UVA1) irradiation and visible light. Photodermatol Photoimmunol Photomed 17:66-70.

el-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW and Vogelstein B. (1992) Definition of a consensusbinding site for p53. Nat Genet 1:45-49.

Ellis LA, Taylor CF and Taylor GR. (2000) A comparison of fluorescent SSCP and denaturing HPLCfor high throughput mutation scanning. Hum Mutat 15:556-564.


54

Ellis LA, Taylor GR, Banks R and Baumberg S. (1994) MutS binding protects heteroduplex DNA fromexonuclease digestion in vitro: a simple method for detecting mutations. Nucleic Acids Res 22:2710-2711.

Ellison J, Dean M and Goldman D. (1993) Efficacy of fluorescence-based PCR-SSCP for detection ofpoint mutations. Biotechniques 15:684-691.

Ellison JS. (1996) Fluorescence-based mutation detection. Single-strand conformation polymorphismanalysis (F-SSCP). Mol Biotechnol 5:17-31.

Emmert-Buck MR, Bonner RF, Smith PD, Chuaqui RF, Zhuang Z, Goldstein SR, Weiss RA and LiottaLA. (1996) Laser capture microdissection. Science 274:998-1001.

Emri G, Wenczl E, Van Erp P, Jans J, Roza L, Horkay I and Schothorst AA. (2000) Low doses of UVBor UVA induce chromosomal aberrations in cultured human skin cells. J Invest Dermatol 115:435-440.

Espinosa JM and Emerson BM. (2001) Transcriptional regulation by p53 through intrinsicDNA/chromatin binding and site-directed cofactor recruitment. Mol Cell 8:57-69.

Evans WE and Relling MV. (1999) Pharmacogenomics: translating functional genomics into rationaltherapeutics. Science 286:487-491.

Fakhrai-Rad H, Pourmand N and Ronaghi M. (2002) Pyrosequencing: an accurate detection platformfor single nucleotide polymorphisms. Hum Mutat 19:479-485.

Fan E, Levin DB, Glickman BW and Logan DM. (1993) Limitations in the use of SSCP analysis.Mutat Res 288:85-92.

Findlay I, Matthews P and Quirke P. (1998) Multiple genetic diagnoses from single cells usingmultiplex PCR: reliability and allele dropout. Prenat Diagn 18:1413-1421.

Fire A and Xu SQ. (1995) Rolling replication of short DNA circles. Proc Natl Acad Sci U S A 92:4641-4645.

Fischer SG and Lerman LS. (1980) Separation of random fragments of DNA according to properties oftheir sequences. Proc Natl Acad Sci U S A 77:4420-4424.

Fisher SG and Lerman LS. (1983) DNA fragments differing by single base-pair substitutions areseparated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci USA80:1579-1583.

Fitzpatrick TB. (1986) Ultraviolet-induced pigmentary changes: benefits and hazards. Curr ProblDermatol 15:25-38.

Ford JM and Hanawalt PC. (1997) Expression of wild-type p53 is required for efficient global genomicnucleotide excision repair in UV-irradiated human fibroblasts. J Biol Chem 272:28073-28080.

Franceschi S, Levi F, Randimbison L and La Vecchia C. (1996) Site distribution of different types ofskin cancer: new aetiological clues. Int J Cancer 67:24-28.

Fu DJ, Tang K, Braun A, Reuter D, Darnhofer-Demar B, Little DP, O'Donnell MJ, Cantor CR andKoster H. (1998) Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass spectrometry. NatBiotechnol 16:381-384.

Fuchs SY, Adler V, Buschmann T, Wu X and Ronai Z. (1998) Mdm2 association with p53 targets itsubiquitination. Oncogene 17:2543-2547.

Fuchs SY, Adler V, Pincus MR and Ronai Z. (1998) MEKK1/JNK signaling stabilizes and activatesp53. Proc Natl Acad Sci U S A 95:10541-10546.

Å Sivertsson

55

Gailani MR and Bale AE. (1997) Developmental genes and cancer: role of patched in basal cellcarcinoma of the skin. J Natl Cancer Inst 89:1103-1109.

Gailani MR, Stahle-Backdahl M, Leffell DJ, Glynn M, Zaphiropoulos PG, Pressman C, Unden AB,Dean M, Brash DE, Bale AE and Toftgard R. (1996) The role of the human homologue of Drosophilapatched in sporadic basal cell carcinomas. Nat Genet 14:78-81.

Ganguly A. (2002) An update on conformation sensitive gel electrophoresis. Hum Mutat 19:334-342.

Ganguly A, Rock MJ and Prockop DJ. (1993) Conformation-sensitive gel electrophoresis for rapiddetection of single-base differences in double-stranded PCR products and DNA fragments: evidencefor solvent-induced bends in DNA heteroduplexes. Proc Natl Acad Sci U S A 90:10325-10329.

Garcia CA, Ahmadian A, Gharizadeh B, Lundeberg J, Ronaghi M and Nyren P. (2000) Mutationdetection by pyrosequencing: sequencing of exons 5-8 of the p53 tumor suppressor gene. Gene253:249-257.

Garvin AM, Holzgreve W and Hahn S. (1998) Highly accurate analysis of heterozygous loci bysinglecell PCR. Nucleic Acids Res 26:3468-3472.

Gatti A, Li HH, Traugh JA and Liu X. (2000) Phosphorylation of human p53 on Thr-55. Biochemistry39:9837-9842.

Gerry NP, Witowski NE, Day J, Hammer RP, Barany G and Barany F. (1999) Universal DNAmicroarray method for multiplex detection of low abundance point mutations. J Mol Biol 292:251-262.

Gharizadeh B, Kalantari M, Garcia CA, Johansson B and Nyren P. (2001) Typing of humanpapillomavirus by pyrosequencing. Lab Invest 81:673-679.

Gharizadeh B, Nordstrom T, Ahmadian A, Ronaghi M and Nyren P. (2002) Long-read pyrosequencingusing pure 2'-deoxyadenosine-5'-O'-(1- thiotriphosphate) Sp-isomer. Anal Biochem 301:82-90.

Giannella C, Zito FA, Colonna F, Paradiso A, Marzullo F, Alaibac M and Schittulli F. (1997)Comparison of formalin, ethanol, and Histochoice fixation on the PCR amplification from paraffin-embedded breast cancer tissue. Eur J Clin Chem Clin Biochem 35:633-635.

Giesendorf BA, Vet JA, Tyagi S, Mensink EJ, Trijbels FJ and Blom HJ. (1998) Molecular beacons: anew approach for semiautomated mutation analysis. Clin Chem 44:482-486.

Glass AG and Hoover RN. (1989) The emerging epidemic of melanoma and squamous cell skincancer. Jama 262:2097-2100.

Glavac D and Dean M. (1993) Optimization of the single-strand conformation polymorphism (SSCP)technique for detection of point mutations. Hum Mutat 2:404-414.

Goldrick MM. (2001) RNase cleavage-based methods for mutation/SNP detection, past and present.Hum Mutat 18:190-204.

Goldrick MM, Kimball GR, Liu Q, Martin LA, Sommer SS and Tseng JY. (1996) NIRCA: a rapidrobust method for screening for unknown point mutations. BioTechniques 21:106-112.

Gorlin RJ. (1995) Nevoid basal cell carcinoma syndrome. Dermatol Clin 13:113-125.

Graeber TG, Peterson JF, Tsai M, Monica K, Fornace AJ, Jr. and Giaccia AJ. (1994) Hypoxia inducesaccumulation of p53 protein, but activation of a G1- phase checkpoint by low-oxygen conditions isindependent of p53 status. Mol Cell Biol 14:6264-6277.

Green A. (1992) Changing patterns in incidence of non-melanoma skin cancer. Epithelial Cell Biol1:47-51.


56

Green A, MacLennan R, Youl P and Martin N. (1993) Site distribution of cutaneous melanoma inQueensland. Int J Cancer 53:232-236.

Griffin TJ, Hall JG, Prudent JR and Smith LM. (1999) Direct genetic analysis by matrix-assisted laserdesorption/ionization mass spectrometry. Proc Natl Acad Sci U S A 96:6301-6306.

Griffin TJ and Smith LM. (2000) Genetic identification by mass spectrometric analysis of single-nucleotide polymorphisms: ternary encoding of genotypes. Anal Chem 72:3298-3302.

Griffin TJ and Smith LM. (2000) Single-nucleotide polymorphism analysis by MALDI-TOF massspectrometry. Trends Biotechnol 18:77-84.

Grossman PD, Bloch W, Brinson E, Chang CC, Eggerding FA, Fung S, Iovannisci DM, Woo S, Winn-Deen ES and Iovannisci DA. (1994) High-density multiplex detection of nucleic acid sequences:oligonucleotide ligation assay and sequence-coded separation. Nucleic Acids Res 22:4527-4534.

Gustafsson AC, Guo Z, Hu X, Ahmadian A, Brodin B, Nilsson A, Ponten J, Ponten F and Lundeberg J.(2001) HPV-related cancer susceptibility and p53 codon 72 polymorphism. Acta Derm Venereol81:125-129.

Gustafsson AC, Kijas JM, Alderborn A, Uhlen M, Andersson L and Lundeberg J. (2001) Screeningand scanning of single nucleotide polymorphisms in the pig melanocortin 1 receptor gene (MC1R) bypyrosequencing. Anim Biotechnol 12:145-153.

Hacia JG, Fan JB, Ryder O, Jin L, Edgemon K, Ghandour G, Mayer RA, Sun B, Hsie L, Robbins CM,Brody LC, Wang D, Lander ES, Lipshutz R, Fodor SP and Collins FS. (1999) Determination ofancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.Nat Genet 22:164-167.

Haff LA and Smirnov IP. (1997) Multiplex genotyping of PCR products with MassTag-labeledprimers. Nucleic Acids Res 25:3749-3750.

Haff LA and Smirnov IP. (1997) Single-nucleotide polymorphism identification assays using athermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry. Genome Res7:378-388.

Hall PA, Meek D and Lane DP. (1996) p53--integrating the complexity. J Pathol 180:1-5.

Hanahan D and Weinberg RA. (2000) The hallmarks of cancer. Cell 100:57-70.

Hansen LL, Justesen J and Kruse TA. (1996) Sensitive and fast mutation detection by solid phasechemical cleavage. Hum Mutat 7:256-263.

Harvey M, Vogel H, Morris D, Bradley A, Bernstein A and Donehower LA. (1995) A mutant p53transgene accelerates tumour development in heterozygous but not nullizygous p53-deficient mice. NatGenet 9:305-311.

Hauser J, Seidman MM, Sidur K and Dixon K. (1986) Sequence specificity of point mutations inducedduring passage of a UV- irradiated shuttle vector plasmid in monkey cells. Mol Cell Biol 6:277-285.

Hawkins GA and Hoffman LM. (1997) Base excision sequence scanning. Nat Biotechnol 15:803-804.

Hawkins GA and Hoffman LM. (1999) Rapid DNA mutation identification and fingerprinting usingbase excision sequence scanning. Electrophoresis 20:1171-1176. [pii].

Hayashi K. (1991) PCR-SSCP: a simple and sensitive method for detection of mutations in thegenomic DNA. PCR Methods Appl 1:34-38.

Hayashi K and Yandell DW. (1993) How sensitive is PCR-SSCP? Hum Mutat 2:338-346.

Å Sivertsson

57

Healy E, Sikkink S and Rees JL. (1996) Infrequent mutation of p16INK4 in sporadic melanoma. JInvest Dermatol 107:318-321.

Herbst RA, Gutzmer R, Matiaske F, Mommert S, Kapp A, Weiss J, Arden KC and Cavenee WK.(1997) Further evidence for ultraviolet light induction of CDKN2 (p16INK4) mutations in sporadicmelanoma in vivo. J Invest Dermatol 108:950.

Higashimoto Y, Saito S, Tong XH, Hong A, Sakaguchi K, Appella E and Anderson CW. (2000)Human p53 is phosphorylated on serines 6 and 9 in response to DNA damage-inducing agents. J BiolChem 275:23199-23203.

Highsmith WE, Jr., Nataraj AJ, Jin Q, O'Connor JM, El-Nabi SH, Kusukawa N and Garner MM.(1999) Use of DNA toolbox for the characterization of mutation scanning methods. II: evaluation ofsingle-strand conformation polymorphism analysis. Electrophoresis 20:1195-1203. [pii].

Hiort O, Wodtke A, Struve D, Zollner A and Sinnecker GH. (1994) Detection of point mutations in theandrogen receptor gene using nonisotopic single strand conformation polymorphism analysis.German Collaborative Intersex Study Group. Hum Mol Genet 3:1163-1166.

Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H, Liu D, Elledge SJ and Mak TW.(2000) DNA damage-induced activation of p53 by the checkpoint kinase Chk2. Science 287:1824-1827.

Hongyo T, Buzard GS, Calvert RJ and Weghorst CM. (1993) 'Cold SSCP': a simple, rapid and non-radioactive method for optimized single-strand conformation polymorphism analyses. Nucleic AcidsRes 21:3637-3642.

Horiguchi M, Masumura KI, Ikehata H, Ono T, Kanke Y and Nohmi T. (2001) Molecular nature ofultraviolet B light-induced deletions in the murine epidermis. Cancer Res 61:3913-3918.

Hoshino S, Kimura A, Fukuda Y, Dohi K and Sasazuki T. (1992) Polymerase chain reaction--single-strand conformation polymorphism analysis of polymorphism in DPA1 and DPB1 genes: a simple,economical, and rapid method for histocompatibility testing. Hum Immunol 33:98-107.

Hovig, Eivind, Smith-Sorensen B, Brogger A and Borrensen AL. (1991) Constant denaturant gelelectrophoresis, a modification of denaturing gradient gel electrophoresis, in mutation detection. MutatRes 263:61.

Hsu IC, Yang Q, Kahng MW and Xu JF. (1994) Detection of DNA point mutations with DNAmismatch repair enzymes. Carcinogenesis 15:1657-1662.

Huang LC, Clarkin KC and Wahl GM. (1996) Sensitivity and selectivity of the DNA damage sensorresponsible for activating p53-dependent G1 arrest. Proc Natl Acad Sci U S A 93:4827-4832.

Huber CG, Premstaller A, Xiao W, Oberacher H, Bonn GK and Oefner PJ. (2001) Mutation detectionby capillary denaturing high-performance liquid chromatography using monolithic columns. J BiochemBiophys Methods 47:5-19.

Humphries SE, Gudnason V, Whittall R and Day IN. (1997) Single-strand conformation polymorphismanalysis with high throughput modifications, and its use in mutation detection in familialhypercholesterolemia. Clin Chem 43:427-435.

Hupp TR, Meek DW, Midgley CA and Lane DP. (1992) Regulation of the specific DNA bindingfunction of p53. Cell 71:875-886.

Hyde J. (1906) On the influence of light in the production of cancer of the skin. Am J Med Sci 131:

Hyman ED. (1988) A new method of sequencing DNA. Anal Biochem 174:423-436.


58

Iacopetta B and Hamelin R. (1998) Rapid and nonisotopic SSCP-based analysis of the BAT-26mononucleotide repeat for identification of the replication error phenotype in human cancers. HumMutat 12:355-360.

Imayama S, Yashima Y, Higuchi R and Urabe H. (1987) A new concept of basal cell epitheliomasbased on the three-dimensional growth pattern of the superficial multicentric type. Am J Pathol128:497-504.

Innis MA, Myambo KB, Gelfand DH and Brow MA. (1988) DNA sequencing with Thermus aquaticusDNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl AcadSci U S A 85:9436-9440.

Iwahana H, Fujimura M, Takahashi Y, Iwabuchi T, Yoshimoto K and Itakura M. (1996) Multiplefluorescence-based PCR-SSCP analysis using internal fluorescent labeling of PCR products.Biotechniques 21:510-514, 516-519.

Iwahana H, Yoshimoto K, Mizusawa N, Kudo E and Itakura M. (1994) Multiple fluorescence-basedPCR-SSCP analysis. Biotechniques 16:296-297, 300-295.

Jackson-Grusby L, Beard C, Possemato R, Tudor M, Fambrough D, Csankovszki G, Dausman J, LeeP, Wilson C, Lander E and Jaenisch R. (2001) Loss of genomic methylation causes p53-dependentapoptosis and epigenetic deregulation. Nat Genet 27:31-39.

Jenkins GJ, Morgan C, Baxter JN, Parry EM and Parry JM. (2001) The detection of mutations inducedin vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay.Mutat Res 498:135-144.

Jiricny J, Su SS, Wood SG and Modrich P. (1988) Mismatch-containing oligonucleotide duplexesbound by the E. coli mutS- encoded protein. Nucleic Acids Res 16:7843-7853.

Jiveskog S, Ragnarsson-Olding B, Platz A and Ringborg U. (1998) N-ras mutations are common inmelanomas from sun-exposed skin of humans but rare in mucosal membranes or unexposed skin. JInvest Dermatol 111:757-761.

Jonason AS, Kunala S, Price GJ, Restifo RJ, Spinelli HM, Persing JA, Leffell DJ, Tarone RE andBrash DE. (1996) Frequent clones of p53-mutated keratinocytes in normal human skin. Proc Natl AcadSci USA 93:14025-14029.

Jones AC, Austin J, Hansen N, Hoogendoorn B, Oefner PJ, Cheadle JP and O'Donovan MC. (1999)Optimal temperature selection for mutation detection by denaturing HPLC and comparison to single-stranded conformation polymorphism and heteroduplex analysis. Clin Chem 45:1133-1140.

Jorde LB, Watkins WS, Bamshad MJ, Dixon ME, Ricker CE, Seielstad MT and Batzer MA. (2000)The distribution of human genetic diversity: a comparison of mitochondrial, autosomal, and Y-chromosome data. Am J Hum Genet 66:979-988.

Karas M and Hillenkamp F. (1988) Laser desorption ionization of proteins with molecular massesexceeding 10,000 daltons. Anal Chem 60:2299-2301.

Kastan MB, Onyekwere O, Sidransky D, Vogelstein B and Craig RW. (1991) Participation of p53protein in the cellular response to DNA damage. Cancer Res 51:6304-6311.

Kawanishi S, Hiraku Y and Oikawa S. (2001) Mechanism of guanine-specific DNA damage byoxidative stress and its role in carcinogenesis and aging. Mutat Res 488:65-76.

Keen J, Lester D, Inglehearn C, Curtis A and Bhattacharya S. (1991) Rapid detection of single basemismatches as heteroduplexes on Hydrolink gels. Trends Genet 7:5.

Kelfkens G, de Gruijl FR and van der Leun JC. (1991) Tumorigenesis by short-wave ultraviolet A:papillomas versus squamous cell carcinomas. Carcinogenesis 12:1377-1382.

Å Sivertsson

59

Kielbassa C and Epe B. (2000) DNA damage induced by ultraviolet and visible light and itswavelength dependence. Methods Enzymol 319:436-445.

Klenow H and Henningsen I. (1970) Selective elimination of the exonuclease activity of thedeoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proc Natl Acad SciU S A 65:168-175.

Kourkine IV, Hestekin CN, Buchholz BA and Barron AE. (2002) High-throughput, high-sensitivitygenetic mutation detection by tandem single-strand conformation polymorphism /heteroduplex analysiscapillary array electrophoresis. Anal Chem 74:2565-2572.

Kozlowski P and Krzyzosiak WJ. (2001) Combined SSCP/duplex analysis by capillary electrophoresisfor more efficient mutation detection. Nucleic Acids Res 29:E71.

Kraemer KH, Greene MH, Tarone R, Elder DE, Clark WH, Jr. and Guerry Dt. (1983) Dysplastic naeviand cutaneous melanoma risk. Lancet 2:1076-1077.

Kricker A, Armstrong BK, English DR and Heenan PJ. (1995) Does intermittent sun exposure causebasal cell carcinoma? a case- control study in Western Australia. Int J Cancer 60:489-494.

Kricker A, Armstrong BK, English DR and Heenan PJ. (1995) A dose-response curve for sun exposureand basal cell carcinoma. Int J Cancer 60:482-488.

Kristensen T, Voss H, Schwager C, Stegemann J, Sproat B and Ansorge W. (1988) T7 DNApolymerase in automated dideoxy sequencing. Nucleic Acids Res 16:3487-3496.

Kruger K, Blume-Peytavi U and Orfanos CE. (1999) Basal cell carcinoma possibly originates from theouter root sheath and/or the bulge region of the vellus hair follicle. Arch Dermatol Res 291:253-259.

Kukita Y, Tahira T, Sommer SS and Hayashi K. (1997) SSCP analysis of long DNA fragments in lowpH gel. Hum Mutat 10:400-407.

Kvam E and Tyrrell RM. (1997) Induction of oxidative DNA base damage in human skin cells by UVand near visible radiation. Carcinogenesis 18:2379-2384.

Lambrinakos A, Humphrey KE, Babon JJ, Ellis TP and Cotton RG. (1999) Reactivity of potassiumpermanganate and tetraethylammonium chloride with mismatched bases and a simple mutationdetection protocol. Nucleic Acids Res 27:1866-1874.

Landegren U, Kaiser R, Sanders J and Hood L. (1988) A ligase-mediated gene detection technique.Science 241:1077-1080.

Lane DP. (1992) Cancer. p53, guardian of the genome. Nature 358:15-16.

Law JC, Facher EA and Deka A. (1996) Nonradioactive single-strand conformation polymorphismanalysis with application for mutation detection in a mixed population of cells. Anal Biochem 236:373-375.

Levine AJ. (1997) p53, the cellular gatekeeper for growth and division. Cell 88:323-331.

Ley RD. (1997) Ultraviolet radiation A-induced precursors of cutaneous melanoma in Monodelphisdomestica. Cancer Res 57:3682-3684.

Li J, Butler JM, Tan Y, Lin H, Royer S, Ohler L, Shaler TA, Hunter JM, Pollart DJ, Monforte JA andBecker CH. (1999) Single nucleotide polymorphism determination using primer extension and time-of-flight mass spectrometry. Electrophoresis 20:1258-1265. [pii].

Lindroos K, Liljedahl U, Raitio M and Syvanen AC. (2001) Minisequencing on oligonucleotidemicroarrays: comparison of immobilisation chemistries. Nucleic Acids Res 29:E69-69.


60

Linke SP, Clarkin KC, Di Leonardo A, Tsou A and Wahl GM. (1996) A reversible, p53-dependentG0/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage.Genes Dev 10:934-947.

Lishanski A, Ostrander EA and Rine J. (1994) Mutation detection by mismatch binding protein, MutS,in amplified DNA: application to the cystic fibrosis gene. Proc Natl Acad Sci U S A 91:2674-2678.

Little DP, Braun A, Darnhofer-Demar B, Frilling A, Li Y, McIver RT, Jr. and Koster H. (1997)Detection of RET proto-oncogene codon 634 mutations using mass spectrometry. J Mol Med 75:745-750.

Little DP, Braun A, O'Donnell MJ and Koster H. (1997) Mass spectrometry from miniaturized arraysfor full comparative DNA analysis. Nat Med 3:1413-1416.

Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F,Bradley A, Donehower LA and Elledge SJ. (2000) Chk1 is an essential kinase that is regulated by Atrand required for the G(2)/M DNA damage checkpoint. Genes Dev 14:1448-1459.

Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN and James CD. (1998) Denaturing high performanceliquid chromatography (DHPLC) used in the detection of germline and somatic mutations. NucleicAcids Res 26:1396-1400.

Livak KJ. (1999) Allelic discrimination using fluorogenic probes and the 5' nuclease assay. Genet Anal14:143-149.

Livak KJ, Flood SJ, Marmaro J, Giusti W and Deetz K. (1995) Oligonucleotides with fluorescent dyesat opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acidhybridization. PCR Methods Appl 4:357-362.

Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC and Ward DC. (1998) Mutation detection andsingle-molecule counting using isothermal rolling-circle amplification. Nat Genet 19:225-232.

Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DE, Housman DE and Jacks T.(1994) p53 status and the efficacy of cancer therapy in vivo. Science 266:807-810.

Lu AL and Hsu IC. (1992) Detection of single DNA base mutations with mismatch repair enzymes.Genomics 14:249-255.

Lysov Iu P, Florent'ev VL, Khorlin AA, Khrapko KR and Shik VV. (1988) [Determination of thenucleotide sequence of DNA using hybridization with oligonucleotides. A new method]. Dokl AkadNauk SSSR 303:1508-1511.

Makino R, Yazyu H, Kishimoto Y, Sekiya T and Hayashi K. (1992) F-SSCP: fluorescence-basedpolymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. PCRMethods Appl 2:10-13.

Makridakis NM and Reichardt JK. (2001) Multiplex automated primer extension analysis:simultaneous genotyping of several polymorphisms. Biotechniques 31:1374-1380.

Mao B, Margulis LA, Li B, Ibanez V, Lee H, Harvey RG and Geacintov NE. (1992) Direct synthesisand identification of benzo[a]pyrene diol epoxide- deoxyguanosine binding sites in modifiedoligodeoxynucleotides. Chem Res Toxicol 5:773-778.

Mao B, Xu J, Li B, Margulis LA, Smirnov S, Ya NQ, Courtney SH and Geacintov NE. (1995)Synthesis and characterization of covalent adducts derived from the binding of benzo[a]pyrene diolexpoxide to a -GGG- sequence in a deoxyoligonucleotide. Carcinogenesis 16:357-365.

Marks R, Foley P, Goodman G, Hage BH and Selwood TS. (1986) Spontaneous remission of solarkeratoses: the case for conservative management. Br J Dermatol 115:649-655.

Å Sivertsson

61

Martincic D and Whitlock JA. (1996) Improved detection of p53 point mutations bydideoxyfingerprinting (ddF). Oncogene 13:2039-2044.

Martincic D and Whitlock JA. (1996) Improved detection of p53 point mutations bydideoxyfingerprinting (ddF). Oncogene 13:2039-2044.

Mashal RD, Koontz J and Sklar J. (1995) Detection of mutations by cleavage of DNA heteroduplexeswith bacteriophage resolvases. Nat Genet 9:177-183.

Matsui MS and DeLeo VA. (1991) Longwave ultraviolet radiation and promotion of skin cancer.Cancer Cells 3:8-12.

Matsumoto H, Takahashi A, Wang X, Ohnishi K and Ohnishi T. (1997) Transfection of p53-knockoutmouse fibroblasts with wild-type p53 increases the thermosensitivity and stimulates apoptosis inducedby heat stress. Int J Radiat Oncol Biol Phys 39:197-203.

Maxam AM and Gilbert W. (1977) A new method for sequencing DNA. Proc Natl Acad Sci U S A74:560-564.

McKay BC, Ljungman M and Rainbow AJ. (1999) Potential roles for p53 in nucleotide excision repair.Carcinogenesis 20:1389-1396.

Meek DW, Simon S, Kikkawa U and Eckhart W. (1990) The p53 tumour suppressor protein isphosphorylated at serine 389 by casein kinase II. Embo J 9:3253-3260.

Melamede RJ, , US Patent No. 1985.

Metzker ML, Raghavachari R, Richards S, Jacutin SE, Civitello A, Burgess K and Gibbs RA. (1994)Termination of DNA synthesis by novel 3'-modified-deoxyribonucleoside 5'-triphosphates. NucleicAcids Res 22:4259-4267.

Miller DL and Weinstock MA. (1994) Nonmelanoma skin cancer in the United States: incidence. J AmAcad Dermatol 30:774-778.

Miller SJ, Sun TT and Lavker RM. (1993) Hair follicles, stem cells, and skin cancer. J Invest Dermatol100:288S-294S.

Miyashita T and Reed JC. (1995) Tumor suppressor p53 is a direct transcriptional activator of thehuman bax gene. Cell 80:293-299.

Monforte JA and Becker CH. (1997) High-throughput DNA analysis by time-of-flight massspectrometry. Nat Med 3:360-362.

Monstein H, Nikpour-Badr S and Jonasson J. (2001) Rapid molecular identification and subtyping ofHelicobacter pylori by pyrosequencing of the 16S rDNA variable V1 and V3 regions. FEMS MicrobiolLett 199:103-107.

Moore L, Godfrey T, Eng C, Smith A, Ho R and Waldman FM. (2000) Validation of fluorescent SSCPanalysis for sensitive detection of p53 mutations. Biotechniques 28:986-992.

Murray V. (1989) Improved double-stranded DNA sequencing using the linear polymerase chainreaction. Nucleic Acids Res 17:8889.

Myers RM, Fischer SG, Lerman LS and Maniatis T. (1985) Nearly all single base substitutions in DNAfragments joined to a GC- clamp can be detected by denaturing gradient gel electrophoresis. NucleicAcids Res 13:3131-3145.


62

Myers RM, Fischer SG, Maniatis T and Lerman LS. (1985) Modification of the melting properties ofduplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gelelectrophoresis. Nucleic Acids Res 13:3111-3129.

Myers RM, Larin Z and Maniatis T. (1985) Detection of single base substitutions by ribonucleasecleavage at mismatches in RNA:DNA duplexes. Science 230:1242-1246.

Nagamine CM, Chan K and Lau YF. (1989) A PCR artifact: generation of heteroduplexes. Am J HumGenet 45:337-339.

Nakamura S, Roth JA and Mukhopadhyay T. (2000) Multiple lysine mutations in the C-terminaldomain of p53 interfere with MDM2-dependent protein degradation and ubiquitination. Mol Cell Biol20:9391-9398.

Nelson WG and Kastan MB. (1994) DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol 14:1815-1823.

Nicholls EM. (1973) Development and elimination of pigmented moles, and the anatomicaldistribution of primary malignant melanoma. Cancer 32:191-195.

Nickerson DA, Kaiser R, Lappin S, Stewart J, Hood L and Landegren U. (1990) Automated DNAdiagnostics using an ELISA-based oligonucleotide ligation assay. Proc Natl Acad Sci U S A 87:8923-8927.

Nikiforov TT, Rendle RB, Goelet P, Rogers YH, Kotewicz ML, Anderson S, Trainor GL and KnappMR. (1994) Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.Nucleic Acids Res 22:4167-4175.

Nilsson M, Krejci K, Koch J, Kwiatkowski M, Gustavsson P and Landegren U. (1997) Padlock probesreveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequencesin human chromosomes 13 and 21. Nat Genet 16:252-255.

Nilsson M, Malmgren H, Samiotaki M, Kwiatkowski M, Chowdhary BP and Landegren U. (1994)Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265:2085-2088.

Nishimura EK, Jordan SA, Oshima H, Moriyama M, Jackson IJ, Barrandon Y, Miyachi Y andNishikawa S. (2002) Dominant role of the niche in melanocyte stem-cell fate determination. Nature416:854-860.

Noguchi S, Motomura K, Inaji H, Imaoka S and Koyama H. (1993) Clonal analysis of fibroadenomaand phyllodes tumor of the breast. Cancer Res 53:4071-4074.

Nordstrom T, Gharizadeh B, Pourmand N, Nyren P and Ronaghi M. (2001) Method enabling fastpartial sequencing of cDNA clones. Anal Biochem 292:266-271.

Nordstrom T, Ronaghi M, Forsberg L, de Faire U, Morgenstern R and Nyren P. (2000) Direct analysisof single-nucleotide polymorphism on double-stranded DNA by pyrosequencing. Biotechnol ApplBiochem 31:107-112.

Nylén P, Bergqvist U, Fischer T, Glansholm A, Hansson J, Surakka J, Söderberg P and Wester U.(2002) Ultraviolett strålning och hälsa - ett kunskapsunderlag. Arbete och Hälsa

Nyren P. (1987) Enzymatic method for continuous monitoring of DNA polymerase activity. AnalBiochem 167:235-238.

Nyren P, Pettersson B and Uhlen M. (1993) Solid phase DNA minisequencing by an enzymaticluminometric inorganic pyrophosphate detection assay. Anal Biochem 208:171-175.

O'Meara D, Ahmadian A, Odeberg J and Lundeberg J. (2002) SNP typing by apyrase-mediated allele-specific primer extension on DNA microarrays. Nucleic Acids Res 30:e75.

Å Sivertsson

63

O'Meara D, Wilbe K, Leitner T, Hejdeman B, Albert J and Lundeberg J. (2001) Monitoring resistanceto human immunodeficiency virus type 1 protease inhibitors by pyrosequencing. J Clin Microbiol39:464-473.

Oefner PJ. (2000) Allelic discrimination by denaturing high-performance liquid chromatography. JChromatogr B Biomed Sci Appl 739:345-355.

Okayama H, Curiel DT, Brantly ML, Holmes MD and Crystal RG. (1989) Rapid, nonradioactivedetection of mutations in the human genome by allele-specific amplification. J Lab Clin Med 114:105-113.

Omholt K, Karsberg S, Platz A, Ringborg U and Hansson J. (2002) Screening of N-ras codon 61mutations in paired primary and metastatic cutaneous melanomas: mutations occur early and persistthroughout tumor progression. Submitted

Orita M, Iwahana H, Kanazawa K, Hayashi K and Sekiya T. (1989) Detection of polymorphisms ofhuman DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad SciUSA 86:2766-2770.

Ortonne JP. (2002) Photobiology and genetics of malignant melanoma. Br J Dermatol 146 Suppl61:11-16.

Orum H, Jakobsen MH, Koch T, Vuust J and Borre MB. (1999) Detection of the factor V Leidenmutation by direct allele-specific hybridization of PCR amplicons to photoimmobilized locked nucleicacids. Clin Chem 45:1898-1905.

Ory K, Legros Y, Auguin C and Soussi T. (1994) Analysis of the most representative tumour-derivedp53 mutants reveals that changes in protein conformation are not correlated with loss of transactivationor inhibition of cell proliferation. Embo J 13:3496-3504.

Oshima H, Rochat A, Kedzia C, Kobayashi K and Barrandon Y. (2001) Morphogenesis and renewal ofhair follicles from adult multipotent stem cells. Cell 104:233-245.

Pastinen T, Kurg A, Metspalu A, Peltonen L and Syvänen AC. (1997) Minisequencing: a specific toolfor DNA analysis and diagnostics on oligonucleotide arrays. Genome Res 7:606-614.

Pastinen T, Partanen J and Syvanen AC. (1996) Multiplex, fluorescent, solid-phase minisequencing forefficient screening of DNA sequence variation. Clin Chem 42:1391-1397.

Perry DJ and Carrell RW. (1992) Hydrolink gels: a rapid and simple approach to the detection of DNAmutations in thromboembolic disease. J Clin Pathol 45:158-160.

Platz A, Ringborg U, Brahme EM and Lagerlof B. (1994) Melanoma metastases from patients withhereditary cutaneous malignant melanoma contain a high frequency of N-ras activating mutations.Melanoma Res 4:169-177.

Polakis P and McCormick F. (1993) Structural requirements for the interaction of p21ras with GAP,exchange factors, and its biological effector target. J Biol Chem 268:9157-9160.

Pollock PM, Yu F, Qiu L, Parsons PG and Hayward NK. (1995) Evidence for u.v. induction ofCDKN2 mutations in melanoma cell lines. Oncogene 11:663-668.

Polyak K, Xia Y, Zweier JL, Kinzler KW and Vogelstein B. (1997) A model for p53-inducedapoptosis. Nature 389:300-305.

Pontén F, Berg C, Ahmadian A, Ren ZP, Nistér M, Lundeberg J, Uhlén M and Pontén J. (1997)Molecular pathology in basal cell cancer with p53 as a genetic marker. Oncogene 15:1059-1067.


64

Pontén F, Berne B, Ren ZP, Nistér M and Pontén J. (1995) Ultraviolet light induces expression of p53and p21 in human skin: effect of sunscreen and constitutive p21 expression in skin appendages. JInvest Dermatol 105:402-406.

Pontén F, Lindman H, Bostrom A, Berne B and Bergh J. (2001) Induction of p53 expression in skin byradiotherapy and UV radiation: a randomized study. J Natl Cancer Inst 93:128-133.

Pontén F, Williams C, Ling G, Ahmadian A, Nistér M, Lundeberg J, Pontén J and Uhlén M. (1997)Genomic analysis of single cells from human basal cell cancer using laser-assisted capture microscopy.Mut Res 382:45-55.

Potten CS. (1981) Cell replacement in epidermis (keratopoiesis) via discrete units of proliferation. IntRev Cytol 69:271-318.

Potten CS and Morris RJ. (1988) Epithelial stem cells in vivo. J Cell Sci Suppl 10:45-62.

Prince JA, Feuk L, Howell WM, Jobs M, Emahazion T, Blennow K and Brookes AJ. (2001) Robustand accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization(DASH): design criteria and assay validation. Genome Res 11:152-162.

Prives C and Manley JL. (2001) Why is p53 acetylated? Cell 107:815-818.

Prober JM, Trainor GL, Dam RJ, Hobbs FW, Robertson CW, Zagursky RJ, Cocuzza AJ, Jensen MAand Baumeister K. (1987) A system for rapid DNA sequencing with fluorescent chain-terminatingdideoxynucleotides. Science 238:336-341.

Qi X, Bakht S, Devos KM, Gale MD and Osbourn A. (2001) L-RCA (ligation-rolling circleamplification): a general method for genotyping of single nucleotide polymorphisms (SNPs). NucleicAcids Res 29:E116.

Ramus SJ and Cotton RG. (1996) Single-tube chemical cleavage of mismatch: successive treatmentwith hydroxylamine and osmium tetroxide. Biotechniques 21:216-218, 220.

Ravnik-Glavac M, Glavac D, Chernick M, di Sant'Agnese P and Dean M. (1994) Screening for CFmutations in adult cystic fibrosis patients with a directed and optimized SSCP strategy. Hum Mutat3:231-238.

Ravnik-Glavac M, Glavac D and Dean M. (1994) Sensitivity of single-strand conformationpolymorphism and heteroduplex method for mutation detection in the cystic fibrosis gene. Hum MolGenet 3:801-807.

Rebel H, Mosnier LO, Berg RJ, Westerman-de Vries A, van Steeg H, van Kranen HJ and de Gruijl FR.(2001) Early p53-positive foci as indicators of tumor risk in ultraviolet- exposed hairless mice: kineticsof induction, effects of DNA repair deficiency, and p53 heterozygosity. Cancer Res 61:977-983.

Rechitsky S, Strom C, Verlinsky O, Amet T, Ivakhnenko V, Kukharenko V, Kuliev A and VerlinskyY. (1998) Allele dropout in polar bodies and blastomeres. J Assist Reprod Genet 15:253-257.

Reeve MA and Fuller CW. (1995) A novel thermostable polymerase for DNA sequencing. Nature376:796-797.

Ren ZP, Hedrum A, Pontén F, Nistér M, Ahmadian A, Lundeberg J, Uhlén M and Pontén J. (1996)Human epidermial cancer and accompanying precursors have identical p53 mutations different fromp53 mutations in adjacent areas of clonally expanded non-neoplastic keratinocytes. Oncogene 12:765-773.

Ren ZP, Pontén F, Nistér M and Pontén J. (1996) Two distinct p53 immunohistochemical patterns inhuman squamous-cell skin cancer, precursors and normal epidermis. Int J Cancer 69:174-179.

Å Sivertsson

65

Ririe KM, Rasmussen RP and Wittwer CT. (1997) Product differentiation by analysis of DNA meltingcurves during the polymerase chain reaction. Anal Biochem 245:154-160.

Ronaghi M. (2001) Pyrosequencing sheds light on DNA sequencing. Genome Res 11:3-11.

Ronaghi M, Karamohamed S, Pettersson B, Uhlen M and Nyren P. (1996) Real-time DNA sequencingusing detection of pyrophosphate release. Anal Biochem 242:84-89.

Ronaghi M, Uhlen M and Nyren P. (1998) A sequencing method based on real-time pyrophosphate.Science 281:363, 365.

Ross P, Hall L, Smirnov I and Haff L. (1998) High level multiplex genotyping by MALDI-TOF massspectrometry. Nat Biotechnol 16:1347-1351.

Rosso S, Zanetti R, Martinez C, Tormo MJ, Schraub S, Sancho-Garnier H, Franceschi S, Gafa L, PereaE, Navarro C, Laurent R, Schrameck C, Talamini R, Tumino R and Wechsler J. (1996) The multicentresouth European study 'Helios'. II: Different sun exposure patterns in the aetiology of basal cell andsquamous cell carcinomas of the skin. Br J Cancer 73:1447-1454.

Ruas M and Peters G. (1998) The p16INK4a/CDKN2A tumor suppressor and its relatives. BiochimBiophys Acta 1378:F115-177.

Saiki RK, Chang CA, Levenson CH, Warren TC, Boehm CD, Kazazian HH, Jr. and Erlich HA. (1988)Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA andnonradioactive allele-specific oligonucleotide probes. N Engl J Med 319:537-541.

Saiki RK, Walsh PS, Levenson CH and Erlich HA. (1989) Genetic analysis of amplified DNA withimmobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A 86:6230-6234.

Samiotaki M, Kwiatkowski M, Parik J and Landegren U. (1994) Dual-color detection of DNAsequence variants by ligase-mediated analysis. Genomics 20:238-242.

Sanger F, Nicklen S and Coulson AR. (1977) DNA sequencing with chain-terminating inhibitors. ProcNatl Acad Sci U S A 74:5463-5467.

Sarkar G, Yoon HS and Sommer SS. (1992) Dideoxy fingerprinting (ddF): a rapid and efficient screenfor the presence of mutations. Genomics 13:441-443.

Schutze K and Clement-Sengewald A. (1994) Catch and move--cut or fuse. Nature 368:667-669.

Schutze K and Lahr G. (1998) Identification of expressed genes by laser-mediated manipulation ofsingle cells. Nat Biotechnol 16:737-742.

Seite S, Moyal D, Verdier MP, Hourseau C and Fourtanier A. (2000) Accumulated p53 protein andUVA protection level of sunscreens. Photodermatol Photoimmunol Photomed 16:3-9.

Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW and Stone EM. (1993) The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions. Genomics16:325-332.

Sheffield VC, Cox DR, Lerman LS and Myers RM. (1989) Attachment of a 40-base-pair G + C-richsequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results inimproved detection of single-base changes. Proc Natl Acad Sci U S A 86:232-236.

Shieh SY, Ikeda M, Taya Y and Prives C. (1997) DNA damage-induced phosphorylation of p53alleviates inhibition by MDM2. Cell 91:325-334.

Siegel J, Fritsche M, Mai S, Brandner G and Hess RD. (1995) Enhanced p53 activity and accumulationin response to DNA damage upon DNA transfection. Oncogene 11:1363-1370.


66

Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E and Kastan MB. (1997) DNA damageinduces phosphorylation of the amino terminus of p53. Genes Dev 11:3471-3481.

Smit ML, Giesendorf BA, Vet JA, Trijbels FJ and Blom HJ. (2001) Semiautomated DNA mutationanalysis using a robotic workstation and molecular beacons. Clin Chem 47:739-744.

Smith J and Modrich P. (1996) Mutation detection with MutH, MutL, and MutS mismatch repairproteins. Proc Natl Acad Sci U S A 93:4374-4379.

Smith LM. (1991) High-speed DNA sequencing by capillary gel electrophoresis. Nature 349:812-813.

Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Dodd C, Connell CR, Heiner C, Kent SB and Hood LE.(1986) Fluorescence detection in automated DNA sequence analysis. Nature 321:674-679.

Soehnge H, Ouhtit A and Ananthaswamy ON. (1997) Mechanisms of induction of skin cancer by UVradiation. Front Biosci 2:D538-D551.

Sommer SS, Groszbach AR and Bottema CD. (1992) PCR amplification of specific alleles (PASA) is ageneral method for rapidly detecting known single-base changes. BioTechniques 12:82-87.

Stern RS and Nichols KT. (1996) Therapy with orally administered methoxsalen and ultraviolet Aradiation during childhood increases the risk of basal cell carcinoma. The PUVA Follow-up Study. JPediatr 129:915-917.

Suarez-Quian CA, Goldstein SR, Pohida T, Smith PD, Peterson JI, Wellner E, Ghany M and BonnerRF. (1999) Laser capture microdissection of single cells from complex tissues. Biotechniques 26:328-335.

Sun X, Ding H, Hung K and Guo B. (2000) A new MALDI-TOF based mini-sequencing assay forgenotyping of SNPS. Nucleic Acids Res 28:E68.

Sweet RW, Yokoyama S, Kamata T, Feramisco JR, Rosenberg M and Gross M. (1984) The product ofras is a GTPase and the T24 oncogenic mutant is deficient in this activity. Nature 311:273-275.

Swerdlow AJ, English JS, MacKie RM, O'Doherty CJ, Hunter JA, Clark J and Hole DJ. (1988)Fluorescent lights, ultraviolet lamps, and risk of cutaneous melanoma. Bmj 297:647-650.

Syvänen AC, K. A-S, Harju L, Kontula K and Söderlund H. (1990) A primer-guided nucleotideincorporation assay in the genotyping of apolipoprotein E. Genomics 8:684-692.

Tabata H, Nagano T, Ray AJ, Flanagan N, Birch-MacHin MA and Rees JL. (1999) Low frequency ofgenetic change in p53 immunopositive clones in human epidermis. J Invest Dermatol 113:972-976.

Tabor S and Richardson CC. (1987) DNA sequence analysis with a modified bacteriophage T7 DNApolymerase. Proc Natl Acad Sci U S A 84:4767-4771.

Tabor S and Richardson CC. (1989) Effect of manganese ions on the incorporation ofdideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I.Proc Natl Acad Sci U S A 86:4076-4080.

Tabor S and Richardson CC. (1995) A single residue in DNA polymerases of the Escherichia coliDNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides.Proc Natl Acad Sci U S A 92:6339-6343.

Tang K, Fu DJ, Julien D, Braun A, Cantor CR and Koster H. (1999) Chip-based genotyping by massspectrometry. Proc Natl Acad Sci U S A 96:10016-10020.

Taranenko NI, Allman SL, Golovlev VV, Taranenko NV, Isola NR and Chen CH. (1998) SequencingDNA using mass spectrometry for ladder detection. Nucleic Acids Res 26:2488-2490.

Å Sivertsson

67

Taylor G, Lehrer MS, Jensen PJ, Sun TT and Lavker RM. (2000) Involvement of follicular stem cellsin forming not only the follicle but also the epidermis. Cell 102:451-461.

Tormanen VT and Pfeifer GP. (1992) Mapping of UV photoproducts within ras proto-oncogenes inUV-irradiated cells: correlation with mutations in human skin cancer. Oncogene 7:1729-1736.

Tornaletti S and Pfeifer GP. (1996) UV damage and repair mechanisms in mammalian cells. Bioessays18:221-228.

Tully G, Sullivan KM, Nixon P, Stones RE and Gill P. (1996) Rapid detection of mitochondrialsequence polymorphisms using multiplex solid-phase fluorescent minisequencing. Genomics 34:107-113.

Tyagi S and Kramer FR. (1996) Molecular beacons: probes that flourescens upon hybridization. NatureBiotechnology 14:303-308.

Tyrrell RM. (1996) Activation of mammalian gene expression by the UV component of sunlight- -frommodels to reality. Bioessays 18:139-148.

Unsal-Kacmaz K, Makhov AM, Griffith JD and Sancar A. (2002) Preferential binding of ATR proteinto UV-damaged DNA. Proc Natl Acad Sci U S A 99:6673-6678.

Urano Y, Asano T, Yoshimoto K, Iwahana H, Kubo Y, Kato S, Sasaki S, Takeuchi N, Uchida N andNakanishi H. (1995) Frequent p53 accumulation in the chronically sun-exposed epidermis and clonalexpansion of p53 mutant cells in the epidermis adjacent to basal cell carcinoma. J Invest Dermatol104:928-932.

van 't Veer LJ, Burgering BM, Versteeg R, Boot AJ, Ruiter DJ, Osanto S, Schrier PI and Bos JL.(1989) N-ras mutations in human cutaneous melanoma from sun-exposed body sites. Mol Cell Biol9:3114-3116.

van Scott EJ and Reinertson RP. (1961) the modulating influence of stromal environment on epithelialcells studied in human autotransplants. Journal of Investigative Dermatology 36:109-117.

Verpy E, Biasotto M, Meo T and Tosi M. (1994) Efficient detection of point mutations on color-codedstrands of target DNA. Proc Natl Acad Sci U S A 91:1873-1877.

Vet JA, Majithia AR, Marras SA, Tyagi S, Dube S, Poiesz BJ and Kramer FR. (1999) Multiplexdetection of four pathogenic retroviruses using molecular beacons. Proc Natl Acad Sci U S A 96:6394-6399.

Vidal-Puig A and Moller DE. (1994) Comparative sensitivity of alternative single-strand conformationpolymorphism (SSCP) methods. Biotechniques 17:490-492, 494, 496.

Vogelstein B and Kinzler KW. (1993) The multistep nature of cancer. Trends Genet 9:138-141.

Wagner T, Stoppa-Lyonnet D, Fleischmann E, Muhr D, Pages S, Sandberg T, Caux V, Moeslinger R,Langbauer G, Borg A and Oefner P. (1999) Denaturing high-performance liquid chromatographydetects reliably BRCA1 and BRCA2 mutations. Genomics 62:369-376.

Wallace RB, Shaffer J, Murphy RF, Bonner J, Hirose T and Itakura K. (1979) Hybridization ofsynthetic oligodeoxyribonucleotides to phi chi 174 DNA: the effect of single base pair mismatch.Nucleic Acids Res 6:3543-3557.

Walter SD, Marrett LD, From L, Hertzman C, Shannon HS and Roy P. (1990) The association ofcutaneous malignant melanoma with the use of sunbeds and sunlamps. Am J Epidemiol 131:232-243.

Wang D, Kreutzer DA and Essigmann JM. (1998) Mutagenicity and repair of oxidative DNA damage:insights from studies using defined lesions. Mutat Res 400:99-115.


68

Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E,Spencer J, Kruglyak L, Stein L, Hsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, MorrisMS, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson TJ, Lander ES and et al. (1998)Large-scale identification, mapping, and genotyping of single- nucleotide polymorphisms in the humangenome. Science 280:1077-1082.

Wartell RM, Hosseini SH and Moran CPJ. (1990) Detecting base pair substitutions in DNA fragmentsby temperature-gradient gel electrophoresis. Nucleic Acids Res 18:2699-2705.

Wasson J, Skolnick G, Love-Gregory L and Permutt MA. (2002) Assessing allele frequencies of singlenucleotide polymorphisms in DNA pools by pyrosequencing technology. Biotechniques 32:1144-1146,1148, 1150 passim.

Weghorst CM and Buzard GS. (1993) Enhanced single-strand conformation polymorphism (SSCP)detection of point mutations utilizing methylmercury hydroxide. Biotechniques 15:396-398, 400.

Weitzman JB and Yaniv M. (1999) Rebuilding the road to cancer. Nature 400:401-402.

Westerdahl J, Olsson H, Masback A, Ingvar C and Jonsson N. (1995) Is the use of sunscreens a riskfactor for malignant melanoma? Melanoma Res 5:59-65.

Westerdahl J, Olsson H, Masback A, Ingvar C, Jonsson N, Brandt L, Jonsson PE and Moller T. (1994)Use of sunbeds or sunlamps and malignant melanoma in southern Sweden. Am J Epidemiol 140:691-699.

Wilkins RJ. (1985) Site-specific analysis of drug interactions and damage in DNA using sequencingtechniques. Anal Biochem 147:267-279.

Williams C, Ponten F, Moberg C, Soderkvist P, Uhlen M, Ponten J, Sitbon G and Lundeberg J. (1999)A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am JPathol 155:1467-1471.

Wolf P, Quehenberger F, Mullegger R, Stranz B and Kerl H. (1998) Phenotypic markers, sunlight-related factors and sunscreen use in patients with cutaneous melanoma: an Austrian case-control study.Melanoma Res 8:370-378.

Wu DY, Ugozzoli L, Pal BK and Wallace RB. (1989) Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A 86:2757-2760.

Wu KJ, Shaler TA and Becker CH. (1994) Time-of-flight mass spectrometry of underivatized single-stranded DNA oligomers by matrix-assisted laser desorption. Anal Chem 66:1637-1645.

Xiao W, Stern D, Jain M, Huber CG and Oefner PJ. (2001) Multiplex capillary denaturing high-performance liquid chromatography with laser-induced fluorescence detection. Biotechniques 30:1332-1338.

Xie T, Ho SL and Ma OC. (1997) High resolution single strand conformation polymorphism analysisusing formamide and ethidium bromide staining. Mol Pathol 50:276-278.

Yagi N, Satonaka K, Horio M, Shimogaki H, Tokuda Y and Maeda S. (1996) The role of DNase andEDTA on DNA degradation in formaldehyde fixed tissues. Biotech Histochem 71:123-129.

Youil R, Kemper B and Cotton RG. (1996) Detection of 81 of 81 known mouse beta-globin promotermutations with T4 endonuclease VII--the EMC method. Genomics 32:431-435.

Youil R, Kemper BW and Cotton RG. (1995) Screening for mutations by enzyme mismatch cleavagewith T4 endonuclease VII. Proc Natl Acad Sci U S A 92:87-91.

Å Sivertsson

69

Young AR, Potten CS, Nikaido O, Parsons PG, Boenders J, Ramsden JM and Chadwick CA. (1998)Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels ofthymine dimers. J Invest Dermatol 111:936-940.

Zanetti R, Rosso S, Martinez C, Navarro C, Schraub S, Sancho-Garnier H, Franceschi S, Gafa L, PereaE, Tormo MJ, Laurent R, Schrameck C, Cristofolini M, Tumino R and Wechsler J. (1996) Themulticentre south European study 'Helios'. I: Skin characteristics and sunburns in basal cell andsquamous cell carcinomas of the skin. Br J Cancer 73:1440-1446.

Zhang W, Remenyik E, Zelterman D, Brash DE and Wikonkal NM. (2001) Escaping the stem cellcompartment: sustained UVB exposure allows p53- mutant keratinocytes to colonize adjacentepidermal proliferating units without incurring additional mutations. Proc Natl Acad Sci U S A98:13948-13953.

Ziegler A, Jonason AS, Leffell DJ, Simon JA, Sharma HW, Kimmelman J, Remington L, Jacks T andBrash DE. (1994) Sunburn and p53 in the onset of skin cancer. Nature 372:773-776.

Documents

Detection and Analysis of Genetic Alterations