Identification and cloning of amplified DNA sequencesby a modified-in-gel renaturation technique: Isolationof an amplified DNA sequence from a human sarcoma.

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Identification and cloning of amplified DNA sequences by a modified in gel renaturation technique: Isolation of an amplified DNA sequence from a human sarcoma

Coccia, Marco Anthony, Ph.D.

The University of Arizona, 1991

Copyright ©1991 by Coccia, Marco Anthony. All rights reserved.

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IDENTIFICATION AND CLONING OF AMPLIFIED DNA SEQUENCES BY A

MODIFIED IN GEL RENATURATION TECHNIQUE: ISOLATION OF AN

AMPLIFIED DNA SEQUENCE FROM A HUMAN SARCOMA

by

Marco Anthony Coccia

Copyright © Marco Anthony Coccia 1991

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY

In Partial Fulfillment of the Requirements

For the Degree of

DOCI'OR OF PHILOSOPHY

In the Graduate College of

THE UNIVERSITY OF ARIWNA

1991

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Final Examination Committee, we certify that we have read

the dissertation prepared by __ ~~~3f~c~o~A~n~ili~o~n~y~C==oc~c~i=a ______________________ __

enti tIed IDENTIFICATION AND CLONING OF AMPLIFIED DNA SEQUENCES BY

A ~ODIFIED IN GEL RENATURATION TECHNIQUE: ISOLATION OF

AMPLIFIED DNA SEQUENCES FRO~ A HUMAN SARCOMA

and recommend that it be accepted as fulfilling the dissertation requirement

for the Degree of Doctor of Philosophy

3115191 Date

3/15/91 Date

3/15/91 Date

3/15/91 Date

3/15/91

Anne Cress Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

3/15/91 Date

2

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made availabe to bor­rowers under the rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or part may be granted by the copyright holder.

3

ACKNOWLEDGEMENT

There are several individuals to whom I am eternally grateful for their help in the completion of this dissertation. First and foremost I am grateful to my advisor Dr. Jeff Trent for giving me the opportunity to pursue my goal to obtain a Ph.D. in molecular biology and for sticking by me when success was not assured. I am equally grateful to Dr. Paul Meltzer, the person who was instrumental in my development as a molecular biologist. My gratitude is also extended to all of my committee members for there advice and guidance. I am especially thankful to committee member Dr. Roger Miesfeld for allowing me to use his laboratory to complete my experiments following the relocation of Dr. Trent and Dr. Meltzer to the University of Michigan.

4

5

TABLE OF CONTENTS

CHAPTER PAGE

LIST OF ILLUSTRATIONS ........................................................... 9

LIST OF TABLES ................................................... , ................... 10

ABSTRACf .............................................................................. 11

I. INTRODUCflON ....................................................................... 12

OVERVIEW ..................................................................... 12

GENE AMPLIFICATION IN CANCER CELLS ........................... 13

MOLECULAR BIOLOGY OF MAMMALIAN GENE AMPLIFICATION ..................................................... 16

REPLICATION-DRIVEN AMPLIFICATION ..................... 19

Onion Skin Model ............................................. 19

Extrachromosomal Double Rolling Circle ModeL ........ 20

Chromosomal-Spiral Model. ................................. 20

SEGREGATION-DRIVEN AMPLIFICATION ................... 21

Deletion-Plus Episome Model. .............................. 21

Unequal Sister Chromatid Exchange Model ............... 21

II. MODIFICATION OF IN GEL RENATURATION .................................. 23

INTRODUCflON .............................................................. 23

RENA TURA TION KINETICS ...................................... 23

IN GEL RENA TURA TION .......................................... 24

RESULTS ........................................................................ 26

DISCUSSION .............................. ; .................................... 29

MATERIALS AND METHODS .............................................. 31

6

Table of Contents, Continued.

CELL CUL WRE ...................................................... 31

HIGH MOLECULAR WEIGHT DNA EXTRACTION .......... 32

IN GEL RENATURATION .......................................... 32

MODIFIED IN GEL RENATURATION ........................... 33

PROBES ................................................................ 34

PROBE LABELLING ................................................. 34

SOUTHERN BLOT HYBRIDIZATION AND AUTORADIOGRAPHY ..................................... 34

III. IN GEL RENATURATION SCREENING OF HUMAN TUMORS AND TUMOR CULTURES ................................................... 35

INTRODUCTION .............................................................. 35

RESULTS ........................................................................ 36

HUMAN TUMOR CULTURES ..................................... 36

ADULT SARCOMAS ................................................. 36

DISCUSSION ................................................................... 36

HUMAN TUMOR CULTURES ..................................... 36

ADULT SARCOMAS ................................................. 42

MATERIALS AND ME11IODS .............................................. 43

CELL CULTURES AND TUMOR SAMPLES .................... 43

MODIFIED IN GEL RENATURATION ........................... 43

PROBES ................................................................ 43

PROBE LABELLING ................................................. 43

SOUTHERN BLOT HYBRIDIZATION AND AUTORADIOGRAPHY ..................................... 43

IV. CLONING OF AMPLIFIED DNA SEQUENCES FROM MFH ST-23987 AND SARCOMA ST-23493 ............................... 44

INTRODUCTION .............................................................. 44

7

Table of Contents, Continued.

RESULTS ........................................................................ 45

IN GEL RENATURATION GENERATED CLONES ............ 45

SOUTIlERN BLOT ANALYSIS OF ADDmONAL SARCOMAS AND HUMAN CULTURES ............... 47

SARCOMA ST-23493 BACTERIOPHAGE LAMBDA GENOMIC CLONES ......................................... 47

ST-23493 Genomic Library Screening ..................... 47

Plasmid Subcloning ........................................... 54

Southern Blot Analysis of Amplified Clones .............. 54

Northern Blot Analysis of Amplified Clones .............. 56

MATERIALS AND ME11IODS .............................................. 59

IN GEL RENATURATION CLONING OF AMPLIFIED FRAGMENTS ................................ 59

PCR AMPLIFICATION OF IN GEL RENATURATION GENERATED CLONES ..................................... 6()

PROBE LABELLING ................................................. 61

SOUTIlERN BLOT ANALYSIS .................................... 61

BACTERIOPHAGE LAMBDA GENOMIC LffiRARY CONSTRUCTION AND SCREENING ................... 62

Preparation of ST-23493 Insert DNA ....................... 62

Ligation, Packaging, and Titering ........................... 62

Plating and Transferring the Bacteriophage Lambda Library ....................................... 63

Screening the Bacteriophage Lambda Library ............. 63

Bacteriophage Lambda DNA Extraction .................... 63

PLASMID SUB CLONING ........................................... 64

NORTIIERN BLOT ANALySIS .................................... 64

RNA Preparation .............................................. 64

8

Table of Contents, Continued.

Northern Blots ................................................. 64

Probe Preassociation .......................................... 65

Hybridization and Autoradiography ......................... 65

V. SUMMARy ...................................................................... 66

LrrERA TURE CrrED ......................................................... 70

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

9

LIST OF ILLUSTRATIONS

PAGE

Renaturation Kinetics of Total Human Nuclear DNA ....................... 25

Band Pattern of High Copy Fragments in HindIII Digested Normal Human Placenta DNA when Subjected to In Gel Renaturation .................................................... 27

Results of In Gel Renaturation Analysis of SCLC H69 .................... 28

Detection of Gene Amplification in Human Tumor Cell Lines by Modified In Gel Renaturation ......................................... 30

Modified In Gel Renaturation Analysis of SCLC UACC735 .............. 38

Southern Blot Analysis of UACC735 ........................................ 39

Modified In Gel Renaturation Analysis of MFH ST-23987 ................ 40

Hybridization Pattern of pMAC895 ........................................... 46

Panel of Amplified Clones Isolated from ST-23987 DNA by In Gel Renaturation ............................................................. 48

DNA Sequence Data for Clones pMAC30 and pMAC895 ................. 49

Panel of Sarcomas Positive for Amplification of pMAC30 ................ 52

Restriction Maps of the Three Unique Bacteriophage Clones Isolated from ST-23493 DNA .................................................. 53

Southern Blot Analysis of Plasmid Subclones p6.0, p5.2, and pIO.5 ................................................................ 55

Northern Blot Analysis with Subclone p5.2 ................................. 57

Table 1.

Table 2.

Table 3.

Table 4.

10

LIST OF TABLES

PAGE

Summary of In Gel Renaturation Analysis of Human Tumor Cultures .................................................................. 37

Panel of Oncogene Probes Used to Analyze Southern Blots of ST-23987 DNA ..................................................... 41

Southern Blot Analysis of Human Tumor Cultures ......................... 50

Southern Blot Analysis of Direct Sarcoma Tissue Samples ................ 51

11

ABSTRACT

A modified in gel renaturation technique has been developed which detects DNA

sequence amplifications of ~10-fold without prior identification of t~e target gene. In order

to determine the sensitivity of detection and applicability of modified in gel renaturation,

primary human tumor cultures and drug resistant human cell lines with identified cellular

gene amplifications were assayed. The modified in gel renaturation technique was then

used to screen 71 human tumor cell lines and direct tumor biopsies for amplified

sequences. Particular emphasis was placed on screening cell lines derived from human

melanoma and direct tumor samples from adult sarcomas. Four of 55 cell lines tested had

amplification ~1O-fold of previously known cellular oncogenes. None of the 25 melanoma

short term cultures tested positive by this assay. One of 16 adult sarcoma samples (a

malignant fibrous histiocytoma (MPH» tested positive for DNA sequence amplification

>1O-fold. The amplified DNA present in this MPH, (termed ST-23987), was not identified

by Southern blot analysis with a panel of oncogene probes previously reported as amplified

in other human malignancies. In order to further characterize the amplified DNA in ST-

23987, DNA was subjected to in gel renaturation cloning procedures followed by PCR

modified cloning. Of 55 clones isolated and analyzed, four cloned inserts (termed

pMAC895, pMACI5, pMAC20, and pMAC30) were amplified between 1O-to-15-fold in

ST-23987DNA. Several other adult sarcomas were probed with these four clones and

three other instances of amplification were identified by Southern blot analysis with probes

generated from pMAC15 and pMAC30 (found in tumors ST-24609, 870141 and ST-

23493). A bacteriophage lambda genomic library was constructed from DNA isolated from

a ST-23493. Four clones representing -34 kb of the amplified domain (designated 493.1,

493.2,493.4, and 493.5) were isolated using clones pMAC15 and pMAC30 as probes.

Plasmid subclones generated from the EcoRI fragments of the amplified bacteriophage

lambda clones (termed p1O.5, p6.0, p5.2, p5.0, p4.7, p2.8, and pO.5) were used as

probes against Northern blots of sarcoma mRNA in an attempt to identify transcribed

sequences within the cloned regions of the amplification unit. The hypothesized target gene

carried within the amplification unit will likely be important to the progression, diagnosis

and treatment of adult sarcoma, irrespective of the target gene identity.

CHAPTER I

INTRODUCTION

OVERVIEW

The primary focus of this dissertation was the cloning of an amplified DNA

sequence directly from human sarcoma tissue. The experimental design developed to

accomplish this goal will be divisible into three Specific Aims:

1.) Modification of an in gel renaturation procedure in order to improve and

simplify the detection sensitivity and specificity.

12

2.) Application of the modified in gel renaturation technique to screen human tumor

cultures and direct adult sarcoma tissue DNAs for DNA sequence amplifications of

>lO-fold.

3.) Cloning of an amplified DNA sequence from MFH ST-23987 and

leiomyosarcoma ST-23493 DNA using an in gel renaturation cloning procedure, a

PCR modified in gel renaturation cloning procedure, and ultimately, conventional

bacteriophage lambda cloning.

Identification of amplified DNA sequences in human tumor cells by in gel

renaturation is of significant interest. In gel renaturation is a new method to detect and to

directly clone amplified DNA sequences which can identify biologically important genes

independently of the target gene DNA sequence or biological function (1-3,4,5). Based

upon the study of other amplified genes (see: Alitalo et al. (1986) for review), the target

gene of an amplified domain may be important in some aspect of the genesis or progression

of the tumor type in which it is identified (6).

This study describes the results of extensive in gel renaturation analysis of human

tumor cultures and direct tumor tissue. Results indicate the existence of a recurring DNA

sequence amplification in adult sarcomas. Amplified clones were isolated from adult

sarcoma tissue by in gel renaturation cloning procedures which employed a PCR

amplification step. These clones were used as probes against a bacteriophage lambda

genomic library which was constructed directly from tissue (leiomyosarcoma ST-23493) in

which these clones are amplified. Approximately 34 kb of the amplification unit was

13

isolated from this genomic library. These amplified clones have been subsequently tested

for transcribed sequences by Northern blot analysis.

Because of the tripart nature of this dissertation, there will be three major sections

for clarity of presentation. The first section will describe the modifications of the in gel

renaturation procedure. The second section presents the results from modified in gel

renaturation screening of human short term cultures and direct tumor tissues for DNA

sequence amplification. The third section will present experiments used to clone sections

of an amplified DNA sequence detected in human sarcomas and to identify transcribed

regions within the isolated amplified fragments.

GENE AMPLIFICATION IN CANCER CELLS

Gene amplification is an important event in several animal systems. It is a way in

which cells can rapidly increase the level of specific gene products in response to demands

of environmental stress (e.g. genes encoding detoxifying genes in insecticide resistant

insects;) [7] or as part of a regulated developmental process (e.g. the chorion genes in

Drosophila;) [8,9].

As described in more detail below, mammalian gene amplification appears to occur

only within transformed cells and amplified genes are commonly found in several human

tumor types including neuroblastoma, breast cancer, and small cell lung cancer (6,10).

Gene amplification is often responsible for drug resistance in mammalian cell lines selected ..

with various agents. In both instances, increased expression of the target gene or genes

within the amplification unit confers a selective advantage resulting in clonal outgrowth of

the selected cell.

Two karyotypic abnormalities are often associated with amplification of cellular

genes, double minute chromosomes (DMs) and homogeneously staining regions (HSRs)

(11). DMs appear in metaphase preparations as small, paired, spherical chromatin bodies

which lack centromeres. HSRs are elongated regions within chromosomes which do not

stain with a normal alternating light and dark banding pattern following G- or Q-banding.

Although the association of DMs and HSRs with amplified DNA was originally made in

cells resistant to the drug methotrexate (MTX), both DMs and HSRs have been found in

the metaphase preparations of freshly isolated tumor cells. Both DMs and HSRs have been

shown to contain amplified DNA by in situ hybridization studies (12-14,90). Therefore,

cytogenetic observation ofDMs or HSRs in tumor cells or cell lines correlates strongly

with DNA sequence amplification.

14

In mammalian cells, gene amplification was first observed in mouse cells made

resistant to MTX (15). Subsequently, amplification of several other drug resistance genes,

most notably MDR1, have been observed in direct tumor biopsies and cell lines selected

with cytotoxic agents (2,16).

Gene amplification, in addition to being an important mechanism of drug resistance

in cultured cell lines and tumors, is a proposed mechanism of activation of cellular

oncogenes (16,17). Oncogenes are normal cellular genes whose alteration or

overexpression is thought to play an important role in the development or progression of

human cancers (18,19). Activation of proto-oncogenes can occur by several mechanisms

including: 1.) gene amplification, 2.) inapprupriate expression resulting from translocations

(or other chromosome rearrangements), 3.) deletion or inactivation of suppressor genes, or

4.) mutations within the coding regions of the proto-oncogenes. Several examples of

oncogene amplification in human malignancies have been extensively studied. Perhaps the

best example is the association of N-myc amplification with the childhood cancer,

neuroblastoma. In this cancer, DMs and HSRs occur in the majority of cases (20,21) and

this observation (as well as the identification of c- myc amplification in the DM containing

colon carcinoma cell line COW320) started the search for a hypothetical amplified

oncogene inneuroblastoma cells. Hybridization of a c-myc second exon probe to an

amplified fragment in neuroblastoma DNA suggested amplification of a gene homologous

to c-myc (22). The eventual cloning and characterization of the amplified sequence

revealed a gene (termed N- myc) which had a high degree of homology to c-myc in amino

acid sequence (32%), structural organization, and hydropathy charts (23-26). In situ

hybridization studies confirmed that N-myc was the amplified sequence carried in the

observed DMs and HSRs. The functional homology of N-myc and c-myc was

demonstrated through cotransformation with activated c-ras. When cotransformed, N-myc

compliments the transforming properties of activated ras in a similar manner to c-myc (27).

N-myc amplifications occur in -40% of neuroblastoma tumors and almost all

neuroblastoma cell lines tested. Amplification ofN-myc in neuroblastoma tumors

correlates well with poor clinical prognosis, and advanced tumor stages (28,29). In one

study, N-myc amplifications were found in 0 of 8 Stage I, 0 of 5 Stage IV-S, 2 of 16 Stage

II, 13 of 20 Stage III and 19 of 40 Stage IV neuroblastoma tumors. Also, increasing N­

myc copy number is associated with a decreasing progression free survival (PFS) rate. At

15

18 months, the PFS rate was 70%, 30%, and 5% for patients with respective N-myc copy

numbers of 1,3-10, and greater than 10. Tumors with amplifications were consistently

more aggressive than those without N-myc amplification is not found in all cases of

neuroblastoma, and therefore, could not be the initiating event in the development of this

tumor. Also, there are examples of patients with single copies of N-myc who clinically do

poorly. However, these patients may overexpress N-myc (or other oncogenes) by a

mechanism unrelated to gene amplification.

The observation of N-myc amplification in neuroblastoma prompted further

research in gene amplification in other types of cancer. Several additional associations have

been identified. Approximately 30% of all breast tumor cell lines have c-erbB-2

amplifications and many overexpress c-erbB-2 mRNA (30). Several studies demonstrated

that the human c-erbB-2 encodes a 138kd protein with the features of a cell surface receptor

molecule similar in structure but completely distinct from and unlinked to c-erbB. Like

epidermal growth factor receptor (EGFR), the c-erbB-2 gene product has an extracellular

domain, a transmembrane domain, and an intracellular tyrosine kinase domain (31). The

degree of homology between c-erbB and c-erbB-2 is greatest at the tyrosine kinase regions

of the two genes (31-33). As with N-myc amplification in neuroblastoma, there is a strong

correlation between amplification level of c-erbB-2 amplification or overexpression and

poor patient prognosis (34).

Small cell lung carcinoma (SCLC) represents 25% of all lung carcinomas. Two

major types of SCLC are recognized. Besides classical SCLC, there is also a

morphological and biochemical variant form known as SCLC-V (35,36). SCLC-V is

highly malignant and responds poorly to chemotherapy (36,37). Similarly, cell lines

derived from SCLC-V tumors have characteristic phenotypes which correspond to those of

the original tumors. For this reason, it is theorized, SCLC may progress to SCLC-V.

Cellular oncogenes c-myc, N-myc or L-myc were shown by Southern blot analysis to be

amplified 20-to 80-fold in 13 of 25 SCLC tumors and 21 of31 SCLC cell lines (38,39).

N-myc and L-myc amplification were detected in only two of the SCLC cell lines analyzed

in these studies (39). Also demonstrated was an association between c-myc amplification

and SCLC-V type cell lines and tumors. More recent data suggested that L-myc and N­

myc amplifications are more often associated with the less aggressive classical SCLC

phenotype (6). Finally, increased c-myc copy number correspond with decreased survival

in SCLC patients whereas increased L-myc and N-myc copy numbers do not.

In summary, oncogene amplification has been reported in several types of human

cancer. In certain oncogene-tumor associations, a correlation between oncogene

amplification level and patient prognosis has been established.

MOLECULAR BIOLOGY OF MAMMALIAN GENE AMPLIFICATION

16

The molecular mechanisms of mammalian DNA amplification are extremely

complex and poorly understood. Unlike lower animals, there is no known instance of a

normal regulated gene amplification event in mammalian cells. Techniques currently

available cannot directly analyze the structure of amplified DNA in a single cell which has

undergone the initial steps of gene amplification and (because there are no normal,

regulated gene amplification events in mammalian cells) synchronous gene amplification in

a mammalian cell population is not available for study. Therefore, the hypothesized

mechanisms of mammalian gene amplification have been inferred from the size, physical

map, and cytologic localization of amplified sequences in clonal outgrowths of cells

carrying the original amplification events.

The vast majority of the data regarding the molecular characteristics of mammalian

gene amplification comes from studies of drug selected cell lines. Drug resistance gene

amplification is most often generated following in vitro stepwise increases in the

concentration of the selective agent. The initial amplification event presumably occurs

randomly in a single cell, the selective agent kills cells with normal copy numbers of the

target drug resistance gene, and clonal expansion of a surviving cell with an increased

expression of the target gene due to increased copy number ultimately yields a resistant

population large enough for molecular analysis. Many studies have been published

reporting cell lines with amplified drug resistance genes carried within amplification units

ranging in size from 200-to- 10,000 kb in length (40-42). Two basic types of amplification

unit organization have been described for such cell lines. The first type is an amplification

unit which contains relatively few rearrangements when compared to germ line organization

and which is homogeneous with respect to other amplification units carried in the same cell.

This type of amplification is most often observed in early stages of amplification, but it has

also been reported to persist after subsequent reamplification events in at least one cell line

(41,43,44). The second type of amplification unit observed in drug resistance gene

amplifications contain significant numbers of rearrangements when compared to germ line

sequences. These amplification units are often heterogeneous to amplification units both

17

within a given cell population and from one independently selected cell line to another

(40,45,46). Heterogeneous amplification units are usually carried in cell lines which have

been passaged many times in the presence of the selective agent (46). Therefore, such cell

lines may represent more advanced stages of gene amplification (46).

Recent data has shown that amplification units are often carried on replicating

submicroscopic extrachromosomal circles (episomes) in both drug resistant cell lines and

cultured tumor cells (58,59). Investigators have demonstrated that the initial event in

episome formation is deletion of a chromosomal sequence that recombines to form a

extrachromosomal circle (60). Should the extrachromosomal circle carry a functional

origin of replication, then the episome can replicates once per cell cycle and is thereby

retained within the nuclei of most cells of a clonal expansion (61). Depending on the nature

of the deletion and recombination events which formed the episome, the resulting structure

can be either a large direct or inverted repeat (60,61). In several cases, episomes have been

shown to be very early molecular products of DNA sequence amplification in drug resistant

cell lines (58,60,62). It has been proposed that episomes are the precursors of DMs (58).

Observation of amplified c-myc genes on episomes in DM carrying cell lines HL60 and

COL0320 supports this theory (59). Finally, episomes have been demonstrated to be

capable of reintegrating into various chromosomal sites within a single cell and, therefore,

may be precursors for HSRs (58).

The sequence heterogeneity and rearrangement observed in advanced stage,

stepwise selected gene amplifications may be manifestations of intrachromosomal

reamplification events. Should the target gene be carried on a episome prior to early

selection steps, then random segregation of the acentric elements during mitosis can lead to

the accumulation of increasing numbers of elements within a small fraction of cells of a cell

popUlation. During initial selection steps, cells which have accumulated greater numbers of

the elements carrying the target gene can survive the selective agent without a rereplication

or recombination event needing to occur within the target gene domain. Therefore, the

amplification units carried within the selected cells remain relatively homogeneous.

However, if the amplified target gene is carried within a chromosome (either at the original

locus or more often following integration of an extrachromosomal element at a secondary

chromosomal location), then intrachromosomal reamplification of the target gene must

occur for a cell to survive the increased selective agent concentration of further selection

steps. Intrachromosomal reamplification events preferentially reamplify specific regions of

the original amplification unit, thereby creating heterogeneous amplification units of higher

18

copy number (47). In such cases, decreasing amounts of flanking DNA may be

coamplified with the target gene and amplified novel joints between sequences from

different regions of the genome are created (47-49). Novel joints can be arranged in two

basic ways. Depending on the mechanism of amplification and the recombinant resolution

of the intermediate structures during the amplification process, the amplification units may

be aligned in head-to-tail (direct) repeats or in head-to-head (inverted) repeats (43,50).

However, novel joints may also arise denovo following single step selection (48). Finally,

simultaneous amplification and formation of inverted novel joints was observed in a cell

line not placed under selective agents (51).

Data regarding molecular structures and rearrangements resulting from de novo

amplification of oncogenes has come mostly from studies on N-myc amplification units in

neruoblastoma cells. As mentioned previously, N-myc is amplified in -40% of

neuroblastoma tumors and almost all neuroblastoma cell lines tested (52). Amplified N­

myc is almost always found localized in DMs in direct preparations of neuroblastoma tumor

cells. In contrast, the amplified copies on N-myc are almost always found localized within

HSRs located at different chromosomal sites than the original N-myc locus in

neuroblastoma cell lines.

The degree ofN-myc amplification in neuroblastoma ranges from 3-to- 3OO-fold

(52). Several studies have shown that the complexity of amplified DNA varies greatly

among cell lines established from tumors of different patients (52-54). Amplification units

carried in HSRs of neuroblastoma cultured cells often contain large amounts of DNA

coamplified with N-myc. N-myc amplification units have been reported to be loo-to- 3000

kb in length, (with an average amplification unit size of-3oo kb) and are characterized by a

high degree of differential recombination. (40,55,56). Amplification units of N-myc

carried in direct tumor preparations have been reported to be more homogeneous in length

and structure. Kinzler et al. (53) showed by in gel renaturation analysis that de novo N­

myc amplification units in 8 different neuroblastoma tumors varied between 290 and 430

kb in length with a relatively high degree of homogeneity. The reasons for the observed

differences in the structures of amplified domains in direct tumor tissues and cell lines are

uncertain. Amler et al. (57) believe that cell lines with lower levels of N-myc amplification

have amplification units of large size and simpler structure. In their study, cell lines HDN-

3, NLF, and IMR-32 were demonstrated to carry both the lowest levels of amplification

and the longest stretches of coamplified flanking DNA without detectable rearrangement

(over 1400 kb in all cases) (57). This result was independent of the number of times the

19

cell lines were passaged in cultured. Almer et al. (57) also showed that N-myc

amplification units carried in the neuroblastoma cell lines they analyzed were almost

exclusively arranged in direct tandem repeats with the N-myc gene located within the core

region of the amplification unit.

The diverse data on the molecular structure of mammalian gene amplification just

described has generated several different mechanistic models to explain the observed

structures. Proposed mechanisms of mammalian gene amplification can be separated into

two classes (56). One class is based on accumulation of extra DNA copies by

overreplication within a single cell cycle (replication-driven mechanisms). In the other

class, unequal segregation of sequence replicated once per cell cycle leads to the

accumulation of extra copies of DNA (segregation-driven mechanisms). None of the

models are necessarily mutually exclusive, and in fact, mammalian gene amplification may

occur by either one or a combination of the proposed mechanisms under different

conditions (56).

REPLICATION-DRIVEN AMPLIFICATION

Onion Skin Model. The general feature of the the onion skin model is a re­

replication of DNA sequences within a single cell cycle (63). This model has been proven

experimentally in the developmentally controlled amplification of chorion genes in

Drosophila follicular cells (64). In mammalian cell amplification, support for this model

comes from experiments in which transient inhibition of DNA synthesis leads to aberrant

replication and occasional gene amplification (65- 67). Vasharvsky et al. (68,69) proposed

that amplifications induced by cytotoxic selection might be caused by several rounds of

illegitimate replication at certain origins of replication during one cell cycle. DNA synthesis

inhibition increases "replicon misfiring" and, consequently, increase the likelihood of gene

amplification. Multiple rounds of misfiring could generate intermediate structures

resembling an onion skin with its many layers (70). Resolution of this structure could

occur through homologous recombination of the many repeated sequences.

Extrachromosomal circles excised during recombination could presumably lead to

formation ofDMs if the target gene and an origin of replication are present (58,71-74).

Recombination could also generate a linear intrachromosomal amplification gradient of

direct or inverted tandemly repeated units which could form an HSR. The centrally located

20

units within the amplified domain would be amplified more than units distal to the origin of

replication, a condition observed to occur in de novo N-myc amplification (55,70).

While the onion skin model is flexible enough to explain many of the cytological

and structural characteristics of gene amplification, observation of amplification units of

10,000 kb appear unlikely to be explained by this model unless unscheduled replication can

occur over a region of approximately 100 replication origins (56). Also, while the onion

skin model can explain the production of intennediate molecular structures which could

resolve into inverted duplications, the recombination event would have to occur early

enough in the process to allow for rereplication of the inverted duplication to high copy

number.

Extrachromosomal Double Rolling Circle Model. This model was initially proposed

to explain the amplification of the 2um yeast plasmid which contains a single origin of

replication located asymmetrically between two inverted duplications (75). Passananti et al.

(76) proposed a model for mammalian gene amplification based on this model. The

amplification event is initiated with an excision of a extrachromosomal circle of DNA by

recombination between newly replicated strands of a replication loop. The liberated

episome contains two identical origins of replication. A double rolling circle could not be

maintained with replication at two origins on one circular element. Therefore, only one of

the two origins can initiate replication for a double rolling circle replication event during the

next cell cycle. The alternative to the undetennined "silencing" of one replication origin

requires that two homologous recombination events occur between the duplicated

sequences which excludes one of the replication origins. Presumably, these structures are

capable of reintegration into chromosomes to produce HSRs.

Chromosomal-Spiral Model. The wide spread occurrence of inverted duplications

in mammalian gene amplification suggested to investigators that a model for rapid gene

amplification events other than the onion skin model was necessary to explain these

structures (50,76).

Hyrien et al. (77) proposed a model explaining both the fonnation of an inverted

duplication and its amplification through a single mechanism that does not include

additional replicon firing or chromosome breakage. An inverted repeat can be fonned if

replication switches strands and proceeds around the replication fork (51). Evidence

supporting this model includes observations of turnaround events within palindromic

21

sequences during prokariotic DNA replication (78). Also, there is a significant amount of

evidence that DNA polymerases behave erratically when traversing template with hairpins

(79,80). The fact that psuedopalindromic sequences which can form hairpin structures are

known to be present within novel joints between inverted repeats suggests that such an

aberrant DNA polymerase turnaround event could occur in conjunction with mammalian

gene amplification (77). A head-to-head novel joint is formed through copy- choice

recombination. A tail-to-tail novel joint is formed if the same event occurs at the opposite

end of the replication bubble, and amplification proceeds by a double rolling circle

replication at two replication forks. The structure can resolve into a tandem array of

inverted repeats with the possible excision of a replicating extrachromosomal circle.

~aGREGA TION-DRIVEN AMPLIFICATION

Deletion-Plus Episome Model. This model proposes that the initial step of

mammalian gene amplification is formation of an extrachromosomal circle with a functional

origin of replication. The episome can be formed by either rereplication or recombination

across a replication loop (61,76,81). Formation of an episome by recombination would

cause a deletion at the chromosomal locus. Experiments have been reported that suggest

that episome formation is coincident with deletion (62). Depending on the recombination

event, the episome can be either a direct repeat or inverted duplication. Amplification can

result from simple unequal segregation at mitosis. As stated earlier, these

extrachromosomal elements may evolve into DMs or reintegrate into chromosomes to

presumably form HSRs.

Unequal Sister Chromatid Exchange Model. The gradual accumulation of amplified

DNA by unequal crossing over between sister chromatids is a proposed explanation for the

evolution of tandem arrays of repeated units in normal eukaryotic DNA. This model is

based on the structure at normal tandemly repeated genes, such as the ribosomal genes of

most vertebrates (82,83). Evidence in support of this theory includes observations of

unequal sister chromatid exchange in many organisms. In Drosophila, unequal sister

chromatid exchanges are believed to be responsible for the number of tandem repeats at the

bar locus and the number of ribosomal genes at the bobbed locus (84). Furthermore, this

process has been suggested as a mechanism of evolution of multigene families, the

magnification-reduction of ribosomal genes in Drosophila, and the evolution of nonnally

repeated DNA sequences (85-87).

22

Unequal sister chromatid exchange has also been proposed as the mechanism of

gene amplification in mammalian cells which have been slowly, step-wise selected for

resistance to specific inhibitors of enzyme activities; such as methotrexate inhibition of

dihydrofolate reductase (86). Evidence in support of this mechanism includes the

consistency between the gradual accumulation of extra copies of the target gene and the

multistep selection procedure necessary to generate them. Such tandemly repeated

structures would be directly repeating units and could undergo excision to generate DMs if

the episome contained an origin of replication (58,62). Also, certain chromosomal regions

appear to be "hot spots" for amplification, perhaps due to a high degree of nonnally

occurring recombination events. In one experiment, Syrian hamster cell lines were

transfonned with a cosmid containing the CAD gene and placed under drug selection.

Depending on the chromosomal integration site, certain clones had as much as 100- fold

higher amplifications. Some clones were observed to have rRNA sequences as the co­

amplified flanking sequences (89,90). However, this mechanism would predict the

coexistence of DMs and HSRs in the same metaphase spreads, a condition rarely observed.

Also, this mechanism alone can not account for developmental amplifications in which

extensive mitosis do not take place (91).

In summary, although several issues regarding mechanisms remain to be resolved,

gene amplification is clearly an important pathway to altered gene expression in human

cancer. To date, spontaneously occurring amplification units in human tumors have always

included a gene likely to be biologically active in maintaining the transfonned phenotype.

At the present time there is only limited data on the frequency and nature of gene

amplification in most human tumors. Therefore, as additional amplification units are

identified in a given tumor, it may be reasonably expected their recognition will contribute

to the identification of genes which are biologically important in that tumor. The remainder

of this dissertation discusses methods for the detection of amplified DNA and their

application to the cloning of an amplified sequence in human sarcomas.

23

CHAPTER II

MODIFICATION OF IN GEL RENATURATION

INTRODUCTION

The initial goal of this dissertation was to improve the sensitivity, specificity, and

reproducibility of the in gel renaturation technique. In gel renaturation is a technique

developed by Roninson and colleagues for the detection of amplified restriction fragments

without prior identification of the target gene within the amplification unit (1,2,4,5).

Unlike other methods which have been used to detect and isolate transforming sequences,

in gel renaturation is independent of assays with biologic endpoints (e.g. assays of the

ability of a sequence to transform Nll-I/3T3 mouse cells). Further, this technique was used

in the identification of drug resistance gene MDRI and the GLI protooncogene (2,3).

Therefore, it appeared likely that in gel renaturation could detect amplified sequences in

tumor cells which were previously unidentified but potentially important in tumor

formation.

As originally described, in gel renaturation was limited by the need to radiolabel

each specimen tested. A recent modification (4,5) eliminates this step simplifying the

procedure and improving its sensitivity and specificity. Initially developed to detect drug

resistance gene amplifications in mouse DNA, this modification has now been applied to

the detection of human gene amplification (5). The technical modifications remained

unpublished until recently. Therefore, the specific methodologic details of this work were

developed independently in order to achieve reliable and reproducible results. Accordingly,

the described protocol differs in several respects to recently published procedures (see

"Materials and Methods" for specific details).

RENATURATION KINETICS. The in gel renaturation procedure is based on

renaturation kinetics of double stranded DNA molecules. Melting of dsDNA secondary

structure is referred to as "denaturation" and reassembly of the secondary structure is

referred to as "renaturation".

The midpoint temperature of denaturation (T m) is effected by several factors.

Increased GCI AT base ratio increases the T m due to increased H-bonds between the

strands. T m is also a function of ionic concentrations and pH.

24

Renaturation is a two step process. The fIrst step for renaturation of complimentary

strands involves nucleation of the strands at very few complimentary bases. The second

step is the zippering together of the remainder of the molecule, a relatively fast event. The

fIrst step is slow and is rate-limiting. Nucleation occurs at a rate proportional to the square

of the specifIc sequence concentration. Thus, in a solution of denatured human genomic

restriction fragments at a given temperature, pH and ionic concentration, renaturation will

be most rapid for those molecules present in greater concentration or copy number. This

concept can be described mathematically:

If cO is the concentration of completely denatured DNA and c

is the concentration of single stranded DNA at time t, then

~ = 1 cO 1+k2cOt

where k2 is the bimolecular constant. clcO is plotted as a

function of cot and is referred to as a "cot curve" (Figure 1).

As in solution, restriction fragments subjected to electrophoresis in agarose and

subsequent alkaline solution denaturation will renature at a rate proportional to the square of

the concentration. Under the renaturing conditions deduced by Roninson, only restriction

fragments in high enough copy number will renature. Fragments present at single copy per

haploid genome will remain single stranded. It is on this principle that the in gel

renaturation procedure is based.

IN GEL RENAIURA TION. In gel renaturation in its original form will be

described below. First, high molecular weight genomic DNA is extracted from the tumor

tissue and is digested with HindIII. A small, quantity of DNA (termed "tracer" DNA) is

end-labelled by T4 DNA polymerase. The end-labelled tracer fragments are mixed with a

o ::::.-- Foldback

"0 ~ 20 as ·0 o en ~ 40 ~ <{

Z 60 c -c Q)

e 80 Q) a.

~ Intermediate

Single copy

100~~--~~--~--~~--~

10-410-310-210-1100 101 102 103

cot

Figure 1. Renaturation Kinetics of Total Human Nuclear DNA. DNA fragments within an amplified domain renature within the intermediate (10-1 to 101 cot value) range. (Reprinted from Zubay, G. (1983). Biochemistry. Addison-Wesley Co.) (134).

25

26

loo-fold excess of unlabelled "driver" DNA from the same DNA source. These tracer­

driver mixtures are electrophoresed in an agarose gel. The gel is then treated with

denaturing solution. The fragments are allowed to renature in a 50% formamide solution.

Only high copy number fragments will renature under these specific conditions, leaving the

vast majority of the fragments single stranded When the gel is subsequently treated with

single stranded DNA specific S 1 nuclease, low copy number, single stranded fragments are

digested The high copy number fragments remain protected from the nuclease by virtue of

their renatured status. The denaturation, renaturation, and S 1 nuclease digestion steps are

repeated, and residual radioactive nucleotides generated by S 1 nuclease digestion are

washed out of the gel. Finally, the gel is dried and autoradiographed (Figure 2). The

pattern of bands produced by placenta control DNA correspond to normally repetitive

fragments and to mitochondrial DNA fragments (1). Additional bands present in

experimental tumor DNA lanes correspond to fragments of a DNA sequence amplifications

of 25-fold or greater (Figure 3).

Although in gel renaturation represented a significant new tool for identification of

amplified sequences, this procedure is extremely time consuming, labor intensive and

requires tedious, expensive (arid frequently inconsistent) restriction fragment end-labelling

by T 4 DNA polymerase. The modification improves the original technique in several

repects described in detail below.

In summary, in gel renaturation in its original form detects DNA sequence

amplification as low as 20-fold without knowledge of the target sequence identity (1). The

modified technique improves sensitivity to -lO-fold amplifications, is simpler to perform,

and is specific for human sequences. It was therefore possible to utilize modified in gel

renaturation to screen multiple human tumor specimens for the presence of amplified DNA.

In this initial section of this dissertation, the utility of the modified in gel renaturation

technique to detect amplification units encoding several different genes was established.

RESULTS

The modified strategy developed differs principally by omitting the T4 DNA

polymerase end-labelling step, and transferring the renatured DNA fragments to a blotting

membrane via Southern blotting following in gel renaturation. HindIII digested DNA was

size fractionated in an agarose gel and SUbjected to two rounds of denaturation,

renaturation, S 1 nuclease digestion and fmal wash exactly as in the original method. Since

-9.7

-6.9

-5.5

-3.0

Figure 2. Band Pattern of High Copy Fragments in HindIII Digested Normal Human Placenta DNA When Subjected to In Gel Renaturation.

27

-9.7

-6.9.

~:,I' -5.5 .. .. ~,..p.

~ .. • .. .. ..

-3.0

Figure 3. Results of In Gel Renaturation Analysis of SCLC H69. DNA from cell line H69, a small cell lung carcinoma with a previously identified c-myc amplification of .... SO­fold (96), was digested with HindIII and subjected to in gel renaturation. All bands not present in control DNA lanes correspond to fragments from the c-myc amplification unit.

28

29

reannealed Alu sequences derived from heterologous restriction fragments were reduced in

size by S 1 nuclease digestion to less than 2 kb, the background hybridization was reduced

by removal of these sequences from the gel by electrophoresis in an electroblot chamber.

Remaining high copy number fragments were transferred to nylon blotting paper by the

alkaline capillary blotting method. These high copy number fragments are detected by

probing the blot human specific SINE sequence BLUR8 (92). In the final analysis, the

number of bands detected in HindIII digested nonnal DNA was reduced to a single strong

-6.5 kb band and two weakly hybridizing weak bands of polymorphic sizes. Therefore, in

the case of an amplified sequence, all other bands detected correspond to restriction

fragments from the amplification unit (Figure 4A).

The applicability of the modified technique for the detection of amplification units

derived from various genes was tested on DNA from a panel of cell lines previously

reported to carry amplified genes. As shown in Figure 4, additional bands hybridizing to

BLUR8 probe were readily detected in lanes derived from cell lines containing amplified

copies of N-myc, c-myc, MDRl, and dihydrofolate reductase (DHFR). The lowest level

of amplification tested was cell line CEM/VLBl00 which carries a MDRI amplification of

-15 fold (93). Careful inspection of the autoradiograph revealed additional bands derived

from an amplified domain. Probing the same blot with a MDRI probe clearly demonstrated

that the -IS-fold amplified MDRI sequences were retained within the gel following the in

gel renaturation procedures (Figure 4B).

DISCUSSION

As stated, this modification to in gel renaturation improved the technique in several

ways. First, the sensitivity of the assay was improved from detection of amplifications of

-25-fold to -IO-fold. Second, the modification specifically detected human sequences.

Third, the modification was simpler, and in laboratory reproducibility was greatly

improved.

Although the original technique and the modification both were based on the same

renaturation kinetics, the modified technique no longer relied on the sensitivity limiting T 4

DNA polymerase end-labelling reaction to detect renatured high copy number fragments.

The T 4 DNA polymerase reaction incorporated 32PdA TP into restriction fragments by

replacement synthesis. Therefore, the distance in from the end of the fragment that the

endonucleolytic reaction was allowed to proceed prior to the resynthesis with

A

! i .. \,.: ;' :':~ !

~I: "

i&~:";

., 1

30

B

2 3 4 5 6

Figure 4. A. Detection of Gene Amplification in Human Tumor Cell Lines by Modified In Gel Renaturation. Human placenta control DNA (Lane 1) demonstrates the single -6.5 kb band detected in all human DNA samples (arrow). Lanes 2 through 6 demonstrate multiple additional bands corresponding to amplified restriction fragments within the amplification units previously reported in these cell lines. N-myc (50-fold) in H69 (96), c­myc (30-fold) in SF-I88 (98); MDR-I (40-fold) in CEMNCR (97); DHFR (-lOO-fold) in KB7A; MDR-I (-IS-fold) in CEMNLBlOO (93). B. The same in gel renaturation blot as in figure 4A was stripped and rehybridized with pMDRl. The bands in the CEMNLBIOO lane correspond to amplified restriction fragments containing the exons of the mdri gene.

31

radionucleotides detennined the copy number of fragments that were detectable by

autoradiography. If the endonucleolytic reaction proceeded too far, then background from

fragments incompletely digested during the S 1 nuclease reaction obscured detection of

amplified fragments. If the endonucleolytic reaction did not proceed far enough,

autoradiographic detection of amplified fragments was prevented due to an insufficient

quantity of radionucleotide incorporation during resynthesis steps. The modifications

described previously overcame this sensitivity limiting obstacle by blotting all fragments

remaining after S 1 nuclease treatment and detecting those fragments with a controllable,

high specific activity BLUR8 probe.

The second major limitation of the original technique overcome by the modification

was human sequence specificity. In the original technique, false positive results were

possible in mycoplasma contaminated cell cultures. If mycoplasma DNA was present in

high enough concentration, then bands corresponding to mycoplasma sequences were

detected by the assay. The detection of high copy number fragments by human specific

repetitive SINE sequence BLUR8 in the modification eliminated this concern.

Reproducibility was also limited by the T4 DNA polymerase reaction. The quality

of tracer fragment labelling varied due to the tedious multistep, time dependent enzymatic

reaction, the large quantities of isotope required, and to inhibitors of enzyme activity in

individual DNA samples. Also, autoradiographic quality of the dried gels diminished

quickly due to short half life of 32PdA TP. Blotting restriction fragments remaining after in

gel renaturation procedures and hybridizing them with BLUR8 probe pennitted consistent

and reproducible results.

As Alu repeats are known to occur in the vicinity of all nonnally single copy

transcribed regions (at an average of once every 3 kb), it is likely that the BLUR8 probe

can detect any gene amplification (92,94). This speculation is supported by my

demonstrated ability to detect amplified sequences derived from five different genes.

Once reproducible results for the complete procedure were obtained for control cell

lines with known gene amplifications, screening of DNA from direct sarcoma tissue and

human tumor cell lines derived from a variety of human malignancies was initiated

MATERIALS AND ME1HODS

CELL CULTURE. Cell culture conditions were as described in Leibovitz, (1986)

(95). Small cell lung carcinoma cell line H69 was kindly provided by Dr. Susan Cole (96).

32

Cell lines CEM/VLB and CEMNCR were kindly provided by Dr. William Beck (96). Cell

line KB7 A was kindly provided by Dr. Yi Cheng. Cell line SF-188 was kindly provided

Dr. Mark Rosenblum (98).

HIGH MOLECULAR WEIGHT DNA EXTRACfIQN. High quality genomic

DNA (DNA which is larger than 50 kb when 0.1 ug is compared to uncut bacteriophage

lambda size marker co-fractionated in 0.3% agarose gel) is essential to the sensitivity of

detection of amplification and great care is taken in its preparation. DNA was isolated by

phenoVchloroform extraction of SDS-proteinase K (Boehringer) treated celllysates (99).

DNA was quantitated by 4,6-Diamidino-2-phenylindole (DAPI) fluorimetric assay (100).

IN GEL RENATURATION. 0.25 ug of HindIII digested genomic DNA fragments

were labelled by replacement synthesis with T4 DNA polymerase (BRL). This tracer

fraction was incubated with 2 U of enzyme in 30 mM Tris-acetate (pH 7.9),65 mM

NaOAce, 10 mM MgOAce, 0.5 mM DTT, and 0.1 mM BSA, (10 ul final volume) for 3

min. The endonucleolytic reaction was stopped by placing the tubes in ice water. For each

reaction 80 ul of 800 uC/mM 32PdCTP (ICN) was lyopholized in an eppendorf tube. The

isotope was redissolved in the resynthesis mixture of 0.33 mM dATP, 0.33 mM dGTP,

0.33 mM dTTP, and 4 mM dCTP (Pharmacia). The T4 DNA polymerase treated samples

were added to 15 ul of the resynthesis mixture and incubated at 370 C for 30 min. The

resynthesis reaction was driven to completion by addition of 2.5 ul of 2 mM dCTP (ICN).

The reaction was stopped by NH40Ace/isopropanol precipitation.

The end-labelled tracer fractions were mixed with 25 ug of unlabelled driver

fragments from respective sources and were electrophoresed in a 1 % agarose gel (FMC)

(32 cm x 18 cm x 2 mm) in Ix TAB at 40 mV for -48 hrs. The gel was EtBr stained,

photographed and transferred to a snug fitting box with a tight lid. The gel was incubated

in 250 ml of prewarmed solutions and was gently rotated in a shaking incubator (25 RPM)

for the following steps. The gel was incubated first in denaturing solution (0.5 M NaOH,

0.6 M NaCl40 mg/l thymol blue) at 450 C for 30 min x 2. The addition of pH indicating

thymol blue dye in the solution stained the gel dark blue. The gel was then incubated in

renaturation solution (50% form am ide, 5x SSPE (pH 7.0) at 450 C. The solution was

changed every 15 mins until the gel was bright yellow. Renaturation of repetitive

fragments occurs when the gel was allowed to incubate in fresh renaturation solution for 2-

to-16 hrs. Following renaturation, the gel was saturated with 370 C, Ix SI nuclease

33

reaction buffer (50 mM NaOAce (PH 4.6), 0.4 M NaCI, 1 mM ZnCI) for 15 mins x 5. The

solution was changed once more and 20k units of S 1 nuclease (Sigma) was added.

Enzymatic digestion of single stranded DNA was allowed to proceed for 2 hrs. The entire

denaturation, renaturation, and SI nuclease steps were repeated. Finally, the gel was

washed for 2 hrs in Ix TBE. 32PdA TP end-labelled high copy number fragments

remained in the gel. The gel was dried on a gel dryer and autoradiographed with X-ray

film (Kodak X-OMA T AR) and an enhancing screen (Dupont Cronex 220059) at -8QOC

overnight.

MODIFIED IN GEL RENATURATION. 25 ug of HindlII digested tumor and

control genomic DNA samples were electrophoresed at 40 m V for 48 hrs in a 1 % agarose

gel (32 cm x 18 cm x 2 mm) in Ix TAE. The gel was EtBr stained, photographed, and

transferred to a snug fitting box with a tight lid. The gel was incubated in 250 ml of

prewarmed solutions and was gently rotated in a shaking incubator (25 RPM) for all the

following steps. The gel was first incubated in denaturing solution (0.4 M NaOH, 0.6 M

NaCI, 40 mg/l thymol blue) at 450 C for 30 min twice. The addition of pH indicating

thymol blue dye in the solution stained the gel dark blue. The gel was then incubated in

renaturation solution (50% formamide, 5x SSPE (pH 7.0» at 450 C. The solution was

changed every 15 mins until the gel was bright yellow. Renaturation of repetitive

fragments occurred when the gel was allowed to incubate in fresh renaturation solution for

2-to-16 hrs. Following renaturation, the gel was saturated with 370 C Ix SI nuclease

reaction buffer (50 mM NaOAce (pH 4.6), 0.4 M NaCl, 1 mM ZnCI) for 15 min x 5. The

solution was changed once more and 20 k units of S 1 nuclease was added. Enzymatic

digestion of single stranded DNA was allowed to proceed for 2 hrs. The entire

denaturation, renaturation, and SI nuclease steps were repeated, and finally the gel was

washed for 2 hrs in 1 x TBE. The gel was then covered with a 1 cm thick layer of 1.5%

agarose in Ix TBE. The gel was electrophoresed in an electroblot chamber (Biorad) at 400

rnA for 3 hrs with the original gel facing the cathode. This step removed reannealed SINE

sequences which survived nuclease digestion while retaining undigested, repetitive

fragments greater than 2 kb within the gel.

All fragments remaining after in gel renaturation were transferred to Zeta-probe

(Biorad) by capillary blotting with 0.4 M NaOH for 40 hrs. The blot was neutralized in

0.5 M Tris-HCI (pH 7.0)/1 M NaCI for 30 mins. The blot was then hybridized with

oligolabelled human specific SINE sequence BLUR8 (Oncor) as described below.

34

PROBES. Human specific SINE sequence BLUR8 was obtained from Oncor, Inc.

(92). All oncogene probes were obtained from the American Type Culture Collection.

PROBE LABELLING. Sequences used to probe Southern blots (including in gel

renaturation blots) were radiolabelled by the Feinberg and Vogel stein oligolabelling method

(101). Plasmid inserts separated by electrophoresis were excised from the 1% low melting

point agarose gel (FMC) in Ix OLRB and were diluted 3-fold with distilled H20. The tube

was boiled and a volume equivalent to SO ngs of DNA was added to a tube containing 10 ul

of Sx oligolabelling buffer, 1 ul 1 mg/ml BSA, 6 ul of 32PdA TP (3000 uC/mM) (ICN) and

distilled H20 to 49 ul. The tube was vortexed and 1 ul (3 U) of large fragment polymerase

(BRL) was added The tube was vortexed again and the reaction was allowed to proceed

overnight. Percent radiolabel incorporation was tested by 10% TCA precipitation of 1 ul of

the reaction volume and counting in a scintillation counter.

SOUTHERN BLOT HYBRIDIZATION AND AUTORADIOGRAPHY. Southern

blots were prehybridized for 4 hrs at 4SoC in prehybridization fluid (SO%formamide, 10%

dextran sulfate, 1 % SDS, 0.06 M Na2P04 (pH 7.0), 1 M NaCI, SX Denhart's solution) in

sealed plastic bags. After prehybridization, labelled probed was added at a specific activity

of-106 cpm/ml and hybridization was allowed to take place for 16 hours. Blots were

washed in 1 % SDS/0.1 x SSC at SSoC for O.S hrs x 4. The blot was then placed in a

cassette with X-ray film (Kodak X-OMAT AR) and an enhancing screen (Dupont Cronex

22ooS9) at -800 C overnight.

CHAPTER III

IN GEL RENATURATION SCREENING OF HUMAN TUMORS

AND TUMOR CULTURES

IN1RODUCflON

Based on the speculation that unidentified genes amplified in human tumor cells

remain to be discovered, the modified in gel renaturation assay was applied to several

human tumor and short term culture DNA samples. The goals of the modified in gel

renaturation survey were to identify 1.) novel amplified DNA sequences and 2.)

uncharacterized amplified oncogene-tumor associations.

35

Identification of novel amplified DNA sequences or uncharacterized amplified

oncogene-tumor type associations is of significant interest. Based on examples of the biologic significance of gene amplification in human cancer cells (discussed in Chapter I), it

is clear that genes important in the genesis or progression of human cancers are carried

within amplified DNA sequences. I speculated that amplified cellular genes important in

the genesis or progression of human malignancies remain undiscovered. The modified in

gel renaturation technique was employed to screen human tumors and short term cultures

for amplified sequences.

Particular emphasis was placed on screening human sarcoma and malignant

melanoma short term cultures. The presence of DMs and/or HSRs is well documented in

both malignancies (102-104). However, only one case of an amplified gene has been

reported for melanoma (105), and no cases of gene amplification have been reported in

adult sarcoma As stated earlier, cytogenetic observation of DMs or HSRs within

metaphase preparations of tumor cells correlates strongly with amplified sequences being

carried within those cells. No known gene has been demonstrated to be commonly

amplified in either of these tumor types. Thus, my hypothesis was that amplified DNA

sequences were indeed carried within melanoma and adult sarcoma cells and that either

novel transcribed sequences or uncharacterized amplified oncogene-tumor type associations

could be identified within the respective amplification units by modified in gel renaturation.

36

RESULTS

HUMAN TUMOR CULTURES. DNA from 55 human tumor cell cultures

established at the University of Arizona Core Culture (UACC) laboratory were analyzed by

modified in gel renaturation (Table 1). The tumor cultures included 25 melanoma, 8

ovarian carcinoma, 6 breast carcinoma, 3 pancreatic carcinoma, 2 colon carcinoma, 2 lung

carcinoma, 2 sarcomas, 2 Wilm's tumors, 1 neuroblastoma, 1 uterine carcinoma, 1

bladder carcinoma, 1 leiomyosarcoma, and 1 small cell lung carcinoma (SCLC). Amplified

DNA was detected in SCLC UACC735 (Figure 5) and 3 of the 6 breast cancers

(UACC732, UACC812, and UACC893). In each of these four cases, the DNA was tested

by Southern blot analysis using the appropriate probe and gene amplification was

confirmed. UACC735 was found to carry a -30-fold amplification of the c-myc gene

(Figure 6), while each of the 3 breast cancer specimen were found to carry amplified copies

of the c-erbB-2 gene. Despite cytogenetic evidence suggesting the existance of amplified

DNA, no evidence of DNA sequence amplification was found in any of the 25 melanomas

studied.

ADULT SARCOMAS. DNA from 16 adult sarcoma samples were analyzed by

modified in gel renaturation. DNA was extracted directly from tumor tissue of 8

liposarcomas, 5 malignant fibrous histiocytomas (MFH), and 3 other adult sarcomas. One

MFH, ST-23987 tested strongly positive for DNA sequence amplification >IO-fold (Figure

7). Southern blot analysis using the panel of oncogene probes previously demonstrated to

amplified in other human malignancies failed to reveal the identity of the amplified

sequences (Table 2; see "Addendum"). Since the amplified sequences remained

unidentified, ST-23987 DNA was selected for in gel renaturation cloning experiments.

DISCUSSION

HUMAN TUMOR CULTURES. The results obtained with modified in gel

renaturation demonstrated that this is a practical approach for screening numerous human

samples for DNA sequence amplification. The results of the survey of human cell lines

were consistent with the previously reported pattern of gene amplification in small cell lung

carcinoma (39) and breast carcinoma (30). In melanoma, DMs or HSRs have been

identified on occasion (102,104), and a single instance of hstl/int-2 amplification has been

Table 1. Summary of in gel renaturation analysis of human tumor cultures.

Tumor type

Melanoma Ovarian carcinoma Breast carcinoma Pancreatic carcinoma Wilm's Tumor Colon carcinoma Lung carcinoma Bladder carcinoma Neuroblastoma SCLC Sarcoma Leiomyosarcoma Uterine carcinoma

Number tested

25 8 6 3 2 2 2 1 1 1 2 1 1

Number positive

o o 3 o o o o o o 1 o o o

SCLC = small cell lung carcinoma. Sarcoma = undiagnosed sarcoma.

Amplified fWne

c-erbB-2

c-myc

37

38

~

,~

~ ~.

~ ~

~ ~

',;

~ I

Figure S. Modified In Gel Renaturation Analysis of SCLC UACC735. The arrow highlights the -6.5 kb band common to all human DNA. Lane 1: UACC735 DNA diluted 3-fold with normal human placental DNA. This dilution would correspond to a -IO-fold c­myc amplification. Several bands are easily detectable at this amplification level. Lane 2: UACC735 DNA diJuted 1:2 with placental DNA. Lane 3: undiluted UACC735 DNA with its -3D-fold c-myc amplification. Lane 4: UACC245, a breast cell line which is negative for> lO-fold gene amplification.

39

1 2 3 4 5

Figure 6. Southern Blot Analysis of UACC735. The blot was probed with a 1.4 kb SstI fragment containing the second exon of c-myc. Lane 1: 5 ug of EcoRI digested normal human placenta DNA. Lanes 2-to-5 contains 5 ugs, 0.5 ug, 0.25 ugs, and 0.16 ug of EcoRI digested UACC735 DNA, respectively. Oncogene c-myc is amplified ..... 30-fold in UACC735 DNA when compared to equal quantities of normal human DNA.

40

Figure 7. Modified In Gel Renaturation Analysis of MFH ST-23987. The dark arrows point out the -6.5 kb repetitive band common to all human DNA. The lighter arrows point out bands corresponding to the respective amplified restriction fragments. Lane 1: UACC735 positive control. Lane 2: an underloaded lane ofUACC460, a leiomyosarcoma short term culture. Lane 3: UACC245, a breast carcinoma cell line which is negative for DNA sequence amplification >lO-fold. Lane 4: Human placenta negative control lane. Lane 5: MFH ST-23987. ST-23987 tests strongly positive for DNA sequence amplification.

Table 2. Panel of Oncogene Probes Used to Analyze Southern Blots of ST-23987 DNA.

Oncogene probe

c-myc N-myc N-ras Ki-ras Ha-ras erbA-l erbB-l erbB-2 int-2 c-sis c-raf c-fos c-myb Hu-ets-l gli

AmplifiedlRearranged in ST-23987 DNA

41

42

recently reported (105). Also reports of amplified repetitive elements derived from

chromosome 15 and an amplified sequence bearing homology to EBV and papillomavirus

type 9 have each been reported in a single case (104,106). In my study there was no

evidence of DNA sequence amplification in the 25 melanoma cell cultures tested It appears

unlikely that gene amplification is frequent in cultured melanoma cells at levels above the

threshold for detection by modified in gel renaturation. However, it should be noted that

biologically significant gene amplification may well be present at lower levels, perhaps

justifying attempts to further optimize this procedure. In addition, we cannot be certain that

the cultures we have studied had not lost extrachromosomal elements carrying genes which

may be maintained under the selective pressure of growth in vivo, but not in vitro. In this

regard, it has been demonstrated that cultured cells may both gain and lose amplified genes

(5). Most studies using in gel renaturation have utilized cultured cells because of the

requirement for high molecular weight DNA which renders this technique sensitive to

minor amounts of degradation. It is likely that this difficulty can be overcome by careful

processing of primary melanoma biopsies.

ADULT SARCOMAS. As with melanoma, little is known of the molecular changes

characterizing adult sarcomas. Liposarcoma represents over 17% of all adult sarcomas.

Malignant fibrous histiocytoma (MFH) tumors represent over 28% of all adult sarcomas

(107). Both tumor types are characterized by large marker chromosomes, DMs, and HSRs

and therefore, are likely to carry amplified DNA (103).

In this study, I demonstrated the presence of amplified DNA sequences in one

MFH tissue sample by modified in gel renaturation. The amplification unit remained

unidentified by a panel of known oncogene probes previously shown to be amplified in

human malignancies. This indicated that the hypothesized target gene(s) carried within the

amplification unit could be either novel transcribed sequences or previously identified

transcribed sequences not known to be amplified in human malignancies. As stated in

detail in Chapter I, amplified DNA is well documented to carry genes important to the

progression, diagnosis and treatment of the particular tumor type in which they are

amplified (6). Therefore, identification of the hypothesized transcribed sequences carried

within the ST -23987 amplification unit would be of significant interest. Experiments

designed to clone large sections of the amplification unit were initiated in an attempt to

identify transcribed sequences hypothesized to be carried within the amplified sequences.

43

MATERIALS AND METIIODS

CELL CULTURES AND TUMOR SAMPLES. Human tumor cultures were

provided by the UACC laboratory, unless specifically noted. Fibrosarcoma cell line HT-

1080 was kindly provided by Dr. Paul Meltzer. Cell culture conditions were as described

in Leibovitz, (1986) (93). Tumor samples were kindly provided by Dr. Avery Sandberg

(ST-23987, ST-23493, ST-24069, ST-23386, 870141, 851184, 850980), Dr. Stanley

Leong (9693, 3938504, 5295464,5273737), Dr. Felix Mitelman (2078,2178) and the

Cooperative Human Tissue Network (88-06- 203, 88-01-039, 88-01-702, 88-12-03, 88-

01-131, 88-03-097, 89-1-277).

MODIFIED IN GEL RENATURA noo. Modified in gel renaturation was

performed exactly as described in "Materials and Methods", Chapter II.

PROBES. Human specific SINE sequence BLUR8 was obtained from Oncor, Inc.

(92). All oncogene probes were obtained from the American Type Culture Collection,

except gli (kind gift of Dr. Ken Kinzler), and int-2 (kind gift of Dr. Bert Vogelstein).

PROBE LABELLING. BLUR8 and oncogene sequences used to probe Southern

blots and in gel renaturation blots were radiolabelled by the Feinberg and Vogelstein

oligolabelling method as previously described in "Materials and Methods", Chapter II.

SOUTHERN BLOT HYBRTDIZA TION AND AUTORADIOGRAPHY. In gel

renaturation blots and Southern blots were prehybridized, hybridized, and

autoradiographed as previously described in "Materials and Methods", Chapter II.

CHAPTER IV

CLONING OF AMPLIFIED DNA SEQUENCES FROM MFH ST-23987

AND SARCOMA ST -23493

IN1RODUCflON

44

As stated, the ultimate aim of this project was to isolate unidentified amplified

sequences in a human cancer. It was my speculation that transcribed sequences important

to sarcoma progression are carried within the MFH ST-23987 amplification unit recognized

by modified in gel renaturation. In order to prove the hypothesis that a biologically

important gene is carried within the amplification unit present in MFH ST-23987, large

sections of the amplification unit had to be cloned for certain identification of the putative

transcribed sequences.

In gel renaturation not only provides a method for detection of an unknown

amplified sequence, it also provides a method to clone fragments of an amplification unit.

Also, as previously mentioned, the in gel renaturation cloning strategy on which this

project was based has been successfully employed in the identification and cloning of the

amplified drug resistance gene MDR 1 (2).

The procedure for cloning a sequence from an amplified domain was based on in

gel renaturation carried out as nonnal except that the DNA was unlabeled and the Sl

nuclease step is omitted. Therefore, restriction fragments remained in the gel, but at full

length with intact "sticky ends". After renaturation, fragments are recovered from the gel

by electroelution in dialysis tubing. High copy number, double stranded fragments are

ligated into a plasmid vector.

Although short, (5-20 kb) in gel renaturation generated amplified clones by

themselves would have clearly verified the existence of an amplification of nonnally single

copy sequences in ST-23987 DNA, the certainty of coamplified DNA made the likelihood

of obtaining in gel renaturation selected clones which recognized transcribed sequences

remote. Further, in gel renaturation cloning alone would not be practical to isolate the large

regions required to ultimately identify the complete transcribed region. Therefore, a

strategy was developed which combined in gel renaturation cloning, peR modified in gel

45

renaturation cloning, and conventional bacteriophage lambda cloning to isolate a significant

quantity of the amplification unit.

RESULTS

IN GEL RENAIURA TION GENERA TED CLONES. Ten insert positive clones

generated by in gel renaturation cloning procedures were isolated. End-labelled RNA

probes generated from the inserts were hybridized to Southern blots of equal quantities ST-

23987 and normal human placenta control DNA. One 4.3 kb clone, pMAC895, hybridized

to a 4.3 kb EcoRI fragment which was 1O-to-15-fold more intense in the ST-23987 DNA

lane when compared to the human control DNA lane (Figure 8). The remainderof the

clones hybridized to repetitive sequences in all lanes of the Southern blots.

Because of the small library size obtained by the in gel renaturation cloning method

(limited by the quantity of DNA recovered after in gel renaturation), a modification on the

basic in gel renaturation cloning technique was developed. The polymerase chain reaction

(peR) technique was used to specifically amplify the numberofinsert positive clones

(112). Briefly, the in gel renaturation cloning procedure was followed through the

recovery steps (as described in "Materials and Methods"). The recovered DNA was

digested with restriction endonuclease MhoI. The DNA was then ligated into plasmid

pKS+ made compatible by EcoRI and BamHI digestion. Insert sequences were amplified

by PCR using defined sequences flanking the cloning site of pKS+ as primers. The PCR

amplified sequences were digested with EcoRI and XbaI. The double digested sequences

were religated into pKS+ also EcoRIIXbaI digested. An aliquot of the ligation reaction was

transformed into competent DH5a cells, and the transformed cells were plated onto

LB/AMP plates. White, insert containing colonies were selected and prepared as described

before. Inserts were screened by hybridizing Southern blots of ST-23987, and human

placenta control DNA with oligolabelled probes generated from the cloned inserts. Clones

which were present as single copy sequences in the control lanes but present in multiple

copies in the ST-23987 lanes were presumed to have been isolated from from the ST-

23987 amplification unit and subjected to further analysis.

Forty seven insert containing clones isolated by this newly developed method were

oligolabelled and hybridized to Southern blots of equal quantities of EcoRI digested ST-

23987 and normal placenta control DNA. Three clones, pMACI5, pMAC20, and

pMAC30 were present as a single copy band in human placenta control DNA but amplified

46

-4.3 kb

Figure 8. Hybridization Pattern ofpMAC895. Lane 1: 5 ug of ST-23987 DNA. Lane 2: 5 ug of human placenta control DNA. The 4.7 kb in gel rentaturation generated clone hybridized to a 4.7 kb EcoRI fragment. pMAC895 is amplified W-to-15-fold in ST-23987 DNA. .... W-to-15-fold in ST-23987 DNA (Figure 9). pMAC15 hybridized to a 10.5 kb fragment, pMAC20 hybridized to a 9.8 kb fragment, and pMAC30 hybridized to a 4.7 kb fragment. Three clones hybridized to single copy fragments in all lanes of the Southern blots and the remaining 41 clones hybridized to repetitive sequences

47

-1O-to-15-fold in ST-23987 DNA (Figure 9). pMAC15 hybridized to a 10.5 kb fragment,

pMAC20 hybridized to a 9.8 kb fragment, and pMAC30 hybridized to a 4.7 kb fragment.

Three clones hybridized to single copy fragments in all lanes of the Southern blots and the

remaining 41 clones hybridized to repetitive sequences.

In total, four in gel renaturation generated clones from the ST- 23987 amplification

unit were isolated. In order to determine if these clones were previously identified

sequences, 77 bases of clone pMAC30 and 145 bases of clone pMAC895 were sequenced

and analyzed for homology to known sequenceg contained in GenBank files (Figure 10).

The sequenced regions of these clones were not homologous to any known human

sequences (including LINE, SINE, 0, Kpn, THE-I, and LRS repetitive sequences)

[92,108-111] nor to any other sequence contained in GenBank files. Since these clones

were clearly human sequences (by virtue of the hybridization to human DNA on Southern

blots) the sequence data was further evidence that pMAC30 and pMAC895 were single

copy human sequences amplified in ST-23987.

SOUTHERN BLOT ANALYSIS OF ADDmONAL SARCOMAS AND HUMAN

CULTURES. Probes generated from the in gel renaturation selected clones were

hybridized to Southern blots of equal quantities of EcoRI digested DNAfrom various

human malignancies and normal tissues in order to further verify the normally single copy

status and to determine if these sequences were commonly amplified in other human

malignancies (Tables 3 and 4). Some or all of the four clones hybridized to single copy

fragments of respective uniform size in 46 of 49 human DNA samples. Clones pMAC30

and pMAC15 hybridized to amplified fragments of respective uniform size in three adult

sarcomas lanes: MFH ST-24069, leiomyosarcoma ST-23493, and liposarcoma 870141

(Figure 11; Data not shown for 870141). Clones pMAC15 and pMAC30 were amplified

5-to-7-fold in ST-24069 and 870141 DNA, and -20-fold in ST-23493 DNA.

SARCOMA ST-23493 BAcrnRIOPHAGE LAMBDA GENOMIC CLONES

ST-23493 Genomic Librruy Screeninl:. A bacteriophage lambda genomic library

was constructed from EcoRI partially digested ST-23987 DNA. A total of 60k plaques

from the library were screened using in gel renaturation generated clones pMAC15 and

pMAC30 as probes. Two clones were isolated which hybridized to clone pMACI5, 493.1

and 493.2 (Figure 12). EcoRI digests of 493.1 and 493.2 were size fractionated in agarose

pMAC15 .10.5kb

pM AC20 .9.8kb i.,

pMAC30 .•

pMAC895 - ... .y .4.3kb

, Hu-ets-1 •••• .. 5.4kb

1 2 3 4

48

Figure 9. Panel of Amplified Clones Isolated from ST-23987 DNA by In Gel Renaturation. Lane 1: normal human blood 1. Lane 2: ST-23987. Lane 3 normal human placenta. Lane 4: normal human blood 2. All lanes were equally loaded with 5 ug of EcoRI digested DNA. pMACI5, a 600 bp EcoRI/MboI clone hybridized to a 10.5 kb EcoRI fragment; pMAC20, a 350 bp EcoRI/MboI clone hybridized to a 9.8 kb EcoRI fragment; pMAC30, a 275 bp EcoRI/MboI clone hybridized to a 4.7 kb EcoRI fragment; and pMAC895, a 4.3 kb EcoRI clone hybridized to a 4.3 kb EcoRI fragment. Hu-ets-l served as a loading control.

49

pMAC30 10 20 30 40 50

CGATGTCACT TTGCCATCTT TCCTCTAAAT CTCGTGACAA CCAAGAGGGG GCTACAGTGA AACGGTAGAA AGGAGATTTA GAGCACTGTT GGTTCTCCCC

60 70 77 ATTGCTTCTT CTTCATATTA CAAGGTA TAACGAAGAA GAAGTATAAT GTTCCAT

pMAC895 10 20 30 40 50

TAGCAAACCC ATATGCAAGG GTAGTAAGCT AGTCATTGGG GATACAGAAA ATCGTTTGGG TATACGTTCC CATCATTCGA TCAGTAACCC CTATGTCTTT

60 70 80 90 100 TGAATAAAAC ATAGTGTTTG CTGCCATAAC GCGATTTGTA ATTGCAAACA ACTTATTTTG TATCACAAAC GACGGTATTG CGCTAAACAT TAACGTTTGT

110 120 130 140 145 AAATAGTTGC AGAATGGCGT GTGTCAGGGA AAACAGTCGC ATAAA TTTATCAACG TCTTACCGCA CACAGTCCCT TTTGTCAGCG TATTT

Figure 10. DNA Sequence Data for Clones pMAC30 and pMAC895.

50

Table 3. Southern Blot Analysis of Human Tumor Cultures.

Sample Tissue number type Amplified clone probes

pMACl~ pMAC3Q pMAC2Q pMAC82~ MHP np CNB nb KNB nb UACC558 mel * * * MNCLl-8 " * * * MMN " * * * UACC97 " * * * UACC827 " * * UACC457 " * * * UACC066 ova * * * UACC188 " * * UACC341 " * * * UACC1053 " * * * UACC326 " * * UACC896 bla * * UACC735 SCLC * * UACCl196 col * * UACC1027 pan * * * UACC732 bre * * * UACC812 " * * * UACC893 " * * * UACC183 " * * * UACC245 " * * * UACC417 lun * * * UACC469 neu * * * UACC289 ade * * *

+ = greater than single copy per haploid genome, - = single copy per haploid genome, * = not determined, nb = normal blood, np = normal placenta, mel = melanoma culture, ova = ovarian carcinoma culture, bla = bladder carcinoma culture, SCLC = small cell lung carcinoma, col = colon carcinoma culture, pan = pancreatic carcinoma, bre = breast carcinoma, lun = non-SCLC lung carcinoma, neu = neuroendocarcinoma, ade = uncertain adenocarcinoma.

51

Table 4. Southern Blot Analysis of Direct Sarcoma Tissue Samples

Sample Sarcoma number type Amplified clone probes

pMAC3Q pMAC15 pMAC2Q pMAC825 ST-23987 MPH + + + + ST-24069 + + 12685 * 07030 * 2078 * 2178 * 3938504 5295464 5273737 II

3086/88 II * * * 88-06-203 II * * * 88-01-039 II * * * 88-01-70 II * * * 88-12-03 II * * * ST-23493 lei + + * 88-01-131 II * * * 88-03-097 II * * * 89-1-277 II * * * 870141 lip + * * 851184 II * * 850980 II * * ST-23386 rha * * HT-1080 fib *

+ = greater than single copy per haploid genome - = single copy per haploid genome * = not determined MFH = malignant fibrous histiocytoma lei = leiomyosarcoma lip = liposarcoma rha = rhabdomyosarcoma fib = fibrosarcoma cell line.

52

• • • 4.7kb

"~ '.~

123456

Figure 11. Panel of Sarcomas Positive for Amplification of pMAC30. Lane 1: MFH ST-23987. Lane 2: MFH 2178. Lane 3: Normal human blood 1. Lane 4: Leiomyosarcoma. ST-23493. Lane 5: Normal human placenta Lane 6. MFH ST-24069. All lanes were loaded with 5 ug of EcoRI digested DNA.

R

493.1

493.2

B

493.5

R I

pMAC15 -

R I

pMAC30 R R

I : I

R I

-8R

jO II He I

- 1 kb scale

R I

R I

R

I

R ,

53

Figure 12. Restriction Maps of the Three Unique Bacteriophage Lambda Clones Isolated from ST-23493 DNA. The scale bar represents 1 kb. R = EcoRI S = SstI Xa = Xbal Xo = XhoI.

54

gels. Both 493.1 and 493.2 contained a 10.5 kb EcoRI insert fragment. Bacteriophage

lambda clone 493.1 contained an additional 6.0 kb flanking fragment and clone 493.2

contained an additional 5.2 kb flanking fragment. Southern blots of the EcoRI digested

lambda clones 493.1 and 493.2 were probed with pMACI5. pMAC15 hybridized

intensely to the 10.5 kb insert fragment, confinning that cloned 10.5 kb EcoRI fragment

was the same 10.5 kb fragment which hybridized to pMAC15 on Southern blots of EcoRI

digested human DNA.

Two lambda clones were isolated by virtue of their hybridization to clone pMAC30

(Figure 12). EcoRI digests of lambda clones 493.4 and 493.5 were size fractionated in

agarose gels. Both clones contained 4.7 kb, 0.5 kb, 2.8 kb, and 5.0 kb EcoRI insert

fragments. Therefore, lambda clones 493.4 and 493.5 were identical clones. pMAC30

was hybridized with Southern blot of EcoRI digested bacteriophage lambda clone 493.5.

pMAC30 hybridized intensely to the 4.7 kb insert fragment, confmning that the cloned 4.7

kb EcoRI fragment was the same EcoRI fragment which hybridized to pMAC30 on

Southern blots ofEcoRI digested human DNA. Bacteriophage lambda clone 493.5 was

mapped by restriction enzyme and Southern blot analysis and shown to be -14 kb of

colinear fragments.

Plasmid Subclonin~. The seven EcoRI fragments contained within the three unique

lambda genomic clones (493.1,493.2, and 493.5) were ligated into EcoRI digested pKS+.

Insert positive plasmid clones were selected. Size fractionated EcoRI digests of the

subclones were used to identify seven subclones with inserts of the same size as the

original bacteriophage lambda clone EcoRI fragments (p1O.5, p6.0, p5.2, p5.0, p4.7,

p2.8, and pO.5). Plasmid subcloning provided a way to produce large quantities of each

EcoRI fragment for subsequent analysis.

Southern Blot Analysis of Amplified Clones. Southern blots of equal quantities of

ST-23493 and nonnal placenta control DNA were hybridized with either end-labelled

probes or oligolabelled probes preassociated with human DNA generated from each of the

subclones (Figure 13). Both the p6.0 and p5.2 subclones recognized single bands of

proper size and were amplified in ST-23493 DNA to the same degree as pMACI5.

Therefore, lambda clones 493.1 and 493.2 represent -19.7 kb of colinear DNA amplified

in ST-23493. Subclones p4.7 and pO.5 hybridized to single bands which were amplified

in ST-23493 DNA to the same degree as all previous amplified clones tested. Repetitive

A 9.0-

6.5-

B

:::~ E I i

I-P10.S

I -p5.2

1 2 3 4 5

Figure 13. Southern Blot Analysis of Plasmid Subclones. A. p6.0 probed and B.p5.2 and pl0.5 probed Southern blot. Lane 1: ST-24069. Lane 2: normal human placenta (MHP). Lane 3: ST-23493. Lane 4: MFH 2178. Lane 5: ST-23987. All lanes were equally loaded with 5 ug ofEcoRI digested DNA.

55

56

DNA free probes could not be generated from sub clones p2.8 and p5.0. and the amplified

status of these fragments could not be verified. Howeverz it is unlikely that bacteriophage

lambda clones 493.4 and 493.5 represent nonlinear, hybrid molecules with EcoRI

fragments of exact equal sizes. p2.8 and p5.0 clearly contain human sequences by virtue

of the intensity of hybridization to repetitive human sequences. Because they are colinear

with EcoRI fragments which are amplified in ST-23493 DNA it was assumed that they too

were amplified. Therefore, the fragments isolated from lambda clones 493.5 and 493.4

represent -14 kb of colinear DNA amplified in sarcoma ST-23493. The three amplified

lambda genomic clones combined represent -33.7 kb ofnonnally single copy sequences

amplified in ST-23493.

Northern Blot Analysis of Amplified Clones. OligolabeUed probes generated from

each of the seven plasmid subclones were preassociated with human DNA hybridized to

Northern blots of total cellular RNA from2 MFH tumors and from four other tumor types.

The p5.2 kb clone hybridized intensely to an approximately 1.4 kb transcript in the MFH

lanes and less intensely in the control lanes (Figure 14). However, due to the large amount

of background hybridization, no conclusive result could be be drawn from this

observation. This result was, however, a strong indication that transcribed sequences are

present within this fragment. There was no detectable hybridization of subclones pO.5 and

p4.7 to the Northern blots, suggesting that the these clones lacked transcribed sequences.

Hybridization with the four remaining plasmid subclone probes provided inconclusive

evidence as to the presence of transcribed sequences. The intensity of background

hybridization with these four subclones made observation of any putative transcribed

sequences undetectable.

DISCUSSION

In gel renaturation cloning procedures provided a practical and rapid method to

directly clone amplified restriction fragments. The vast majority of single and low copy

number fragments recovered from renaturation gels remained single stranded following the

renaturation step, and therefore were unable to produce viable insert-plasmid ligations.

High copy number and amplified fragments regained double stranded status during the

renaturation step and produce viable ligation products. However, due to the constraints of

the library size generated by in gel renaturation cloning and the insufficient amount of

-285

-185 ',!' -1.4kb

1 2 3 4 5 6

57

Figure 14. Northern Blot Analysis with Subclone p5.2. Lane 1: RNA size marker (not shown). Lane 2: Panc 1, a pancreatic carcinoma cell line. Lane 3: HT-1080, a fibrosarcoma cell line (Both kindly provided by Dr. Paul Meltzer). Lane 4: CEM, a human T-cell leukemia cell line (kindly provided by William Beck). Lane 5: MFH 8901. Lane 6: MFH 9693. 20 ug of total cellular RNA was loaded per lane. The preassociated p5.2 probe hybridized to a -1.4 kb transcript in the MFH lanes.

additional ST-23987 tissue available for further cloning gels, development of the PCR

modified protocol was necessary to insure sufficient numbers of amplified clones for

additional experiments. The PCR modified method proved to be an effective method to

maximize the data that was generated from the remaining quantity of ST-23987 DNA.

58

Southern blot analysis of additional adult sarcoma DNA samples using the in gel

renaturation generated amplified clones as probes demonstrated that the amplification event

originally detected in MPH ST-23987 was a recurring feature of adult sarcomas. This was

perhaps the most significant observation of this study. The occurrence of an individual

case of DNA sequence amplification would itself be a interesting observation. The

observation of a recurring amplification in four different adult sarcoma specimens validated

the hypothesis that transcribed sequences important in sarcoma progression are carried

within the amplification unit. It would be unlikely that a DNA sequence amplification

would spontaneously arise within several sarcoma specimen without transcribed sequences

that are advantageous to sarcoma progression being carried within the amplification unit.

Two in gel renaturation generated probes, pMAC15 and pMAC30, were amplified in all of

the amplification positive sarcomas in which they were tested. Since pMAC15 and

pMAC30 were always amplified, these clones are likely to be relatively close to the core of

the amplification unit. As stated in Chapter I, the amplified target genes have been shown

to be located within the core region of amplification units (57). Clones pMAC20 and

pMAC895 were more likely to be a significant distance from the core sequences and

therefore, were excluded from further analysis.

In gel renaturation generated clones clearly demonstrated that a specific DNA

sequence amplification was a recurring event in adult sarcomas. For the reasons outlined in

Chapter I, large stretches of the amplified domain need to be cloned in order to identify the

hypothesized transcribed sequences carried within the amplification unit. The sarcoma with

the highest level of DNA sequence amplification detected was leiomyosarcoma ST-23493.

A EcoRI partially digested genomic library was constructed from ST-23493 based on the

this high grade amplification and on the quantity of material available for the library

construction and further analysis. The number of clones necessary to screen in order to

find amplified clones decreases proportionately with increasing levels of amplification. The

three unique bacteriophage lambda clones isolated using pMAC15 and pMAC30 clearly

contained colinear EcoRI fragments carried within the ST-23493 amplification unit. These

clones represent -34 kb of the amplification unit.

59

Northern blot analysis with plasmid subclones generated from the lambda genomic

clones provided inconclusive results. 34 kb may represent only a small fraction of the

entire amplification unit and, therefore, it is was unlikely that I would find the target gene

within the cloned sequences. However, there was a strong indication of transcribed

sequences within the subclone p5.2. The amount of background hybridization due to the

quantity of repetitive sequences within the clone obscured any conclusive result. There

were clearly no transcribed sequences within pO.5, p4.7 kb and p6.0 subclones. The

absence of repetitive sequences in these clones allowed conclusive determination of this

observation. The remaining clones (plO.5, p2.8 and p5.0) provided inconclusive data due

to the intensity of hybridization of the repetitive sequences carried within the subclones.

Further experimentation beyond the scope of this dissertation will be required to verify the

speculation that transcribed sequences are present within the p5.2 subclone. Future

experiments are discussed fully in Chapter V.

MATERIALS AND ME1HODS

IN GEL RENATURATION CLONING OF AMPLIFIED FRAGMENTS. The

cloning technique was based on the same principles as in gel renaturation amplification

detection (1). 100 ug of EcoRI digested tumor DNA was size fractionated in 4 lanes of a

1 % GTG agarose gel (FMC) in Ix TAE at 40 mV for .... 48 hrs. HindIII digested

bacteriophage lambda size markers were co-fractionated on either side of the 4 lanes. The

gel was EtBr stained and photographed The region of the gel containing genomic

fragments ranging from .... 5-25 kb was isolated and placed in a box with a tight lid. In gel

renaturation steps (as previously described in "Materials and Methods", Chapter II) were

followed up to the SI nuclease digestion step. The gel was then washed with Ix TAE for

15 min x 4. The gel was cut to fit in a prepared dialysis bag, the bag was filled with 5 ml

of Ix TAB buffer and electrophoresed at 40 V/cm overnight. The current was reversed for

2 min at 80 V /cm to remove DNA from the inside surface of the dialysis bag. The Ix TAB

buffer containing the electroeluted DNA fragments was removed from the bag, the inside of

the bag was thoroughly rinsed with 5 ml of Ix TAE and the fractions were pooled This

solution was phenoVchloroform/isoamyl alcohol (25:24:1) extracted three times and the

solution was concentrated by sec-butanol to .... 0.5 ul volume. 1 ul of 95% ethanol was

added, the solution was mixed and placed at -200C for 60 min. The tube was

microcentrifuged for 15 min, the pellet was washed with 70% ethanol and air dried. The

pellet was resuspended in 10 ul of TE and was quantitated by DAPI fluorescence (as

previously described in "Materials and Methods", Chapter II). This recovered DNA was

mostly single stranded fragments. Double stranded fragments which reannealed due to

high copy number were suitable for ligation within a plasmid vector.

60

Ligations were performed in 50 ul volumes with 0.5 ug of EcoRI digested calf

intestinal phosphatase treated pKS+ (Stratagene), 0.1 ug of recovered amplification unit

enriched insert DNA, 5 ullOx ligase buffer supplied by the manufacturer, 10 mM ATP,

and 3 ul (3U) ofT4 DNA ligase (Promega). The reaction was incubated at 40C overnight.

S ul of the ligation reaction was transformed into 100 ul of DHSa competent cells

(BRL) according to the manufactures protocol. 50 ul of2% X-gal (Fischer) and 100 ul of

100 mM IPTG was spread onto the surface of 150 mm LB/Amp plates 30 min prior to

plating. 200 ul of the final 1 ml volume of transformed cells was plated per plate. The

plates were incubated at 370C overnight. White, insert containing colonies were selected

from among the background blue colonies. The selected clones were grown in 2 ml

volumes in LB/Amp broth at 37 0C overnight. A 500 ul aliquot was saved and mixed with

500 ul of 50% glycerol for a stock culture. The remainder was used for plasmidextraction

performed according to the protocol presented with the Geneclean kit (Bio 101).

PCR AMPLIFICATION OF IN GEL RENATURATION GENERA TED

CLONES. Procedures for "In Gel Renaturation Cloning Procedures" (above) were

followed through the DNA recovery steps. 1 ug of recovered DNA was digested with

MboI restriction enzyme in 20 ul of the appropriate Ix buffer overnight at 37oC. The

reaction was phenol/chloroform/isoamyl alcohol (25:24:1) extracted and then

NaOAce/isopropanol precipitated The tube was microcentrlfuged for 15 min. The pellet

was washed with 70% ethanol and air dried. The pellet was resuspended in 10 ul of

distilled H2O. 1 ul was added to a ligation reaction (50 ul final volume) with 0.5 ug

EcoRIlBamHI digested pKS+, Ix ligase buffer, 10 mM ATP, and 1 ul (3 U). The reaction

was incubated at 40C overnight. The reaction was diluted to 100 ul. The small percentage

of plasmids with inserts was enriched by selective amplification by the polymerase chain

reaction (112). 2 ul of the diluted ligation reaction was added to a 20 ul reaction solution

containing 2 mM dA TP, 2 mM dGTP, 2 mM dCTP, 2 mM dTTP, 0.1 pM/ml of M13

2Omer/KS+ 18mer primers, 0.1 pM/ml reverse primer, Ix ABC Taq polymerase buffer (50

mM KCI, 1.5 mM MgCI2, 10 mM Tris-HCI (pH 8.3), 0.2% gelatin, and 1 U of Taq

polymerase (Cetus). The reaction was allowed to cycle 30 times in a thermocycler (MJ

61

Research) using 550 C for 30 sec as the annealing conditions, 750 C for 40 sec as extension

conditions, and 950 C for 40 sec as the denaturing conditions. The reaction was added to a

200 ul XbaI restriction enzyme reaction containing 20 ul of lOx XbaI restriction buffer, 10

ul (80 U)XbaI enzyme (BRL), and 70 ul distilled H20. The tube was vortexed and the

reaction was incubated at 370 C for 4 hrs. The DNA was phenoVchlorofonnlisoamyl

alcohol extracted, NaOAce/isopropanol precipitated, and resuspended in 10 ul of H2O.

0.5 ug of the EcoRIlXbaI digested DNA was added to a 50 ulligation reaction with

1 ug of EcoRIlXbaI cut pKS+, Ix ligation buffer, 10 mM ATP, and 3 U ofT4 DNA ligase

and incubated at 40 C overnight. The ligation reaction was digested with restriction enzyme

SmaI in order to reduce insert lacking background colonies following subsequent

transformation and plating steps. 5 ul of the Smal digested ligation reaction was used to

transform 100 ul of DH5a competent cells (BRL) according to the accompanying protocol.

White, insert containing colonies are selected from among reduced blue background

colonies. Plasmid DNA was isolated from insert containing colonies according to the

Geneclean (BiolOl) rapid boiling protocol, N,\OAce/isopropanol precipitated and

resuspended in 10 ul ofTE. Cloned insert size was determined by electrophoresis of

EcoRIIXbaI double digested plasmids in 3% NuSieve low melting point agarose (FMC)

using Mbol digested pBR322 (BRL) as a comparative size marker.

PROBE LABELLING. Plasmid clones isolated by the in gel renaturation cloning

procedure were Mbol digested and labelled by synthesis of complimentary radioactive

RNA transcripts using 32pCTP (ICN) and the TIm RNA polymerase system provided by

the manufacturer (Stratagene).

Clones generated from the PCR modified in gel renaturation cloning procedure

were digested with EcoR! to liberate the cloned insert andthe digested sequences were

electrophoresed in 1 % low melting point gel in Ix OLRB (40 mm Tris-Ace (PH 8.0), 1

mM EDTA, 5 mM NaOAce). The insert fragments were excised from the gel, diluted 3-

fold with distilled H20, and labelled by the oligolabelling method (as previously described

in "Materials and Methods", Chapter IT).

SOUTHERN BLOT ANALYSIS. Southern blots of tumor experimental DNA and

normal human control DNA were prehybridized, hybridized with oligolabelled or RNA

probes, washed, and autoradiographed (as previously described "Materials and Methods",

Chapter IT).

SEOUENCING. In gel renaturation clones were sequenced by the 17 DNA

polymerase dideoxynucleotide sequencing method developed by Tabor et al. (113). The

protocol followed was according to the manufacturer of Sequenase 2.0 T7 DNA

polymerase sequencing system (USB). Sequences were analyzed by comparison with

sequences stored within GenBank sequence files (illI).

BAcmRIOPHAGE LAMBDA GENOMIC LffiRARY CONSTRUCTION AND

SCREENING

62

Preparation of ST-23493 Insert DNA. DNA extracted from sarcoma ST- 23493

was partially digested with EcoR! restriction enzyme so that most of the DNA fragments

fractionated between the 23 kb and 9.5 kb fragments of Hindill digested bacteriophage

lambda DNA when electrophoresed in a 0.3% agarose gel in Ix TBE. The digestion

conditions were determined by time point assays with aliquots of genomic DNA and

enzyme and then scaled up to a reaction volume sufficient to partially digest 10 ug of ST-

23493 (a quantity sufficient for subsequent cloning experiments). 10 ug of DNA was

incubated in a volume reaction of 200 ul (Ix EcoRI restriction enzyme buffer (BRL), 1 mM

spermidine, 1 U of EcoR! restriction enzyme at 370 C overnight. The EcoR! enzyme was

deactivated by incubation of the reaction at 650C for 10 minutes. The amount of digestion

was assessed by size fractionating a 1 ug aliquot in a 0.3% agarose gel in Ix TBE with

Hindill digested bacteriophage lambda size marker DNA to be certain digestion was limited

to the intended fragment sizes. The reaction was subsequently phenoVchloroform

extracted, precipitated with 132 ul of 5 M NaOAce and 335 ul of isopropanol, and

resuspended in a 20 ul ofTE. The partially digested DNA was CIP treated (Boehringer) in

a 50 ul reaction (20 mM Tris-HCI, pH 8.0, 1 mM MgCI2, 1 mM ZnCl2 and 1 U CIP) for

30 min at 370 C. The reaction was stopped by incubation of the reaction at 750 C for 10

min.

Ligation. Packaging. and Titering. 1 ug of EcoRI digested lambda Dash cloning

vector (Stratagene) was incubated with 0.25 ug of CIP treated ST -23493 DNA, Ix ligation

buffer (Promega), 10 mM ATP, and 2 U ofT4ligase (Promega) in a reaction volume of 10

ul overnight at 40 C. 2 ul of the ligation reaction was packaged with Gigapak Gold II

packaging extract according to the manufacturers protocol (S tratagene). The titer of the

packaging reaction was determined by transfecting 200 ul of 0.D.600=O.5 P2392 E.coli

63

cells (Stratagene) (grown to saturation in LB, 10 mM MgS04, and 0.2% maltose)

resuspended in 10 mM MgS04 with 1 ul of the 500 ul packaging reaction with at 370 C for

15 min, adding 3 ml of 0.7% agarose in LB and plating the cells on 82 mm LB plates. The

plates were incubated overnight at 370 C overnight and the titer determined by counting the

plaques.

Platin~ and Transferrin~ the Bacteriopha~e Lambda Librruy. Plating and

nitrocellulose transfer of the bacteriophage lambda genomic library was based on the

protocol of Arber et al. (114). A volume of the packaging reaction corresponding to 20k

pfu was incubated with 0.5 ml of O.D.600=O.S P2392 cells (grown as before) and

resuspended in 10 mM MgS04 for 15 min. 7 ml of 0.7% agarose dissolved in LB was

added and the cells were plated onto five 150 mm LB plates. The plates were incubated

overnight at 37oC. Each plate of plaques was transferred to two 137 mm S&S NC

nitrocellulose discs (Schleicher and Schuell) for 5 mins, and air dried for 10 mins. The

discs were subsequently treated to denaturing conditions (0.2 M NaOH/l.S M NaCl) for 2

min, buffering conditions (0.4 M Tris-HCI (pH 7.6)/2x SSC) for 2 mins, washing

conditions (2x SSC) for 2 min and finally, were UV crosslinked for 30 sec (UV

Statalinker, Stratagene) according to the manufacturers recommendations.

SCreenin~ the Bacteriopha~e Lambda Library. The plaque lifts were prehybridized,

hybridized with oligolabelled pMAClS and pMAC30 probes, washed, and

autoradiographed exactly as previously described above for Southern blot analysis (as

previously described in "Materials and Methods", Chapter m. Both replica lifts from the

same plate wereassayed simultaneously to be certain that hybridization to any given lift was

not a false positive result. Plaques which hybridized to the probes were picked, replated

and secondarily screened in order to isolate an individual plaque.

BacteriQPha~e Lambda DNA Extraction. Bacteriophage lambda clones which

hybridized to the pMAClS and pMAC30 and were grown to confluence on several 82 mm

LB plates ~md the DNA was isolated by the DE-52 resin (Whatman) method developed by

Meese et al. (115). Southern blots of EcoRI digests of these clones were prehybridized,

hybridized to oligolabelled pMAC15 and pMAC30 probes, washed and autoradiographed

(as previously described in "Materials and Methods", Chapter II).

64

PLASMID SUBCLQNING. Plasmid subclones of all 7 EcoRI bacteriophage

lambda clone fragments were generated by incubating 0.1 ug of digested bacteriophage

lambda clone DNA with 1 ug of EcoRI digested, CIP treated pKS+ (Stratagene), Ix

ligation buffer in a 10 ul volume. Transformation of the ligation reaction into competent

DHSa cells as described in the manufactures protocol (BRL). Insert positive clones were

selected, EcoRI digested, and size fractionated in 1 % agarose gels in Ix TBE. Clones with

inserts of the proper sizes were hybridized with Southern blots of ST-23493 DNA to

ascertain that they were from the amplification unit

NORTHERN BLOT ANALYSIS

RNA Preparation. Total cellular RNA was prepared with baked glassware and

solutions prepared with DEPC-treated H20. Tumor tissue was frozen in liquid nitrogen

and pulverized with a hammer. 1 gm of powdered tissue was added to 10 ml of tissue

guanidinium solution consisting of 50 mM Tris-HCL (pH 7.5), 10 mM EDTA, 50 ml/l b­

mercaptethanol, and 5 M guanidinium isothyocyanate. The viscosity was lessened by

drawing the lysate in and out of a 20-gauge needle. 1.5 ml of a 5.7 M CsCI solution was

placed in a 13 x 51 mm ultracentrifuge tube. 3.5 ml of cell lysate was layered on top of the

CsCI cushion. The samples were centrifuged at 35k rpm in a Beckman SW- 28 rotor

overnight at 180 C. The supernatant was carefully removed and the RNA pellet was

resuspended in 360 ul TES (10 mM Tris-HCI (pH 7.4), 5 mM EDTA and 1 % SDS)

solution. 40 ul of 3 M NaOAce was added. The pellet was precipitated with 1 ml of

ethanol and placed in a dry ice/ethanol bath for 30 min. The RNA was pelleted,

resuspended in 360 ul of H20 and reprecipitated as before. The pellet was resuspended in

H20 and quantitated by spectrophotometry at A260 and A280.

Northern Blots. Total RNA was size fractionated in agarose/formaldehyde gels and

transferred to nitrocellulose based on the methods described by Lehrach et al. (116). 20 ug

of precipitated and dried RNA samples were resuspended in 5.5 ul diethylpyrocarbonte

(DEP) treated water, 1 ull0x MOPS (0.37 gms Na2EDTA, 0.82 gms NaOAce, 9.25 gm

3(-[N-Morpholino]propanesolfonic acid), 90 uls DEP treated water, pH 7.0),3.5 u137%

formaldehyde, and 10 ul formamide. The samples were denatured at 650 C for 10 min and

2 ul of lOx RNA loading buffer and 0.5 ul of ethidium bromide were added. The samples

were loaded into 14 cm x 12 cm gels consisting of 1 gm agarose, 72 ul ofDEP treated

65

water, 10 ul of lOx MOPS, and 17.9 ul offonnaldehyde. The gels were electrophoresed at

25 m V overnight and photographed the next day. The size fractionated RNA was

transferred to nitrocellulose by capillary blotting in lOx SSC overnight. The RNA was

then crosslinked to the nitrocellulose blot by UV irradiation in a Stratalinker UV

Crosslinker according to the manufacturers specifications (Stratagene).

Probe Preassociation. Probes generated from plasmid subclones of the amplified

bacteriophage lambda genomic clones were labelled by the oligolabelling method (as

previously described in "Materials and Methods", Chapter n). These labelled probes were

preassociated with total human DNA according to the method of Sealey et al. (94) in order

to allow detection of transcripts in the subsequent Northern blot hybridization steps.

Hybridization and Autoradiography. Northern blots were prehybridized,

hybridized, washed and autoradiographed exactly as for Southern Blot analysi3 (as

previously described in "Materbls and Methods", Chapter ll).

66

CHAPTER V

SUMMARY

This dissertation was based on the theory that acquisition of genetic instability

generates variants which have the potential to become a cancer cell, and that continued

genetic instability facilitates its continual progression (117). Cancer cells are characterized

by genetic alterations including translocation, rearrangement, deletion, aneuploidy, and

amplification (118,119). These characteristic genetic alterations suggest a significant

difference in the genetic stability of tumor cells as compared to normal diploid cells. This

inherent genetic instability of cancer cells is manifest in tumor cell heterogeneity and the

generation of variant cell lines with altered phenotypes. Tumor cell heterogeneity extends

to progressively increasing karyotypic, morphologic, and molecular diversity as the tumor

becomes more advanced (120-122). Clonal expansions of cells whose genetic alterations

provide a selective advantage through altered cellular growth, differentiated state, drug

resistance, or metastatic potential predominate within the primary tumor or at secondary

metastatic tumor sites. Activation of dominant oncogenes and inactivation of tumor

suppressor genes are two types of genetic alteration which are clearly important in the

progression of cancer cells (123).

An important manifestation of genetic instability acquired by cancer cells is

amplification potential. In mammalian cells, gene amplification is a genetic event believed

to be unique to tumor cells or transformed cell lines (10). Overexpression of oncogenes

and drug resistance genes by gene amplification are well documented events in human

cancer cells (6). Cancer cells within a tumor have varying degrees of amplification

potential and this potential is a selectable phenotype (124). In fact, individual subclones

have been selected from a parental tumor cell line which have vastly differing amplification

potentials (124). Specific trans-acting factors which convey this selectable phenotype have

yet to be identified or isolated. Cell Survival Molecular System (CSMS) enzymes have

been postulated to be involved in this unique alteration process (125).

Based on the just described theory of genetic alteration and cancer progression, I

speculated that biologically important amplified sequences remained undiscovered. The

intent of this dissertation was to identify and isolate amplified DNA sequences important to

the progression of human cancer. In pursuit of this goal, a large body of work was

67

accomplished and several highly significant observations were reported. First, the

practicality of using modified in gel renaturation to screen several human tumors or cell

lines for gene amplification was clearly established. Second, this study represents the most

comprehensive analysis of gene amplification in malignant melanoma and in adult sarcoma

to date. Third, DNA sequence amplification was identified in adult sarcoma. Fourth, large

segments of the adult sarcoma amplification unit were cloned Fifth, and perhaps most

significant, DNA sequence amplification was demonstrated to be a recurring phenomenon

in adult sarcoma.

The study of DNA sequence amplification in adult sarcoma requires continued

analysis. The clones which I have isolated from the sarcoma amplification unit may be

useful to continued experimentation. These clones could be used as probes for Southern

blot analysis of adult sarcoma tissue. Continued screening experiments will determine the

frequency of amplification in adult sarcoma and the types of sarcoma that carry the

amplified sequences. Identification of more amplification positive sarcoma samples will

also provide additional material for continued experimentation. In situ hybridization of .-

florescence labelled cloned sequences to both normal and amplification carrying adult

sarcoma cells will identify the chromosomal location of the normal and amplified

sequences. The amplified clones I have isolated may also be used as probes to screen

bacteriophage lambda or cosmid genomic libraries constructed from amplification positive

sarcoma tissue. Identification of large clones selected by hybridization to probes generated

from the cloned amplified sequences will allow for chromosome walking experiments and

the eventual successful identification of the amplified target gene carried within the sarcoma

amplification unit. Large clones from the amplification unit can be quickly screened for

potential exons by probing cross species Southern blots and selecting clones which contain

highly conserved sequences (by virtue of hybridization to specific fragments of divergent

species DNA) (126). Positive clones can be more finely analyzed for transcribed

sequences by making several radiolabeledprobes from repetitive DNA free sub-fragments

and hybridizing them with Northern blots of adult sarcoma RNA. Finally, sub-fragments

of genomic clones which are shown to contain transcribed sequences by Northern blot

analysis may be used as probes against cDNA library constructed from an amplification

positive sarcoma sample in order to isolate full length transcripts of the gene which is

hypothesized to be carried within the sarcoma amplification unit. Isolation of fuUlength

transcripts will allow for the characterization of the target gene carried within the sarcoma

amplification unit. Comparisons of the amplified sarcoma target gene with sequences of

known genes can be used to predict the cellular function of the target gene product. This

infonnation will generate continued speculation and experimentation to detennine the

biological significance of the identified gene product in cancer and nonnal cellular

functions.

68

I speculate that the amplified gene carried within the sarcoma amplification unit will

prove to be a growth factor or growth factor receptor gene specific to the mesenchymal cell

lineage. I base this speculation on several points. I have shown by Southern blot analysis

this novel gene is not commonly amplified in cancer cells derived from all cell lineages is

amplified in at least three different types of sarcoma (see: "Results", Chapter N).

Therefore, it appears that the gene product of the hypothesized target gene amplified in the

sarcoma tumors must be involved in growth or differentiation pathways specific to cell

derived from a mesenchymal lineage. Growth factors and their receptors are well

documented to function in a cell lineage or tissue specific manner (127-129). Amplification

of growth factor receptor genes is a well documented event in several other tumor types

(6,130). Oncogene erbB-l (epidermal growth factor receptor) amplifications are known to

be carried in glioblastoma cells (6). Oncogene erbB-2 (HER2/neu) is commonly amplified

in breast carcinoma cells (30). Interestingly, c-sis (Platelet-derived growth factor-(3) has

been shown to be expressed in several types of human sarcoma (131,132). Expression of

the PDGF receptor family in nonnal cells is known to occur a variety of tissues derived

from a mesenchymal lineage (133). However, Southern blot analysis ofMFH ST-23987

with a c-sis probe did not identify the amplification unit (see: "Results", Chapter Ill). It is

possible that the hypothesized target gene carried within the amplification unit of adult

sarcoma is one of the two identified PDGF receptors. A literature search revealed no

reports of amplification of PDGF receptor genes in cancer cells. Other possibilities are that

the amplified target gene encodes a novel growth factor, or a novel growth factor receptor.

Although genes from the other classes of protooncogenes are amplified in other human

tumors, these are usually genes whose products are nuclear proteins or proteins involved in

intracellular signaling pathways on levels which have not as yet been shown to be specific

to a particular cell lineage. Such oncogenes have been shown to be activated by gene

amplification and other mechanisms in various cancer cells derived from several cell types

(Le. ras and myc genes) (117).

In conclusion, the essential observation of this dissertation was the recuning DNA

sequence amplification in biopsies of adult sarcomas. I suggest that the hypothesized target

gene carried within the amplification unit will prove to be important to the genesis or

progression of adult sarcomas.

69

70

LITERATURE CITED

1. Roninson, I. (1983). Detection and mapping of homologous, repeated, and amplified DNA sequences. Nucl Acids Re.s,11, 5413-5431.

2. Roninson, I., Abelson, H.T., Housman, D.E., Howell, N., and Vasharvsky, A. (1984). Amplification of specific DNA sequences correlates with multidrug resistance in Chinese hamster cells. Nature, .3.Q2, 626-628.

3. Kinzler K., Bigner, S., Bigner, D., Trent, J.M., Law, M., Obrien, S., Wong, A., and Vogel stein, B. (1984). Identification of an amplified, highly expressed gene in human glioma. Science, 2JQ, 70-73.

4. Fukumoto, M., and Roninson, I. (1986). Detection of amplified sequences in mammalian DNA by in-gel renaturation and SINE hybridization. Somatic Cell and Mol Genet, 12, 611-623.

5. Fukumoto, M., and Roninson, I. (1988). Analysis of gene amplification in human cell lines. Proc Nat Acad Sci, ~, 6846-6850.

6. Alitalo, K., and Schwab, M. (1986). Oncogene amplification in tumor cells. Adv in Cancer Res, 41, 235-281.

7. Mouches, C., Pasteur, N., Berge', J.B., Hyrien, 0., Raymond, M., Robert de St.Vincent, B., de Silvestri, M., and Georghiou, G.P. (1986). Amplification of an esterase gene is responsible for insecticide resistance in a Culex mosquito. Science, m, 778-780.

8. Spralding, AC., and Mahowald, AP. (1980). Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster. Proc Nat Acad Sci, TI, 1096-2002.

9. Scott, G.K., Davies, P.L., Kao, M.H., and Fletcher, G.L. (1988). Differential anlplification of antifreeze genes in the pleuronectinea J Mol Evol, 21, 29-35.

10. Tylsty, T.D. (1990). Normal diploid human rodent cells lack a detectable frequency of gene amplification. Proc Nat Acad ScLll: 3132-3136.

11. Biedler, J.L., and Spengler, M.B. (1976). Metaphase chromosome anomaly: as:.ociation with drug resistance and cell specific products. Science, 12.1, 185-187.

12. Hamkalo, B.A., Farnham, P.J., Johnston, R.,and Schimke, R.T. (1985). Ultrastuctural features of minute chromosomes in a methotrexate resistant mouse 3T3 cell line. Proc Nat Acad Sci, .81, 1126-1130.

71

13. Kovacs, G., Freund, M., Poliwoda, H., Leithauser, H., and Thiel, E. (1986). Homogeneously staining region in a case of erythroleukaemia. Scand J Haematol, JQ, 232-237.

14. Schwab, M., Alitalo, K., Varmus H.E., Bishop, M., and George, D. (1983). A cellular oncogene (c-Ki-ras) is amplified, overexpressed and located within karyotypic abnormalities in mouse adrenocortical tumor cells. Nature, J.Q3., 497-501.

15. Alt, F.W., Kellems, R.E., Bertino, I.R., and Schimke, R.T. (1978). Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J BioI Chern, ID, 5852-5860.

16. Schimke, R.T. (1982). Gene Amplification. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.

17. Collins, S.I., and Groudine, M.T. (1982). Amplification of endogenous myc-related DNA sequence in a human myeloid leukemia cell line. Nature, .22..8, 679-682.

18. Brodeur, G.M. (1987). The involvement of oncogenes and suppressor genes in human neoplasia. Adv Pediatr • .3.4, 1-44.

19. Bishop, J.M. (1983). Cellular oncogenes and retroviruses. Annu Rev Biochem,.52.. 301-354.

20. Biedler, J.L., Meyers, M.B., and Spengler, B.A. (1983). Homogeneously staining regions and double minute chromosomes are prevalent cytogenetic abnormalities of neuroblastoma cells. Advances in Cellular Neurobiology,~, New York: Academic Press, 268-301.

21. Brodeur, G.M., Green, A.A., Hayes F.A., Williams, K.J., Williams, D.L., and Tsiatis, A.A. (1981). Cytogenetic features of a neuroblastoma and neuroblastoma cell lines. Cancer Res, 41, 4678-4686.

22. Schwab, M., Alitalo, K., Klempnauer, K.H., Varmus, H.E., Bishop, I.M., Gilbert, F., Brodeur, G.M., Goldstein, M., and Trent, J. (1983). Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumor. Nature,~, 245-248.

23. Battey, I., Moulding, C., Taub, R., Murphy, W., Stewart, T., Potter, H., Lenoir, G., and Leder, P. (1983). The human c-myc oncogene: structural consequences of translocation into the IgH locus in Burkitt's lymphoma. !&ll,.3.4, 779-787.

24. Michitsch, R.W., Montgomery, K.T., and Melera, P.W. (1984). Expression of the amplified domain in human neuroblastoma. Mol Cell BioI, ~, 2370-2380.

25. Kohl, N.E., Kanda, N., Schreck, R.R., Bruns, G., Latt, S.A., Gilbert, F., and Alt, F.W. (1983). Transposition and amplification of oncogene related sequences in human neuroblastoma . .Qill,~, 359-367.

26. Stanton, L.W., Schwab, M., and Bishop, J.M. (1986). Nucleotide sequence of the human N-myc gene. Proc Nat Acad Sci,.8..l, 1772-1776.

27. Yancopoulos, G.D., Nisen, P.D., Tesfaye, A., Kohl, N.E., Goldfarb, M.P., and Alt, F.W. (1985). N-myc can cooperate with ras to transform normal cells in culture. Proc Nat Acad Sci,,RZ., 5455-5459.

28. Seeger, R.C., Brodeur, G.M., Sather, H., Dalton, A., Siegel, S., Wong, K., and Hammond, D. (1985). Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med, ill, 1111-1116.

29. Brodeur, G.M., Seeger., R.C., Schwab, M., Varmus, H.E., and Bishop, J.M. (1984). Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science, 224, 1121-1124.

30. Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A., and McGuire, W.L. (1987). Human breast cancer: Correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science, 2ll, 177-182.

72

31. Schecter, A.L., Hung, M., Vaidzanather, L., Wienberg, A., Yang-Feng, T.L., Franke, V., Ullrich, A., and Coussens, L. (1985). The neu gene: an erbB­homologous gene distinct from and unlinked to the gene encoding the epidermal growth factor receptor. Science, 229, 974-976.

32. Bargmann, C.I., Hung, M., and Weinberg, R.A. (1986). The neu oncogene encodes an epidermal growth factor-related protein. Nature, 1l.2, 226-230.

33. Yamamoto, T., Ikawa, S., Akiyama, T., Semba, K., Nomura, N., Miyajima, N., aito, T., and Toyoshima, K. (1986). Similarity of protein encoded by the human c-erbB-2 gene to the epidermal growth factor receptor. Nature,1l.2, 230-235

34. Seshadri, R., Matthews, C., Dobrovic, A., and Horsfall, D.J. (1989). The significance of oncogene amplfication in primary breast cancer. lot J Cancer, ~, 270-272.

35. Minna, J.D., Higgins, G.A., and Glatstein, EJ. (1982). Principles and Practice of Qncolo~ (DeVita, Hellman, and Rosenberg, eds.) Philidelphia: Lippincott, 396-474.

36. Radice, P.A., Matthews, M.J., Ihde, D.C., Gazder, A.F., Carney, D.N., Bunn P.A., Cohen, M.H., Fossieck, B.E., Makuch, R.W., and Minna, J.D. (1982). The clinical behavior of "mixed" small cell/large cell bronchogenic carcinomas compared to "pure" small cell subtypes. Cancer, ~, 2894-2902.

37. Gazdar, A.F., Carney, D.N., Guccion, lG., and BayIin, S.B. (1981). Small Cell Lune Cancer. New York: Grone and Stratton, 145-175.

38. Little, C.D., Nau, M.M., Carney, D.N., Gazdar, A.F., and Minna, J.D. (1983). Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature, JUQ, 194- 196.

73

39. Nau, M.M., Carney, D.N., Battey, J., Johnson, B., Little, C., Gazdar, A.F., and Minna, J.D. (1984). Amplification, expression, and rearrangement of c-myc and N-myc oncogenes in human lung cancer. Curr Top Microbiol Immunol, ill, 172- 177.

40. Hamlin, J.L., Milbrandt, J.D., Heintz, N.H., and Azizkhan, J.C. (1984). DNA sequence amplification in mammalian cells. Int Rey Cytol, 20, 31-82.

41. Guilotto, E., Saito, I., and Stark, G.R. (1986). Structure of DNA formed in the first step of CAD gene amplification. EMBO J, j" 2115-2121.

42. Schimke, R.T. (1988). Gene amplification in cultured cells. J BioI Chern, 2Ql, 5989-5992.

43. Looney, J .E., amd Hamlin, J .L. (1987). Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells. MQL Cell BioI, 1, 569-577.

44. Montoya-Zala, M., and Hamlin, J.L. (1985). Similar 150-kilobase DNA sequences are amplified in independently derived methotrexate-resistant Chinese hamster cells. Mol Cell BioI, j" 619-627.

45. Federspiel, N.A., Beverley, J.W., Schilling, J.W., and Shimke, R.T. (1984). Novel DNA rearrangements are associated with dihydrofolate reductase gene amplification. J BioI Chern, 252,9127-9140.

46. Stark, G.R., and Wahl, G.M. (1984). Gene amplification. Annu Rey Biochem,~, 447-491.

47. Debatisse, M., Hyrien, 0., Petit-Koskas, E., Robert de Saint Vincent, B., and Buttin G. (1986). Segregation and rearrangement of coamplified genes in different lineages of mutant cell that overproduce adenyl ate deaminase. Mol Cell BioI, ,n, 1776-1781.

48. Saito, I., Groves, R., Guilotto, E., Rolfe, M., and Stark, G.R. (1989). Evolution and stability of chromosomal DNA coamplified with the CAD gene. Mol Cell BioI, ,2, 2445-2452.

49. Ma, C., Looney, J.E., Leu, T.H., and Hamlin, J.L. (1988). Origin and genesis of dihydrofolate reductase amplicons in the genome of a methorexate-resistant Chinese hamster ovary cell line. Mol Cell BioI,.8., 2316-2327.

50. Ford, M., and Fried, M. (1986). Large inverted duplications are associated with gene amplification. Qill, ~, 425-430.

51. Nalbantoglu, J., and Mueth, M. (1986). DNA amplification-deletion in a spont~.neous mutation of the hamster aprt locus: structure and sequence of the novel joint. NYcJ. Acids Res, 14, 8361-8371.

52. Shiloh, Y., Korf, B., Kohl, N.E., Sakai, K., Brodeur, G.M., Harris, P., Kanda, N., Seeger, R.C., Alt, F.W., and Latt, S.A. (1986). Amplification and rearrangement of DNA sequences from the chromosomal region of 2p24 in human neuroblastomas. Cancer Res,~, 5297-5301.

74

53. Kinzler, K.W., Zehnbauer, B.A., Brodeur, G.M., Segger, R.C., Trent, J.M., Meltzer, P.S., and Vogelstein, B. (1986). Amplification units containing human N-myc and c-myc genes. Froc Nat Acad Sci,.8..3" 1031-1035.

54. Zehnbauer, B.A., Small D., Brodeur, G.M., Seeger, R.C., and Vogelstein, B. (1988). Characterization ofN-myc amplification units in human neuroblastoma cells. Mol Cell BioI, ,8, 522-530.

55. Kanda, N., Schreck, R., Alt, F., Bruns, G., Baltimore, D., and Latt, S.A (1983). Isolation of amplified DNA sequences from IMR-32 human neuroblastoma cells: facilitation by fluoresence-activated flow sorting of metaphase chromosomes. Proc Nat Acad Sci,.8Q, 4068-4074.

56. Stark, G.R., Debatisse, M., Giulotto, E., and Wahl, G.M. (1989). Recent progress in understanding mechanisms of mammalian DNA amplification. Qill, j2, 901-908.

57. Almer, L.C., and Shwab, M. (1989). Amplified N-myc in human neuroblastoma cells is often arranged as clustered tandem repeats of differentially recombined DNA. Mol Cell BioI, 2,4903-4913.

58. Carroll, S.M., Gaudray, P., Derose, M.L., Emery. J.F., Meinkoth, J.L., Nakkim, E., Subler, M., Vohn Hoff, D.D., and Wahl, G.M. (1987). Characterization of an episome produced in hamster cells that amplify a transfectted CAD gene at high frequency: functional evidence for a mammalian replication origin. Mol Cell BioI, 1, 1740-1750.

59. Vohn Hoff, D.D., Needham-VanDevanter, D.R., Yucel, J., Windle, B.E., and Wahl, G.M. (1988). Amplified human MYC oncogenes localized to replicating submicroscopic circular DNA molecules. Proc Nat Acad Sci,~, 4804-4808.

60. Ruiz, J.C., and Wahl G.M. (1988). Formation of an inverted duplication can be an intial step in gene amplification. Mol Cell BioI, ,8, 4302-4313.

61. Wahl, G.M. (1989). The importance of circular DNA in mammalian gene amplification. Cancer Res, 42, 1333-1340.

62. Carroll, S.M., DeRose, M.L., Gaudray, P., Moore, C.M., Needham- Vandervanter, D.R., Vohn HoffD.D", and Wahl, G.M. (1988). Double minute chromosomes can be produced from precursors derived from a chromosomal deletion. Mol Cell B.iQl,.8" 1525-1533.

63. Johnston, R.N., Beverly, S.M., and Schimke, R.T. (1983). Rapid spontaneous dihydrofolate reductase gene amplification shown by fluorescence cell sorting. Proc Nat Acad Sci, .8Q, 3711- 3715.

64. Kofatos, F.C., Orr, W., and Delidikas, C. (1985). Developmentally regulated gene amplification. Trends Genet,l, 301-~06.

65. Laughlin, T.J., and Taylor, J.H. (1979). Initiation of DNA replication in chromosomes of Chinese hamster ovary cells. Chromosoma,12, 19-35.

75

66. Lavi, S. (1981). Carcinogen-mediated amplification of viral DNA sequences in simian virus 40-transformed Chinese hamster embryo cells. Proc Nat Acad Sci, 18., 6144-6148.

67. Woodcock, D.M., and Cooper, I.A. (1981). Evidence for double replication of chromosomal DNA segments as a general consequence of DNA replication. Cancer Res, 41,2483-2490.

68. Vasharvsky, A. (1981a). Phorbol ester dramatically increases incidence of methotrexate-resistant mouse cells: Possible mechanisms and relevance to tumor promoters . .cen,~, 561- 572.

69. Vasharvsky, A. (1981b). On the possiblity of metabolic control ofreplicon "misfiring": Relationship to emergence of malignant phenotypes in mammalian cell lineages. Proc Nat Acac Sci,18., 3673-3677.

70. Roberts J.M., Buck, L.B., and Axel, R. (1983). A structure for amplified DNA . .cen, n, 53-63.

71. Bullock, P., and Botchan, M. (1982). Gene Amplification. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 215-230.

72. Pelligrini, S., Dailey, L., and Basilico, C. (1984). Amplification and excision of integrated polyoma DNA sequences requires a functional origin of replication . .cen, .3..Q, 943-949.

73. Brookwell, R., and Hunt, F.A. (1988). Formation of double minutes by breakdown of a homogeneously staining region. Cancer Genet Cyto~enet,.J.1, 47-52.

74. Inanazawa, J., Abe, T., Inoue, K., and Nishigaki, H. (1989). Simultaneous existence of double minute chromosomes and a homogeneously staining region in a retinoblastoma cell line. Cancer Genet Cytogenet, n., 133-137.

75. Futcher, A.B. (1986). Copy number amplification of the 2um circular plasmid of Saccharomyces cervisiae. J Theor BioI,.l.l2, 197-204.

76. Passananti, C., Davies, B., Ford, M., and Fried, M. (1987). Structure of an inverted duplication formed as a first step in a gene amplification event: implications for a model of gene amplifcation. EMBO J, Q, 1697-1703.

77. Hyrien, 0., Debatisse, M., Buttin, G., and Robert de Saint Vincent, B. (1988). The multicopy appearance of a large inverted duplication and the sequence at the inversion joint suggest a new model for gene amplification. EMBO J, Q, 2401-2408.

76

78. Backman, K., Betlach, M., Boyer, H.W., and Yanofsky, S. (1978). Genetic and physical studies on the replication of ColE I-type plasm ids. Cold Spring Harbor Quant BioI,~, 69-76.

79. lkoku, A.S., and Hearst, E. (1981). Identification of a structural hairpin in the filamentous chimeric phage M13Gori1. J Mol Bioi, ill, 245-259.

80. Kaguni, L.S., and Clayton, D.A. (1982). Template-directed pausing in in vitro DNA synthesis by DNA polymerase a from Drosophila meanogaster embryos. ~ Nat Acad Sci, 79,983- 987.

81. Schimke, R.T., Sherwood, S.W., Hill, A.B., and Johnston, R.N. (1986). Evolution and stability of chromosomal DNA coamplified with the CAD gene. Proc Nat Acad Sci, ~, 2157- 2161.

82. Brown, D., and Blacker, A. (1972). Gene amplification proceeds by a chromosome copy mechanism. J Mol Biol,~, 75-83.

83. Tartoff, K. (1975). Redundant genes. Ann Rev Genet, 2,355-387.

84. Petersen, H.M., and Laughnan, J.R. (1963). Intrachromosomal exchange at the bar locus in Drosophila. Proc Nat Acad Sci, jQ, 126-133.

85. Smith, G.P. (1973). Unequal crossover and the evolution of multi gene families . .Qllil Spring Harbor Symp Qyant Biol,~, 507-513.

86. Tartoff, K.D. (1973). Unequal mitosic sister chromatid exchange and disproportionate replication as mechanisms regulating ribosomal RNA gene redundancy. Cold Spring Harbor Symp Quant Biol,lB., 491-500.

87. Smith, G.P. (1976). Evolution of repeated DNA sequences by unequal crossover. Science, 12.1, 528-535.

88. Alt, F.W., Kellems, R.E., Bertino, J.R., and Schimke R.T. (1978). Selective multiplication of dihydrofolate reductase genes in methotrexate-resistant variants of cultured murine cells. J Bioi Chern, ill, 1357-1370.

89. Wahl, G.M., Robert de Saint Vincent, B., and DeRose, M.L. (1984). Effect of chromosomal position on amplification of transfected genes in animal cells. Nature, NI, 516-520.

90. Robert de Saint Vincent, B, and Wahl, G.M. (1983). Homologous recombination in mammalian cells mediates formation of a functional gene from two overlapping gene fragments. Proc Nat Acad Sci, .till, 2002-2006.

91. Schimke, R.T., Brown, P.C., Kaufman, R.J., McGrogan, M., and Slate, D.L. (1981). Chromosomal and extrachromosomal localization of amplified dihydrofolate reductase genes in cultured mammalian cells. Cold Spring Harbor Symp Quant Biol,,45., 785-797.

77

92. Jelinek, W.R., Toomey, T.P., Leinwand, L., Duncan, C.H., Biro, P.A., Choudary, P.V., Weissman, S.M., Rubin, C.M., Houck, C.M., Deininger, P.L., and Schmid, C.W. (1980). Ubiquitous, interspersed repeated sequences in mammalian genomes. Proc Nat Acad Sci, TI, 1398-1402.

93. Hill, A.B., Beck, W.T., and Trent, J.M. (1988). Cytogenetic and molecular characterization of tumors in nude mice derived from a multidrug-resistant human leukemia cell line. Cancer Res, 4R, 393-398.

94 Sealey, P.G., Whittaker, P.A., and Southern, E.M. (1985). Removal of repeated sequences from repetitive probes. Nuel Acids Res, li, 1905-1922.

95. Leibovitz, A. (1986). Development of tumor cell lines. Cancer Genet Cyto~enet, 12, 11-19.

96. Gadzar, A.F., Carney, D.N., Russell, E.K., Sims, H.L., Baylin, S.B., Bunn, P.A., Guccion, J.G., and Minna, J.D. (1980). Establishment of continuous, elonable cultures of small-cell carcinoma of the lung which have amine precursor uptake and decarboxylation cell properties. Cancer Res,.4Q, 3502-3507.

97. Beck, W.T., Mueller, T.J., and Tanzer, L.R., (1979). Altered surface membrane glycoproteins in Vinca alkaloid-resistant human leukemic lymphoblasts. Cancer Re..s.,,32,, 2070-2076.

98. Rutka, J.T., Giblin, J.R., Hoifodt, H.K., Dougherty, D.V., Bell, C.W., McCulloch, J.R., Davis, R.L., Wilson, C.B., and Rosenblum, M.L. (1986). Establishment and characterization of a cell line from a human gliosarcoma. Cancer Res, 42, 5893-5902.

99. Gross-Bjelland, M., Oudet, M., and Chambon, P. (1973). Isolation of high molecular weight DNA from mammalian cells. Eur J Biochem, .JQ, 32-38.

100. Kapuscinski, J., and Skoczylas, B. (1977). Simple and rapid fluorometric method for DNA micro assay. Anal Biochem,.8.,3, 252-257.

101. Feinberg, A., and Vogelstein, B. (1984). A technique for labelling DNA restriction fragments to high specific activity. Anal Biochem, 14,66-67.

102. Trent, J.M., and Thompson, F.H. (1984). Evidence for selection of homogeneously staining regions in a melanoma cell line. Cancer Res, .14,233-237.

103. Mandahl, N., Heim, S., Willen, H., Rydholm, A., Eneroth, M., Nilbert, M., Kreicbergs, A., and Mitelman, F. (1989). Characteristic anomalies identify subtypes of malignant fibrous histiocytoma. Genes Chromo Cancer, 1, 9-14.

104. Simmons, M.C., Maxwell, J., Haliotis, T., and Higgins, M.J. (1984). Amplified KpnL repetitive DNA sequences in homogeneously staining regions of a human melanoma. J Nat Cancer Inst, 11,801-808.

78

105. Adelaide, J., Mattei, M.G., Marics, I., and Raybaud, F. (1988). Chromosomal localization of the hst oncogene and its co- amplification with the int-2 oncogene in a human melanoma. Qnco~ene, ~, 413-416.

106. Klingel, R., Mincheva, A., Kahn, T. Gissmann, L., Dippold, W., Meyer zum Bushenfelde, K.H., and zur Hausen, H. (1987). An amplification unit in human melanoma cells showing partial homology with sequences of human papillomavirus type 9 and with nuclear antigen 1 of the Epstein-Barr virus. Cancer Res, ~, 4485-4492.

107. McGuire, G.M. (1989). Treatment of soft tissue sarcomas. Boston: Kluwer Academic Publications.

108. Fanning T.G., and Singer, M.F. (1987). LINE-I: a mammalian transposable element. Biochim Biophys Acta, 2.li!, 203-212.

109. Sun, L., Paulson., K.E., Deka, N., Schmid, C.W., Kadyk, L., and Leinwand, L. 1984). Non-alu family interspersed repeats in human DNA and their transcriptional activity. Nucl Acids Res, 12,2669-2690.

110. Paulson, K.E., Deka, N., Schmid, C.W., Misra, R., Schindler, C.W., Kadyk, L., and Leinwand, L. (1985). A transposon-like element in human DNA. Nature, ill, 359-361.

111. Law, M.L., Xu, Y.S., Berger, R., and Tung, L. (1987). Molecular analysis of human repetitive sequence family and its use as a genetic marker. Somat Cell Mol Genet. ll381-389.

112. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Hom, G.T., Mullis, K.B., and Erlich, H.A. (1988). Primer directed enzymatic amplificaiton of DNA with a thermostable DNA polymerase. Science, 22, 487-491.

113. Tabor, S., and Richardson, C.C. (1989). J BioI Chern, 264, 6647-6658.

114. Arber, W., Enquist, L., Hohn, B., Murray, N., and Murray, K. (1983). Lambda II. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 433-466.

115. Meese, E., Qlson, S., Leis, L., and Trent, J.M. (1990). Quick method for high yields of lambda bacteriophage DNA. Nucl Acids Res,,lli, 1923.

116. Lehrach, H., Diamond, D., Wozney, J.M., and Boedtker, H. (1977). RNA molecular weight determinations by gel electrophoresis under denaturing conditions: a critical reexamination. Biochemistry, lQ, 4743.

117. Nowell, P.C. (1976). The clonal evolution of tumor cell populations. Science, .1.21, 23-28.

118. Callahan, R. (1989). Genetic alterations in primary breast cancer. Breast Cancer Res, 1.3., 191-203.

79

119. Vogelstein, B., Fearon, E.R., Hamilton, S.R., Kern, S.E., Preisinger, A.C., Leppert, M., Nakamura, Y., White, R., Smits, A.M.M., and Bos, J.L. (1988). Genetic alterations during colo-rectal tumor development. New En~l J Med, ill, 525-532.

120. Rowley, J.D., and Ultmann, J.E. (1983). Chromosomes and Cancer. New York: Academic Press.

121. Shtivelman, E., Lifshitz, B., Gale, R.P., and Cananni, E. (1985). Fused transcripts of abl and bcr genes in chronic myelogenous leukaemia. Nature, ill, 550-554.

122. Nicolson, G.L. (1987). Tumor cell instability, diversification, and progression to the metastatic phenotype: from oncogene to oncofetal expression. Cancer Res, £i, 1473-1487.

123. Klein, G. (1988). Oncogenes and tumor suppressor genes. Acta Oncol, 21, 427-437.

124. Giulotto, E., Knights, C., and Stark, G.R. (1987). Hamster cells with increased rates of DNA amplification, a new phenotype. EMBO J,~, 2115-2121.

125. Tonini, G.P. (1988). Molecular mechanisms involved in DNA repair, in gene rearrangement and in gene amplification may be considered as an integrated system in maintaining cellular homeostasis and cell survival. Anticancer Res, ,8., 881-884.

126. Rommens, J.M., Iannuzzi, M.C., Kerem, B., Drumm, M.L., Melmer, G., Dean, M., Rozmahel, R., Cole, J.L., Kennedy, D., Hidaka, N., Zsiga, M., Buchwald, M., Riordan, J.R., Tsui, L.-C., Collins, F.S. (1989). Identification of the cystic fibrosis gene: chromososme walking and jumping. Science, W, 1059- 1065.

127. Nicola, N.A. (1989). Hemopoietic cell growth factors and their receptors. Annu Rev Biochem, 28., 45-77.

128. Wiedermann, C.J. (1989). Bombesin-like peptides as growth factors. Wien Klin Wochenschr, .lQl, 435-440.

129. Springer, J.E. (1988). Nerve growth factor receptors in the central nervous system. Exp Neurol, lQ2, 354-365.

130. Shimizu, N., Kondo, I., Gamou, S.M., Behzadian, M.M., and Shimizu, Y. (1984). Genetic analysis of hyperproduction of epidermal growth factor receptor in human epidermoid carcinoma A431 cells. Somatic Cell and Mol Genet, lQ, 45-53.

131. Eva, A., Robbins, K.C., Andersen, P.R., Srinivasan, A., Tronick, S.R., Reddy, E.P., Ellmore, N.W., Galen, A.T., Lautenberger, J.A., Papas, T.S., Westin, E.H., Wong-Staal, F., Gallo, R.C., and Aaronson, S.A. (1982). Cellular genes analogous to retroviral one genes are transcribed in human tumour cells. Nature, 22,5., 275-277.

132. Versnel, M.A., Hagemeijer, A., Bouts, M.I., van der Kwast, T.H., and Hoogsteden, H.C. (1985). Expression of c-sis (PDGF-B chain) and PDGF-A chain genes in ten malignant mesothelioma cell lines derived from primary and metastatic tumors. Oncogene,,2, 601-605.

80

133. lohnsson, A., Betsholtz, C., Heldin, C.H., and Westermask, B. (1985). Antibodies against platelet-derived growth factor inhibit acute transformation by simian sarcoma virus. Oncoeene.l, 79-85.

134. Zubay, G. (1984). Biochemistry. Menlo Park: Addison-Wesley Publishing Co.

135. Pinkel, D., Straume, T., and Gray, I.W. (1986). Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Nat Acd Sci, . .8.3., 2934-2938.

136. Roberts, W.M., Douglas, E.C., Peiper, S.C., Houghton, P.I., Look, A.T. (1989). Amplification of the gli gene in chilhood sarcomas. Cancer Res, 42,5407-5414.

137. Bigner, S.H., Burger, P.C., Wong, A.I., Werner, M.H., Hamilton, S.R., Muhlbaier, L.H., Vogelstein, B., and Bigner, D.D. (1988). Gene amplfication in malignant human gliomas: clinical and histopathologic aspects. Journal of Neuropath and Exp NeumJ.,~, 191-205.