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35 Cancer- part 2
Lecture Outline, 11/30/05
• Finish Cancer genetics– Review Oncogenes and proto-oncogenes– Tumor Suppressor genes
• Normally inhibit cell growth. • Allow cell growth when damaged or deleted.
– Mutator genes– The multi-step model of cancer
• Cloning a cancer gene: BRCA1
Changes in growth properties of cancer cells
Cell Cycle Regulators and Cancer
Oncogenes• All are involved in positive control of cell growth
and division. – About 100 different oncogenes have been identified
• Can be various kinds of proteins:– Growth factors, regulatory genes involved in the control
of cell multiplication.– Protein kinases, add phosphate groups to target
proteins, important in signal transduction pathways.
• “Proto-oncogenes” – Normal form of the gene that is involved in positive
regulation of the cell cycle
Receptor tyrosine kinases can activate ras
ras is a monomeric G-protein“molecular switch”
You’ve seen RAS before . . .
Oncogenes act cooperatively in tumor-induction
Tumor Suppressor Genes
• Normally inhibit cell growth
• Example: retinoblastoma– RB protein normally blocks a transcription
factor, E2F
Somatic 2nd hit
• Heterozygous carrier cell just before mitosis• 1. Mutations affecting coding region• 2. Deletion of chromosomal region including RB1 gene
Mutantallele
wildtypeallele1.
2.
p53 Gene
• Detects DNA damage • The “Last Gatekeeper”
– Involved in 50% of cancers – Often not malignant despite other cancer-causing
mutations until p53 is inactivated by mutation.
• Two possible responses to DNA damage:– 1) Acts as a Transcription Factor to activate
expression of p21, which inhibits CDK/G1 cyclin to halt the cell cycle; then activates DNA repair.
– 2) Triggers Apoptosis (programmed cell death) if damage can’t be repaied.
Apoptosis = programed cell death
Reduced cell death can also lead to cancer
Particular “executioner” proteins (caspases) break down the cell
http://www.cell-research.com/20014/20014cover.htm
Apoptosis pathways
Oncogenes vs Tumor Suppressors
• Oncogenes are dominant mutations
• Tumor Suppressors are recessive
• Why?
Mutator genes
• Cancer is caused by mutations, so factors that increase mutation rate will increase cancer rate.– What kinds of genes would increase mutation
rate?– Example: BRCA1 and BRCA2
• Many environmental factors (carcinogens) also cause DNA damage or mutations, that can lead to cancer
Colon
1 Loss oftumor-suppressorgene APC (orother)
2 Activation ofRas oncogene
3 Loss oftumor-suppressorgene DCC
4 Loss oftumor-suppressorgene p53
5 Additionalmutations
Colon wall
Normal colonepithelial cells
Small benigngrowth (polyp)
Larger benigngrowth (adenoma)
Malignant tumor(carcinoma)
A multistep model for the development of colorectal cancer
Figure 19.13
(1) The clonal origin of tumors: each individual cancer is a clone that arises from a single cell.The progeny cells have growth advantage over the surrounding normal cells.
(2) Cancer development is a multi-step process. Multiple mutations accumulated over periods of many years ----“multi-hit” model.
Changes in growth properties of cancer cells
Incidence of Cancers in Females
Case Study: BRCA1
Narod, Steven A. BRCA1 and BRCA2: 1994 and Beyond. Nature Reviews (2004), 670.
Probably involved in DNA repair pathways
Would this be a tumor suppressor or an oncogene?
BRCA1: DNA Repair
Kennedy, Richard D. The Role of BRCA1 in the Cellular Response to Chemotherapy. Journal of National Cancer Institute (2004), 1660.
Finding the Cancer Gene BRCA1
• 1980’s: found several families that were predisposed to breast cancer
• Studied 23 breast cancer families– Early onset– Frequent bilateral disease– Male relatives with breast cancer
• 1990: linked the disease to a marker on Chromosome 17q21– D17S74 - 183rd marker used! – Initial candidate region spanned half the chromosome
(hundreds of possible genes . . .)
12
4
8
2 , 8 4 , 8 1 , 2
1 , 8 2 , 4
Linkage study
Loci far apart
AB
ab
AB
Ab
aB
ab
Recombinants: Ab and aB
Loci close together
AB
ab
AB
ab
AB
ab
No recombinants between A and B
• Even when a disease gene has not yet been cloned an abnormal allele can be diagnosed with reasonable accuracy if a closely linked RFLP marker has been found
Figure 20.15
RFLP markerDNA
Restrictionsites
Disease-causingallele
Normal allele
Restriction enzymes cut DNA at particular sequences
• Two alleles of a gene may produce restriction fragments with different lengths.
Figure 20.9
Normal -globin allele
Sickle-cell mutant -globin allele
175 bp 201 bp Large fragment
DdeI DdeI DdeI DdeI
DdeI DdeI DdeI
376 bp Large fragment
DdeI restriction sites in two alleles of the-globin gene.
Electrophoresis shows that the fragments have different lengths
Normalallele
Sickle-cellallele
Largefragment
201 bp175 bp
376 bp
Dde1 cuts at the sequence
C|TNAG
GANT|C
DNA + restriction enzyme Restriction
fragments I II III
I Normal-globinallele
II Sickle-cellallele
III Heterozygote
Preparation of restriction fragments
Gel electrophoresis
Blotting: transfer to a nylon membrane
Gel
Sponge
Alkalinesolution
Nitrocellulosepaper (blot)
Heavyweight
Papertowels
1 2 3
Figure 20.10
Radioactivelylabeled probefor is addedto solution ina plastic bag
Probe hydrogen-bonds to fragmentscontaining the complementary DNA sequence
Fragment fromsickle-cell-globin allele
Fragment fromnormal -globinallele
Paper blot
Film overpaper blot
Hybridization with radioactive probe.
Autoradiography.
I II IIII II III
4 5
How would you make the probe?
Linkage study
Disease Allele “A”*
DNA probe
Normal Allele “B”DNA probe
AA AB BB
What next?
Identifyrecombinants
Try moremarkers
Test more families
Recombination
246
453
121
864
243
864
Marker 1Marker 2Marker 3
Occasionally there is a crossover during meiosis
To find those rare crossovers, they needed many families with inherited breast cancer
This individual shows that it is not near Marker3
Mapping BRCA1
• Larger study• 214 breast cancer families
– Region narrowed to 8 cM• That is still a 600,000 nucleotide region
• Step 2: Positional cloning
Figure 20.3
Restriction site
DNA 53 5
3G A A T T CC T T A A G
Sticky endFragment from differentDNA molecule cut by thesame restriction enzyme
One possible combination
Recombinant DNA molecule
G
C T T A AA A T T C
G
A A T T C
C T T A AG
G
G GA A T T C A A T T C
C T T A A G C T T A A G
Using a restriction enzyme and DNA ligase to make recombinant DNA
Cut DNA with Restriction enzyme, leaving overhanging ends
1
Base pairing of sticky ends produces various combinations.
2
DNA ligaseseals the strands.
3
Transform the recombinant plasmid into E. coli
To produce a “library” of different DNA fragments
Order and Sequence the clones
Contig construction
1 Probe a large insert library to identify a clone containing the marker linked to the trait. sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
2 Probe a large insert library to identify clones containing the sequence of the ends of the first clone
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
3 These clones must overlap the first clone. ie they have some of the same DNA - and hopefully also some not in the first clone
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
4 Again, probe the large insert library to identify clones containing the sequence of the ends of these clones.
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
4 Again, these clones must overlap the existing clones. ie they have some of the same DNA - and hopefully also some new sequence
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
In this way we build up a CONTIG - a series of overlapping clones centred on our region of interest.
Contig construction
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
Results of sequencing
– Found 65 expressed genes– Looked for sequence differences between family
members with and without cancer
BRCA1 found in 1994Science. 1994 Oct 7;266(5182):66-71.
A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1.Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harshman K, Tavtigian S, Liu Q, Cochran C, Bennett LM, Ding W, et al.Department of Medical Informatics, University of Utah Medical Center, Salt Lake City 84132.A strong candidate for the 17q-linked BRCA1 gene, which influences susceptibility to breast and ovarian cancer, has been identified by positional cloning methods. Probable predisposing mutations have been detected in five of eight kindreds presumed to segregate BRCA1 susceptibility alleles.
How would you make the probe?