1. 35 Cancer- part 2

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

3. Changes in growth properties of cancer cells

4. Cell Cycle Regulators and Cancer

5. 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

6. Receptor tyrosine kinases can activate ras
ras is a monomeric G-protein
“molecular switch”
You’ve seen RAS before . . .

7. Oncogenes act cooperatively in tumor-induction

8. Tumor Suppressor Genes
Normally inhibit cell growth
Example: retinoblastoma
RB protein normally blocks a transcription factor, E2F

10. Somatic 2nd hit Heterozygous carrier cell just before mitosis
1. Mutations affecting coding region
2. Deletion of chromosomal region including RB1 gene
wildtype
allele 1. 2.
Mutant
allele

11. 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.

12. Apoptosis = programed cell death
Particular “executioner” proteins (caspases) break down the cell
Reduced cell death can also lead to cancer

13. Apoptosis pathways

15. Oncogenes vs Tumor Suppressors
Oncogenes are dominant mutations
Tumor Suppressors are recessive
Why?

16. 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

17. A multistep model for the development of colorectal cancer
(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.
Colon
(2) Cancer development is a multi-step process. Multiple mutations accumulated over periods of many years ----“multi-hit” model.
1 Loss of
tumor-suppressor
gene APC (or
other)
2 Activation of
Ras oncogene
4 Loss of
tumor-suppressor
gene p53
Colon wall
3 Loss of
tumor-
suppressor
gene DCC
5 Additional
mutations
Normal colon
epithelial cells
Small benign
growth (polyp)
Larger benign
growth (adenoma)
Malignant tumor
(carcinoma)
Figure 19.13

18. Changes in growth properties of cancer cells

19. Incidence of Cancers in Females

21. Case Study: BRCA1 Probably involved in DNA repair pathways
Would this be a tumor suppressor or an oncogene?
Narod, Steven A. BRCA1 and BRCA2: 1994 and Beyond. Nature Reviews (2004), 670.

22. BRCA1: DNA Repair
Kennedy, Richard D. The Role of BRCA1 in the Cellular Response to Chemotherapy. Journal of National Cancer Institute (2004), 1660.

23. 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
D17S rd marker used!
Initial candidate region spanned half the chromosome (hundreds of possible genes . . .)

24. Linkage study
1 , 8
2 , 4
2 , 8
4 , 8
1 , 2 1 2 4 8

25. Loci far apart A a B b A A a a B b B b
Recombinants: Ab and aB

26. Loci close together No recombinants between A and B A a B b A a A a B

27. 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 marker
DNA
Restriction
sites
Disease-causing
allele
Normal allele

28. Restriction enzymes cut DNA at particular sequences

29. Two alleles of a gene may produce restriction fragments with different lengths.
Normal  -globin allele
175 bp
201 bp
Large fragment
DdeI
DdeI
DdeI
DdeI
DdeI restriction sites in two alleles of the-globin gene.
Sickle-cell mutant -globin allele
376 bp
Large fragment
DdeI
DdeI
DdeI
Dde1 cuts at the sequence
C|TNAG
GANT|C
Normal allele
Sickle-cell allele
Electrophoresis shows that the fragments have different lengths
Large fragment
376 bp
201 bp 175 bp
Figure 20.9

31. DNA + restriction enzyme
Heavy weight
Restriction
fragments
Nitrocellulose
paper (blot) I II
III
Gel
Sponge
Paper towels
I Normal
-globin
allele
II Sickle-cell
allele
III Heterozygote
Alkaline solution 1
Preparation of restriction fragments 2
Gel electrophoresis 3
Blotting: transfer to a nylon membrane
Figure 20.10

32. How would you make the probe?
Probe hydrogen-
bonds to fragments
containing the complementary DNA sequence I II
III I II
III
Radioactively
labeled probe
for is added
to solution in
a plastic bag
Fragment from
sickle-cell
-globin allele
Film over
paper blot
Fragment from
normal -globin
allele
Paper blot 4 5
Autoradiography.
Hybridization with radioactive probe.
How would you make the probe?

33. Linkage study * Disease Allele “A” DNA probe Normal Allele “B”
AA AB BB

34. What next?
Test more families
Try more
markers
Identify
recombinants

35. Recombination Occasionally there is a crossover during meiosis
Marker 1
Marker 2
Marker 3 1 2 8 6 4 2 4 6 4 5 3
To find those rare crossovers, they needed many families with inherited breast cancer
This individual shows that it is not near Marker3 2 4 3 8 6 4

36. 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

37. Using a restriction enzyme and DNA ligase to make recombinant DNA
Restriction site
DNA 5 3
G A A T T C
Cut DNA with Restriction enzyme, leaving overhanging ends 3
C T T A A G 5 1 G
A A T T C
C T T A A G
Sticky end
Fragment from different DNA molecule cut by the same restriction enzyme 2
Base pairing of sticky ends produces various combinations.
A A T T C G G
C T T A A G
A A T T C G
A A T T C C
T T A A G
C T T A A G
One possible combination
DNA ligase seals the strands. 3
Figure 20.3
Recombinant DNA molecule

39. Transform the recombinant plasmid into E. coli
To produce a “library” of different DNA fragments

40. Order and Sequence the clones

42. 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/

43. Contig construction
2 Probe a large insert library to identify clones containing the sequence of the ends of the first clone
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

44. Contig construction
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
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

45. Contig construction
4 Again, probe the large insert library to identify clones containing the sequence of the ends of these clones.
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

46. Contig construction
4 Again, these clones must overlap the existing clones. ie they have some of the same DNA - and hopefully also some new sequence
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

47. Contig construction
In this way we build up a CONTIG - a series of overlapping clones centred on our region of interest.
sphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/

48. Results of sequencing Found 65 expressed genes
Looked for sequence differences between family members with and without cancer

49. BRCA1 found in 1994
Science 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
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.

51. How would you make the probe?