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
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 . . .
10. Somatic 2nd hit
• Heterozygous carrier cell just before mitosis
• 1. Mutations affecting coding region
• 2. Deletion of chromosomal region including
RB1 gene
Mutant
allele
wildtype
allele
1.
2.
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.
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. Colon
1 Loss of
tumor-suppressor
gene APC (or
other)
2 Activation of
Ras oncogene
3 Loss of
tumor-
suppressor
gene DCC
4 Loss of
tumor-suppressor
gene p53
5 Additional
mutations
Colon wall
Normal colon
epithelial cells
Small benign
growth (polyp)
Larger benign
growth (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.
21. 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?
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
– D17S74 - 183rd marker used!
– Initial candidate region spanned half the
chromosome (hundreds of possible genes . . .)
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
29. • 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
Normal
allele
Sickle-cell
allele
Large
fragment
201 bp
175 bp
376 bp
Dde1 cuts at the
sequence
C|TNAG
GANT|C
30.
31. DNA + restriction
enzyme Restriction
fragments I II III
I Normal
-globin
allele
II Sickle-cell
allele
III Heterozygote
Preparation of
restriction
fragments
Gel
electrophoresis
Blotting: transfer to a
nylon membrane
Gel
Sponge
Alkaline
solution
Nitrocellulose
paper (blot)
Heavy
weight
Paper
towels
1 2 3
Figure 20.10
32. Radioactively
labeled probe
for is added
to solution in
a plastic bag
Probe hydrogen-
bonds to fragments
containing the
complementary DNA
sequence
Fragment from
sickle-cell
-globin allele
Fragment from
normal -globin
allele
Paper blot
Film over
paper blot
Hybridization with
radioactive probe.
Autoradiogra
phy.
I II III
I II III
4 5
How would you
make the probe?
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. Figure 20.3
Restriction site
DNA 5
3 5
3
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
One possible combination
Recombinant DNA molecule
G
G
G
G
G G
A 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 ligase
seals the strands.
3
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. 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/
44. 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/
45. 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/
46. 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/
47. 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/
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. 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.
