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
wildtype
allele
• 2. 1.Deletion of chromosomal
region including
RB1 gene
2.
Mutant
allele
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
Apoptosis pathways
http://www.cell-research.com/20014/20014cover.htm
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
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.
Loss of
tumor-suppressor
1
Colon wall gene APC (or
Loss of
tumor-suppressor
gene p53
4
Activation of
Ras oncogene
2
other)
Loss of
tumorsuppressor
gene DCC
3
Normal colon
epithelial cells
Figure 19.13
Small benign
growth (polyp)
5 Additional
mutations
Larger benign
growth (adenoma)
Malignant tumor
(carcinoma)
Changes in growth properties of cancer cells
Incidence of Cancers in
Females
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.
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 . . .)
Linkage study
1,8
2,8
1
2
4
8
4,8
2,4
1,2
Loci far apart
a
b
A
B
A
B
A
b
a
B
a
b
Recombinants: Ab and aB
Loci close together
A
B
A
B
a
b
a
b
A
B
a
b
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
RFLP marker
DNA
Restriction
sites
Disease-causing
allele
Normal allele
Figure 20.15
Restriction enzymes cut DNA at particular sequences
• Two alleles of a gene may produce
restriction fragments with different
lengths.
Normal  -globin allele
201 bp
175 bp
DdeI
DdeI restriction sites in
two alleles of theglobin gene.
DdeI
DdeI
DdeI
Sickle-cell mutant -globin allele
Large fragment
376 bp
DdeI
DdeI
DdeI
Normal
allele
Electrophoresis
shows that the
fragments have
different lengths
Large fragment
Dde1 cuts at the
sequence
Sickle-cell
allele
C|TNAG
GANT|C
Large
fragment
376 bp
201 bp
175 bp
Figure 20.9
DNA + restriction
enzyme
Restriction
fragments
I
II
III
Nitrocellulose
paper (blot)
Heavy
weight
Gel
Sponge
I Normal
-globin
allele
1
Alkaline
solution
II Sickle-cell III Heterozygote
allele
Preparation of
restriction
fragments
Figure 20.10
2
Gel
electrophoresis
3
Paper
towels
Blotting: transfer to a
nylon membrane
Radioactively
labeled probe
for is added
to solution in
a plastic bag
I
II
III
I
Fragment from
sickle-cell
-globin allele
Paper blot
4
Probe hydrogenbonds to fragments
containing the
complementary DNA
sequence
Hybridization with
radioactive probe.
How would you
make the probe?
Fragment from
normal -globin
allele
II
III
Film over
paper blot
5
Autoradiogra
phy.
Linkage study
*
Allele “A”
Disease
DNA probe
Allele “B”
Normal
DNA probe
AA
AB
BB
What next?
Test more
families
Try more
markers
Identify
recombinants
Recombination
Occasionally there is
a crossover during
meiosis
Marker 1
Marker 2
Marker 3
1
2
1
This individual
shows that it is not
near Marker3
8
6
4
2
4
3
2
4
6
8
6
4
4
5
3
To find those rare
crossovers, they
needed many families
with inherited breast
cancer
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
Using a restriction enzyme and DNA
ligase to make recombinant DNA
Restriction site
DNA 5
with 3
1
3
5
GAATTC
CTTAAG
Cut DNA
Restriction
enzyme, leaving
overhanging ends
G
G
Sticky end
2
Base pairing of sticky
ends produces various
combinations.
G AATT C
C TTAA G
3
DNA ligase
seals the strands.
Figure 20.3
G
G AATTC
CTTAA G
One possible combination
Recombinant DNA molecule
G
Fragment from different
DNA molecule cut by the
same restriction enzyme
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/
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/
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/
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/
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/
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/
Results of sequencing
– Found 65 expressed genes
– Looked for sequence differences between family
members with and without cancer
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.
How would you
make the probe?