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?