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 wildtype allele 1. 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 Particular “executioner” proteins (caspases) break down the cell Reduced cell death can also lead to cancer

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. 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

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 , 4 2 , 8 4 , 8 1 , 2 1 2 4 8

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

Loci close together No recombinants between A and B A a B b A a A a 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 marker DNA Restriction sites Disease-causing allele Normal allele

Restriction enzymes cut DNA at particular sequences

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

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

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?

Linkage study * Disease Allele “A” DNA probe Normal Allele “B” 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 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

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 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

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?