2 Lecture Outline, 11/30/05 Finish Cancer genetics Review Oncogenes and proto-oncogenesTumor Suppressor genesNormally inhibit cell growth.Allow cell growth when damaged or deleted.Mutator genesThe multi-step model of cancerCloning a cancer gene: BRCA1
5 OncogenesAll are involved in positive control of cell growth and division.About 100 different oncogenes have been identifiedCan 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 region2. Deletion of chromosomal region including RB1 genewildtypeallele1.2.Mutantallele
11 p53 Gene Detects DNA damage The “Last Gatekeeper” Involved in 50% of cancersOften 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 cellReduced cell death can also lead to cancer
15 Oncogenes vs Tumor Suppressors Oncogenes are dominant mutationsTumor Suppressors are recessiveWhy?
16 Mutator genesCancer 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 BRCA2Many 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 oftumor-suppressorgene APC (orother)2 Activation ofRas oncogene4 Loss oftumor-suppressorgene p53Colon wall3 Loss oftumor-suppressorgene DCC5 AdditionalmutationsNormal colonepithelial cellsSmall benigngrowth (polyp)Larger benigngrowth (adenoma)Malignant tumor(carcinoma)Figure 19.13
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 RepairKennedy, 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 cancerStudied 23 breast cancer familiesEarly onsetFrequent bilateral diseaseMale relatives with breast cancer1990: linked the disease to a marker on Chromosome 17q21D17S rd marker used!Initial candidate region spanned half the chromosome (hundreds of possible genes . . .)
25 Loci far apartAaBbAAaaBbBbRecombinants: 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 foundFigure 20.15RFLP markerDNARestrictionsitesDisease-causingalleleNormal allele
28 Restriction enzymes cut DNA at particular sequences
29 Two alleles of a gene may produce restriction fragments with different lengths. Normal -globin allele175 bp201 bpLarge fragmentDdeIDdeIDdeIDdeIDdeI restriction sites in two alleles of the-globin gene.Sickle-cell mutant -globin allele376 bpLarge fragmentDdeIDdeIDdeIDde1 cuts at the sequenceC|TNAGGANT|CNormal alleleSickle-cell alleleElectrophoresis shows that the fragments have different lengthsLarge fragment376 bp201 bp 175 bpFigure 20.9
31 DNA + restriction enzyme Heavy weightRestrictionfragmentsNitrocellulosepaper (blot)IIIIIIGelSpongePaper towelsI Normal-globinalleleII Sickle-cellalleleIII HeterozygoteAlkaline solution1Preparation of restriction fragments2Gel electrophoresis3Blotting: transfer to a nylon membraneFigure 20.10
32 How would you make the probe? Probe hydrogen-bonds to fragmentscontaining the complementary DNA sequenceIIIIIIIIIIIIRadioactivelylabeled probefor is addedto solution ina plastic bagFragment fromsickle-cell-globin alleleFilm overpaper blotFragment fromnormal -globinallelePaper blot45Autoradiography.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 familiesTry moremarkersIdentifyrecombinants
35 Recombination Occasionally there is a crossover during meiosis Marker 1Marker 2Marker 312864246453To find those rare crossovers, they needed many families with inherited breast cancerThis individual shows that it is not near Marker3243864
36 Mapping BRCA1 Larger study 214 breast cancer families Region narrowed to 8 cMThat is still a 600,000 nucleotide regionStep 2: Positional cloning
37 Using a restriction enzyme and DNA ligase to make recombinant DNA Restriction siteDNA53G A A T T CCut DNA with Restriction enzyme, leaving overhanging ends3C T T A A G51GA A T T CC T T A AGSticky endFragment from different DNA molecule cut by the same restriction enzyme2Base pairing of sticky ends produces various combinations.A A T T CGGC T T A AGA A T TCGA A T T CCT T A AGC T T A AGOne possible combinationDNA ligase seals the strands.3Figure 20.3Recombinant DNA molecule
42 Contig construction1 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 construction2 Probe a large insert library to identify clones containing the sequence of the ends of the first clonesphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
44 Contig construction3 These clones must overlap the first clone. ie they have some of the same DNA - and hopefully also some not in the first clonesphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
45 Contig construction4 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 construction4 Again, these clones must overlap the existing clones. ie they have some of the same DNA - and hopefully also some new sequencesphere.bioc.liv.ac.uk:8080/bio/studyweb/ modules/BIOL315/
47 Contig constructionIn 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 1994Science 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 CityA 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.