1. Tumor Suppressors Versus Oncogenes

2. Retinoblastoma is a Cancerous Disease Hereditary childhood cancer: bilateral tumors in 25-30% of cases unilateral tumors in 10-15% of all cases 1/20,000 children; 300 per year Average age is 18 months Sporadic cancer in 55-65% of all cases Sporadic cancers are typically unilateral Treatment: enucleation = eye removal Prognosis is good after enucleation over 90% survival with early detection and treatment Secondary cancers can occur, including bone, skin, muscle and brain. Leukocoria or “white pupil” Retinoblastoma “seeds” on the iris

3. It is also highly penetrant Hereditary Retinoblastoma is Transmitted as an Autosomal Dominant Trait +/++/- +/+ +/-

4. The Knudson “Two Hit” Hypothesis for the Generation of RB Alfred Knudson, PNAS 68:820 (1971)

5. Retinoblastoma is Associated with Loss of Heterozygosity (LOH) at the RB Locus ~40% of the time the Wild type allele is mutated 4% of these are deletions

6. (nondisjunction) ~30% of the time Mitotic Nondisjunction Causes (LOH) at the RB Locus

7. Mitotic Recombination Causes LOH at the RB Locus ~30% of the time

8. The RB Gene is Located on Chromosome 13

9. Wen-Hwa Lee et al Science, 235:1394 (1987) The RB Gene was Cloned by Chromosome Walking

10. Fung et al Science 236: 1657 (1987) Wen-Hwa Lee et al Science, 235:1394 (1987) Lane 7 = normal fetal retina Other lanes are RB tumors Lane 1,2 normal cells Other lanes are RB tumors RB mRNA Expression in Retinoblastomas

11. Dr. David Abramson, RB expert at New York Hospital (ca. 1986, According to Natalie Angier) “I believe that in fifteen years, at the outside, we’ll be able to stop retinoblastoma before it begins. I’m so sure that I’ve already given the drug a name. I call it retino-revert, or retino-prevent. The drug will be an analogue of the natural protein that is missing in retinoblastoma cells … We’ll be able to diagnose a child prenatally and start giving this retino-revert to the mother to prevent retinoblastomas from growing as the fetus is developing. I know I’m going out on a limb with this one, but … Come back to me in 2001 and tell me if I wasn’t right.” Bold Predictions, Further Work

12. Huang et al Science, 242:1563-6 (1988) Saos-2 cells Saos-2 cells infected with RB retrovirus Expression of the RB Gene in RB Mutant Cells Corrects the Cancer No virus RB virus Control virus Parental = WERI-Rb27 cells Lux = control virus infected Rb = RB virus infected Tumor formation assayed in “nude” mice

13. Forward versus reverse genetics Forward Genetics: isolate mutants with a specific phenotype and then clone the gene Reverse Genetics: clone the gene and then make a mutant to determine phenotype

14. Culturing Pluripotent ES Cells

15. Formation of ES Cells Carrying a Knockout Mutation

16. Positive and Negative Selection of Recombinant ES Cells

17. Making the Mutant Mouse

18. The Phenotype of RB Mutant Mice Homozygous embryos die at day 13.5 massive apoptosis in the CNS anemia caused by lack of blood cells made by the fetal liver therefore RB is not essential for cell division Heterozygous animals are viable they develop pituitary tumors with near 100% penetrance these tumors lose the wild type RB allele also develop thyroid adenomas therefore the mouse is not a good model for retinoblastoma but it is a good model for tumor suppressors