Lecture 12. Transgenic KO Conditional
p53-alterations and aging Review: Attardi LD, Donehower LA. Probing p53 biological functions through the use of genetically engineered mouse models. Mutat Res. 2005 Aug 25;576(1-2):4-21
p53-mutant mice First mice were transgeniccarried an additional mutated copy of p53 Dominant-negative effect (dimerization domain of p53- p53 acts a tetramer, DD does not have transactivation domain, sequesters p53 in inactive complexes) 20-30% of these mice developed tumors by 18 months of age Tumorigenic effect only in the presence of wt but not the mutant allele Transgenic mice with different mutations in p53 were made-some conferred susceptibility to tumors, some did not-complex regulation of p53 Knock-out mice HR to disrupt p53 Mice-viable Early onset of tumors-within 2-9 months of birth p53+/- also tumor-prone but longer latency Cancer incidence was strain-dependent Knock-in p53 mice explain Generate particular mutant, expressed from endogenous promoter PICT
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In addition enhanced existing predisposition of mouse strains: 129/Sv mice-are prone to testicular cancer-p53 KO on 129 background-even more BALB/c mice-suseptible to mammary cancer, KO most common cancer-mammary cancer (half of female mice affected) Mice with p53-/+ are a model for Li-Fraumeni syndrome Human familial cancer syndrome Patients are prone to several types of cancer breast cancer and soft tissue sarcomas, brain tumors, osteosarcoma, leukemia, melanoma etc The diverse tumor types in family members characteristically develop at unusually early ages (children), and multiple primary tumors are frequent. By segregation analysis, Strong et al. (1987) demonstrated that the observed cancer distribution in families best fit a rare autosomal dominant gene model. The model also predicted that in families at risk the probability of developing any invasive cancer (excluding carcinomas of the skin) reaches almost 50% by age 30, when only 1% of the general population has developed cancer. • several kinds of cancer are involved, • cancer often strikes at a young age, and • cancer often strikes several times throughout the life of an individual with the syndrome. Conditional KO Mice p53 -/- die very young from lymphomasTherefore to study effect on other types of cancer-make conditional KO p54 flanked by two loxP sites, mice are crossed with transgenic mice expressing Crerecombinase under tissue specific promoter p53-Brca2 mammary-specific KO-increase in mammary cancer, but not in Brca2 alone
p53 KO mice were crossed with telomerase KO – mice Normally mice do not have cancers of epithelial origin-carcinomas, which are the most common in human Human carcinomas-high genomic instability-translocations-it is not observed in mice-long telomeres
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Telomerase-KO-telomere shortening-start to develop carcinomas p53 prevents growth of cells with critically short telomeres p53 mutation in human contribute to genomic instability that causes epithelial tumors But p53 mutation also causes tumors in mice with long telomeres p53 mice as a test for carcinogens Carcinogens produce earlier tumors in p53 KO -/- too high background +/- 6 months old approved by FDA to replace the 2 yr old carcinogenicity mouse tests From what we know-p53 protects from cancer If we increase activity of p53-eliminate cancer and increase survival and longevity Attempts to generate transgenics that globally overexpressed p53 failed-problems in development One mouse model with hyperactive p53 was generated “by mistake” L. Donehower Tyner SD, Venkatachalam S, Choi J, Jones S, Ghebranious N, Igelmann H, Lu X, Soron G, Cooper B, Brayton C, Hee Park S, Thompson T, Karsenty G, Bradley A, Donehower LA. p53 mutant mice that display early ageing-associated phenotypes. Nature. 2002 Jan 3;415(6867):45-53.
Aberrant gene targeting event-partially deleted copy PICT Mice were tumor resistant TABLE But-short-lived PICT Senescent phenotypes Slow wound healing Slow hair growth Examined p53 responses p53-seems more stable after DNA damage Increased trans-activation activity EXPLAIN-luciferase under p53-inducible promoter How do we explain this paradox-mice are cancer resistant but age prematurely? Higher apoptosis-or senescence-that leads to exhaustion of stem cell reserves-early loss of tissue function
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Donehower LA. p53: guardian AND suppressor of longevity? Exp Gerontol. 2005 Jan-Feb;40(1-2):7-9. Review.
PICT1-relationship of p53 function with cancer resistance and fitness PICT2-stem cell based model Serrano Garcia-Cao I, Garcia-Cao M, Martin-Caballero J, Criado LM, Klatt P, Flores JM, Weill JC, Blasco MA, Serrano M. "Super p53" mice exhibit enhanced DNA damage response, are tumor resistant and age normally. EMBO J. 2002 Nov 15;21(22):6225-35.
Mice with two extra genomic copies of p53, inside a large chunk of original chromosomeThis p53 behaves as a functional replica of endogenous p53 (Normal p53, not truncated) Resistance to tumors-chemically induced, probably some resist to spontan. But aged normally It is feasible to increase p53 function slightly without detrimental effects First model-constitutively activated-here-normal activity Potentially possible to increase tumor resistance without affecting ls More recent papers will be presented today: p53 activity was modulated by playing with MDM2 or Arf (regulators of p53). The p53 itself was functional. Mice were more resistant to tumors, and had even slightly increased ls. However, increased level of apoptosis. This may be OK if you are a mouse, no telomere shortening. IN humans may be more problematic. In addition too much apoptosis-waste energy. Human polymorphism study van Heemst D, Mooijaart SP, Beekman M, Schreuder J, de Craen AJ, Brandt BW, Slagboom PE, Westendorp RG; Long Life study group. Variation in the human TP53 gene affects old age survival and cancer mortality. Exp Gerontol. 2005 Jan-Feb;40(1-2):11-5. Review.
Polymorpism in human population for p53 codon 72, 72 Arg-more prevalent - 70% in caucasians 72 Pro - 30% in Caucasians (G to C single nucleotide substitution) 72 Pro homozygotes –increased modest increase in cancer incidence p53 72 Pro is less robust in antiproliferative function, weak p53 poorer prognosis during anticancer treatment However, is not associated with reduced ls Look at individuals 85 and older -4-
PICT Despite higher risk of cancer Pro/Pro individuals-had higher relative survival Death from frailty 21% Arg/Arg and only 6% of Pro/Pro In old age Pro/Pro provides significant survival benefit for those who escaped cancer Antagonistic pleiotropy: p53-positive effect on longevity by its tumor suppressor function, but also has negative effect at very old age
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