Genetic Susceptibility to Loss of Tumor Suppressor Gene Function

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The genetic variants of cancer susceptibility genes between species and isogenic strains of these animal models have not been investigated. Critical insights leading to translational research may be gained using this paradigm. A dominant feature of human carcinogen induced tumors in this mouse model is the loss of the Trp53 wildtype allele and genome wide loss of heterozygosity often associated with loss of tumor suppressor genes. The pattern of LOH suggests significant changes in gene copy number variation (CNV; gene deletions and gene duplications). Together, these preliminary results implicate loss of regulation of DNA strand break repair. We propose to use F1 hybrids that are haploinsufficient for p53 on the B6 genetic background to define the utility of this model and perform haplotype-phenotype segregation studies for quantitative trait loci analysis. Preliminary genetic analysis of DNA strand break repair and loss of heterozygosity in p53 deficient and wildtype hematopoietic stem cells suggest haplotype dependent efficiency of resolution of strand breaks (repair). Misrepair of carcinogen induced DNA damage may lead to the loss of heterozygosity (LOH) and changes in gene copy number variation (CNV) associated with the loss of tumor suppressor genes leading to cancer. Coordination of non-homologous end joining (NHEJ) and homologous recombination (HR) pathway repair is dependent on the phase of the cell cycle, DNA replication, and expression of the p53 tumor suppressor protein. We have reported that carcinogens (ionizing radiation, benzene, melphalan, etc.) rapidly induce tumors with LOH of the wild type Trp53 allele in B6.129-Trp53tm1Brd heterozygous N5 mice carrying an inherited p53 null mutation and a functioning wildtype allele. The LOH phenotype observed in ionizing radiation (IR) induced tumors is highly penetrant in the B6.129-Trp53tm1Brd N12 heterozygous mouse strain and appears low in the D2B6.129F1-Trp53tm1Brd N12 strain. IR induced Trp53 sequence loss is the greatest in B6.129>C3B6.129F1>D2B6.129F1. D2 alleles appear to suppress both the magnitude of LOH and tumor prevalence. LOH in these tumors show a pattern of non-random loss of chromosome 11 C3 specific alleles(maternal SSLP markers) as well as C11 and genome wide CNV consistent with NHEJ/HR misrepair of strand breaks presenting as intervals of gene specific CNV (gain or loss). Using long-term primary cultures of initiated hematopoietic stem cells (HSC), B6.129-Trp53 deficient as well as wildtype p53 B6 and D2 isogenic mouse hematopoietic stem cells (HSC), we have observed significant differences between strains in the abundance of DNA repair gene transcripts up to 3 h post-irradiation. Preliminary data on terminal deoxynucleotidyl transferase (TdT) or gama-H2AX fluorescence for quantification of DNA strand breaks and the time required to resolve breaks (as estimated by the slope of the curve) are significantly different between these strains. Maximum TdT fluorescence associated with breaks occurred within minutes and the time required for the resolution of TdT fluorescence was strain dependent. The data suggest that misrepair of strand breaks and LOH is a quantitative trait (polygenic) dependent upon the DNA damage repair capacity. These data warrant an investigation to identify the genetic variants associated with these distinct phenotypes. Analysis of NHEJ repair gene haplotypes by similarity matrices indicates that allelic diversity across isogenic strains in some genes is significant but many components of this repair pathway are identical by descent. In order to identify the allelic variants (haplotypes) of genes associated with this strand break repair and LOH phenotype additional phenotyping and functional analysis of multiple isogenic strains are required. Determination of the allelic variants of genes causally related to DNA damage and repair with altered function is critical in order to understand the differences in risk due to exposure to environmental mutagens. Individual genetic susceptibility to cancer from environmental exposure to carcinogens is a complex trait based upon inheritance of allelic variants of multiple genes that increase susceptibility. Inheritance of a combination of minor allelic variants of genesmay significantly increase susceptibility and the risk for cancer. Thus, experimental genetic models for cancer and carcinogen identification must also used to increase genome wide genetic variation in order to determine the genetic basis for cancer incidence and identification of the genes and their allelic variants that modify outcome in response to environmental carcinogens. Hypothesis: The following null hypothesis will be tested: (1) Exposure of isogenic heterozygous AB6.129-Trp53tm1Brd F1, BB6.129-Trp53tm1Brd F1, C3B6.129-Trp53tm1Brd F1, D2B6.129-Trp53tm1Brd F1, and FB6.129-Trp53tm1Brd F1 p53 haploinsufficient hybrid mice to 6 Gy ionizing radiation will not result in a different tumor spectrum and tumor incidence for each F1 hybrid strain. (2) Exposure of cohorts of these F1 p53 haploinsufficient hybrid mice to 6-Gy ionizing radiation will not result in isogenic strain differences between biomarkers of exposure. (3) Exposure of stromal and hematopoietic progenitor cell (HPC) cultures to 6-Gy ionizing radiation will not result in isogenic strain differences between quantitative rates for DSB resolution. Allelic variation introduced by outcross of female isogenic mice (A/J, BALB/cByJ, C3H/HeJ, DBA/2, and FVB/NJ) to B6.129-Trp53tm1Brd N12 deficient is expected to modify tumor incidence and the magnitude of genomic loss of heterozygosity based upon preliminary evidence. Suppression and/or repair of DNA strand breaks in hematopoietic stem cells may be modified by allelic variants of DNA strand break repair protein complexes and enzymes. Failure to suppress and/or alter the efficiency of carcinogen induced DNA strand break (DSB) repair pathways is expected to result in loss of tumor suppressor gene function associated with genome wide loss of heterozygosity and tumorigenicity. These studies are in progress.