Studies

Transgenic Carcinogenesis Group

Genetic susceptibility to the loss of tumor suppressor gene function in environmental carcinogenesis

Individuals that develop cancer at an early age usually have inherited a mutated allele of a tumor suppressor gene, e.g. the p53 tumor suppressor gene (human TP53) as observed in the Li-Fraumeni Syndrome. Tumors arising in individuals with an inherited TP53 loss of function mutation often show an increased frequency for loss of heterozygosity (LOH) of the wild type TP53 allele (highly penetrant; 104/160 observed to date in the IARC TP53 database). The complex genetic basis for this susceptibility to the loss of tumor suppressor gene function by LOH in environmental carcinogenesis is not understood and has not been investigated.

Mechanisms for LOH: Mis-segregation and Aneuploidy.

The TC Group has observed that human carcinogens (ionizing radiation, benzene, melphalan, etc.) rapidly induce tumors with LOH of the wild type p53 allele in p53 haploinsufficient mice carrying an inherited p53 null mutation and a functioning wildtype allele. The LOH phenotype associated with ionizing radiation induced lymphohematopoietic cancer is highly penetrant in the C57BL/6 (B6) mouse strain, and appears low in the DBA/2 (D2) strain (B6>B6C3F1>C3H>B6D2F1>D2) that are p53 haploinsufficient. The group’s hypothesis is that susceptibility to LOH in lymphohematopoietic tumors is a quantitative trait dependent upon the DNA damage repair capacity of hematopoietic stem cells. If so, allelic variants (strain dependent haplotypes) and their modifier genes may be the basis for susceptibility or resistance to this trait. Using quantitative analysis of gene expression and simple sequence repeat (strain specific) polymorphic and/or single nucleotide polymorphic marker profiles the group aims to determine quantitative trait loci (QTL) and the highly penetrant genes involved that confer susceptibility or resistance, as well as the low penetrance modifier genes that affect DNA repair and the frequency of LOH resulting in tumor suppressor gene loss. Currently, mouse (up to 25 selected strains) and human long-term cultures of bone marrow derived stromal and multipotential hematopoietic stem cells are being phenotyped and compared for dose-related DNA damage and repair gene expression profiles and repair capacity in response to ionizing radiation exposure. Using haplotype association these results will be compared with short-term cancer studies in inbred (B6 and D2), F1 intercross, (B6D2F1), and the F2 intercross (B6D2F2) mice (p53 null mutant and wild type co-isogenic siblings) to determine and confirm the penetrance of the strain-dependent phenotype associated with B6 and D2 alleles, and to identify the quantitative trait loci (QTL). Use of the p53 haploinsufficient mice show decreased time to tumor without affecting strain penetrance or target tissues. Identification of associated QTL can be further refined using selected recombinant inbred B6D2F2 mice with B6 or D2 alleles at the specific loci under investigation identified by statistical association between haplotype and phenotype based on DNA repair gene transcription profiling. By narrowing the location of the putative QTL, positional cloning methods and bioinformatics may be used to identify the quantitative trait genes responsible for the phenotype.

Mechanisms for LOH: DSB Break Repair and Recombination.

Bcl2 promotes both resistance to apoptosis and suppresses tumorigenesis

Bcl2 is another gene critical to and often involved in cancers (e.g., leukemia and lymphoma) that show a dysfunction and/or loss of p53 and Bax function. In addition to promoting apoptosis in response to DNA damage, overexpression of Bcl2 in the absence of p53 function appears to suppress tumorigenesis. The TC Group is investigating changes to cell cycle control, anchorage independent growth, and decreased rate of cell proliferation and in vivo tumorigenicity in PT67 (NIH 3T3) cells with comparison to wild type and engineered mutant Bcl2 PT67 and other cell lines. Preliminary research suggests that overexpression of Bcl2 results in several functional changes, including altered adhesion of the cell to cellular substratum. This observation might indicate that Bcl2 overexpression inhibits or dysregulates cell adhesion function, in addition to or as a component of altering signaling pathways that affect cell proliferation and tumorigenicity. To test this hypothesis, the group is determining the consequence of Bcl2 overexpression properties in mouse and human cell lines.

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