Alkylating Agent-Induced Carcinogenesis in Xiphophorus Genetic Hybrids

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DESCRIPTION: (Applicant's Description) Project 2 has three goals: (1) Experimental development of N-methyl-N-nitrosourea (MNU)-induced tumor models; (2) Determination of DNA repair capabilities for parental stocks and hybrid animals for correlation of DNA repair capability with induced tumorigenesis; and (3) Comparison of MNU-induced tumorigenesis with UV-induced tumorigenesis in these genetic susceptibility models. In Project 2, we will use both classical genetics and molecular biological approaches to analyze MNU-induced tumors in Xiphophorus backcross hybrid (BC1) fish produced from 3 interspecific matings. Xiphophorus backcross hybrid (BC1) fish produced from 3 interspecific matings. BC1 animals from these crosses are predisposed to MNU-induced melanoma, fibrosarcoma, or neuroblastoma. Parental stocks utilized in these crosses are highly inbred and carry more than 70 polymorphic genetic markers allowing classical linkage analyses of marker inheritance with tumor induction. Accordingly, the Specific Aims of Project 2 are: (1) To investigate the kinetics of MNU-induced tumor formation (melanoma and neuroblastoma) in three Xiphophorus hybrid models. BC1 animals from each cross will be exposed to doses optimized for tumor incidence in order to perform linkage analyses aimed at identification of chromosomal regions associated with tumor susceptibility; (2) To investigate co-segregation and expression of specific target genes associated with tumor induction. The influence of tumor suppressor genes, specific oncogenes, and epigenetic methylation patterns will be analyzed to determine if gene expression is modulated in tumors and/or tumors susceptible fish hybrids; (3) To investigate the potential relationships between MNU-induced DNA damage, DNA repair and tumorigenicity in parental, F1 and BC1 hybrid fishes and fish tissues. Both 06-methylguanine DNA methyltransferase (06MGT) repair and base excision DNA repair (BER) capabilities and rates in parental and hybrid animals and in tumor and non-tumor tissues, will be determined and compared to DNA damage distribution data derived from in situ methodologies which will employ damage specific antibodies in radioimmunoassays to provide the sensitivity required to assess DNA damage and repair in very small animals and tissue or tumor samples. Concurrent use of parallel methodologies for determination of DNA damage and repair will allow us to thoroughly investigate the organismal and cellular distributions of alkylating agent-induced DNA damage and to directly determine damage tolerance in normal tissues and tumors.