HMG-1/Y AND NEOPLASTIC TRANSFORMATION

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DESCRIPTION: (adapted from the investigator's abstract) Normal cellular proliferation is initiated by growth factors that bind to their specific receptors and ultimately results in the sequential expression of genes important in regulating cell growth. Although a number of cell cycle regulatory genes have been identified, further elucidation of this genetic program will enhance our understanding of normal and neoplastic cell growth. Immediate-early genes are expressed within minutes of growth factor stimulation and include the c-myc proto-oncogene; delayed-early genes are activated hours after growth factor stimulation, but before DNA synthesis, and include the HMG-I/Y gene. To better understand the mechanisms by which delayed-early genes are activated in the G1/S transition of the cell cycle, we have been studying the regulation of the HMG-I/Y gene. Their laboratory has recently shown that HMG-I/Y is a gene target of c-Myc. Their preliminary results also indicate that the HMG-I isoform alone causes neoplastic transformation in an experimental cell line. Because expression of both c-myc and HMG-I/Y are correlated with cellular proliferation and neoplastic transformation, we hypothesize that HMG-I/Y is an important c-Myc target gene required by c-Myc for the regulation of normal and neoplastic cell growth. In addition, HMG-I/Y expression is increased in cancerous cell lines not characterized by elevated c-myc expression. They, therefore, propose that HMG-I may also lead to neoplastic transformation, independent of c-Myc. Thus, the major focus of this research proposal will be to define the role of HMG-I/Y in neoplastic transformation. The specific aims and basic experimental approaches outlined in this proposal include: 1) Evaluate the role of HMG-I/Y in transformed cell lines characterized by increased c-myc and/or HMG-I/Y expression. Antisense and potential dominant-negative HMG-I/Y vectors will be transfected into appropriate cell lines and subjected to soft agar transformation assays to address this aim. 2) Determine if HMG-I/Y plays a role in apoptosis induced by c-Myc. Antisense and potential dominant-negative HMG-I/Y vectors will also be used. In addition, we will determine if ectopic expression of HMG-I/Y leads to apoptosis. 3) Investigate the mechanisms by which HMG-I leads to neoplastic transformation. By mutagenesis analysis, we will identify the domains of the HMG-I protein required for cell transformation in soft agar assays. Target genes regulated by HMG-I/Y will be determined using representational difference analysis from an inducible HMG-I/Y cell line. 4) Explore the role of Mad, Mxi, and AP-2 in the regulation of HMG-I/Y. Additional co-transfection experiments and mutagenesis analysis using the HMG-I/Y promoter constructs will be performed. Insight gained from these studies should advance our understanding of malignancies associated with increased c-myc and or HMG-I/Y expression and may lead to new treatment strategies.