Application): This proposal investigates expression of genes related to the process of transformation induced in human keratinocytes by infection with the oncogenic virus, SV40. Previously the investigator has demonstrated that viral-infected keratinocytes in long-term culture (late stage transformants) exhibit transformed properties which distinguish them from cells prior to the crisis period that marks the establishment of permanent cell lines. The goal of the experiments described in this proposal is to detail changes in gene expression, which arise at the early versus late stages of transformation. In one set of experiments, novel-transformation related genes from cDNA libraries derived from late passaged SV40-transformed human keratinocytes will be cloned and identified by: (1) colony hybridization using a cDNA probe made from the polyA+ RNAs from normal and transformed keratinocytes to select clones carrying transformation- related cDNAs, and (2) by cloning back cDNA sequences which have been retained in cells transfected with a cDNA expression library after periods of growth Western blots and immunoprecipitation will be used to study time-dependent changes in the intermediates of two pathways involved in the regulation of cell growth, the ras-dependent signal transduction pathway and the cyclin/cyclin-dependent kinase (cdk) regulatory elements of the cell cycle. A construct which places expression of the SV40 early genes under the control of a dexamethasone- inducible promoter will be used to determine changes in gene expression that maintain dependence on expression of the SV40 T antigen and to identify viral independent changes in gene expression may emerge after long-term culture.
Showing the most recent 10 out of 94 publications
