Phorbol ester tumor promoters induce the expression of certain proliferation-associated genes by a pathway that involves activation of the Ca2+ and phospholipid-dependent protein kinase C. We have shown that activation of protein kinase C by the phorbol ester TPA induces the expression of VL30, a proliferation-associated gene which can also be induced by EGF. Structurally, VL30 resembles both retroviruses and eukaryotic transposable elements in the possession of long terminal repeats (LTR) which appear to be required for the insertional mutagenesis caused by integration of retroviruses. Retroviral LTRs contain sequences which function as promoters and enhancers of gene transcription. Activation of cellular oncogenes following the insertion of retroviruses may reflect the replacement of normal cellular regulatory elements with the retroviral control elements. A major goal of this proposal is the characterization of the putative regulatory elements within the VL30 LTR, particularly in identifying those sequences within the VL30 LTR which respond to protein kinase C activation. This will be accomplished by detailed study of two cell lines subcloned from Rat-1 cells which had been infected with VL30-containing psuedovirions. Both cell lines express VL30 RNA in response to TPA treatment. Structural techniques will be used to identify the VL30 sequences which have become integrated in these subclones, and the ability of these sequences to act as transcriptional promoters or enhancers will be studied by inserting these sequences into expression vectors. Once these sequences have been identified, we will attempt to identify proteins in nuclear extracts which bind to these DNA sequences. It will be particularly relevant to our understanding of carcinogenesis if can identify DNA-binding proteins whose abundance or activity are modified by treatment with phorbol ester tumor promoters. Expression of retroviral genes has been correlated with increased rates of transposition in other systems. We will alter the expression of VL30RNA by a variety of means, including long-term exposure to TPA or EGF or transfection of cells with known oncogenes in order to determine the relationship between gene expression and translocation in this system. We will also investigate the oncogenic potential of VL30 elements by monitoring the transformation rate in cells which have been infected with VL30. We will simultaneously monitor the translocation of VL30 elements by using specific probes derived from our structural studies of the VL30 integrants and the flanking host sequences. These studies will allow us to determine whether transposition and transformation are correlated in the VL30 system, and whether agents which increase VL30 expression can act as carcinogens.
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