MECHANISMS OF TUMOR PROMOTION AND CARCINOGENESIS

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Determining the mechanisms that allow neoplastic cells to undergo uncontrolled proliferation is crucial to understanding carcinogenesis. Recent studies indicate that protein phosphatase, like protein kinases, play important and specific roles in cell-cycle regulation; thus alterations in the expression or regulation of these proteins may affect cell-cycle progression and could even allow cells to undergo unregulated proliferation. A role for protein phosphatase in the tumor promotion process is also emerging from studies with okadaic acid, microcystin-LR, and calyculin A, three highly specific and potent inhibitor of certain serine/threonine protein phosphatase. We have identified, purified and characterized a novel serine/threonine protein phosphatase belonging to a new class of protein phosphatase, designated as type 3 (PP3). PP3, like type 1 (PP1) and type 2A (PP2A), is potently inhibited by okadaic acid and calyculin A, two potent tumor promoters in mouse skin. PP3 is also potently inhibited by microcystin- LR, a tumor promoter in liver. This suggests that these three classes of protein phosphatase may have a role in the tumor promotion process. This proposal is designed to test the hypothesis that protein phosphatase play an important role in cell cycle progression; thus, the interference of their activity may contribute to the aberrant proliferative behavior of neoplastic cells. These studies will focus around the following specific aims: Aim 1. Clone PP3. Once clones are identified, the cDNA will be sequenced and the amino acid sequence of PP3 will be deduced. The cDNA will then be used to study the expression of PP3 during cell cycle progression and carcinogenesis. Aim 2. Characterize the mechanisms regulating the activity of PP3. The mechanisms influencing the activity of PP3 will be investigated with the use of specific inhibitors (i.e. okadaic acid, microcystin-LR, nodularin, methyl okadaate, calyculin A and tautomcyin), type specific antibodies, and cDNA probes. Aim 3, Characterize the role of PP# in EGF stimulated cell proliferation and the relationship between serine/threonine protein phosphatase and TGFBeta1 mediated growth arrest in CCL64 cells. Specific inhibitors, type-specific cDNA probes and antisense phosphorothioate oligodeoxynucleotides will be employed to determine the role of PP1 and PP3 in TGFBeta1-mediated growth arrest of CCL64 cells.