Apoptotic cell death plays an important role in multistage carcinogenesis, tumor growth and the pathogenesis of renal tubular epithelial damage following low dose chemical exposure. Although many factors have been associated with apoptosis, factors that regulate the apoptotic response are unclear. It is proposed that phosphorylation-dependent activation of mitogen activated protein kinase (MAPK) cascades results in phosphorylation of transcription factors that regulate renal epithelial cell susceptibility to apoptosis. I addition, regulation of protein phosphatase activity will define a physiologically relevant pathway by which environmental toxins can activate the MAP-kinase cascade. We will use a well characterized, okadaic acid -induced renal epithelial cell model of apoptosis to specifically: (1) Examine the role of cellular proliferation/differentiation markers such as MAPK~s p53, bcl-2, c- jun/AP-1 and cell cycle inhibitory proteins in okadaic acid-induced apoptosis of renal epithelial cells. We have found that transformation of renal epithelial cells with ras-oncogene increases sensitivity of renal epithelial cells to the cytotoxic effects of okadaic acid. It is expected that ras-oncogene transformation increases apoptotic sensitivity by activating molecular targets employed during apoptosis. Therefore, studies are proposed to (2) examine the effect of ras-activation and transformation on cellular factors involved in the okadaic acid apoptotic signaling pathway. For this aim, all studies will be carried out using a ras-transformed, tumorigenic renal epithelial cell line. Furthermore, since PP2A activity is dependent on phosphorylation of the phosphatase, ras-transformation may have an affect on protein phosphatase 2A (PP2A) activity. Therefore, studies will be carried out to (3) Compare the phosphorylation state and activity of PP2A in ras-transformed and non-transformed renal epithelial cells. Elucidation of apoptotic mechanisms in renal epithelium may identify signaling mechanisms pivotal to cellular entry into a death or proliferative pathway. Knowing the regulatory factors within this signaling pathway may provide molecular markers that aid in predicting the long term effect(s) of chemical mixtures or chronic exposures on an organ. Furthermore, since these studies will be significant in the elucidation of mechanisms for induction of apoptosis, they may provide a means by which tumor cells expressing ras oncogene can be selectively targeted.
