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Abstract

Protein phosphorylation plays an indispensable role in cellular regulation of mitosis, metabolism, differentiation, and death. We previously reported that the protein phosphatase inhibitor okadaic acid (OKA) induces apoptosis in renal epithelial cells in culture. In the present study, we examined the role of phosphotidylinositol 3 (PI3) kinase signaling in okadaic acid-induced apoptosis by pre-treating normal rat kidney renal epithelial cells expressing human bcl-2 with the PI3 kinase inhibitors, LY294002 and wortmannin, followed by apoptosis-inducing concentrations of okadaic acid. Given the reported cell survival activity of PI3 kinase signaling mostly attributed to Akt kinase activation, we hypothesized that inhibition of PI3 kinase would enhance okadaic-induced apoptosis. Surprisingly, our data show that pretreatment with LY294002, but not wortmannin, attenuated okadaic acid-induced apoptosis. In contrast, to LY294002, wortmannin enhanced apoptosis. Interestingly, we also found that LY294002 treatment increased bcl-2 protein levels in normal rat kidney epithelial cells expressing bcl-2 (NRK-bcl-2). In untreated cells, bcl-2 appeared to be mainly perinuclear, coincident with the nuclear membrane, or in the cytosol. In OKA treated cells that were pre-treated with Ly294002, bcl-2 was highly co-localized with mitochondria, but in cells treated with okadaic acid alone, bcl-2 was associated with fragmented chromatin. In this model, it appears that LY294002 may exert anti-apoptotic effects by a previously unreported treatment related increase in bcl-2. Although it is widely accepted that bcl-2 protein can inhibit apoptosis, we propose that the subcellular location of bcl-2 is an important determinant in whether bcl-2 effectively inhibits apoptosis.

Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

“…The pathways regulated by PI3K and PI3KR1 are, however, quite complex and we do not yet have a satisfying mechanistic explanation for the radiomitigation. It is noteworthy that LY294002 has been shown to inhibit apoptosis induced by the phosphatase inhibitor okadaic acid in normal renal epithelial cells (31). This antiapoptotic effect of LY294002 was associated with an increase in the antiapoptotic protein Bcl-2 (31).…”

“…It is noteworthy that LY294002 has been shown to inhibit apoptosis induced by the phosphatase inhibitor okadaic acid in normal renal epithelial cells (31). This antiapoptotic effect of LY294002 was associated with an increase in the antiapoptotic protein Bcl-2 (31). While we found NCCIT cells expressed basal levels of Bcl-2, we were unable to detect any increase in Bcl-2 after treatment of cells with LY249002 (data not shown).…”