Ras mutation, irrespective of cell type and p53 status, determines a cell's destiny to undergo apoptosis by okadaic acid, an inhibitor of protein phosphatase 1 and 2A

Okadaic acid (OA), a toxin from the black sponge Halicondria okadai, is a s pecific inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A ( PP2A). OA is a tumor promoter but also induces apoptosis in some tumor cell lines. In this study, we determined whether ras mutation and/or p53 status are characteristics associated with the cell's sensitivity to the inductio n of apoptosis by OA. Several cell lines that differed in ras and p53 mutat ions were treated with OA (10 - 100 nM). At 24 to 48 h after treatment, the percentage of cells undergoing apoptosis was quantitated. The cell lines w ith mutations in either H-ras (human bladder carcinoma cell line T24 and mo use keratinocyte cell line 308), or K-ras (human colon carcinoma cell lines DLD-1 and HCT116; human prostate cancer cell lines LNCaP and PC-3; human l ung cancer cell lines Calu-6 and SKLU-1; and human pancreatic cancer cell l ine MIAPaCa2) were more sensitive to OA-induced apoptosis (3- to 10-fold) t han the cell lines that lacked the ras mutation (mouse epidermal cell lines C50 and JB6; murine fibroblast cell line NIH3T3; human colon cancer cell l ine HT29; human kidney epithelial cell line Hs715.K; and human pancreatic c ancer cell line Bx-PC3). Similarly, using isogenic cell lines we found that overexpression of mutated H-ras in NIH3T3 and in SV40 immortalized human u roepithelial cells (SVHUC) enhanced their sensitivity to undergo apoptosis in response to OA treatment. The T24, DLD-1, SKLU-1, Calu-6, and MIAPaCa2 c ell lines express mutated p53. The SVHUC as well as their ras-transfected c ounterparts have inactive p53 due to complex formation between large "T" an tigen and p53. Taken together, these results imply that OA-induced apoptosi s may involve a p53-independent pathway. The transfectants (NIH3T3-ras and SVHUC-ras), which express mutated H-ras, have up-regulated PP2A activity. O A treatment inhibited in vivo the levels of PPI and PP2A activity, and indu ced apoptosis in SVHUC-ras and other cell lines. We conclude that OA-induce d cell death pathway in ras-activated cell lines may involve a cross talk b etween PPI and PP2A and ras signaling pathways. In light of the present res ults, the current theory that OA promotes mouse skin tumor formation by sel ective expansion of initiated cells that harbor ras mutations needs reevalu ation.