Abstract
Ras oncogene activation is a key genetic event in several types of human cancer, making its signal pathways an ideal target for novel therapies. We previously showed that expression of mutant ras sensitizes human thyroid epithelial cells to induction of cell death by treatment with phorbol 12-myristate 13-acetate (PMA) and other phorbol esters. We have now investigated further the nature and mechanism of this cell death using both primary and cell line models. The cytotoxic effect of PMA could be blocked by bisindolylmaleimide (GF 109203X), a well-characterized inhibitor of c and n protein kinase C (PKC) isoforms, and by prior down-regulation of PKC, indicating that it is mediated by acute stimulation, rather than down-regulation. Western analysis identified two candidate isoforms--alpha and epsilon--both of which showed PMA-induced subcellular translocation, either or both of which may be necessary for PMA-induced cell death. Immunofluorescence showed that PMA induced a rapid nuclear translocation of p42 MAP kinase of similar magnitude in the presence or absence of mutant ras expression. Cell death exhibited the microscopic features (chromatin condensation, TdT labelling) and DNA fragmentation typical of apoptosis but after a surprising lag (4 days). Taken together with recent models of ras-modulated apoptosis, our data suggest that activation of the MAPK pathway by PMA tips the balance of pro- and anti-apoptotic signals generated by ras in favour of apoptosis. The high frequency of ras mutations in some cancers, such as cancer of the pancreas, which are refractory to conventional chemotherapy, together with the potential for stimulating PKC by cell-permeant pharmacological agents, makes this an attractive therapeutic approach.
Highlights
Induced subcellular translocation, either or both of which may be necessary for phorbol 12myristate 13-acetate (PMA)-induced cell death
Given the potential therapeutic implications of these findings. we have investigated the mechanism of PMA-induced toxicity further. in particular to distinguish whether the toxic effect is a result of stimulation or of down-regulation of protein kinase C (PKC). to identify the role of specific PKC isoforms and to analyse the 'mode' of PMA-induced cell death
In addition. to account for the possibility that GF 109203X might block toxicity by non-specific inhibition of pathways other than those operating via PKC. we tested the effect of down-regulation of PKC by prolonged pretreatment with PMA. before the induction of ras
Summary
Monoclonal antibodies against PKC a yD P . 6. 0. USA) was diluted from a 34 mm stock in the appropriate culture medium as indicated. Cells were grown in RPMI 1640 medium (Flow Laboratories) supplemented with 10% FCS and 0.4 mg ml-' G418 (Gibco). Cultures were re-fed with non-selective medium 24 h after infection and maintained for 3 days to permit viral integration and expression They were passaged into three 60-mm dishes with medium containing 10% FCS and. R18 cells were seeded at 106 per 150-mm dish and incubated for 5 days before the preparation of protein fractions. After blocking non-specific binding sites with 10% FCS in phosphate-buffered saline (PBS), cells were incubated for 1 h with rabbit polyclonal anti-p42 MAPK antibody (1:500) diluted in PBS/0.6% bovine serum albumin (BSA) (Sigma), followed, after washing, by goat antirabbit-TRITC antibody (1:50) (Southern Biotech) for 1 h. Agarose/TBE gel containing ethidium bromide and was visualized on an ultraviolet transilluminator
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