Leukemia Cells to Apoptosis by a Protein Kinase C-dependent Selectively Promotes Sensitivity of Myeloid RAS Mutant

RAS mutations arise at high frequency in human malignancy and have been shown to play a role in the disruption of both normal differentiation and proliferation. In addition, RAS influences a number of intracellular signaling pathways, which impinge on proteins that regulate programmed cell death. In this study, we have examined whether this oncogene can influence the activation of the apoptotic process induced by a range of therapeutic agents used to treat leukemia, and we have identified the downstream targets of RAS mediating the observed changes in sensitivity. Using myeloid leukemia cells (P39) retrovirally transduced with mutant II-Ä.1.S, we found that the influence of this oncogene was highly dependent on the inducer used: whereas RAS had no significant effect on spontane ous apoptosis or on the response to the cytotoxic drugs (doxorubicin or 1-ß-arabinofuranosylcytosine), P39-R4S cells showed a strongly aug mented response to all-frans-retinoic acid (ATRA) in both the induction of apoptosis and differentiation. Because, under some circumstances, RAF has been associated with promoting apoptosis, we examined whether the activation of this kinase by mutant RAS could be responsible for the augmented response to ATRA. However, constitutive activation of RAF did not alter the apoptotic sensitivity of these cells, making it unlikely that RAS promotes apoptosis by stimulating this kinase. Nor did we find that BCL-2 was differentially down-regulated in P39-RAS cells. Rather, we found that the activation of protein kinase C (PKC) by low-dose phorbol ester could almost entirely recapitulate transformation by RAS, in terms of promoting both apoptosis and differentiation after treatment with ATRA. Moreover, the /M.S'-imlurcd phenotype could be completely abol ished by a specific inhibition of PKC under conditions that had no effect on the response of control cells. In conclusion, we have shown that mutant RAS promotes differentiation-associated cell death in P39 cells by stimu lating the activity of PKC, which is itself an important regulator of myeloid differentiation. PKC activation, in turn, powerfully synergizes with the PKC-independent action of ATRA. This work identifies a possi ble explanation for the ability of this oncogene to promote myeloid dif ferentiation of hematopoietic cells. Clinically, it raises the possibility that although leukemias expressing mutant RAS may not show an altered response to cytotoxic agents, they may show enhanced sensitivity to dif ferentiation therapy with ATRA.