Sensitisation of HL60 human leukaemic cells to cytotoxic drug-induced apoptosis by inhibition of PI3-kinase survival signals

Abstract

Drug resistance remains a serious limiting factor in the treatment of acute myeloid leukaemia (AML) either at initial presentation or following primary or subsequent relapses. Using specific kinase inhibitors, this study has investigated the contribution of the Ras/PI3-kinase regulated survival pathways to drug resistance and suppression of apoptosis in a cell line derived from AML (HL60). Inhibition of the Raf/MAP-kinase (ERK) pathway with a specific MAP-kinase inhibitor, apigenin did not sensitise HL60 cells to drug-induced apoptosis, indicating a lack of involvement in chemoresistance. In contrast, the PI3-kinase inhibitors, LY294002 and wortmannin, did induce a significant increase in apoptosis in combination with cytotoxic drugs. The contribution of downstream mediators of PI3-kinase, p70S6-kinase and PKB/Akt were then investigated. While inhibition of p70S6-kinase with rapamycin did not increase drug-induced apoptosis, PI3-kinase inhibition resulted in notable dephosphorylation of PKB, suggesting that the PI3-kinase/PKB survival pathway may play a major role in chemoresistance in AML. This pathway has been reported to mediate heterodimer interactions with the proapoptotic regulator, Bad. In contrast to previous studies, we found no evidence of Bad binding to anti-apoptotic Bcl-2, Bcl-X L or Mcl-1, or of alterations in Bax heterodimers. This suggests that alternative targets of PI3-kinase/PKB, distinct from the Bcl-2 family may be responsible for contributing to survival factor-mediated drug resistance in AML. Leukemia (2000) 14, 602-611.

Molecular signals in anti-apoptotic survival pathways

“…50 A study investigating this possibility demonstrated that inhibition of PI3-kinase survival Leukemia signals, with concomitant inhibition of PKB, in the acute myeloid leukaemia (AML)-derived cell line, HL60, which endogenously expresses an activated N-Ras mutation, greatly increased sensitivity to cytotoxic drug-induced apoptosis. 42 Similar findings were reported by a second group, where PI3-kinase inhibition accelerated daunorubicin-induced apoptosis in the monocytic cell line, U937. 59 Deregulated PKB is evident in a variety of tumours, due to PKB overexpression, constitutive activation by oncogenic Ras, or following inactivation of the tumour suppressor, PTEN.…”

“…Likewise, no specific inhibitor of either PKB or JNK has been identified to date. A role for PI3-kinase in chemoresistance in vitro has only recently been reported 42,59 and the potential (and toxicity) of the PI3-kinase inhibitors LY294002 and wortmannin have not yet been clinically evaluated.…”

“…PI3-kinase inhibition sensitises myeloid leukaemia cells to drug-induced apoptosis, independently of alterations in Bad phosphorylation status or binding to antiapoptotic Bcl-2 family members. 42 Recently, additional targets of PKB have been described, which presumably, in time, will be confirmed as participants in PI3-kinase/PKB-mediated Leukemia drug resistance.…”

“…However, the contribution of ERK to survival under certain circumstances, was not found to extend to protection from cytotoxin-mediated apoptosis. 42 Interestingly, it is the upstream components of this pathway which appear to have an increased capacity for promoting survival. As previously mentioned, oncogenic Ras up-regulates expression of the anti-apoptotic molecules Bcl-2 and Bcl-XL.…”

“…Following reports establishing PI3-kinase as a contributor of consequence to cytotoxic drug resistance in leukaemia cell lines, most likely through PKB signalling, 42,59 we have speculated upon the potential contribution of known PKB substrates to this process (Figure 2). The importance of PKB signalling to survival is underlined by the finding that PKB is also cleaved and inactivated by caspase action during apoptosis.…”