Abstract
Prior studies indicate that leukemias expressing high levels of the p21WAF1 cell cycle inhibitor have a poorer prognosis than p21WAF1-negative leukemias. Although p21WAF1 is upregulated by p53 in the setting of DNA damage, the prognostic significance of p21WAF1 is independent of p53 status. The molecular basis of the negative prognostic effect of p21WAF1 remains obscure, but it is believed to result from decreased apoptosis of p21WAF1-expressing leukemias. We studied the effects of p21WAF1 on apoptosis of K562 leukemic cells, which lack wild-type p53 and do not express endogenous p21WAF1. An inducible p21WAF1 system was used and the effect of p21WAF1 induction on susceptibility to etoposide-mediated apoptosis was measured. p21WAF1 decreased apoptotic death of K562 leukemic cells in response to etoposide. Analysis of intermediaries in the apoptotic pathway indicated that p2 WAF1 had no effect on cytochrome c release or cleavage of procaspase-3. In contrast, p21WAF1 was protective against cleavage of caspase targets poly(ADP-ribose)polymerase (PARP), retinoblastoma protein (Rb), and lamin. The expression of the inhibitor of apoptosis protein c-IAP1, which inhibited the function of executioner caspases 3 and 7, was studied. c-IAP1 protein expression was found to be present in a majority of leukemic blasts from untreated patients, but absent in normal differentiating myeloid progenitor cells. In K562 cells, treatment with etoposide in the absence of p21WAF1 induction resulted in post-transcriptional down-modulation of c-IAP1 levels. c-IAP1 loss involved proteasomal, rather than caspase, degradation pathways. Expression of p21WAF1 sustained c-IAP1 protein levels in the presence of etoposide. Etoposide-mediated apoptosis involves down-modulation of the anti-apoptotic protein c-IAP1. Our findings support the hypothesis that p21WAF1 contributes to leukemic chemoresistance by stabilizing c-IAP1 levels in the presence of chemotherapy.
Highlights
The cell cycle inhibitor p21WAF1, is reported to be a negative prognostic factor in leukemia, associated with leukemic chemoresistance [1]
We examined whether PARP cleavage was altered by p21WAF1 induction
Peptide aldehyde proteasome inhibitors (LNLL) and (MG132) both enhanced the expression of c-IAP1 in K562 cells. c-IAP1 expression was enhanced markedly by incubation of cells for 24 hr in the presence of the specific proteasome inhibitor lactacystin. These findings indicated that c-IAP1 degradation may be mediated through the proteasome
Summary
The cell cycle inhibitor p21WAF1, is reported to be a negative prognostic factor in leukemia, associated with leukemic chemoresistance [1]. Several reports indicate that exogenous or induced p21WAF1 neither protects nor sensitizes cells to apoptosis [16,17,18,19]. Anti-apoptotic effects of p21WAF1 could derive from its ability to inhibit cdk, causing cells to arrest in a G1 cell cycle phase, in which they are relatively insensitive to the effects of pro-apoptotic agents that target S-phase mechanisms. Pro-apoptotic effects of p21WAF1 occur in settings of growth arrest [11], so resistance to apoptosis may not be an epiphenomenon of cell cycle arrest [8,22]. The recruitment of p21WAF1 into pro- or anti-apoptotic pathways may depend upon the availability of specific p21-WAF1-interacting proteins. The induction of p21WAF1 stabilized levels of (c-IAP1), an inhibitor of distal apoptotic mediators, which was otherwise down-modulated by etoposide
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