Selective modulation of cell death is important for rational chemotherapy. By depleting Hsp90-client oncoproteins, geldanamycin (GA) and 17-allylamino-17-demethoxy-GA (17-AAG) (heat-shock protein-90-active drugs) render certain oncoprotein-addictive cancer cells sensitive to chemotherapy. Here we investigated effects of GA and 17-AAG in apoptosis-prone cells such as HL60 and U937. In these cells, doxorubicin (DOX) caused rapid apoptsis, whereas GAinduced heat-shock protein-70 (Hsp70) (a potent inhibitor of apoptosis) and G1 arrest without significant apoptosis. GA blocked caspase activation and apoptosis and delayed cell death caused by DOX. Inhibitors of translation and transcription and siRNA Hsp70 abrogated cytoprotective effects of GA. Also GA failed to protect HL60 cells from cytotoxicity of actinomycin D and flavopiridol (FL), inhibitors of transcription. We next compared cytoprotection by GA-induced Hsp70, caspase inhibitors (Z-VAD-fmk) and cell-cycle arrest. Whereas cell-cycle arrest protected HL60 cells from paclitaxel (PTX) but not from FL and DOX, Z-VAD-fmk prevented FLinduced apoptosis but was less effective against DOX and PTX. Thus, by inducing Hsp70, GA protected apoptosis-prone cells in unique and cell-type selective manner. Since GA does not protect apoptosis-reluctant cancer cells, we envision a therapeutic strategy to decrease side effects of chemotherapy without affecting its therapeutic efficacy.

Damage-induced G1 checkpoint in mammalian cells involves upregulation of p53, which activates transcription of p21Waf1 (CDKN1A). Inhibition of cyclin-dependent kinase (CDK)2 and CDK4/6 by p21 leads to dephosphorylation and activation of Rb. We now show that ectopic p21 expression in human HT1080 fibrosarcoma cells causes not only dephosphorylation but also depletion of Rb; this effect was p53-independent and susceptible to a proteasome inhibitor. CDK inhibitor p27 (CDKN1B) also caused Rb dephosphorylation and depletion, but another CDK inhibitor p16 (CDKN2A) induced only dephosphorylation but not depletion of Rb. Rb depletion was observed in both HT1080 and HCT116 colon carcinoma cells, where p21 was induced by DNA-damaging agents. Rb depletion after DNA damage did not occur in the absence of p21, and it was reduced when p21 induction was inhibited by p21-targeting short hairpin RNA or by a transdominant inhibitor of p53. These results indicate that p21 both activates Rb through dephosphorylation and inactivates it through degradation, suggesting negative feedback regulation of damage-induced cell-cycle checkpoint arrest.Oncogene ( Keywords: p21; Rb; p27; damage response p53-inducible cyclin-dependent kinase (CDK) inhibitor p21 (CDKN1A) is the key mediator of damage-induced cell-cycle arrest. p21 interacts with different cyclin/CDK complexes and other regulators of transcription and signal transduction, exerting broad effects on cell survival, gene expression and morphology (Roninson, 2002). p21 effects are partially mediated by Rb, which is inactivated in proliferating cells through phosphorylation by CDK2 and CDK4/6, both of which are inhibited by p21. As a result, p21 induction leads to Rb dephosphorylation and activation, with ensuing G1 arrest.Whereas p21 activates Rb by dephosphorylation, several oncoproteins inactivate Rb by degradation via the proteasome. Proteasome-mediated Rb degradation is promoted by Mdm2 (Sdek et al., 2005) and gankyrin (Higashitsuji et al., 2000), E7 of papilloma virus (Boyer et al., 1996) and Tax of HTLV1 (Kehn et al., 2005). Oncoprotein-induced proteasomal degradation of Rb is one of the mechanisms for Rb inactivation in carcinogenesis (Ying and Xiao, 2006), but Rb degradation has not been described in DNA damage response.Changes in Rb phosphorylation are most commonly detected by immunoblotting through changes in the protein's electrophoretic mobility. Examination of numerous Rb immunoblots published by different groups showed that in many (but not all) cases Rb dephosphorylation, which results from drug treatment, cell senescence or ectopic p21 expression, is associated with a reduction in the Rb protein signal. In the present study, we have asked (i) whether a decrease in the Rb signal in response to p21 reflects protein degradation or merely altered immunoreactivity of dephosphorylated Rb, (ii) if p53 plays a p21-independent role in the decrease in Rb, (iii) whether such decrease can be induced by other CDK inhibitors that induce Rb dephosphorylation and (iv) if the decrease...

Apoptotic resistance of cancer cells may be overcome by the combination of treatments that activate the two major apoptotic pathways: (i) the death receptor pathway activated by death ligands and (ii) the DNA damage pathway activated by chemotherapy. We have previously shown that mesothelioma cells, resistant to most treatments, are sensitive to the combination of the death ligand tumor necrosis factor-related apoptosis inducing ligand (TRAIL/Apo2L) plus chemotherapy. We investigated a possible role for c-Jun N-terminal kinase (JNK) in the synergistic effect, knowing that JNK can be activated separately by TRAIL and by DNA damage. We chose to study the M28 and REN human mesothelioma cell lines, which are p53-inactivated, to avoid an interaction between p53 and JNK. We showed that JNK was activated by TRAIL and by etoposide and that the activation was enhanced by the combination of the two treatments. We found this activation to be caspase-independent. To inhibit the JNK pathway, we used either dominant-negative constructs of JNK1 and JNK2 (compared with dominant-negative caspase 9) or a chemical inhibitor of the JNK pathway (SP600125). In cells treated with TRAIL plus etoposide, JNK inhibition increased cell survival and decreased apoptosis significantly. In transfected M28 cells, the effect of JNK inhibition was as great as that of the dominant-negative caspase 9 construct. We conclude that JNK contributes to the synergistic effect of TRAIL combined with DNA damage by mediating signals independent of p53 leading to apoptosis.

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