Triggering Senescence Programs Suppresses Chk1 Kinase and Sensitizes Cells To Genotoxic Stresses

Abstract

Depletion of the major heat shock protein Hsp72 leads to activation of the senescence program in a variety of tumor cell lines via both p53-dependent and p53-independent pathways. Here, we found that the Hsp72-depleted cells show defect in phosphorylation and activation of the protein kinase Chk1 by genotoxic stresses, such as UVC irradiation or camptothecin. Under these conditions, phosphorylation of Rad17 was also suppressed, whereas phosphorylation of p53 at Ser 15 was not affected, indicating a specific defect in phosphorylation of a subset of the ATR kinase substrates. Similarly, suppression of Chk1 activation was seen when senescence signaling was triggered by direct stimulation of p53, depletion of Cdc2, or overexpression of the cell cycle inhibitors p21 or p16. Thus, defect in Chk1 activation was not a consequence of the chaperone imbalance, but rather a downstream effect of activation of the senescence signaling. Inhibition of Chk1 was associated with inefficient inter-S phase checkpoint, as Hsp72 depleted cells failed to halt cell cycle progression upon UVC irradiation. Accordingly, sensitivity of cells to genotoxic stimuli after Hsp72 depletion was significantly enhanced. Thus, activation of the senescence signaling causes a defect in the DNA damage response manifested in increased sensitivity to genotoxic stresses.

HSP72 depletion suppresses γH2AX activation by genotoxic stresses via p53/p21 signaling

“…To address this question, we synchronized control and sh72 HCT116 cells in S-phase by arresting cells in mitosis by nocodazole treatment followed by release into fresh medium for 10 h as described earlier (Gabai et al, 2008). We observed that g-IR or CPT treatment of Hsp72-depleted cultures resulted in similar inhibition of H2AX phosphorylation in unsynchronized and S-phasesynchronized cells (Figure 3a), indicating that cell cycle does not affect suppression of gH2AX.…”

“…Earlier, we reported that downregulation of the major heat shock protein Hsp72 promotes aneuploidy, reduces DNA repair and increases sensitivity of cancer cells to UVC irradiation or CPT (campthotecin) (Gabai et al, 2008). Here, we investigated the basis of the observed DNA instability and sensitization of transformed cells toward genotoxic treatments.…”

“…Such inhibition of the Chk1 signaling resulted in inefficient S-phase checkpoint, and led to sensitization of cells to UVC radiation and campthotecin (Gabai et al, 2008). However, we noticed that on exposure to distinct DNAdamaging agents, like g-radiation (g-IR) or doxorubicin (Dox), Chk1 was not activated and there was no suppression of cell-cycle checkpoints.…”

“…Earlier, we have shown that knockdown of Hsp72 in human tumor cell lines triggers senescence programs, leading to impairment of Chk1 activation on UVC radiation and campthotecin (Gabai et al, 2008). Such inhibition of the Chk1 signaling resulted in inefficient S-phase checkpoint, and led to sensitization of cells to UVC radiation and campthotecin (Gabai et al, 2008).…”

“…To distinguish between these two possibilities, we tested whether direct stimulation of p53 pathway causes similar defect in DDR. HCT116 cells were treated with a specific chemical activator of p53, nutlin-3, which binds p53 and inhibits its interaction with the ubiquitin ligase MDM2, resulting in p53 accumulation and activation (Efeyan et al, 2007;O'Callaghan-Sunol et al, 2007;Vassilev, 2007;Gabai et al, 2008). Following 2 days incubation with nutlin-3, cells were exposed to various doses of g-IR.…”