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.
Highlights
Treatment of various types of tumors often relay on the efficiency of radiation and chemotherapy
Loss or inhibition of Chk1 leads to inability to shutdown the cell cycle progression and, allows cells to progress into mitosis before completion of DNA repair [5]
HEK293 and HeLa cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS); HCT116 cells were grown in McCoy 5Â medium with 10% FBS; PC-3 were grown in RPMI 1640 supplemented with 10% FBS; MCF10A were cultivated in DMEM/F12 medium supplemented with 5% horse serum, hydrocortisone (500 ng/mL), insulin (10 Ag/mL), and epidermal growth factor (20 ng/mL); IMR90 cells were cultivated in MEM with 15% FBS
Summary
Treatment of various types of tumors often relay on the efficiency of radiation and chemotherapy. Because most of anticancer drugs represent genotoxic compounds, cellular DNA repair systems counteract anticancer activities of these drugs [1]. Response to genotoxic insults involves sensing of DNA damage by a class of protein kinases, including ATM, ATR, and DNA-PK, followed by activation of Chk and Chk kinases that cause temporal cell cycle arrest, as well as promote assembly of DNA repair complexes at the damaged sites at chromosomes Chk1/Chk2-mediated temporal cell cycle arrest is critical to allow successful completion of DNA repair [3, 4]. Loss or inhibition of Chk leads to inability to shutdown the cell cycle progression and, allows cells to progress into mitosis before completion of DNA repair [5].
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