Cisplatin-induced DNA damage activates replication checkpoint signaling components that differentially affect tumor cell survival

Abstract

Cisplatin and other platinating agents are some of the most widely used chemotherapy agents. These drugs exert their antiproliferative effects by creating intrastrand and interstrand DNA cross-links, which block DNA replication. The cross-links mobilize signaling and repair pathways, including the Rad9-Hus1-Rad1-ATR-Chk1 pathway, a pathway that helps tumor cells survive the DNA damage inflicted by many chemotherapy agents. Here we show that Rad9 and ATR play critical roles in helping tumor cells survive cisplatin treatment. However, depleting Chk1 with small interfering RNA or inhibiting Chk1 with 3-(carbamoylamino)-5-(3-fluorophenyl)-N-(3-piperidyl)thiophene-2-carboxamide (AZD7762) did not sensitize these cells to cisplatin, oxaliplatin, or carboplatin. Moreover, when Rad18, Rad51, BRCA1, BRCA2, or FancD2 was disabled, Chk1 depletion did not further sensitize the cells to cisplatin. In fact, Chk1 depletion reversed the sensitivity seen when Rad18 was disabled. Collectively, these studies suggest that the pharmacological manipulation of Chk1 may not be an effective strategy to sensitize tumors to platinating agents.

Fig. 1.

Rad9 and ATR but not Chk1 are important for tumor resistance to platinating agents. A, Rad9(-/-) murine ES cells stably transfected with empty vector or expressing wild-type Rad9 (inset) were exposed to the indicated cisplatin concentrations for 24 h and analyzed for clonogenic survival. B to F, HeLa cells were transfected with luciferase (Con), ATR, Chk1, or Rad9 siRNAs, and 48 h later, lysates were sequentially immunoblotted (B) for ATR, Rad9, Chk1, and β-actin. The remainder of the cells were treated with the indicated concentrations of cisplatin (C), gemcitabine (D), oxaliplatin (E), or carboplatin (F) for 24 and analyzed for clonogenicity.

Fig. 2.

Chk1 inhibition and Chk2 codepletion do not affect cisplatin cytotoxicity. A and B, HeLa cells were treated with the indicated concentrations of dimethyl sulfoxide (DMSO) or AZD7762 (AZD) simultaneously with the indicated concentrations of gemcitabine or cisplatin for 24 h and analyzed for clonogenicity. C and D, HeLa cells were transfected with luciferase (Luc), Chk1, Chk2, or Chk1 and Chk2 siRNAs and 48 h later were analyzed for Chk1, Chk2, and heat shock protein 90 expression (Hsp90; A) and for clonogenic survival (B) after 24-h treatment with the indicated concentrations of cisplatin.

Fig. 3.

Cisplatin induces activating Chk1 phosphorylation and causes Chk1-dependent S-phase arrest. A, HeLa cells were exposed to vehicle (-) or the indicated concentrations of cisplatin or gemcitabine for 4 or 24 h. Cell lysates were then sequentially immunoblotted for phospho-Ser³⁴⁵-Chk1 (P-Chk1), total Chk1, and Cdc25A. ^*, nonspecific band. B, HeLa cells transfected with luciferase (Control) or Chk1 siRNAs were treated with indicated concentrations of cisplatin for 24 h, stained with propidium iodide, and analyzed by flow microfluorometry.

Fig. 4.

Chk1 depletion does not sensitize HCT-116 and U2OS cells to cisplatin. A to D, HCT-116 cells (A and B) or U2OS (C and D) transfected with luciferase (Control), ATR, or Chk1 siRNAs were treated with the indicated concentrations of cisplatin or gemcitabine for 24 h, and clonogenic assays were performed.

Fig. 5.

Chk1 depletion does not further sensitize cells with defects in pathways that repair cisplatin-induced DNA lesions. A to E, HeLa cells transfected with siRNAs to FancD2 (A), Rad51 (B), BRCA1 (C), BRCA2 (D), or Rad18 (E), alone or in combination with Chk1 siRNA, were treated for 24 h with the indicated concentrations of cisplatin and analyzed for clonogenicity.