Decarbamoyl mitomycin C (DMC) activates p53-independent ataxia telangiectasia and rad3 related protein (ATR) chromatin eviction

Abstract

Interstrand crosslinks induce DNA replication fork stalling that in turn activates the ATR-dependent checkpoint and DNA repair on nuclear chromatin. Mitomycin C (MC) and Decarbamoyl Mitomycin C (DMC) induce different types of DNA crosslinks with DMC being a more cytotoxic agent. We previously reported that the novel DMC induced β-interstrand DNA crosslinks induce a p53-independent form of cell death. The p53-independent DMC cytotoxicity associates with the activation, and subsequent depletion, of Chk1. In this study we further dissect the novel DMC signal transduction pathway and asked how it influences chromatin-associated proteins. We found that treatment with DMC, but not MC, stimulated the disassociation of ATR from chromatin and re-localization of ATR to the cytoplasm. The chromatin eviction of ATR was coupled with the formation of nuclear Rad51 foci and the phosphorylation of Chk1. Furthermore, DMC but not MC, activated expression of gadd45α mRNA. Importantly, knocking down p53 via shRNA did not inhibit the DMC-induced disassociation of ATR from chromatin or reduce the activation of transcription of gadd45α. Our results suggest that DMC induces a p53-independent disassociation of ATR from chromatin that facilitates Chk1 checkpoint activation and Rad51 chromatin recruitment. Our findings provide evidence that ATR chromatin eviction in breast cancer cells is an area of study that should be focused on for inducing p53-independent cell death.

Keywords: ATR; ATR, Ataxia Telangiectasia and Rad3 Related; Cdc25, cell division cycle 25.; ChIP, chromatin immunoprecipitation; Chk1, checkpoint serine/threonine protein kinase 1; DDR, DNA damage response; DMC, Decarbamoyl Mitomycin C; DOX, doxycycline; HR, homologous recombination; ICLs, interstrand cross-links; MC, Mitomycin C; NER, nuclear excision repair; PCNA, Proliferating Cell Nuclear Antigen; RPA, replication protein A; TNBCs, triple negative breast cancers; breast cancer; cell death; chromatin; interstrand crosslink; mitomycin C; p53.

Figure 1.

DMC induces Chk1 phosphorylation at ser 345, p38MAPK phosphorylation, ATR chromatin eviction and chromatin recruitment homologous recombination protein Rad51. (A) Cytoplasmic and nuclear proteins were extracted from MCF7 cells, untreated (Con) or treated with 10 μM of MC or DMC for 4 hours. 50 μg of cytoplasmic or nuclear extracts were loaded on a 4–12% SDS-PAGE gel, immunoblotted with p-ser345 Chk1, Chk1, p38MAPK, pT-180 pY182 p38 MAPK and Actin antibodies. Densitometry analysis of p-ser345 Chk1 or pT-180 pY182 p38 MAPK was performed using NIH ImageJ, and values were normalized to total Chk1 or p38 MAPK. Number represents average of 2 experiments. (B) MCF7 cells, untreated or treated with 10 μM MC or DMC for 4 hours were fixed and double stained with ATR (red) and Lamin (green) antibodies. Nuclear DNA was stained with DAPI (blue). ATR staining is representation of 3 experiments. (C) MCF7 cells, untreated or treated with 10 μM MC or DMC for 4 hours were fractionated into cytosol, nuclear soluble and chromatin bound fractions. 50 μg of cytosolic or chromatin-bound fractions were resolved by SDS/PAGE and immunobloted with RPA, ATR, Chk1, p53 and Histone 3 antibodies. (D) Immunofluoresence analysis of Rad51 foci formation after 10 μM MC or DMC for 4 hours treatment of MCF7 cells. Rad51 signals are shown in green and DNA was counter-stained with DAPI (blue). Rad51 staining is representation of 2 experiments. The RAD51 foci are quantified in 4 hours 10 μM of MC or DMC treated MCF7 cells.

Figure 2.

DMC treatment inhibits the interaction of proteins with the DNA. (A) Nuclei were isolated from MCF7 cells and treated with MNase to obtain mononucleosome fragmented DNA (150 bp). Mononucleosomal DNA was extracted and labeled with [³²P]. DNA-binding activity of nuclear proteins of untreated, 10 μM MC or DMC treated for 4 hours was analyzed by EMSA. (B) The cell cycle profile was checked by Fluorescence activated cell sorting (FACS). MCF7 cells left untreated, or treated with 10 μM MC or DMC for 4 hours and fixed in 30% ethanol and cellular DNA were stained with propidium iodide. (C) Chromatin immunoprecipitation (ChIP) was carried out in untreated, 10 μM MC or DMC treated for 4 hours and UV (50J/m²) irradiated MCF7 cells. ChIP was performed in 400 μg of cross-linked and sonicated cell lysates and antibody against ATR. Immunoprecipitated DNA was amplified by real-time quantitative PCR with primers for p53 intron 7 gene. Non-specific IgG was used to subtract the background. Values were normalized to IgG and inputs, followed by normalization to the untreated samples.

Figure 3.

p53-independent activation of gadd45 by DMC. (A and B) MCF7 cells, untreated or treated with 10 μM MC or DMC for 4 hours and RNA (A) or protein (B) were extracted. The DNA damage response genes, gadd45α, puma and p21 mRNA, were analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction PCR (qRT-PCR). Results were normalized to untreated samples and gapdh values. Graphs show means and standard errors of 2 independent experiments. Western blot analysis of p53 and Actin protein levels from 50 μg of whole cell lysates in response to MC or DMC treatment. (C and D) The MCF7 cell line with inducible p53.shRNA was either treated with 2 μg/ml doxycycline (DOX) for 7 d to induce shRNA expression or left without DOX treatment. Cells were then treated with 10 μM of MC or DMC for 4 hours. Gadd45α mRNA levels were analyzed by qRT-PCR. Results were normalized to untreated samples and gapdh values. Graphs show means and standard errors of 2 independent experiments. Western blot analysis shows p53 and Actin protein levels from 50 μg of whole cell lysates in response to MC or DMC treatment.

Figure 4.

DMC induced p53-independent cell death in MCF7 cells. MCF7 cells were left either untreated or treated with 1, 5, 10, 50 and 100 μM MC or DMC for 48 hours. (A) Phase contrast microscopy image of MCF7 cells treated with MC and DMC as indicated concentrations at ×20 magnification. (B) Quantification of cell viability to MC or DMC treatment was carried out by Guava ViaCount. (C) The MCF7 cell line with inducible p53.shRNA was either treated with 2 μg/ml doxycycline (DOX) for 5 d to induce shRNA expression or left without DOX treatment, followed by treatment with 1, 5, 10, 50 and 100 μM DMC for 48 hours. (D) Isogenic MCF7 cell lines with p53.shRNA or control vector were treated with 2 μg/ml doxycycline (DOX) for 7 d to induce shRNA expression. 5000 cells were plated on the E-plate for real-time growth assay. After 6 hours the cells were treated or untreated with 10 μM DMC. The growth of untreated or 10 μM DMC treated MCF7 cells with either p53 shRNA or control vector were monitored for a total of 26 hours by xCELLigence System. Arrow indicates the 4 hours DMC treatment.

Figure 5.

DMC-mediated ATR dissociation from chromatin is p53-independent. (A) MCF7 cell lines with either p53.shRNA or control vector were either treated with 2 μg/ml doxycycline (DOX) for 7 d or left DOX untreated. Cells were then treated with 10 μM of MC or DMC for 4 hours. The cell fractionation protocol was carried out to separate cellular proteins. 50 μg of chromatin bound proteins were resolved by SDS/PAGE and immunoblotted with ATR, p53 and Histone H3 antibodies. Densitometry analysis of chromatin bound ATR was performed using NIH ImageJ, and values were normalized to untreated samples (Con). (B) A simplified scheme is shown to model the signaling pathways for DMC-activated cell death through eviction of ATR from chromatin and activation of both Chk1 and gadd45 pathways.