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01.12.2012 | Research | Ausgabe 1/2012 Open Access

Molecular Cancer 1/2012

MutS homologue hMSH5: role in cisplatin-induced DNA damage response

Zeitschrift:
Molecular Cancer > Ausgabe 1/2012
Autoren:
Joshua D Tompkins, Xiling Wu, Chengtao Her
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1476-4598-11-10) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JT carried out most of the experiments and participated in the preparation of the manuscript. XW participated in reagent preparation, interpretation of critical data, and manuscript preparation and revision. CH conceived of the study and participated in its design and coordination as well as manuscript preparation. All authors read and approved the final manuscript.

Abstract

Background

Cisplatin (cis-diamminedichloroplatinum (II), CDDP) and its analogues constitute an important class of anticancer drugs in the treatment of various malignancies; however, its effectiveness is frequently affected by mutations in genes involved in the repair and signaling of cisplatin-induced DNA damage. These observations necessitate a need for a better understanding of the molecular events governing cellular sensitivity to cisplatin.

Results

Here, we show that hMSH5 mediates sensitization to cisplatin-induced DNA damage in human cells. Our study indicates that hMSH5 undergoes cisplatin-elicited protein induction and tyrosine phosphorylation. Silencing of hMSH5 by RNAi or expression of hMSH5 phosphorylation-resistant mutant hMSH5Y742F elevates cisplatin-induced G2 arrest and renders cells susceptible to cisplatin toxicity at clinically relevant doses. In addition, our data show that cisplatin promotes hMSH5 chromatin association and hMSH5 deficiency increases cisplatin-triggered γ-H2AX foci. Consistent with a possible role for hMSH5 in recombinational repair of cisplatin-triggered double-strand breaks (DSBs), the formation of cisplatin-induced hMSH5 nuclear foci is hRad51-dependent.

Conclusion

Collectively, our current study has suggested a role for hMSH5 in the processing of cisplatin-induced DSBs, and silencing of hMSH5 may provide a new means to improve the therapeutic efficacy of cisplatin.
Zusatzmaterial
Additional file 1: Figure S1 Representative images of CDDP-induced hMSH5 foci formation at 1, 6, and 24 hrs post treatment. Cells were treated with 10 μM CDDP for 2 hrs and hMSH5 foci formation was analyzed at indicated time points after treatment. Figure S2. Representative images of cisplatin-induced γ-H2AX foci formation in 293T, 293T/f-hMSH5, 293T/f-hMSH5Y742F, and 293T hMSH5 RNAi cells (48 hrs post transfection with pmH1P-Bsd/hMSH5 sh-2). (A) Untreated cells were examined in parallel to establish the basal levels of γ-H2AX signal in these cell lines. (B) Cells were treated with 10 μM cisplatin for 2 hrs followed by γ-H2AX foci analysis at 24 hrs after cisplatin removal. Nuclei are counterstained with DAPI and merged images are provided. Figure S3. Representative images of CDDP-induced γ-H2AX, hRad51, and hMSH5 foci formation in 293T and A549 cells. (A) Analysis of CDDP-triggered γ-H2AX and hMSH5 foci formation. (B) Analysis of CDDP-triggered γ-H2AX and hRad51 foci formation. (C) Analysis of CDDP-triggered hRad51 and hMSH5 foci formation. Consistent with hMSH5 cytoplasmic and nuclear distribution patterns, CDDP-induced hMSH5 foci appear to be present in both cytoplasm and nucleus, whereas CDDP-induced γ-H2AX and hRad51 foci are predominately nuclear. Arrows indicate potential overlaps of two different signals. Figure S4. Effects of hMSH5 Tyr742-to-Phe mutation on its interaction with hMSH4 and c-Abl. (A) Co-IP analysis of the interaction between hMSH5 cp-1 Y742F and hMSH4. The results indicated that hMSH5 cp-1 Y742F could interact with hMSH4 as efficient as hMSH5 cp-1. (B) Co-IP analysis of the interaction between hMSH5Y742F and c-Abl. hMSH5Y742F interacted with c-Abl in the same manner as hMSH5 did. (PDF 6 MB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 5
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Authors’ original file for figure 6
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