Background
Despite being members of the MMR protein family, the MSH5 homologues have not been demonstrated to function in MMR. Instead, studies in mice,
C. elegans, and
S. cerevisiae have shown that MSH5 plays an array of diverse functions ranging from meiotic recombinational DSB repair, maintenance of chromosome integrity, to DNA damage response [
1‐
6]. Purified hMSH4-hMSH5 protein complexes have been shown to possess binding activities towards recombination intermediate structures including the Holliday junction [
7], and endogenous hMSH5 has been shown to interact with a Holliday junction binding protein [
8]. In addition, hMSH5 forms chromosomal foci in human fetal oocytes at different stages of meiotic prophase I [
9].
Coherent with a conjectured role in recombinational DSB repair, hMSH5 has been reported to interact with several proteins related to DSB sensing and repair, including the c-Abl tyrosine kinase and HR protein hRad51 [
10,
11]. It is observed that RAD51 silencing in MSH5-deficient
C. elegans oocytes can result in chromosome fragmentation [
6], suggesting that MSH5 and RAD51 may play a synergistic role in DSB processing at least during meiosis in
C. elegans. In addition, interaction between endogenous hMSH5 and hMRE11 has been observed in human alveolar basal epithelial derived lung adenocarcinoma A549 cells [
8]. Studies performed with mouse models and human patient samples have also suggested a role for hMSH5 in class switch recombination during B and T cell development, whereas hMSH5 deficiency associates with long microhomologies at Ig switch joints [
12]. These observations have raised the possibility that, through interacting with various DSB repair proteins, hMSH5 could exert multiple roles in DNA damage surveillance and DSB repair. Although the link between hMSH5 mutation and diseases in humans has not been explored, a genome-wide association study has designated the
hMSH5 locus at 6p21.33 as a high risk factor for lung cancer development [
13]. In addition to its potential role in DNA repair, hMSH5 interplays with c-Abl in mediating apoptotic response in cells treated with ionizing radiation--a process involved with the activation of p73 and caspase-3 [
14].
In the present study, we have investigated the role of hMSH5 in cisplatin-induced DNA damage response. Cisplatin is the drug of choice for combination chemotherapy of testicular cancers [
15], and hMSH5 is known to be expressed abundantly in the testis [
16]. The cytotoxicity of cisplatin is mainly caused by its ability to form adducts with DNA. The major types of biologically active cisplatin adducts are 1,2-intrastrand crosslinks between guanines or between guanine and adenine, and, to a lesser extent, interstrand DNA crosslinks [
17]. These DNA distortions can effectively block the progression of DNA replication and activate cell cycle checkpoint [
18‐
20]. Cisplatin-induced DNA intrastrand crosslinks can be effectively removed by nucleotide excision repair (NER) [
17,
21]; however, the removal of cisplatin-induced interstrand crosslinks requires the HR pathway [
22]. The importance of recombinational repair in resolving cisplatin-induced DNA damage has also been suggested by the observation that cisplatin increases the rate of recombination, presumably attributing to the formation of DSB [
23,
24]. In addition, the single-strand breaks generated from the processing of cisplatin-induced DNA lesions by NER can also be converted into one-ended DSBs when single-strand breaks collide with the replication forks [
25]. Nevertheless, it becomes increasingly clear that HR plays an essential role in the repair of cisplatin-induced DSBs that may arise from stalled replication forks. In addition, HR-deficient cells are highly sensitive to DNA damaging agents that induce DNA cross-links, due to replication blockade.
Discussion
Our current study has demonstrated an important role for hMSH5 in the processing of cisplatin-elicited S phase-dependent DSBs. Like other HR repair proteins, hMSH5 is predominantly expressed during S and G2/M phases of the cell cycle. Given the observation that silencing of hRad51 can significantly compromise the formation of cisplatin-induced hMSH5 foci, it is conceivable that hMSH5 acts downstream of hRad51 in the DSB repair process. Disruption of hMSH5 function either by RNAi or by the over-expression of a phosphorylation-deficient mutant significantly increased the number of cells displaying γ-H2AX foci at 24 hrs after cisplatin treatment, indicating an increase in the retention of cisplatin-induced DSBs. In addition, disruption of hMSH5 enhances a sustained cisplatin-triggered G2 arrest, thereby rendering cells more sensitive to cisplatin toxicity. It appears that the reduction of clonogenic survivability correlates well with the levels of γ-H2AX foci in 293T, 293T/f-hMSH5, and 293T/f-hMSH5
Y742F cells at 24 hrs after cisplatin challenge. This observation is consistent with previous reports showing that the level of γ-H2AX foci retention 24 hrs after treatment is a useful indicator for cisplatin-mediated cell killing [
28,
34]. Although the precise role of hMSH5 in this process remains to be delineated, the current evidence collectively supports a scenario by which hMSH5 functions downstream of hRad51 in recombinational repair of cisplatin-induced DSBs.
Due to its radio-sensitizing activity, cisplatin has been frequently used in combination chemoradiation therapy of human malignancies [
35]. Presently, however, the mechanisms underlying the effects of this radiosensitizer are still being studied. It is suggested that cisplatin adducts can block the repair of ionizing radiation-induced DSBs by the nonhomologous end-joining pathway [
35,
36]. However, the results of our current study have raised another possibility that cisplatin-triggered hMSH5 induction may potentially contribute to the effectiveness of cisplatin combination chemoradiation therapy. It is known that higher levels of hMSH5 promote IR-induced apoptosis [
14]. Thus, in spite of the fact that cisplatin-triggered hMSH5 induction can facilitate the repair of cisplatin-induced DSBs, the higher levels of hMSH5 could promote a robust apoptotic response to IR during cisplatin combination chemoradiation therapy.
In addition to hMSH5, other MMR proteins are also involved in cisplatin-triggered DNA damage repair and response. In fact, the roles of several other MMR family members in mediating cellular responses to cisplatin-induced DNA lesions have been studied [
37‐
46]. As a whole, these studies highlight two opposite effects of individual MMR proteins on cellular responses to cisplatin toxicity. By functioning in the recognition and signaling of cisplatin-induced DNA lesions, MMR proteins can promote cell killing. On the other hand, they can directly participate in the processing of cisplatin-induced DSBs, thereby exerting a protective effect. It has been shown that hMSH2 recognizes cisplatin-induced DNA lesions through direct binding of DNA-cisplatin adducts [
41,
42]. Cells defective in the expression of hMSH2 or hMLH1 often exhibit a 2- to 4-fold increase in resistance to cisplatin in comparison to corresponding controls [
37,
39,
40,
44‐
46], indicating that hMSH2 and hMLH1 are involved in mediating cisplatin-triggered DNA damage signaling. Accordingly, cisplatin-resistant cells derived from repetitive drug selection are frequently associated with defective hMSH2 or hMLH1 expression [
47‐
49]. However, the effects of hMSH2 or hMLH1 in cellular sensitization to cisplatin have not been observed in a few other studies, reflecting the complex nature of cellular response to cisplatin-induced DNA damage (reviewed in ref. [
19]). It is conceivable that the roles of hMSH2 and hMLH1 may be regulated differently in the processes of repair and DNA damage signaling in different cell types. In addition, difference in cell cycle regulation may be another important factor in controlling various levels of cellular sensitivity to cisplatin. In fact, it is demonstrated recently that disruption of RPA's role in cell cycle regulation synergistically enhances the cytotoxic effects of cisplatin [
50].
In contrast to aforementioned observations, a recent study has revealed an important role for hMSH3 in the repair of cisplatin-induced DSBs [
38]. Using an isogenic HCT116-derived cell line in which the expression of hMSH3 can be controlled, Goel and colleagues [
38] demonstrated that hMSH3 deficiency sensitizes cells to both cisplatin and oxaliplatin toxicity, and this effect of hMSH3 is not dependent on the canonical MMR pathway. In addition, in response to oxaliplatin treatment, hMSH3-deficient cells sustain a higher level of γ-H2AX, suggesting that hMSH3 plays an important role in DSB repair [
38]. Intriguingly, the role of hMSH5 in mediating cellular response to cisplatin-induced DSBs bears a resemblance to that reported for hMSH3. In spite of using different cell lines and different ways to disrupt gene expression, cells deficient in hMSH5 or hMSH3 show comparable levels of reduction in clonogenic survivability in response to the same doses of cisplatin (Figure
6C) [
38]. Although the timing for the elevation of treatment-induced γ-H2AX appears to be different in cells subjected to RNAi-mediated silencing of hMSH5 or hMSH3 (Figure
5C) [
38], these observations warrant future studies to determine whether these two MutS homologues act in the same repair process of cisplatin-induced DSBs.
Intuitively, fertility preservation in male cancer patients undergoing chemotherapy is highly desired, and the relatively high levels of hMSH5 expression in the testis would be expected to provide a protection against cispaltin-induced DSBs. However, this effect of hMSH5 undoubtedly requires coordinated actions from a network of proteins involved in the repair process, and the efficiency of this pathway in various cell types in the testis is presently unknown. Since cisplatin represents a main treatment choice for testicular cancers [
15], it would be interesting to investigate the relative expression levels of hMSH5 in testicular tumors and matched normal testicular tissues. This information will be useful for assessing the value of using hMSH5 as a prognosis biomarker. Finally, our study has implicated that combining hMSH5 disruption with cisplatin treatment might be an alternative strategy for enhancing the therapeutic effects of cisplatin.
Conclusion
In summary, our study has demonstrated a role for hMSH5 in protecting cells from cisplatin-induced DNA damage. Inactivation of hMSH5 by RNAi or by expressing a phosphorylation-deficient hMSH5 mutant elevates cisplatin-induced G2 arrest and renders cells susceptible to cisplatin toxicity.
Collectively, our data is compatible with the idea that hMSH5 is involved in HR repair of cisplatin-induced DSBs.
School of Molecular Biosciences, Mail Drop 64-7520, College of Veterinary Medicine, Washington State University, Pullman, WA 99164, USA
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.