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. 2008 Sep 12;4(9):e1000189.
doi: 10.1371/journal.pgen.1000189.

Human MLH1 protein participates in genomic damage checkpoint signaling in response to DNA interstrand crosslinks, while MSH2 functions in DNA repair

Qi Wu  1 , Karen M Vasquez
Affiliations

Human MLH1 protein participates in genomic damage checkpoint signaling in response to DNA interstrand crosslinks, while MSH2 functions in DNA repair

Qi Wu et al. PLoS Genet. .

Abstract

DNA interstrand crosslinks (ICLs) are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA) is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSalpha and MutSbeta mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Sensitivity of MLH1-proficient or MLH1-deficient cells to PUVA treatment.
(A) Viability curves are shown for MLH1+ (A2780) or MLH1− (A2780/cp70) cells treated with PUVA. The bars represent the standard errors of the means. **p<0.001, *p<0.01. (B) Results from clonogenic assays are shown for the same cells treated with PUVA.
Figure 2
Figure 2. Caspase-3/7 activation in MLH1-deficient or MSH2-deficient human cells in response to PUVA treatment.
(A) MLH1+ (A2780), MLH1− (A2780/cp70); (B) MSH2+ (HEC59+Chr2), MSH2− (HEC59) cells. The relative level of caspase-3/7 activity is shown for cells 48 hours after PUVA treatment. Caspase-3/7 activation was determined by cleavage of a caspase-3/7 substrate and performed in triplicate. The bars represent the standard errors of the means.
Figure 3
Figure 3. FACS Analysis of apoptotic cells after PUVA treatment in MLH1-deficient or MSH2-deficient human cells.
(A) MLH1+ (A2780), MLH1− (A2780/cp70); (B) MSH2+ (HEC59+Chr2), and MSH2− (HEC59) cells were treated with 1×10−6 M HMT+UVA at 1.8 J/cm2. Forty-eight hours later, cells were first stained with annexin V-FITC and PI, then subjected to fluorescence-activated cell sorter analyses to identify apoptotic cells. The x-axis represents the staining level of annexin; the y-axis represents the staining level of PI. The lower right and upper right quadrants contain cells with annexin positive cells indicating the apoptotic cell population. The cell lines used in this study are indicated on the left of the figure and the treatment conditions are listed on top of the figure.
Figure 4
Figure 4. PUVA-induced checkpoint signaling in MLH1-proficient or MLH1-deficient cells.
Lysates from MLH1-proficient (A2780) and MLH1-deficient (A2780/cp70) cells 1 hour following control (no treatment) or PUVA treatment (1×10−6 M HMT+1.8 J/cm2 UVA) were probed for phosphorylation of ATR (at Ser428), CHK1 (at Ser345), ATM (at Ser1981), CHK2 (at Thr68), total CHK1, total CHK2, and β-actin by western blotting. Lane 1: MLH1-proficient cells with no treatment; lane 2: MLH1-deficient cells with no treatment; lane 3: MLH1-proficient cells with PUVA treatment; lane 4: MLH1-deficient cells with PUVA treatment.
Figure 5
Figure 5. Psoralen ICL-induced mutagenesis in MLH1-proficient or MLH1-deficient human cell lines.
MLH1-proficient (A2780) and MLH1-deficient cell lines (A2780/cp70) were transfected with the pSupFG1 mutation reporter plasmid and mutations in the supF reporter gene were measured 72 hours later. +UVA represents plasmid in the presence of UVA irradiation only at 1.8 J/cm2; pAG30+UVA represents pSupFG1 plasmid treated with the specific psoralen-modified TFO (pAG30) and then UVA irradiated at 1.8 J/cm2; and pSCR30 represents plasmid that was incubated with the psoralen-modified control oligonucleotide and UVA irradiated at 1.8 J/cm2. The bars represent the standard errors of the means of three independent experiments. The absolute mutation frequency is listed above each bar.
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