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J. Biol. Chem., Vol. 283, Issue 9, 5533-5541, February 29, 2008

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A Sensitive Flow Cytometry-based Nucleotide Excision Repair Assay Unexpectedly Reveals That Mitogen-activated Protein Kinase Signaling Does Not Regulate the Removal of UV-induced DNA Damage in Human Cells^*

From the Department of Immunology/Oncology, Maisonneuve-Rosemont Hospital Research Center, Faculty of Medicine, University of Montreal, Montreal, Quebec H1T 2M4, Canada

Received for publication, July 30, 2007 , and in revised form, December 17, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38/β become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38/β to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38 mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Nucleotide excision repair (NER)³ is the only pathway available to human cells for the removal of helix-distorting (replication- and transcription-blocking) "bulky" DNA adducts generated by a multitude of environmental carcinogens. Among these adducts is the highly promutagenic UV-induced cyclobutane pyrimidine dimer (CPD), which lies at the origin of sunlight-associated mutagenesis and skin cancer development (1). The physiological importance of NER is highlighted by xeroderma pigmentosum (XP), a rare genetic disorder characterized by defective removal of bulky DNA adducts, UV hypersensitivity, and striking predisposition to skin cancer (2). Furthermore, NER status of tumors in cancer patients has been identified as a major determinant in the clinical response to UV-mimetic chemotherapeutic agents, such as cisplatin, which exert antineoplastic effects via the induction of bulky DNA adducts (3).

NER is composed of two distinct subpathways (i.e. global genomic-NER (GG-NER) removes bulky adducts from the genome overall, whereas transcription-coupled NER (TC-NER) removes such adducts exclusively from the transcribed strands of active genes) (see Ref. 4 for an excellent overview). These subpathways differ only in the mechanism of lesion recognition. GG-NER is triggered when the UV-DDB1/UV-DDB2 heterodimer recognizes and binds the helical distortion created by bulky adducts, which is followed by recruitment of the XPC-hHR23B complex. On the other hand, TC-NER is initiated uniquely by blockage of RNA polymerase II and subsequent recruitment of the CS-A and CS-B gene products. Thereafter, in the case of either GG-NER or TC-NER, the common "core NER pathway" is recruited to faithfully restore the integrity of the DNA through sequential steps of localized strand unwinding, incision of the DNA on either side of the adduct, excision of the adduct leaving a small single-stranded gap, and, finally, gap filling and ligation using normal DNA replication factors and the intact complementary strand as template.

Since its initial discovery over 40 years ago, the GG-NER pathway has been extensively studied and fully reconstituted in vitro (5). Nonetheless, relatively little is known about the potential roles in this repair process of various preeminent mutagen-responsive cellular signaling cascades. It has, however, been shown in many human cell lines treated with the model mutagen 254-nm UV (hereafter UV) that the p53 tumor suppressor, a critical stress-induced regulator of apoptosis and cell cycle checkpoints, is strictly required for efficient GG-NER (6, 7). In support of this, up-regulation of the GG-NER-specific proteins UV-DDB2 and XPC were shown to depend upon the presence of functional p53 (8, 9). Furthermore, p53 up-regulates Gadd45 and binds the histone acetyltransferase p300, events required to stimulate chromatin relaxation, which in turn facilitates access of GG-NER recognition proteins to damaged heterochromatin within the genome overall (10, 11).

Following exposure to diverse genotoxic agents, the canonical mitogen-activated protein kinases (MAPKs), including extracellular signal-related kinase (ERK1/2), c-Jun N-terminal kinase (JNK1/2), and p38/β kinase, become rapidly phosphorylated and go on to activate a plethora of transcription factors that regulate apoptosis and/or growth arrest (12). Of particular interest here, it has been demonstrated in UV-irradiated human cells that all three MAPKs phosphorylate p53 on multiple amino acid residues and that this has functional consequences for both p53 stabilization and p53-dependent apoptosis (13–17). This substantial level of cross-talk between the MAPK and p53 pathways strongly suggests a priori that the former pathway may also play a role in p53-dependent GG-NER. Although previous investigations have supported such a role in human cells, this important issue has still not been conclusively addressed (see "Discussion"). We thus employed human primary and tumor cell strains wherein MAPK signaling was abrogated using highly specific small molecule inhibitors, shRNA targeting, and/or expression of dominant negative mutant protein. A sensitive flow cytometry-based NER assay recently optimized and validated in our laboratory was then used to directly determine, in each UV-irradiated human strain, whether or not individual MAPKs modulate the efficiency of DNA photoproduct removal via GG-NER.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture—Primary human diploid skin fibroblasts (HDSFs), including the wild type strain (GM01652B) and XPA-deficient counterpart (GM01630), were purchased from the Coriell Institute. Primary human diploid lung fibroblasts (HDLFs) were provided by Dr. J. Sedivy (Brown University). HDLF-E6 (i.e. a p53-deficient isogenic derivative stably expressing the HPV-E6 oncoprotein) was described previously (18). Low passage HDSFs and HDLFs were cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics (Wisent, Montreal, Canada). HCT116/p53^+/+ human colon carcinoma cells and the isogenic p53-deficient counterpart (HCT116/p53^-/-) (a gift of Dr. B. Vogelstein, The Johns Hopkins University) were cultured in McCoy's 5A supplemented with 10% fetal bovine serum, L-glutamine, and antibiotics. Human U2OS osteosarcoma cells were grown in Dulbecco's minimal essential medium containing 10% fetal bovine serum, L-glutamine, and antibiotics.

UV Irradiation—Cell monolayers were washed twice with PBS, covered with 2 ml of PBS, and irradiated with UV using a Philips G25T8 germicidal lamp at a fluence of 0.2 J/m²/s.

Pharmacological Inhibition of MAPK Activity in Human Primary and Tumor Cell Strains—HDSFs, HDLFs, or HCT116 colon carcinoma cells were pretreated for 2 h with Me₂SO (carrier) or with one among the highly specific chemical MAPK inhibitors 10 µM U0126, 30 µM SP600125, or 20 µM SB202190 (Cell Signaling Technology, Inc.) to abrogate signaling through ERK1/2, JNK1/2, and p38/β, respectively. Following UV or mock irradiation, fresh medium containing either Me₂SO or inhibitor was added during subsequent incubations.

shRNA-mediated Knockdown of ERK1/2, JNK1/2, or p53 in U2OS Osteosarcoma Cells—shRNA constructs, cloned into the pLKO.1-puro vector and targeting ERK1 (clone TRCN0000006150), ERK2 (clone TRCN0000010050), JNK1 (clone TRCN0000010580), or JNK2 (clone TRCN0000001012), were purchased from Sigma. ERK1 and ERK2 or JNK1 and JNK2 shRNA constructs were pooled (4 µg of each) and transiently transfected into U2OS osteosarcoma cells using Lipofectamine 2000 according to the manufacturer's directions (Invitrogen). Immediately following antibiotic selection (i.e. for 3 days in 2 µg/ml puromycin), transfected cells were incubated for 3–5 h in complete medium without antibiotic prior to treatment with UV. Cells transfected with pLKO.1 expressing a scrambled shRNA were used as control.

For stable knockdown of p53 protein in U2OS cells, an shRNA targeting nucleotides 1095–1115 of this tumor suppressor was chosen (GenBank^TM accession number AF307851). This shRNA was cloned into the pSuper-retroviral vector (Oligo-Engine), and infectious retroviral particles were produced following transfection of PT67 packaging cells. Retroviral transductions were carried out as previously described (19). A polyclonal U2OS population stably expressing p53-targeting shRNA was obtained following selection in 2 µg/ml puromycin (Sigma). The efficiencies of ERK1/2, JNK1/2, and p53 knockdowns were monitored by Western blotting (see below).

Abrogation of p38/β Signaling in HDLFs via Stable Expression of the Dominant Negative Mutant p38 (AGF)—Total RNA from HDLFs was reverse transcribed using oligo(dT) primers (Invitrogen). Four µl of the reaction was used for PCR amplification of full-length human p38 wild type cDNA (GenBank^TM accession number NM_001315.1) using cloned pfu polymerase (Stratagene) in conjunction with the forward and reverse primers (5'-GCTGGAAAATGTCTCAGGAGA-3' and 5'-CTCAGGACTCCATCTCTTCTT-3'), respectively. The wild type p38 cDNA was cloned into the retroviral vector pMSCVretro (Clontech). The dominant negative p38 (AGF) mutant (20) was then produced using site-directed mutagenesis according to the manufacturer's protocol (QuikChange site-directed mutagenesis kit; Stratagene). Point mutations were introduced at each of the p38 kinase activation sites ((Thr¹⁸⁰ (ACA) Ala¹⁸⁰ (GCA) and Tyr¹⁸² (TAC) Phe¹⁸² (TTC)) using the following forward/reverse primer sets (mutations underlined): 5'-C ACA GAT GAT GAA ATG GCA GGC TAC GTG GCC-3' and 5'-GGC CAC GTA GCC TGC CAT TTC ATC ATC TGT G-3';5'-GAT GAA ATG GCA GGC TTC GTG GCC ACT AGG TGG-3' and 5'-CCA CCT AGT GGC CAC GAA GCC TGC CAT TTC ATC-3'). A polyclonal HDLF population stably expressing p38 (AGF) was isolated by retroviral transduction as mentioned above. Inhibition of p38/β signaling was monitored by Western blotting (see below).

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Activation and pharmacological abrogation of MAPK signaling in UV-exposed primary human skin fibroblasts. A, normal HDSFs were harvested at the indicated times post-UV and analyzed by Western blotting for specific phosphorylated MAPKs using primary antibodies directed against the threonine/tyrosine activation sites of ERK1/2, JNK1/2, and p38/β. B, the ability of phosphorylated ERK1/2, JNK1/2, and p38/β to activate the specific downstream effectors GST-Elk-1, GST-Jun, or GST-ATF2, respectively, was assessed at the indicated times post-UV using solid-phase kinase assays (see "Materials and Methods") in conjunction with Western analysis. C–E, to evaluate pharmacological inhibition of MAPK activity, HDSFs were isolated (i) with no treatment (No UV) or (ii) at 1 h post-UV in the presence of either carrier (Me2SO; DMSO) or of a specific MAPK inhibitor, as indicated. Detection of phospho-ERK1/2 and -Elk-1 (C), phospho-JNK1/2 and -c-Jun (D), and phospho-MK2 and -ATF-2 (E) were accomplished as described under "Materials and Methods." For A–E, actin was used as protein loading control with the following exceptions. (i) For detection of GST-phospho-c-Jun, total JNK1/2 was first immunoprecipitated (IP) and then probed with anti-phospho-c-Jun antibody. In this case, total JNK1/2 can be, and was, used as loading control. (ii) For detection of phospho-Elk-1 or phospho-ATF2, no loading control was possible, since these effectors were directly immunoprecipitated in the phosphorylated forms.

Flow Cytometry-based Determination of GG-NER Kinetics—Normal or XPA-deficient HDSFs were maintained at confluence for 4 days prior to irradiation (yielding > 95% synchronization in G₀) and maintained at confluence during post-UV incubation. In the case of HDLFs or colon carcinoma cells, exponentially growing monolayers were synchronized in G₀/G₁ by serum starvation (0.5%) for 3 days, and 3 h before treatment fresh complete medium was added to restimulate proliferation. U2OS cells transiently expressing shRNAs could not be synchronized by either confluence or serum starvation. As such, immediately following UV exposure, 200 ng/ml nocodazole was added to the medium to block cell division.

At various times post-UV, cells were washed with PBS plus 50 mM EDTA, trypsinized, resuspended in 1 ml of PBS plus 50 mM EDTA, and fixed by the addition of 3 ml of ice-cold 100% ethanol. 1 x 10⁶ fixed cells were then washed with PBS plus 50 mM EDTA, resuspended in either 0.5% Triton X-100 plus 2 N HCl (for CPD detection) or 0.5% Triton X-100 plus 0.1 N HCl (for 6-4 photoproduct (6-4PP) detection), and incubated for 20 min at 22 °C. Cells were washed with 0.1 M Na₂B₄O₇ (pH 9.0) and then with PBS and resuspended in 300 µl of RNase (100 µg/ml in PBS) for 1 h at 37 °C followed by washing with PBS-TB (1% bovine serum albumin plus 0.25% Tween 20 in PBS). Cells were resuspended in PBS-TB containing a primary monoclonal antibody against either CPDs or 6-4PPs (Kamiya Biomedical Company) for 1 h at room temperature, washed with PBS-TB, and then resuspended in 300 µl of fluorescein isothiocyanate-conjugated rabbit anti-mouse secondary antibody for 45 min at room temperature. Pellets were washed twice with PBS-TB and resuspended in 300 µl of PBS containing 5 µg/ml propidium iodide (Molecular Probes, Inc.), and repair kinetics were monitored for populations gated in G₁ using a flow cytometer fitted with an argon laser and CellQuestPro software (BD Biosciences).

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Activation and pharmacological abrogation of MAPK signaling in UV-exposed primary human lung fibroblasts and HCT116p53+/+ colon carcinoma cells. Phosphorylation and pharmacological inhibition of specific MAPKs in exponentially growing HDLFs (A and B) or in human HCT116p53+/+ (C and D) were evaluated essentially as described in the legend to Fig. 1 for HDSFs. DMSO, Me2SO; IP, immunoprecipitation.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
UV-induced Activation and Pharmacological Abrogation of MAPK Activity in Human Cells—Replicate cultures representing three human strains (i.e. to stringently control for cell type-specific effects that are highly characteristic of MAPK function) were irradiated with 10 J/m² UV (yielding 5–10% relative clonogenic survival in each strain; data not shown) in the presence of either carrier (Me₂SO) or well characterized, highly specific chemical MAPK inhibitors (i.e. U0126, SP600125, and SB202190 to abrogate signaling through ERK1/2, JNK1/2, and p38/β, respectively) (21, 23, 24). At various times post-UV, cultures were evaluated for levels of MAPK phosphorylation. In the case of HDSFs, phospho-JNK1/2 and phospho-p38/β peaked at early times (0.5–1 h) and diminished rapidly thereafter, whereas phospho-ERK1/2 peaked at 0.5 h and remained elevated for at least 24 h (Fig. 1A). This correlated temporally with in vitro phosphorylation by phospho-ERK1/2, -JNK1/2, and -p38/β of the specific downstream effectors Elk-1, c-Jun, and ATF2, respectively (Fig. 1B). Furthermore, incubation of HDSFs with chemical inhibitors resulted in strong diminishment (>85%) of ERK1/2, JNK1/2, or p38/β activity at 1 h post-UV (Fig. 1, C–E). Results similar to those described immediately above for MAPK activation/abrogation in HDSFs were obtained for UV-irradiated HDLFs (Fig. 2, A and B). However in the case of HCT116/p53^+/+, although phospho-JNK1/2 peaked at 1 h post-UV as anticipated, ERK1/2 appeared to be constitutively phosphorylated (Fig. 2, C and D), and no phospho-p38/β could be detected within 48 h despite the presence of nonphosphorylated forms (not shown).

Optimization and Validation of a Flow Cytometry-based DNA Repair Assay and Its Application to Examine the Potential Role of MAPK Signaling in Human GG-NER—In parallel with the above MAPK activation/abrogation studies, cultures were set aside at various times post-UV to evaluate the role of individual MAPKs in GG-NER using a flow cytometry-based DNA repair assay recently developed in our laboratory. This assay directly detects CPDs in partially denatured double-stranded DNA in intact permeabilized cells using highly specific fluorescently labeled anti-CPD antibodies. Although a similar method was previously reported over a decade ago (25), it was subsequently only very rarely used to address biological mechanisms. We have now optimized this type of assay (i) to be rapid, reproducible, and extremely sensitive, detecting CPDs induced by UV doses as low as 1 J/m² and (ii) to exhibit a linear dose response for CPD induction up to 40 J/m² (Fig. 3A). Our optimized NER assay was initially applied to confluent UV-exposed HDSFs treated or not with MAPK inhibitors. (For all repair analyses described in this study, cells were synchronized by various approaches (see "Materials and Methods") to prevent cell division, which would otherwise dilute the CPD-specific immunofluorescence signal, thereby interfering with accurate determination of repair kinetics.) Representative raw data are shown for normal HDSFs and NER-deficient XPA HDSFs (Fig. 3, B and C), and the complete experiment in HDSFs is graphically illustrated in Fig. 4A. Relative to Me₂SO-treated controls, incubation with any of U0126, SP600125, or SB202190 had virtually no effect on CPD removal over a 48-h period in normal HDSFs, whereas GG-NER-deficient XPA HDSFs (negative control), in line with expectations, exhibited almost complete abrogation of CPD repair. For HDLFs, precisely the same trends with respect to CPD repair kinetics were observed (Fig. 4B). Also, repair of another UV-induced bulky adduct, i. e. the 6-4PP (induced 5-fold less frequently than CPDs), was evaluated in HDLFs after irradiation with 25 J/m² UV using a specific anti-6-4PP antibody. 6-4PPs are characteristically removed much more rapidly relative to CPDs via GG-NER in human and murine cells (26). In accord with this, we observed nearly 100% 6-4PP removal by 8 h post-UV in HDLFs; moreover, incubation of HDLFs with MAPK inhibitors engendered virtually no effect on repair of 6-4PP (Fig. 4C). In addition, as fully expected, HPV-E6-expressing HDLFs and XPA-deficient HDSFs (negative controls) manifested significant reductions in the removal of CPD and 6-4PP, respectively (Fig. 4, B and C). Finally, toward characterizing the potential influence of MAPKs on GG-NER in human tumor cells, pharmacological inhibition of signaling via ERK1/2 or JNK1/2 in HCT116p53^+/+ had no significant impact on CPD repair kinetics (Fig. 4D). On the other hand, isogenic control p53-null HCT116/p53^-/- cells characteristically exhibited significantly reduced GG-NER efficiency relative to its isogenic p53-proficient counterpart.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. Validation of a novel flow cytometry-based NER assay. A, CPD induction in normal HDSFs as a function of dose. B and C, representative raw profiles depicting CPD repair kinetics for normal HDSFs and for NER-deficient XPA HDSFs, following UV exposure. The arrows indicate small artifactual shoulders in the flow cytometry curves, ostensibly generated by minor populations of doublet cells, which were routinely excluded from repair analysis.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Flow cytometry-based evaluation of NER kinetics in UV-exposed human primary and tumor cell lines treated with chemical MAPK inhibitors. A, kinetics of CPD removal in UV-exposed normal HDSFs and in UV-exposed XPA-deficient HDSFs (negative control), treated or not with MAPK inhibitors. B, kinetics of CPD removal in UV-exposed HDLFs and in isogenic UV-exposed HDLF-E6 cells (negative control), treated or not with MAPK inhibitors. C, kinetics of 6-4PP removal in UV-exposed HDLFs and in UV-exposed XPA-deficient HDSFs (negative control), treated or not with MAPK inhibitors. D, kinetics of CPD removal in UV-exposed human HCT116 p53+/+ colon carcinoma cells and in UV-exposed p53-deficient isogenic counterparts (HCT116 p53-/-; negative control), treated or not with MAPK inhibitors. Each time point represents the mean ± S.E. of at least three independent experiments. DMSO, Me2SO.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Flow cytometry-based evaluation of NER kinetics in UV-exposed U2OS osteosarcoma cells following shRNA-mediated knockdown of ERK1/2 or JNK1/2 protein or in UV-exposed HDLFs following knockdown of p38/β signaling via expression of dominant negative p38 (AGF). A, activation profiles of ERK1/2 and JNK1/2 in UV-exposed U2OS cells. B, shRNA-mediated knockdown of p53, ERK1/2, and JNK1/2 in U2OS cells. C, kinetics of CPD removal in UV-exposed U2OS cells transiently transfected with shRNAs targeting ERK1/2 or JNK1/2. U2OS stably expressing shRNA targeting p53 is used as negative control. D, top, Western blot of phospho-MK2 induction in HDLFs irradiated with UV and expressing or not the p38 (AGF) dominant negative mutant; bottom, kinetics of CPD removal in HDLFs expressing or not the p38 (AGF) dominant negative mutant. HDLF-E6 cells are used as negative control. Each time point represents the mean ± S.E. of at least three independent experiments.

In addition to the shRNA experiments in U2OS cells described above, HDLFs were stably transduced with a retroviral vector expressing the dominant negative p38 (AGF) mutant. Expression of this mutant, compared with HDLFs expressing an empty vector, resulted in a 5-fold reduction in the ratio of phosphorylated to unphosphorylated forms of the unique p38/β substrate MK2, (Fig. 5D, top). Despite this indication that p38/β signaling had been substantially reduced, no significant effect on the kinetics of GG-NER was observed (Fig. 5D, bottom), although HDLF-E6 cells, as expected, manifested a significant reduction in GG-NER capacity.

Effects of MAPK Inactivation on Cell Death and Proliferation in Primary Human Lung Fibroblasts—Having observed no effects on GG-NER as above, it became necessary to provide assurance that in our hands abrogation of MAPK signaling nonetheless generates some anticipated phenotypic outcomes. We therefore undertook to characterize the effects of MAPK inhibitors on cell death and proliferation in HDLFs, either unstressed or exposed to UV. No reports to our knowledge have previously addressed such MAPK-dependent phenomena in primary human cells from the lung. In this sense, it should be emphasized that in general DNA damage responses elicited by MAPKs are subject to high variability (e.g. although JNK1/2 activation is most often associated with induction of apoptosis, it is now becoming increasingly clear that this kinase can mediate either proapoptotic or antiapoptotic effects, or no apoptotic effects, depending upon stimulus, dose, and/or cell type) (27, 28). Specifically in the case of HDLFs irradiated with 10 J/m² UV, SP600125 treatment significantly stimulated apoptosis at 48 and 72 h post-UV as evaluated by either sub-G₁ peak analysis or annexin V staining (p < 0.05; two-tailed unpaired t test) (Fig. 6, A and B). These data support an antiapoptotic role for JNK1/2 in UV-exposed HDLFs, in line with prior investigations in some other cultured cell strains (29, 30). On the other hand, perhaps not surprisingly based on a dearth of previous studies to the contrary, no effect on UV-induced apoptosis was noted here upon ERK1/2 inactivation in HDLFs (Fig. 6, A and B). Finally, we observed a moderate but significant decrease in annexin V-positive staining at 72 h post-UV in SB202190-treated HDLFs (Fig. 6B), supporting a proapoptotic role for p38/β in UV-irradiated HDLFs, as previously observed in various cell lines (31). In addition, sub-G₁ peak analysis consistently revealed a slight reduction in UV-induced apoptosis in SB202190-treated cells, although this was not statistically significant (Fig. 6A).

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Modulation of cell death and proliferation by MAPK inhibitor treatment in mock- versus UV-irradiated (10 J/m2) primary lung fibroblasts. A, HDLFs were stained with propidium iodide at various times post-UV, and the percentage of the population in sub-G1 was then evaluated by flow cytometry. B, quantification of annexin-V-positive cells (early apoptotic fraction) post-UV. C, effect of MAPK inhibition on clonogenic survival in HDLFs, either mock-irradiated or irradiated with 10 J/m2 UV. D, HDLFs were processed for determination of cellular proliferation by MTT assay at 48 h and 96 h post-UV as described under "Materials and Methods." For A–D, results represent an average of at least three independent experiments ± S.E. *, p < 0.05; two-tailed unpaired t test. DMSO, Me2SO.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The current study provides solid evidence that abrogation of ERK1/2, JNK1/2, or p38/β activity in UV-exposed human primary and tumor cells via treatment with highly specific chemical inhibitors, although significantly influencing cell death and proliferation, has no effect whatsoever on the efficiency of GG-NER. This conclusion is strongly reinforced by experiments showing that repair is similarly unaffected following (i) transient shRNA-mediated knockdown of ERK1/2 or JNK1/2 in U2OS osteosarcoma cells or (ii) stable expression of a dominant negative p38 mutant in HDLFs. We strongly emphasize that the observed lack of participation of MAPK signaling in human GG-NER is unexpected in light of previous reports. For example, UV-irradiated transformed mouse embryonic fibroblasts, either genetically null for the JNK1/2 effector c-fos or treated with SP600125, were shown to be deficient in GG-NER as determined by Southwestern blotting analysis using a specific anti-CPD antibody (29); furthermore, interference with c-Fos-mediated up-regulation of XPF (i.e. a structure-specific endonuclease required to incise DNA at damaged sites during NER) may underlie the observed effect (36). The apparent discrepancy between these data in a murine system and ours in humans may be attributable to species-specific effects. In fact, rodent cells, relative to human, generally exhibit profoundly reduced expression of the GG-NER-specific protein UV-DDB2 and are consequently much less proficient in GG-NER (37), a significant caveat that may complicate interspecies comparisons.

Regarding previous reports in human cells, interference with JNK1/2 signaling was shown to engender cellular sensitivity to the UV-mimetic agent cisplatin as well as decreased post-treatment recovery of PCR amplification efficiency (DNA polymerase stop assay) (35, 38, 39). Although it was legitimately concluded that this might be attributable to deficient removal of cisplatin-induced DNA adducts, the affected repair pathway(s) could not be identified via the indirect polymerase stop assay employed. Indeed although cisplatin induces replication-blocking DNA intrastrand cross-links that are repaired by NER, this agent also generates a highly significant yield of replication-blocking DNA interstrand cross-links. The latter type of cross-link represents a distinct class of adduct that requires (in addition to NER) the concerted action of multiple DNA repair pathways for efficient removal (i.e. homologous recombination, DNA mismatch repair, and DNA translesion synthesis) (40), any of which may be influenced by MAPK signaling. On the other hand, herein we have monitored DNA adduct repair in intact cells by direct quantification of CPDs or 6-4PPs, both of which are clearly removed exclusively by NER.

It is important to highlight another recent report that contributes to the burgeoning perception that MAPKs participate in the NER process. Specifically, ERK1/2 signaling was firmly linked to regulation of the essential NER core pathway protein ERCC1 in UV-exposed human hepatoma cells, and additionally, abrogation of ERK1/2 activation with U0126 was shown to possibly reduce the efficiency of UV DNA photoproduct removal as evaluated by a host cell reactivation assay (41). However, again (i.e. analogous to the situation for the aforementioned DNA polymerase stop assay) such an experimental approach, which measures post-UV recovery of plasmid-borne reporter gene expression, only indirectly reflects repair of RNA polymerase II-blocking lesions. Other caveats associated with the above study include the following: (i) DNA repair kinetics were not evaluated (i.e. recovery of transcription was examined only at 24 h post-UV), and curiously (ii) the putative effect of ERK1/2 on NER was observed following irradiation with 80 J/m² UV but not with 40, 60, or 120 J/m².

It should be noted that although MAPKs apparently do not influence repair of CPDs within the genome overall via GG-NER in human cells, such an influence on repair along the transcribed strands of active genes via TC-NER (which is not measured by our GG-NER assay) cannot be entirely ruled out. Indeed, it is possible that MAPKs participate in TC-NER but not GG-NER by regulating the unique lesion recognition step of the former NER subpathway, involving, for example, activation of the TC-NER-specific proteins CS-A or CS-B and/or removal of stalled RNA polymerase II at damaged sites and subsequent recruitment of the core NER pathway.

The MAPKs comprise a preeminent mutagen-inducible cascade, which, by all previous indications, appeared to play a significant role in GG-NER. Nonetheless, here we have employed a highly sensitive DNA repair assay to directly demonstrate that MAPKs play no such role in cultured human cells. These data thus reorient our mechanistic perception of a critical antineoplastic DNA repair pathway. We also emphasize that aberrant MAPK signaling has been firmly implicated in diverse pathologies, including cancer, inflammation, and cardiovascular disorders; furthermore, MAPK inhibitors are currently being evaluated in therapy for such diseases (42, 43). The revelations provided by our study would ostensibly constitute an important consideration when designing treatment protocols that target the MAPK pathway.

	FOOTNOTES

 
^* This study was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society and by the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Maisonneuve-Rosemont Hospital Research Center, 5415 Boul. de l'Assomption, Montreal, Quebec H1T 2M4, Canada. Tel.: 514-252-3400 (ext. 4665); Fax: 514-252-3430; E-mail: elliot.drobetsky{at}umontreal.ca.

³ The abbreviations used are: NER, nucleotide excision repair; CPD, cyclobutane pyrimidine dimer; XP, xeroderma pigmentosum; GG-NER, global genomic-NER; TC-NER, transcription-coupled NER; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; HDSF, human diploid skin fibroblast; HDLF, human diploid lung fibroblast; PBS, phosphate-buffered saline; shRNA, short hairpin RNA; GST, glutathione S-transferase; XPC, XP complementation group C; XPA, XP complementation group A; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PP, photoproduct.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge Drs. Dindial Ramotar and Eric Milot for critically reading the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Melnikova, V. O., and Ananthaswamy, H. N. (2005) Mutat. Res. 571, 91-106[Medline] [Order article via Infotrieve]
2. Kraemer, K. H., Patronas, N. J., Schiffmann, R., Brooks, B. P., Tamura, D., and DiGiovanna, J. J. (2007) Neuroscience 145, 1388-1396[CrossRef][Medline] [Order article via Infotrieve]
3. Olaussen, K. A., Dunant, A., Fouret, P., Brambilla, E., Andre, F., Haddad, V., Taranchon, E., Filipits, M., Pirker, R., Popper, H. H., Stahel, R., Sabatier, L., Pignon, J. P., Tursz, T., Le Chevalier, T., and Soria, J. C. (2006) N. Engl. J. Med. 355, 983-991[Abstract/Free Full Text]
4. Cleaver, J. E. (2005) Nat. Rev. Cancer 5, 564-573[CrossRef][Medline] [Order article via Infotrieve]
5. Aboussekhra, A., Biggerstaff, M., Shivji, M. K., Vilpo, J. A., Moncollin, V., Podust, V. N., Protic, M., Hubscher, U., Egly, J. M., and Wood, R. D. (1995) Cell 80, 859-868[CrossRef][Medline] [Order article via Infotrieve]
6. Mathonnet, G., Leger, C., Desnoyers, J., Drouin, R., Therrien, J. P., and Drobetsky, E. A. (2003) Proc. Natl. Acad. Sci. 100, 7219-7224[Abstract/Free Full Text]
7. Ford, J. M., and Hanawalt, P. C. (1995) Proc. Natl. Acad. Sci. 92, 8876-8880[Abstract/Free Full Text]
8. Adimoolam, S., and Ford, J. M. (2002) Proc. Natl. Acad. Sci. 99, 12985-12990[Abstract/Free Full Text]
9. Hwang, B. J., Ford, J. M., Hanawalt, P. C., and Chu, G. (1999) Proc. Natl. Acad. Sci. 96, 424-428[Abstract/Free Full Text]
10. Smith, M. L., Ford, J. M., Hollander, M. C., Bortnick, R. A., Amundson, S. A., Seo, Y. R., Deng, C. X., Hanawalt, P. C., and Fornace, A. J., Jr. (2000) Mol. Cell Biol. 20, 3705-3714[Abstract/Free Full Text]
11. Rubbi, C. P., and Milner, J. (2003) EMBO J. 22, 975-986[CrossRef][Medline] [Order article via Infotrieve]
12. Hildesheim, J., and Fornace, A. J., Jr. (2004) DNA Repair 3, 567-580[Medline] [Order article via Infotrieve]
13. Bulavin, D. V., Saito, S., Hollander, M. C., Sakaguchi, K., Anderson, C. W., Appella, E., and Fornace, A. J., Jr. (1999) EMBO J. 18, 6845-6854[CrossRef][Medline] [Order article via Infotrieve]
14. She, Q. B., Chen, N., and Dong, Z. (2000) J. Biol. Chem. 275, 20444-20449[Abstract/Free Full Text]
15. Huang, C., Ma, W. Y., Maxiner, A., Sun, Y., and Dong, Z. (1999) J. Biol. Chem. 274, 12229-12235[Abstract/Free Full Text]
16. She, Q. B., Ma, W. Y., and Dong, Z. (2002) Oncogene 21, 1580-1589[CrossRef][Medline] [Order article via Infotrieve]
17. Buschmann, T., Potapova, O., Bar-Shira, A., Ivanov, V. N., Fuchs, S. Y., Henderson, S., Fried, V. A., Minamoto, T., Alarcon-Vargas, D., Pincus, M. R., Gaarde, W. A., Holbrook, N. J., Shiloh, Y., and Ronai, Z. (2001) Mol. Cell Biol. 21, 2743-2754[Abstract/Free Full Text]
18. Therrien, J. P., Drouin, R., Baril, C., and Drobetsky, E. A. (1999) Proc. Natl. Acad. Sci. 96, 15038-15043[Abstract/Free Full Text]
19. Loignon, M., and Drobetsky, E. A. (2002) Carcinogenesis 23, 35-45[Abstract/Free Full Text]
20. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) J. Biol. Chem. 270, 7420-7426[Abstract/Free Full Text]
21. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler, J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., and Young, P. R. (1994) Nature 372, 739-746[CrossRef][Medline] [Order article via Infotrieve]
22. Clifton, A. D., Young, P. R., and Cohen, P. (1996) FEBS Lett. 392, 209-214[CrossRef][Medline] [Order article via Infotrieve]
23. Bennett, B. L., Sasaki, D. T., Murray, B. W., O'Leary, E. C., Sakata, S. T., Xu, W., Leisten, J. C., Motiwala, A., Pierce, S., Satoh, Y., Bhagwat, S. S., Manning, A. M., and Anderson, D. W. (2001) Proc. Natl. Acad. Sci. 98, 13681-13686[Abstract/Free Full Text]
24. Favata, M. F., Horiuchi, K. Y., Manos, E. J., Daulerio, A. J., Stradley, D. A., Feeser, W. S., Van Dyk, D. E., Pitts, W. J., Earl, R. A., Hobbs, F., Copeland, R. A., Magolda, R. L., Scherle, P. A., and Trzaskos, J. M. (1998) J. Biol. Chem. 273, 18623-18632[Abstract/Free Full Text]
25. Berg, R. J., de Gruijl, F. R., Roza, L., and van der Leun, J. C. (1993) Carcinogenesis 14, 103-106[Abstract/Free Full Text]
26. Mitchell, D. L., Haipek, C. A., and Clarkson, J. M. (1985) Mutat. Res. 143, 109-112[CrossRef][Medline] [Order article via Infotrieve]
27. Liu, J., and Lin, A. (2005) Cell Res. 15, 36-42[CrossRef][Medline] [Order article via Infotrieve]
28. Gururajan, M., Chui, R., Karuppannan, A. K., Ke, J., Jennings, C. D., and Bondada, S. (2005) Blood 106, 1382-1391[Abstract/Free Full Text]
29. Christmann, M., Tomicic, M. T., Aasland, D., and Kaina, B. (2007) Carcinogenesis 28, 183-190[Abstract/Free Full Text]
30. Potapova, O., Basu, S., Mercola, D., and Holbrook, N. J. (2001) J. Biol. Chem. 276, 28546-28553[Abstract/Free Full Text]
31. Jinlian, L., Yingbin, Z., and Chunbo, W. (2007) J. Biomed. Sci. 14, 303-312[CrossRef][Medline] [Order article via Infotrieve]
32. Du, L., Lyle, C. S., Obey, T. B., Gaarde, W. A., Muir, J. A., Bennett, B. L., and Chambers, T. C. (2004) J. Biol. Chem. 279, 11957-11966[Abstract/Free Full Text]
33. Manke, I. A., Nguyen, A., Lim, D., Stewart, M. Q., Elia, A. E., and Yaffe, M. B. (2005) Mol. Cell 17, 37-48[CrossRef][Medline] [Order article via Infotrieve]
34. Ussar, S., and Voss, T. (2004) J. Biol. Chem. 279, 43861-43869[Abstract/Free Full Text]
35. Hayakawa, J., Depatie, C., Ohmichi, M., and Mercola, D. (2003) J. Biol. Chem. 278, 20582-20592[Abstract/Free Full Text]
36. Christmann, M., Tomicic, M. T., Origer, J., Aasland, D., and Kaina, B. (2006) Nucleic Acids Res. 34, 6530-6539[Abstract/Free Full Text]
37. Hanawalt, P. C. (2001) Environ. Mol. Mutagen. 38, 89-96[CrossRef][Medline] [Order article via Infotrieve]
38. Potapova, O., Haghighi, A., Bost, F., Liu, C., Birrer, M. J., Gjerset, R., and Mercola, D. (1997) J. Biol. Chem. 272, 14041-14044[Abstract/Free Full Text]
39. Gjerset, R. A., Lebedeva, S., Haghighi, A., Turla, S. T., and Mercola, D. (1999) Cell Growth Differ. 10, 545-554[Abstract/Free Full Text]
40. Nojima, K., Hochegger, H., Saberi, A., Fukushima, T., Kikuchi, K., Yoshimura, M., Orelli, B. J., Bishop, D. K., Hirano, S., Ohzeki, M., Ishiai, M., Yamamoto, K., Takata, M., Arakawa, H., Buerstedde, J. M., Yamazoe, M., Kawamoto, T., Araki, K., Takahashi, J. A., Hashimoto, N., Takeda, S., and Sonoda, E. (2005) Cancer Res. 65, 11704-11711[Abstract/Free Full Text]
41. Andrieux, L. O., Fautrel, A., Bessard, A., Guillouzo, A., Baffet, G., and Langouet, S. (2007) Cancer Res. 67, 2114-2123[Abstract/Free Full Text]
42. Sebolt-Leopold, J. S., and Herrera, R. (2004) Nat. Rev. Cancer 4, 937-947[CrossRef][Medline] [Order article via Infotrieve]
43. Johnson, G. L., and Nakamura, K. (2007) Biochim. Biophys. Acta 1773, 1341-1348[Medline] [Order article via Infotrieve]

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