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Cancer Research 66, 11554, December 15, 2006. doi: 10.1158/0008-5472.CAN-06-1935
© 2006 American Association for Cancer Research
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Ataxia Telangiectasia Mutated Down-regulates Phospho-Extracellular Signal-Regulated Kinase 1/2 via Activation of MKP-1 in Response to Radiation

Mukesh K. Nyati1, Felix Y. Feng1, Divya Maheshwari1, Sooryanarayana Varambally2, Steven P. Zielske1, Aarif Ahsan1, Patrick Y. Chun1, Vinay A. Arora1, Mary A. Davis1, Mira Jung4, Mats Ljungman1, Christine E. Canman3, Arul M. Chinnaiyan2 and Theodore S. Lawrence1

Departments of 1 Radiation Oncology, 2 Pathology, and 3 Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan and 4 Department of Radiation Medicine, Georgetown University Medical School, Washington, District of Columbia

Requests for reprints: Theodore S. Lawrence, Department of Radiation Oncology, University of Michigan Medical Center, UH-B2C490, Box 0010, 1500 East Medical Center Drive, Ann Arbor, MI 48109-0010. Phone: 734-647-9955; Fax: 734-763-7371; E-mail: tsl{at}med.umich.edu.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Ataxia telangiectasia mutated (ATM) kinase plays a crucial role in the cellular response to DNA damage and in radiation resistance. Although much effort has focused on the relationship between ATM and other nuclear signal transducers, little is known about interactions between ATM and mitogenic signaling pathways. In this study, we show a novel relationship between ATM kinase and extracellular signal-regulated kinase 1/2 (ERK1/2), a key mitogenic stimulator. Activation of ATM by radiation down-regulates phospho-ERK1/2 and its downstream signaling via increased expression of mitogen-activated protein kinase phosphatase MKP-1 in both cell culture and tumor models. This dephosphorylation of ERK1/2 is independent of epidermal growth factor receptor (EGFR) activity and is associated with radioresistance. These findings show a new function for ATM in the control of mitogenic pathways affecting cell signaling and emphasize the key role of ATM in coordinating the cellular response to DNA damage. (Cancer Res 2006; 66(24): 11554-9)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The cellular response to radiation-induced DNA damage is a complex process requiring the integration of a wide array of signal transduction pathways. Many studies have shown that the ataxia telangiectasia mutated (ATM) kinase is a central coordinator of this response. ATM is located predominantly within the nucleus and responds to double-stranded DNA breaks by phosphorylating downstream effectors, resulting in the activation of cell cycle checkpoints (1). Although much effort has focused on the relationship between ATM and other nuclear signal transducers, little is known about the cytoplasmic molecules regulated by ATM in response to DNA damage.

We hypothesized that there might be a link between ATM and the mitogen-activated protein kinase (MAPK) pathway, which control many aspects of cellular physiology, including transcription of various factors that regulate DNA synthesis, cell cycle entry, cell growth, differentiation, and apoptosis in response to growth factors and environmental stress (2). One of the key regulators in the MAPK pathway is the extracellular signal-regulated kinase 1/2 (ERK1/2), which is a prime signaling molecule involved in the regulation of cell cycle progression (3) and has been shown to be affected by radiation (4). We hypothesized that as part of a coordinated response to DNA damage, ATM activation would decrease ERK1/2 activity, and that this response might act to preserve cell survival. Conversely, (inappropriate) continued activation of ERK1/2 after radiation might drive the cell through S phase in the face of DNA damage, leading to radiation-resistant DNA synthesis and decreased clonogenic survival.

Therefore, we decided to investigate whether radiation affected ERK1/2 activity. When we found that radiation decreased ERK1/2 activity in an ATM-dependent manner, we decided to investigate the mechanism by which ATM activation could down-regulate ERK1/2 activity. We focused on the MAPK phosphatase 1 (MKP-1; CL100 or DUSP1), which is known to inactivate ERK by dephosphorylating two critical sites in the activation loop (5) and to be highly inducible by oxidative stress (6). Finally, we extended these cell culture studies into nude mice bearing tumor xenografts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Reagents. Phospho-epidermal growth factor receptor (pEGFR; Y845), phospho-ERK (pERK; T202/Y204) and total ERK antibodies, and; U0126 were purchased from Cell Signaling Technology (Beverly, MA). EGFR antibody (Sc-03) was acquired from Santa Cruz Biotechnology (Santa Cruz, CA). MKP-1 and calcineurin antibodies were from Upstate (Charlottesville, VA); ß-actin antibody (clone AC-15), okadaic acid, and calyculin-A were from Sigma (St. Louis, MO); phospho-ATM antibody was from Rockland (Gilbertsville, PA), and glyceraldehyde-3-phosphate dehydrogenase antibody was purchased from Abcam (Cambridge, MA). RNA interference smart pools were purchased from Dharmacon (Lafayette, CO) and used as suggested by the manufacturer.

Cell culture. All the cell lines were purchased from the American Type Culture Collection (Manassas, VA) unless noted. The human squamous cell carcinoma cell line UMSCC-1 was a gift from Dr. Thomas E. Carey (University of Michigan, Ann Arbor, MI). AT fibroblasts (AT3BIVA, AT4BIVA, and AT5BIVA) were derived from individuals with AT and were immortalized by transfection with SV40 large T antigen. Stable clones of AT5BIVA expressing full-length ATM (AT5BIVA-AT reconstituted), which show nearly normal ATM activity, ATM kinase-dead (AT5BIVA-AT KD), or vector pCDNA3 (AT5BIVA-PCDNA3), were generated as described previously (7). All the cell lines were grown in RPMI 1640 supplemented with 10% cosmic calf serum (Hyclone, Logan, UT) except AT cells that were supplemented with 10% non–heat-inactivated fetal bovine serum (Hyclone). All experiments were conducted in serum-containing media. For all in vitro experiments, cells were released from flasks using PBS containing 0.01% trypsin and 0.20 mmol/L EDTA, and 6 x 105 cells were plated onto 100-mm culture dishes 2 days before any treatment. Cultures were between 30% and 50% confluency at the time of harvest.

Immunoblotting and immunofluorescence. Standard techniques were employed to perform immunoblotting and immunofluorescence as described previously (8). For quantification of relative protein levels, immunoblot films were scanned and analyzed using ImageJ 1.32j software (NIH, Bethesda, MD). Unless otherwise indicated, the relative protein levels shown represent a comparison to untreated controls.

Clonogenic cell survival assay. Clonogenic assays were done using standard techniques (9). Immediately after irradiation, cells were subcultured at clonal density. Cell survival curves were fitted using the linear quadratic equation, and the mean inactivation dose was calculated according to the method of Fertil et al. (10). The radiation enhancement ratio was calculated by dividing the mean inactivation dose under control conditions by the mean inactivation dose of U0126-treated cells.

Thymidine incorporation. The AT5BIVA-AT reconstituted and kinase-dead cells, at passages between 5 and 15, were plated onto 100-mm culture dishes at about 6 x105 per dish. After 2 days of growth, medium containing 0.01 µCi/mL [14C]thymidine (specific activity = 50 µCi/mL; Amersham Biosciences, Piscataway, NJ) was added to all cultures, and incubation was continued for 24 hours. To analyze the effect of U0126 on radioresistant DNA synthesis, half the plates were exposed to 10 µmol/L U0126 for an hour. Then, cells were simultaneously exposed to 4 Gy of ionizing radiation (300 kV X-rays, about 3 Gy/min). Immediately after irradiation, all the plates were washed, and fresh medium containing 1 µCi/mL [3H]thymidine (specific activity = 1 mCi/mL; Amersham Biosciences) was added for 10, 20, 30, or 60 minutes. After this period, the medium was removed, and the cells were washed twice with ice-cold PBS and scraped using ice-cold 10% trichloroacetic acid. The cell suspension was filtered through Glass Microfibre Filters (Whatman International Ltd., Maidstone, England). The filters were rinsed with ice-cold PBS, dried overnight, and assayed for radioactivity in a liquid scintillation spectrometer. The resulting 3H/14C ratios were measures of the specific activity of DNA and, therefore, of the rate of DNA synthesis.

MKP-1 dominant-negative cells. The MKP dominant-negative (MKPdn) mutant plasmid (a gift from Françoise Carlotti, Leiden University, The Netherlands) was subcloned into a lentiviral vector as a bicistronic expression cassette containing the GFP gene (MKPdn-IRES-GFP) and under the control of a cytomegalovirus promoter (11). AT5BIVA-AT reconstituted and AT5BIVA-AT KD cells were transduced with vector, and the proportion of transduced cells was estimated by GFP fluorescence microscopy at the initiation of experiments to be >95%.

RNA isolation and quantitative PCR. After cells were harvested in TRIzol (Invitrogen Co., Carlsbad, CA), RNA was extracted and reverse transcribed into cDNA. Quantitative PCR was then done using SYBR Green dye on an Applied Biosystems 7300 Real-time PCR system (Applied Biosystems, Foster City, CA). All oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). The primers used in this study for ERK-2 (12) and MKP-1 (13) were described previously.

In vivo studies. A431 tumor cells (5 x 106) were transplanted into the flanks of nu/nu CD-1 nude mice. When tumors reached an average volume of 100 mm3, the mice were randomized into two groups, and then tumors were radiated with a single 4-Gy dose. Animals were sacrificed at various intervals; tumors were harvested and either snap-frozen for immunoblotting or fixed in formalin for 24 hours before being stored in 70% ethanol for immunofluorescence studies. Animals were handled according to the established procedures of the University of Michigan Laboratory Animals Maintenance Manual.

Statistics. Results are presented as mean ± SE of at least three experiments. Student's t test was used to assess the statistical significance of differences. A significance level threshold of P ≤ 0.05 was used in this study.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
To investigate the effect of ATM activation on ERK1/2 activity in response to radiation in proliferating normal cells and tumors, we used logarithmically growing normal human skin fibroblasts and AT fibroblasts (and, in studies described below, tumor cells and xenografts). Normal fibroblasts irradiated with 4 Gy displayed a marked down-regulation of the phosphorylation of ERK1/2. The dephosphorylation of ERK1/2 became detectable within 5 minutes after irradiation and persisted for up to 1 hour (Fig. 1A ). In contrast, in four different ATM fibroblasts, pERK1/2 levels remained unchanged after radiation (Fig. 1B; Supplementary Fig. S1).


Figure 1
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  		Figure 1. Effect of ionizing radiation on ERK1/2 phosphorylation in normal and AT cells. ERK1 and ERK2 in all blots are as labeled in (A). A, logarithmically growing normal human skin fibroblasts were exposed to 4 Gy or to sham irradiation. Immunoblot analyses show decreased levels of pERK1/2 and RSK, a downstream effector of ERK1/2 after radiation. Total ERK1/2 (tERK) protein levels were unchanged. The intensities of both ERK1 and ERK2 were quantitated on the immunoblot to measure the degree of dephosphorylation and are represented numerically. B, pERK1/2 levels were unaffected in AT5BIVA, AT-KD, and vector PCDNA3-transfected fibroblasts. However, ERK1/2 dephosphorylation upon irradiation was restored in AT-reconstituted cells stably transfected with full-length ATM (7, 27). C, knockdown of ATM levels in AT5BIVA-AT reconstituted cells reduced ERK1/2 dephosphorylation after irradiation. Cells were oligofected with 100 nmol/L siRNA smart pool specific for ATM (ATM RNAi) or with 100 nmol/L nonspecific pool siRNA control and were irradiated 48 hours after transfection. Cells were then harvested and immunoblotted with antibodies against pERK1/2, total ERK1/2, ATM, or ß-actin as a loading control. D, treatment with calyculin-A (20 nmol/L), CPD-5 (20 µmol/L), or okadaic acid (1 µmol/L) for 1 hour before radiation blocked ERK1/2 dephosphorylation induced by radiation compared with the control treatment (DMSO). E, an increase in MKP-1 (the specific phosphatase for both ERK1/2) levels is responsible for ERK1/2 dephosphorylation upon radiation. Unrelated phosphatase calcineurin (PP2BA {alpha}) levels remained unaffected in all the cell lines. Neither MKP-1 levels nor pERK1/2 levels were affected by radiation in AT5BIVA-AT KD cells (bottom). Equal loading was confirmed by immunoblotting for ß-actin.

 
To confirm the role of ATM in ERK1/2 dephosphorylation, we employed full-length AT-reconstituted AT5BIVA cells (AT-recon) and AT-KD AT5BIVA (AT-KD) cells (7). In contrast to vector only and AT-KD cells, AT-reconstituted cells showed pERK1/2 down-regulation resembling that of normal skin fibroblasts (Fig. 1B). This suggested that the decrease in ERK1/2 phosphorylation after exposure to radiation is dependent on ATM activity. These results were further corroborated by the use of small interfering RNA (siRNA) to knockdown ATM in normal fibroblasts, which eliminated ERK1/2 down-regulation after radiation. These findings show that ATM mediates transient dephosphorylation of pERK1/2 after radiation (Fig. 1C).

Because ERK1/2 phosphorylation was reduced within 5 minutes of irradiation, whereas total ERK1/2 remained unaffected, we hypothesized that down-regulation is due to the rapid activation of an ATM-dependent phosphatase. To investigate this possibility, normal skin fibroblasts were pretreated with either calyculin-A, okadaic acid (PP-2A and PP-1 phosphatase inhibitors), or CPD-5 (a PTPase inhibitor that constitutively activates ERK1/2 phosphorylation; ref. 14) before radiation. These agents inhibited the radiation-induced ERK1/2 dephosphorylation (Fig. 1D). This suggested that radiation was indeed activating a phosphatase. Because the MKP-1 gene is known to be highly inducible by oxidative stress and heat shock in human skin cells (6), we hypothesized that expression of MKP-1, a specific phosphatase for pERK1/2 (15), would be enhanced by ionizing radiation, and that this change in expression is mediated by ATM. Consistent with this hypothesis, we found that radiation had no effect on MKP-1 expression in AT-KD cells, whereas in normal skin fibroblasts and AT-reconstituted fibroblasts, MKP-1 expression increased with a time course that corresponded with that of dephosphorylation of ERK1/2 (Fig. 1E). The exact mechanism of this rapid increase in MKP-1 protein levels has not yet been determined. It may be due to posttranslational modification, such as phosphorylation, that might increase protein stability, as we have ruled out a transcriptional change in the MKP-1 RNA level at the early time point (see Supplementary Fig. S2). The levels of another phosphatase, calcineurin (PP2B), remained unchanged, suggesting that MKP-1 induction is specific to radiation therapy.

Because ERK1/2 phosphorylation can also be affected by upstream signaling from EGFR, we wished to determine whether any of the effects we observed were secondary to radiation-induced changes in EGFR activity. We found no effect of radiation on pEGFR levels (Fig. 2A ) in logarithmically growing cells, although it has been reported previously (4) and confirmed by us (Fig. 2B), that pEGFR does increase after radiation in confluence arrested serum-starved cells. In addition, the use of siRNA to knockdown EGFR in AT5BIVA-AT reconstituted cells (Fig. 2C), normal skin fibroblasts, and A431 cells (Fig. 3A and B ) had no effect on ERK1/2 dephosphorylation. Finally, we documented that pERK1/2 levels decreases in a variety of cell lines with widely varying EGFR status, including cells lacking detectable EGFR, pEGFR, and EGFR message (ref. 9; Fig. 2D). Thus, the effect of radiation on ERK1/2 dephosphorylation seems to be independent of EGFR. In contrast, the importance of the activation of MKP-1 and its dependence on ATM activity was confirmed by siRNA specific for ATM in normal skin fibroblasts and A431 cell lines. In both the cell lines, we found that functional ATM is required for MKP-1 up-regulation and ERK1/2 dephosphorylation (Fig. 3A and B).


Figure 2
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  		Figure 2. Effect of ionizing radiation on EGFR levels in log-phase cultured cells. EGFR, RAS, and BRAF, which are molecules upstream of ERK1/2, do not affect ERK1/2 dephosphorylation upon irradiation. A, both phosphorylation at Y845 and total levels of EGFR were unaffected in normal fibroblasts after radiation. B, we confirmed that radiation enhances EGFR phosphorylation in confluence arrested fibroblasts. Total EGFR protein levels were unchanged. C, reduction of EGFR levels by siRNA in AT5BIVA-AT reconstituted cells also had no effect on ERK1/2 dephosphorylation. D, ERK1/2 was dephosphorylated in various cell lines independent of EGFR, RAS, or BRAF status. A431 skin epidermoid carcinoma cells (which contain an EGFR amplification and overexpress EGFR), UMSCC-1 (which are head and neck carcinoma cells that overexpress EGFR), and SW620 (EGFR null) colorectal carcinoma cells all showed a similar pattern of ERK1/2 dephosphorylation after irradiation. The total levels of unphosphorylated ERK1/2 were unchanged in these cell lines.

 

Figure 3
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  		Figure 3. Effect of ATM and EGFR levels on radiation-induced ERK1/2 dephosphorylation. Fibroblasts (A) and A431 (B) cells were oligofected with 100 nmol/L siRNA specific for either ATM or EGFR, or with nonspecific siRNA used as a control. Forty-eight hours after transfection, cells were irradiated with 4 Gy and then harvested at different time intervals for immunoblotting. A, knockdown of ATM in normal fibroblast cells prevented the radiation-induced increase in MKP-1 levels and the corresponding ERK1/2 dephosphorylation shown previously (Fig. 1E). Knockdown of EGFR in the same cell line did not affect MKP-1 and pERK1/2 levels. B, similar effects were observed in the A431 cell line.

 
We next determined the physiologic significance of the observed ATM-mediated decrease in ERK1/2 phosphorylation by assessing the role of pERK1/2 in radiation resistant DNA synthesis and radiation-induced decrease in clonogenic survival. Normal cells show a transient decrease in DNA synthesis after radiation (16). A hallmark of the AT phenotype is "radioresistant DNA synthesis," defined as continuation of DNA synthesis after radiation (17), which can be reversed by ectopic expression of ATM (18). We hypothesized that pERK1/2 plays a role in ongoing DNA synthesis, and that, in normal cells, ATM activation after radiation leads to down-regulation of ERK activity, leading to decreased DNA synthesis. Conversely, AT cells may be unable to down-regulate ERK1/2 after radiation. This (inappropriate) continued activation of ERK1/2 in AT cells after radiation would lead to radioresistant DNA synthesis and decreased clonogenic survival. If this were the case, U0126, a specific inhibitor of MAP/ERK kinase 1/2, would be anticipated to decrease radiation-resistant DNA synthesis. Indeed, we found that radiation-resistant DNA synthesis was decreased in both AT5BIVA-AT reconstituted and AT5BIVA-AT KD cells upon pretreatment with U0126, which specifically and potently inhibited ERK1/2 phosphorylation (Supplementary Fig. S3A). This phenomenon suggests a role for ERK1/2 activity in radioresistant DNA synthesis. To further assess the role of ERK1/2 dephosphorylation in radiation-induced cell death, we determined the effect of U0126 on the clonogenic survival of AT-reconstituted and AT-KD cells. We hypothesized that activated ERK1/2 might decrease clonogenicity by stimulating radiation-resistant DNA synthesis and that inhibition of ERK1/2 activity might be protective. In fact, U0126 treatment did cause a significant enhancement in cell survival after radiation in AT-reconstituted cells, but it had no effect on the survival of AT-KD cells (Fig. 4A ; Supplementary Fig. S3B). This suggests that other cell cycle checkpoint defects in cells lacking ATM function prevent AT-KD cells from being protected by pERK inhibition.


Figure 4
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  		Figure 4. Effect of ERK1/2 modulation on clonogenic survival. A, logarithmically growing AT5BIVA-AT reconstituted and AT5BIVA-AT KD cells were either pretreated for 1 hour with U0126 (10 µmol/L) or left untreated. The cells were then irradiated with 4 Gy. Treatment with U0126 resulted in significant enhancement in clonogenic survival in AT-reconstituted cells. B and C, effect of MKP-1 dominant-negative expression on radiation-induced cell death. Stable expression of MKPdn was achieved in AT5BIVA-AT reconstituted and AT-KD cells by infecting them with lentivirus carrying either empty vector or a MKPdn mutant gene under control of a cytomegalovirus promoter. B, the expression of MKPdn gene was confirmed by an increase in ERK phosphorylation. Total ERK levels remained unaffected, and MKP-1 protein levels were enhanced moderately. C, MKPdn cells were radiated as described in Fig. 1 and immunoblotted for pERK, total ERK, and MKP-1. The intensity of both ERK1 and ERK2 were quantitated on the immunoblot to measure the degree of dephosphorylation and is represented numerically. D, vector only or MKPdn cells were irradiated with 5 Gy. Clonogenic survival of MKPdn–expressing cells was decreased significantly compared with vector only cells. Points, average (n = 3); bars, SE.

 
To confirm a functional link between the activation of MKP-1 in irradiated cells and ERK dephosphorylation, we constructed MKP-1 dominant-negative cell lines. We hypothesized that ERK would become constitutively active in MKP-1 dominant-negative cells as shown previously (5, 19, 20), and that radiation would not induce dephosphorylation of ERK. This would result in an enhanced level of ERK1/2 phosphorylation at the time of radiation compared with cells with only wild-type MKP-1 and would sensitize cells to radiation as cells would no longer halt DNA synthesis. We first confirmed MKP-1 dominant-negative expression by analyzing ERK phosphorylation status, which was increased >2-fold compared with vector only cells (Fig. 4B). Total ERK levels remained unaffected and MKP-1 protein levels were enhanced moderately. We then established that ERK dephosphorylation after radiation was partially inhibited in AT-reconstituted MKPdn cells (Fig. 4C) compared with AT-reconstituted cells (see Fig. 1B), and that there was no effect on pERK1/2 levels in AT-KD MKPdn cells (Fig. 4C). Finally, we compared the effects of irradiation with 5 Gy on clonogenic survival of AT-reconstituted cells and AT-KD cells transduced with MKPdn. We found a significant decrease in clonogenic survival in cell containing MKPdn, suggesting an important role of MKP-1 in cell survival in irradiated cells (Fig. 4C).

We then carried out experiments to determine if ERK1/2 dephosphorylation occurred after radiation in a tumor xenograft model. As was the case in vitro, we observed a decrease in pERK1/2 levels in tumors, and this decrease was well correlated with the increase in MKP-1 expression (Fig. 5A and B ). Interestingly, the time course of the response in vivo is much slower than in vitro, with peak changes occurring 6 hours after radiation, rather than 20 minutes after radiation, as was seen in vitro. Although we are uncertain as to why these time courses differ, it is possible that this is secondary to the fact that, compared with in vitro conditions, the in vivo conditions include the presence of stroma, slower tumor cell growth rate, and the potential presence of tumor hypoxia.


Figure 5
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  		Figure 5. Radiation-induced changes in pERK1/2 and MKP-1 levels in A431 tumor xenografts. Athymic nude mice bearing A431 s.c. xenografts were irradiated (4 Gy), and tumors were harvested at the indicated times. Tumors were then homogenized in lysis buffer before immunoblotting or processing for immunofluorescence. A decrease in pERK1/2 levels corresponded with an increase in MKP-1 levels at 6 hours after radiation, as shown using immunoblotting (A) and immunofluorescence (B). C, model summarizing the role of ATM in pERK activation. Radiation-induced DNA damage activates phospho-ATM (pATM), which activates MKP-1, leading to dephosphorylation and inactivation of pERK. Activated pERK tends to drive radioresistant DNA synthesis and decrease cell survival; dephosphorylation of ERK by ATM is a protective mechanism that decreases radioresistant DNA and increases survival. Whether there is a causal relationship between radioresistant DNA synthesis and survival is still uncertain (28).

 
ATM is the major cellular mechanism for enforcing checkpoint control, as evidenced by the fact that a host of factors regulating the response to DNA double-strand breaks through DNA binding, such as breast cancer–associated 1, p53-binding protein 1, and checkpoint kinase 2, are substrates for ATM. Through these interactions, ATM can mediate the G1 and G2 checkpoints. It is also capable of interacting with the Mre11-Rad50-Nbs1 complex and thus regulates S phase (21, 22). Our findings, which are summarized by the model presented in Fig. 5C, suggest that ATM can carry out checkpoint control not only through interaction with DNA-binding proteins but by down-regulating mitogenic signaling pathways, such as the ERK1/2 pathway. Under physiologic conditions, ERK1/2 activation stimulates transcription leading to cell cycle progression and proliferation (23) by activated growth factor signaling, either in response to appropriate environmental stimuli, or inappropriately, in the case of constitutively activated growth factor receptors associated with a variety of malignancies (24). The ability of ATM to activate the phosphatase MKP-5 (25) and MKP-1 (this report), and thereby down-regulate ERK1/2, seems to give ATM an even tighter rein on the cell cycle in proliferating cells after DNA damage than was previously appreciated. Future studies to identify a possible role of other DNA damage repair molecules that form the ATM complex might provide a clearer picture for the role of ATM in ERK dephosphorylation upon irradiation. In addition, as ERK1/2 inhibitors are being investigated as a cancer treatment (26), it may be important to separate their administration from irradiation in future clinical trials to avoid radioprotection of the tumor.


   Acknowledgments
 
Grant support: NIH through the University of Michigan Head and Neck Specialized Program of Research Excellence grant 1 P50 CA97248 and University of Michigan Cancer Center support grant 5 P30 CA46592.

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.

We thank Dr. Brian Carr (University of Pittsburgh) for CPD-5, Dr. Françoise Carlotti (Leiden University, The Netherlands) for the MKP-1 dominant-negative mutant lentivirus, Steven Kronenberg for help in making figures for this article, and the microscopy laboratory at the Department of Cell and Developmental Biology at the University of Michigan.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

F.Y. Feng, D. Maheshwari and S. Varambally contributed equally to this work.

Received 5/26/06. Revised 10/27/06. Accepted 11/ 1/06.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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