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Originally published In Press as doi:10.1074/jbc.M304012200 on May 6, 2003

J. Biol. Chem., Vol. 278, Issue 32, 29394-29399, August 8, 2003
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Dominant-negative cAMP-responsive Element-binding Protein Inhibits Proliferating Cell Nuclear Antigen and DNA Repair, Leading to Increased Cellular Radiosensitivity*

George P. Amorino, Ross B. Mikkelsen, Kristoffer Valerie and Rupert K. Schmidt-Ullrich {ddagger}

From the Department of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298

Received for publication, April 16, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Selective inhibition of the epidermal growth factor receptor or mitogen-activated protein kinase (MAPK) results in radiosensitization of cancer cells. One potential mechanism involves cAMP-responsive element-binding protein, which is activated by radiation via the epidermal growth factor receptor/MAPK pathway and which regulates synthesis of proliferating cell nuclear antigen (PCNA), a protein involved in repair of ionizing radiation-induced DNA damage. To test for a role of CREB in cellular radiosensitivity, CHO cells were transfected with plasmids expressing dominant-negative CREB mutants (CR133 or KCREB), and various end-points were measured 48 h later. Basal levels of PCNA-CAT reporter construct activity were reduced by 60 and 40% following expression of CR133 and KCREB, respectively; similar decreases were observed in PCNA protein levels. Pulsed-field gel electrophoresis measurements showed that CR133 inhibited the repair of radiation-induced DNA double-strand breaks, and this effect was reversed by over-expression of PCNA; dominant-negative CREB also significantly inhibited split-dose recovery. Clonogenic assays were used to determine surviving fraction; the dose enhancement ratios for dominant-negative CREB-expressing cells compared with control (vector alone) were 1.5 and 1.3 for CR133 and KCREB, respectively. Importantly, co-transfection of mutant CREB and a construct constitutively expressing PCNA protein restored radiosensitivity of CHO cells back to wild-type levels. Moreover, cells expressing either CREB mutant showed no significant cell cycle redistribution. These data demonstrate that genetic disruption of CREB results in radiosensitization, and that this effect can be explained by a mechanism involving decreased PCNA expression and inhibition of DNA repair.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The activation of epidermal growth factor receptor (EGFR)1 by ionizing radiation induces in autocrine growth regulated tumor cells a strong cytoprotective response (1). We and others have demonstrated that inhibiting radiation-induced EGFR Tyr autophosphorylation with specific EGFR kinase inhibitors or by over-expressing a dominant-negative (DN) EGFR mutant radiosensitizes human carcinoma and malignant glioma cells (2, 3). Similar radiosensitization is observed upon inhibiting a downstream target of EGFR, MAPK, with the MAPK kinase inhibitor PD98059 (4, 5). The underlying mechanism of EGFR/MAPK radiosensitization remains to be defined. We have focused on a potential transcriptional role for MAPK acting through p90S6 kinase (RSK) (6, 7) as an effector on multiple transcription factors. We have examined a panel of 8 transcription factors; of these, the radiation-induced activation of CREB, early growth response 1, ETS2, and STAT3 totally depends on signals from EGFR, RAS, and MAPK. Inhibiting any of these signaling components completely ablates these transcription factor responses (8). These results expand on previous studies with other cell types demonstrating by gel-shift analysis radiation-induced stimulation of CREB binding (9). Other transcription factors, such as CCAATT/enhancer-binding protein-{beta} and STAT1, only partially depend on signals from the EGFR/MAPK cascade (8).

Among the radiation-responsive, EGFR-dependent transcription factors, CREB is of interest because it mediates transcription of genes potentially involved in radiosensitivity (8). A mechanistic relationship between CREB expression/activity and DNA repair has been established by the finding that the proliferating cell nuclear antigen (PCNA) is transcriptionally controlled by CREB (10–12). PCNA is required for DNA excision repair (13) and is involved in the repair of ionizing radiation-induced DNA damage (14–18). Significant increases in nuclear PCNA immunofluorescence (14, 18) and mRNA levels (15) have been observed between 15 min and 2 h after irradiation. Because PCNA also functions in the replication of DNA (and thus, progression through the S-phase of the cell cycle) (19–20), depletion of PCNA could potentially modify cellular radiosensitivity through both repair inhibition and cell cycle redistribution mechanisms. The radiation effects on CREB and PCNA support their role as determinants of cellular radiosensitivity. We have, therefore, investigated the mechanism of DN-CREB-mediated radiosensitization by defining the effects of CREB on PCNA function and expression.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Unless specified otherwise, all reagents were obtained from Sigma. RPMI 1640 medium and LipofectAMINE PLUS reagent were obtained from Invitrogen, and fetal bovine serum was purchased from HyClone (Logan, UT). The DN-CREB constructs, pCMV-CREB133 (CR133) and pCMV-KCREB (KCREB), and pCRE-Luc reporter plasmids were purchased from Clontech (Palo Alto, CA). Luciferase assay kits and the empty vector, pCMV (which carries the CMV promoter without any downstream gene) were obtained from Promega (Madison, WI). Bradford protein assay reagents and broad-range molecular weight markers were obtained from Bio-Rad (Hercules, CA). Primary antibodies against PCNA and {beta}-actin and horseradish peroxidase-linked secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell Treatments and Irradiation—Chinese hamster ovary (CHO.K1) cells from American Type Tissue Collection (Rockville, MD) were cultured in RPMI 1640 medium + 5% fetal bovine serum + penicillin/streptomycin. Cells were plated for 2 days in 10- or 3.5-cm dishes to achieve 50–90% confluency the day of transfection. Two days after transfection, cells were exposed to ionizing radiation at a dose rate of 1.4–1.6 Gy/min (depending on the month of calibration) using a 60Co source, and incubated for colony formation. Alternatively, cells were lysed 2 days after transfection for reporter gene assays or fixed in ethanol for flow cytometry.

DNA Transfections—Plasmid DNA containing the wild-type PCNA promoter with a chloramphenicol acetyl transferase (CAT) reporter gene (PCNA-87 CAT) was generously provided by Dr. Michael Mathews (10); PCNA-87 CAT is simply referred to as PCNA-CAT throughout this paper. A construct expressing the human PCNA gene, pCMV-PCNA, was obtained from ATTC. The {beta}-galactosidase reporter plasmid, RSV{beta}galBSH, was described previously (21). Plasmids were co-transfected using optimum LipofectAMINE PLUS conditions described by the manufacturer. When two plasmids were co-transfected and compared with data with only one active construct, vector DNA was co-transfected in equal amounts such that the total amount of DNA was the same for all transfections. Three hours after transfection in serum-free, antibiotic-free medium, complete medium was added, and dishes were incubated at 37 °C for 48 h.

Reporter Gene Assays—CAT and {beta}-galactosidase assays were performed as previously described (21). Briefly, equal amounts of whole-cell lysates (without protease inhibitors) were incubated with CAT mixture containing 125 mM Tris, pH 7.5, 1 nCi/µl 14C-chloramphenicol, and 670 µM Acetyl-CoA. Reactions were extracted with ethyl acetate and spotted onto TLC plates. Chromatograms were developed with 95% chloroform-5% methanol for 45 min, and exposed overnight to x-ray film. For {beta}-galactosidase assays, equal amounts of protein and 150 µM resorufin in PBS + 1 mM MgCl2 were added to wells, and {beta}-galactosidase activity was measured using a fluorimeter. CAT activity, calculated by densitometry as % (converted)/(converted + unconverted), was then normalized for transfection efficiency based on {beta}-galactosidase assays.

Luciferase assays were performed according to the manufacturer's instructions. Cells were co-transfected with 0.5 µg of pCRE-Luc and 0.5 µg of DN-CREB (or vector control), and cells were lysed 48 h later. Equal amounts of protein (20 µg) were added to tubes, and a luciferin substrate was injected. The luminescence of the product, oxyluciferin, was monitored using a Berthold luminometer.

Western Blot Analyses—Two days after transfection, cells were rinsed with ice-cold PBS and snap-frozen on dry ice. Cells were scraped into a lysis buffer (25 mM Tris, pH 7.4, 50 mM {beta}-glycerophosphate, 1.5 mM EGTA, 0.5 mM EDTA, 5% glycerol, 1% Triton-X) containing protease inhibitors (1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM benzamidine, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 100 g/ml phenylmethylsulfonyl fluoride) and passed 5 times through a 20-gauge needle and syringe. Samples were centrifuged at 11,000 x g for 10 min at 4 °C, and supernatant proteins were determined by the Bradford assay. For Western blotting, 5x loading buffer (50 mM NaPO4, 5% SDS, 0.25% bromphenol blue, 12.5% {beta}-mercaptoethanol, and 10% glycerol) was added to samples. Equal amounts of protein were fractionated on SDS-polyacrylamide gels and protein transferred electrophoretically onto nitrocellulose membranes. Membranes were probed with primary antibodies against PCNA or {beta}-actin and horseradish peroxidase-linked secondary antibody, according to the manufacturer's instructions. Blots were analyzed by chemiluminescence detection and densitometry.

DNA Double-strand Break Measurements—Cells were transfected with CR133, vector, and/or pCMV-PCNA as described above. After 48 h, log-phase cells were trypsinized and resuspended in PBS at 4 x 106 cells/ml. Cells were mixed with an equal volume of 1% InCert Agarose/PBS and plugs were cast. Plugs were then equilibrated in growth medium for 1 h at 37 °C. Plugs were irradiated at 20 Gy, and repair processes were terminated at various timepoints by submersion of plugs into ice-cold digestion buffer (0.5 M EDTA, pH 8.0, 1% Sarkosyl, 1 mg/ml proteinase K) for 2 h. Plugs were incubated in digestion buffer overnight at 50 °C, rinsed 3 times with 0.5x Tris/EDTA buffer for 1 h, and stored in 0.5 M EDTA at 4 °C. Plugs were inserted into the wells of a 0.5% agarose, 0.5x Tris/boric acid/EDTA gel and electrophoresed for 16 h using a BioRad CHEF DRII pulsed-field gel electrophoresis system, according to manufacturer's specifications. Gels were stained with ethidium bromide, and inverted ultraviolet images were acquired and analyzed using densitometry. The fraction of DNA released from plugs was calculated from densitometry using a Bio-Rad Molecular Imager as DNA released/(DNA in plug + DNA released), and normalized by defining the ratio at t = 0 as unity.

Clonogenic Assay—Cells were trypsinized, counted, diluted, and plated at numbers appropriate for each treatment, then incubated at 37 °C for 8 days after plating. Colonies were fixed with methanol and stained with 0.5% crystal violet. Colonies were counted by eye, with a cut-off of 50 viable cells. Surviving fractions for transfected cells were normalized to the respective plating efficiencies for transfection alone. The dose enhancement ratio was calculated as the dose (Gy) for radiation alone divided by the dose (Gy) for radiation plus transfection (normalized for transfection toxicity), at a surviving fraction of 0.37. Error bars represent the standard error of the mean for three independent experiments, each plated in quadruplicate. Survival curve parameters D0 and n were fitted to the multi-target single hit equation: S = 1 – (1 – e–D/D0)n. The survival curve parameters {alpha} and {beta} were fitted to the {alpha}-{beta} model: S = exp(–{alpha}D – {beta}D2).

Cell Cycle Analysis—Two days after transfection, cells were trypsinized and 2 x 106 cells were fixed by slowly adding ice-cold 70% ethanol in PBS while vortexing. Fixed cells were stained overnight at 4 °C with 50 µg/ml propidium iodide + 40 units RNase in PBS, and analyzed using a Coulter flow cytometer. Cell cycle analysis was performed using ModFit software.

Statistical Analysis—A two-tailed Student's t test was used to determine statistical significance for n = 3 independent experiments. p values of less than 0.05, as calculated using Sigma-Plot software, were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Dominant-negative CREB Constructs and Their Effect on CREB Transcriptional Activity—We have shown that transfection or adenoviral transduction of a DN EGFR mutant results in radiosensitization of human cancer cells (2, 3). Similarly, we have demonstrated that selective inhibition of the MAPK pathway potentiates cell killing by ionizing radiation (4, 5). The purpose of this study was to determine whether genetic inhibition of CREB, which is activated by the same pathways (6–9), can influence subsequent molecular events that are involved in cellular radiosensitivity. For these initial studies we used CHO cells because they are well defined radiobiologically and are readily manipulated genetically by transfection (efficiency > 90%). DN-CREB constructs were utilized to inhibit the transcriptional activity of CREB in this cellular system. Two DN-CREB mutants with different mechanisms of CREB inactivation were used: CR133, which expresses a CREB protein with a mutation in the Ser-133 phosphorylation site; and KCREB, encoding a CREB protein with a mutation in the DNA binding domain. To verify inactivation of basal CREB-induced transcription, a luciferase-based CREB reporter system was used (Fig. 1). When cells were co-transfected with pCRE-Luc and CR133 or KCREB, 5.6- and 2.6-fold decreases in luciferase activity were observed within 48 h, respectively.



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  		FIG. 1.
Reduction in basal levels of CREB-mediated transcription by DN-CREB expression. CHO cells were co-transfected with the CREB reporter construct, pCRE-Luc, and either empty vector (pCMV), pCMV-CREB133, or pCMV-KCREB; these plasmids are labeled in all figures as vector, CR133, and KCREB, respectively. Cells were lysed 48 h after transfection, and luciferase assays were performed. Data are normalized for {beta}-galactosidase transfection efficiencies. Error bars represent the ± S.E. of three independent experiments.

 

Reduction of PCNA Reporter Activity and PCNA Protein Levels by DN-CREB—Because the PCNA gene is transcriptionally activated by CREB (10–12), we tested whether inhibition of CREB would reduce PCNA promoter activity. When CHO cells were co-transfected with CR133 and PCNA-CAT reporter (10) and assayed 48 h later (same protocol as with pCRE-Luc), a 60% decrease in basal PCNA-CAT activity was observed (Fig. 2). Co-transfection of PCNA-CAT and KCREB resulted in a modest but still significant (p < 0.05) 41% decrease in basal PCNA-CAT levels. Co-transfection with the empty vector (pCMV) did not change PCNA-CAT activity. A plasmid encoding an unrelated downstream gene ({beta}-galactosidase) was co-transfected instead of vector DNA, and this also had no effect on PCNA-CAT promoter activity (data not shown).



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  		FIG. 2.
Effect of DN-CREB expression on basal levels of PCNA-CAT reporter activity. Cells were co-transfected with either CR133 or KCREB and the PCNA-CAT reporter construct. Total cellular protein was isolated after 48 h, and CAT assays were performed. Normalized CAT activity represents the percent conversion from CAT assays, normalized by the {beta}-galactosidase transfection efficiency (n = 3 independent experiments).

 

To determine whether the effects of DN-CREB on PCNA promoter activity were accompanied by a corresponding change in PCNA protein levels, the same protocol was followed except that Western blotting was used as an end point. As was found with PCNA promoter activity, expression of CR133 or KCREB resulted in 62 and 38% decreases, respectively, in PCNA protein levels (Fig. 3). To test whether over-expression of PCNA could modulate this effect, a plasmid was used that expresses the human PCNA gene (pCMV-PCNA). Transfection of pCMV-PCNA resulted in a 2-fold increase (p < 0.05) in PCNA expression above basal levels of vector control cells, and recovery of PCNA protein levels to near normal in CR133-expressing cells.



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  		FIG. 3.
Effect of DN-CREB expression and PCNA over-expression on PCNA protein levels. Cells were transfected with either CR133, KCREB, or pCMV-PCNA + CR133. Total cellular protein was isolated 48 h after transfection and subjected to Western blot analysis against PCNA. Equal amounts of DNA were transfected by using vector DNA. Fold-changes represent the mean of n = 3 independent experiments. Equal protein loading was verified by stripping and re-probing for {beta}-actin.

 

Modulation of DNA Double-strand Break Repair and Split-dose Recovery by DN-CREB—Because PCNA is required for DNA double-strand break (DSB) repair (22, 23), pulsed-field gel electrophoresis was used to measure the induction and repair of DSBs. A dose of 20 Gy was used to induce double-strand breaks, because repair kinetics could not be adequately resolved with lower doses (data not shown). When cells were transfected with CR133 and irradiated 48 h later, a statistically significant (p < 0.05) decrease in the rate of DSB repair was observed at each time point, compared with cells transfected with the pCMV vector (Fig. 4). The repair half-times of vector-transfected and CR133-transfected cells were 38 min and 60 min, respectively. Cells transfected with CR133 and pCMV-PCNA exhibited repair kinetics nearly that of control levels. Transfection of pCMV-PCNA alone did not influence repair kinetics.



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  		FIG. 4.
Repair kinetics of radiation-induced DNA double-strand breaks after expression of CR133 and/or pCMV-PCNA. Cells transfected with the plasmids were cast into agarose plugs, irradiated with 20 Gy, and analyzed by pulsed-field gel electrophoresis. A, inverted images of ethidium bromide-stained gels. B, DSB repair kinetics calculated by densitometry. Error bars represent the ± S.E. of three independent experiments. {square}, vector; •, pCMV-PCNA + vector; {blacktriangleup}, CR133 + vector; {circ}, CR133 + pCMV-PCNA.

 

Split-dose recovery is the operational term for the increase in cell survival that is observed if a given radiation dose is split into two fractions separated by a certain time interval; DSB repair-proficiency is required for split-dose recovery (24–27). Therefore, experiments were performed to test the role of CREB in split-dose recovery. Cells were transfected with DN-CREB mutants and irradiated as described above, except that two separate doses of 2 Gy were delivered to cells with varied time intervals (0–5 h) between doses. Clonogenic assays were then performed. Transfection of CR133 resulted in almost complete inhibition of split-dose recovery, and KCREB expression caused intermediate but still significant (p < 0.05) inhibition of split-dose recovery (Fig. 5).



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  		FIG. 5.
Effect of DN-CREB expression on split-dose recovery. Cells were transfected and irradiated 48 h later with two 2 Gy doses, separated by various time intervals. Surviving fraction values are normalized to reflect the fold-increase above the zero time point for each treatment group (which represents two continuous 2 Gy doses, or 4 Gy). Error bars represent the ± S.E. of three independent experiments, each plated in quadruplicate. {circ}, vector; {blacksquare}, CR133; {blacktriangleup}, KCREB.

 

Radiosensitization by Transfection of DN-CREB—Because the capacity for split-dose recovery, correlating with DSB repair capacity, is a major determinant of cellular radiosensitivity (26, 27), the effects of DN-CREB expression on the survival of CHO cells were evaluated. Cells were transfected with either construct, irradiated 48 h later, and cell survival was determined using a clonogenic assay. The mean surviving fraction for cells transfected with DN-CREB constructs (without radiation) are 0.60 and 0.77 for CR133 and KCREB, respectively. Survival curves (Fig. 6) are normalized for this transfection-derived toxicity. Transfection of the empty vector (pCMV) did not cause any significant toxicity or changes in radiation survival (p > 0.05). The dose enhancement ratios (dose enhancement ratio at surviving fraction = 0.37) for the curves shown in Fig. 6 were 1.5 and 1.3 for CR133 and KCREB, respectively. Statistically significant decreases (p < 0.05) in surviving fraction were observed at both 2 and 4 Gy for either DN-CREB construct relative to control vector. A 2-fold decrease in CR133 survival was measured at 4 Gy. Although the shoulder regions (n value) of these survival curves are reduced when cells are transfected with DN-CREB, the final slopes (D0) did not change (Table I). Using the {alpha}/{beta} model (26), a 20-fold increase in the {alpha} value was observed with transfection of CR133, whereas the {beta} value decreased by a factor of 2.5. KCREB caused a 10-fold increase and a 1.5-fold decrease in the {alpha} and {beta} values, respectively. Taken together, these curve fitting parameters demonstrate that only the shoulder region of the survival curves is changed by expression of DN-CREB. This is consistent with the inhibition of split-dose recovery by DN-CREB, because the size of the shoulder region is directly related to the capacity of cells to undergo split-dose recovery (26).



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  		FIG. 6.
Ionizing radiation survival curves for cells expressing DN-CREB. Cells were transfected with either CR133, KCREB, or vector, irradiated 48 h later, and clonogenic assays were performed. Survival curves are normalized by the respective plating efficiencies for each treatment group. Error bars represent the ± S.E. of three independent experiments, each plated in quadruplicate. •, vector; {circ}, CR133; {triangleup}, untransfected; {blacksquare}, KCREB.

 

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  		TABLE I
Curve-fitting parameters and dose enhancement ratios (DER) corresponding to the survival curves in Figs. 6 and 7

 



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  		FIG. 7.
Effect of PCNA gene transfection on radiosensitization by pCMV-CREB133. Cells were transfected with vector, CR133, pCMV-PCNA, or CR133 + pCMV-PCNA. Equal amounts of total DNA were transfected by using vector DNA. Curves are normalized for the respective plating efficiencies for each treatment. Error bars represent the ± S.E. of three independent experiments, each plated in quadruplicate. •, vector; {circ}, CR133 + vector; {triangleup}; pCMV-PCNA + vector; {blacksquare}, pCMV-PCNA + CR133.

 
Reversal of DN-CREB-mediated Radiosensitization by PCNA Over-expression—DN-CREB expression reduced PCNA protein levels and inhibited DNA repair, effects that were reversed by over-expression of PCNA, suggesting a role of PCNA in DN-CREB-induced radiosensitization. When cells were co-transfected with CR133 and pCMV-PCNA, radiosensitivity was also restored to control levels (Fig. 7). Transfection of pCMV-PCNA alone or empty vector did not affect radiation survival curves. Thus, PCNA over-expression overrides the radiosensitizing effect of DN-CREB, but does not cause radioresistance by itself.

Because the radiation response of mammalian cells varies with the phase of the cell cycle (26), and both CREB and PCNA contribute to proliferation/S-phase progression (6, 7, 19–20), the possible effect of DN-CREB expression on cell cycle distribution was quantified. Flow cytometric analysis of propidium iodide stained cells (Table II) demonstrated no significant (p > 0.05) cell cycle redistribution at the time of maximum CR133 or KCREB expression (48 h after transfection); similar results were obtained when pCMV-PCNA and CR133 were co-transfected. Overall, this demonstrates that radiosensitization by DN-CREB expression is at least in part mediated by PCNA and its effect on DNA repair.


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  		TABLE II
Flow cytometric cell cycle analysis of CHO cells transfected with DN CREB constructs and/or pCMV-PCNA

Values represent the Mean ± SEM of 3 independent experiments.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
These studies were initiated because we have previously shown that the transcription factors CREB, early growth response, ETS, and STAT3 are activated by ionizing radiation via the EGFR/MAPK pathway (8), and inhibition of either EGFR tyrosine phosphorylation or MAPK activity radiosensitizes a variety of human tumor cells (2–5). Because PCNA is both transcriptionally regulated by CREB (10–12) and involved in repair of radiation-induced DNA damage (14–18), we examined the effects of DN-CREB on PCNA promoter activation and expression of PCNA protein. These experiments show that DN-CREB expression significantly reduces basal levels of both PCNA-CAT reporter construct activity and PCNA protein, affirming the role of CREB on PCNA gene expression. The finding that PCNA over-expression reversed the repair-inhibiting and radiosensitizing effects of DN-CREB supports the involvement of PCNA in repair of radiation-induced DNA damage.

Using the same PCNA-CAT reporter construct, it was previously shown that CREB (but not ATF-1) mediates transactivation of the PCNA promoter, and that the transcriptional co-activator, CREB-binding protein (CBP), is also involved in this process (10). Others have shown that the promoter activity of the PCNA gene is associated with inducible CRE-binding proteins in interleukin-2-stimulated T-lymphocytes (11), and that rapamycin can inhibit the binding of CREB/ATF transcription factors to CRE elements in the PCNA promoter (12). Based upon these previous studies, CREB inhibition by DN-CREB probably has a direct effect on basal PCNA expression because both PCNA promoter activity and PCNA protein levels are reduced to the same extent by DN-CREB. Further work is necessary to test whether CREB serves as the primary activator of the PCNA promoter in our system or whether other molecules, such as p53, act as secondary effectors (28–30).

We have shown that expression of DN-CREB inhibits the repair of radiation-induced DNA DSBs, and that this effect is reversed by transfection of pCMV-PCNA. This result is consistent with the established roles for PCNA in the repair of radiation-induced DNA damage (14–18). PCNA is involved in both nucleotide excision repair and base excision repair (BER) (13, 28). Evidence also suggests that the BER pathway is involved in the PCNA-dependent repair of radiation-induced DNA damage (14, 31) and that BER plays an important role in cellular radiosensitivity (32). Importantly, PCNA has been shown to be an indispensable component in the process of DSB repair (22, 23), critical for cell survival following exposure to ionizing radiation (26). Our results demonstrating that split-dose recovery is inhibited using either DN-CREB construct are important in this regard. Split-dose recovery is a probable consequence of DSB repair because cell lines deficient in DSB repair are incapable of split-dose recovery (24, 25, 27).

Previously, it was shown that selective inhibition of MAPK abolishes induction by radiation of the ERCC1 and XRCC1 repair genes, inhibits BER, and radiosensitizes human prostate cancer cells (33). Thus, our present study is consistent with the finding that molecules downstream of the MAPK pathway are involved in DNA repair. Because the EGFR/MAPK pathway activates CREB (6–8), these experiments present another line of evidence that EGFR signaling is linked to DNA repair processes. CREB has been shown to be required for activation of other genes known to be involved in DNA repair, such as BRCA1 and DNA polymerase-{beta} (34, 35). However, this report is the first to describe a direct mechanistic link between CREB function and DNA repair.

Importantly, we provide experimental evidence that CREB contributes to the radiosensitivity of mammalian cells, because disruption of CREB activity leads to increased cell killing by radiation. Only a few previous studies have demonstrated radiosensitization by disruption of transcription factor activity (36–38). Inhibition of NF-{kappa}B by drugs has been reported to result in radiosensitization (36, 37), but these effects are attributed primarily to inhibition of anti-apoptotic signaling. Herein, we suggest that radiosensitization by DN-CREB may be due to inhibition of DNA repair processes. In addition to our measurements of PCNA expression and DSB repair, the survival curves also support the hypothesis of repair inhibition. A reduction in the shoulder region (n value) of radiation survival curves (without a change in the D0 value) can be interpreted as repair inhibition, when the repair model is considered (39).

Although transfection of pCMV-PCNA alone does not increase cell survival, co-transfection of pCMV-PCNA and DN-CREB restores radiosensitivity to control levels. These findings suggest that increases in PCNA above physiological levels do not contribute further to cellular repair capabilities with effects on radiosensitivity. The exact role of PCNA in radiation sensitivity of mammalian cells is difficult to assess, because genetic disruption of PCNA results in cellular lethality (28). Although constitutive levels of PCNA in different cell lines have been correlated with their capacity for adaptive survival response to low-dose radiation (17), a direct link between PCNA levels and radiosensitivity has not been previously established. Because partial modulation of PCNA levels by DN-CREB can influence cellular radiosensitivity, our results demonstrate a new mechanistic relationship between cellular PCNA levels and cell survival following exposure to radiation.

We also show that DN-CREB does not alter the cell cycle distribution 48 h after transfection. Although antisense PCNA oligonucleotides can prevent cells from entering the S-phase of the cell cycle (40), this phenomenon occurs in cells that are destined to die due to complete loss of PCNA and the resulting lack of DNA synthesis. In contrast, the majority of cells transfected with DN-CREB survive the transfection process, indicating that DNA synthesis still occurs in these cells. The lack of a % S-phase decrease with DN-CREB is also supported by evidence indicating that PCNA has separate roles in DNA synthesis and DNA repair (41). DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication (41). Thus, a 60% decrease in PCNA levels may not significantly change the percentage of cells undergoing DNA synthesis, but this reduction is apparently sufficient to inhibit the repair of radiation-induced DNA damage.

In summary, we have demonstrated that two different forms of DN-CREB can radiosensitize CHO cells. The mechanisms of radiosensitization include a reduction of basal PCNA levels, resulting in decreased DNA repair. We have identified CREB and PCNA as downstream targets of MAPK and have established CREB as an important link between mitogenic signaling and radiosensitivity as a function of DNA repair. Because of the previously established EGFR/MAPK dependence of CREB activity, CREB may be the downstream effector that, at least in part, modulates cellular radiosensitivity and could be considered an alternative molecular target for radiosensitization.


   FOOTNOTES
 
* This work was supported by Public Health Service Grants P01 CA72955 and R01 CA65896 (to R. S.-U.), R01CA90881 (to R. M.), and by the Florence and Hyman Meyers Head and Neck Cancer Research Fund. 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. Back

{ddagger} To whom correspondence and reprint requests should be addressed: Dept. of Radiation Oncology, Medical College of Virginia, Virginia Commonwealth University, P.O. Box 980058, 401 College St., Richmond, VA 23298-0058. Tel.: 804-828-7238; Fax: 804-828-6042; E-mail: rullrich{at}hsc.vcu.edu.

1 The abbreviations used are: EGFR, epidermal growth factor receptor; DN, dominant-negative; MAPK, mitogen-activated protein kinase; CREB, cAMP-responsive element-binding protein; STAT, signal transducer and activator of transcription; PCNA, proliferating cell nuclear antigen; CHO, Chinese hamster ovary; CMV, cytomegalovirus; CAT, chloramphenicol acetyl transferase; Luc, luciferase; DSB, double-strand break; BER, base excision repair; Gy, gray; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We thank Elizabeth Rosenberg for teaching pulsed-field gel electrophoresis, Francis White for performing flow cytometry and cell cycle analysis, and Dr. Michael B. Matthews for providing the PCNA-CAT reporter construct.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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