©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phorbol Ester Abrogates Up-regulation of DNA Polymerase by DNA-alkylating Agents in Chinese Hamster Ovary Cells (*)

Deepak K. Srivastava , Teresa Y. Rawson , Stephen D. Showalter (1), Samuel H. Wilson (§)

From the (1)Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1068 and Bio-Molecular Technology, Inc., Frederick, Maryland 21701

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Mammalian DNA polymerase (-pol), a DNA repair polymerase, is known to be constitutively expressed in cultured cells, but treatment of cells with the DNA-alkylating agents MNNG or methyl methanesulfonate has been shown to up-regulate -pol mRNA level. To further characterize this response, we prepared a panel of monoclonal antibodies and used one of them to quantify -pol in whole cell extracts by immunoblotting. We found that treatment of Chinese hamster ovary cells with either DNA-alkylating agent up-regulated the -pol protein level 5-10-fold. This induction appeared to be secondary to DNA alkylation, as induction was not observed with a genetically altered cell line overexpressing the DNA repair enzyme O-methylguanine-methyltransferase. We also found that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of wild type Chinese hamster ovary cells increased expression of -pol protein (10-fold). Any interrelationship between this TPA response and the DNA-alkylation response was studied by treatment with combinations of MNNG and TPA. The -pol up-regulation observed with MNNG treatment was abrogated by TPA, and conversely the up-regulation observed with TPA treatment was abrogated by MNNG.


INTRODUCTION

DNA polymerase (-pol)()is involved in DNA repair in mammalian cells, based upon inhibitor studies with cultured cells and permeabilized cell systems (Miller and Chinault, 1982; Smith and Okumoto, 1984; Dresler and Kimbro, 1987; DiGiuseppe and Dresler, 1989). The catalytic specificity of -pol in vitro is consistent with a role in gap-filling synthesis in vivo, to restore double-stranded DNA at short-gapped intermediates produced during excision repair (Wang and Korn, 1980; Mosbaugh and Linn, 1983; Randahl et al., 1988; Singhal and Wilson, 1993). -Pol has been shown to be responsible for the single-base gap-filling synthesis step in base-excision repair in several mammalian crude nuclear extract in vitro systems (Wiebauer and Jiricny, 1990; Dianov et al., 1992; Singhal et al., 1995). -Pol is considered a constitutively expressed enzyme in vertebrates, and in most cells and tissues (Chang and Bollum, 1972). -Pol enzymatic activity and mRNA are expressed at a relatively low level and are independent of cell growth and cell cycle stage (Chang and Bollum, 1972; Zmudzka et al., 1988; Nowak et al., 1989). However, -pol mRNA and enzymatic activity are regulated in a tissue-specific fashion in rodents (Hirose et al., 1989; Nowak et al.,1989), and -pol mRNA is induced by treatment of cells with some DNA-damaging agents, but not by others. For example, in Chinese hamster ovary (CHO) cells, treatment with the DNA-alkylating agents MMS or MNNG causes an induction in -pol mRNA level (Fornace et al., 1989).

The promoters of the human, bovine, and rodent -pol genes have been cloned and characterized. The promoters are G + C-rich and have binding cis-elements for initiation site-binding protein(s) and two well known transcriptional activators, Sp1 and ATF/CREB, within a core promoter of 100 base pairs 5` of the mRNA start site (Widen et al., 1988; Widen and Wilson, 1991; Chen et al., 1995). These promoters lack typical TATA or CCAAT elements. In the human promoter, the palindromic sequence GTGACGTCAC at -40 to -49 upstream of the transcription start site is a typical activating transcription factor/cAMP-response element (ATF/CRE). This element, which was found to be essential for human -pol promoter activity in transient expression experiments (Widen and Wilson, 1991), mediates protein kinase A-dependent stimulation of the cloned promoter in CHO cells (Englander and Wilson, 1992a). The element also mediates stimulation of the cloned promoter after treatment of CHO cells with MNNG (Kedar et al., 1991), and this up-regulation was not seen in genetically altered CHO cells deficient in protein kinase A activity, indicating that this particular signal transduction pathway is required for the response to MNNG (Englander and Wilson, 1992a, 1992b).

The -pol core promoter is up-regulated by expression of the activated Harvey p21 protein (Kedar et al., 1990), consistent with the idea that activation of the protein kinase C signal transduction pathway may also be important in -pol gene expression. However, the cloned -pol promoter does not have functional binding sites for the transcriptional activator AP-1, a principal protein kinase C pathway target for transcriptional regulation (Widen et al., 1988). This is in contrast to the proposed mechanism of up-regulation of the so-called DNA damage-inducible and oxidative stress-responsive genes by protein kinase C stimulation with TPA (Macfarlane and Manzel, 1994; Stevenson et al., 1994) and subsequent activation of AP-1. In this case, treatment of cells with TPA stimulates the DNA binding activity of AP-1 by dephosphorylation of the c-Jun subunit; thus, protein kinase C activation appears to lead to elevation of a protein phosphatase activity and dephosphorylation of c-Jun's phosphorylation sites at residues 227 and 252 (Boyle et al., 1991). It also has been found that TPA treatment can directly stimulate members of the ATF/CREB superfamily of transcriptional activator; for example, ATF-2 (Zu et al., 1993) is stimulated due to phosphorylation by protein kinase C at Ser-340 and Ser-367, within the DNA-binding domain (Sakurai et al., 1991).

The present investigation was undertaken to examine the idea that the response of a mammalian DNA repair gene (-pol) to DNA alkylation damage may be regulated by the growth factor/protein kinase C/mitogen-activated protein kinase cascade. Several growth factors, including platelet-derived growth factor BB (Graves et al., 1993) and epidermal growth factor (Arteaga et al., 1994), are known to activate protein kinase C and the mitogen-activated protein kinase cascade in their signal transduction process. The growth factor/protein kinase C/mitogen-activated protein kinase cascade has recently been found to be antagonistic against the protein kinase A signal transduction system in CHO cells, rat adipocytes (Sevetson et al., 1993), and in human aortic smooth muscle cells (Graves et al., 1993). Thus, protein kinase C involvement in regulating -pol transcription could have implications for the DNA damage-induced response in -pol level. We found an induction of -pol level in CHO cells after treatment with either MNNG or TPA alone. These two agents, however, when used in combination were mutually antagonistic. These interesting results are discussed in the context of DNA damage-induced regulation of a DNA repair gene and the potential for modulation of this regulation by the protein kinase C signal transduction system.


MATERIALS AND METHODS

Isolation and Purification of -Pol

Recombinant rat -pol was purified from Escherichia coli RR1 (pRK 248clts, pRC-R1) as described by Kumar et al.(1990). The molecular mass of the -pol protein was 39 kDa, as expected from the open reading frame in the cDNA from mammalian sources (SenGupta et al., 1986; Zmudzka et al., 1986). The concentrations of stock solutions of -pol were calculated from ultraviolet absorbance values using the molar absorption coefficient = 2.12 10M cm.

Controlled Proteolysis with Trypsin

Large-scale digestion of -pol with trypsin was carried out in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride by the procedure of Kumar et al.(1990). This digestion generated 31-, 27-, and 8-kDa fragments of -pol. By changing the concentration of trypsin to a substrate:enzyme ratio of 10:1 (w/w), two more fragments of 10- and 12-kDa were produced from the 27-kDa domain.()These fragments, along with overexpressed 16/18-kDa peptides of -pol, were used to determine the epitope for several monoclonal antibodies (mAbs) raised to the whole protein.

Generation of Anti--pol Monoclonals

Immunization, Hybridization, Selection, and Cloning

Eight-week old BALB/c mice were inoculated intraperitoneally with purified bacterial recombinant -pol (75 µg) in a 0.5-ml emulsion with complete Freund's adjuvant. Two weeks later, the animals were boosted with a 0.5-ml subcutaneous inoculation of purified -pol (75 µg) with incomplete Freund's adjuvant. The following week, serum from the mice was tested by ELISA against a highly purified antigen preparation to determine the level of specific anti--pol activity. Two weeks following the second injection, the most reactive mice were given an intravenous injection of 0.1 ml containing 100 µg of highly purified -pol, and their spleens were harvested for fusion 4 days later. The general procedures used for cell fusion, selection, cloning, and propagation have been previously described (Köhler et al., 1976; Showalter et al., 1981).

Beginning about day 10, fluids from actively growing cells were tested by ELISA for anti--pol activity. By controlled washing of antigen-antibody complexes on the plate, this test was designed to select for antibodies that would also give strong immunoblot signals. Cells from positive wells were expanded into 24-well plates and cloned by limiting dilution in 96-well plates on a feeder layer of compatible thymocytes. Cloned cells were passaged in ascites tumors by intraperitoneal injection of 3 10 cells into adult BALB/c mice primed 2 weeks previously with 0.5 ml of pistrane (2,6,10,14-tetramethylpentadecane; Aldrich) injected intraperitoneally. The ascitic fluids resulting 7-10 days later were harvested, clarified, and the immunoglobulin purified to homogeneity for further use. Cell lines were designated as 4S, 6S, 9S, 10S, 14S, 15S, 16S, 17S, 18S, 22S, 23S, 30S, 35S, 38S, 40S, and 41S, respectively.

Analysis and Characterization

Antibody subclasses of all monoclonals obtained were determined using a commercially available isotyping kit (Amersham). The kit is essentially a sandwich immunoblot using specific anti-subtype reagents. Supernatant fluid from cultures of each line as tested for immunoglobulin class by ELISA and immunoblot assays using secondary antibodies specific for IgG (-chain) and IgM (µ-chain). Six cell lines (10S, 14S, 18S, 22S, 35S, and 38S) were found to produce IgG antibodies with the subclass of IgG, IgG, IgG, IgG, IgG, and IgG, respectively. The remaining 10 cell lines (4S, 6S, 9S, 15S, 16S, 17S, 23S, 30S, 40S, and 41S) produced IgM. Large-scale isolation of some of the mAbs (10S, 17S, 18S, 22S, 35S, and 38S) was accomplished; these were tested by ELISA and immunoblot assays against various domain peptides of rat -pol. In a subsequent experiment, we compared the specificity of each mAb for rat and human -pols. mAbs 6S, 18S, 22S, 35S, 40S, and 41S gave stronger reaction against rat -pol than human -pol, while mAbs 10S, 17S, 23S, and 38S were stronger against human -pol than rat -pol. The mAbs 4S, 14S, and 30S shared almost equal avidity for human and rat -pol; mAb 9S, 15S, and 16S show relatively less reactivity with -pol, as compared to the other mAbs.

The epitopes for mAbs 14S and 30S were in the 31-kDa domain(87-335), as they gave cross-reactivity with all domains tested except the 8-kDa domain(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80) . The epitopes for mAbs 17S and 4S were in the region between residues 75 and 154, as these mAbs reacted with -pol, 31-, 27-, 18-, and 10-kDa fragments. Epitopes for the other mAbs were found between amino acids 140 and 335. In the cases of 23S, 40S, and 41S, we detected no reaction with the 8-kDa domain, 16- or 18-kDa fragments. The epitopes of these mAbs may be contributed by different regions of -pol, resulting in the cross-reactivity to the 31-, 27-, 12-, and 10-kDa domains. The mAbs 6S and 38S have epitopes in the 12-kDa domain (230-335).

Cell Culture

CHO-K1 cells were purchased from the American Type Culture Collection (Rockville, MD). These cells were grown per ATCC recommendations. GC-1 cells (gift from Dr. S. Mitra, Sealy Center for Molecular Science, Galveston) were maintained as described (Dunn et al., 1991). Actively dividing CHO cells were exposed to different effectors, e.g. MNNG (10 and 30 µM) for 4 h or the indicated time periods; TPA (500 nM), dibutyryl cAMP (100 µM), and forskolin (1 µM) for 4 h. Different concentrations of TPA (10, 100, 200, 500, and 1,000 nM), dibutyryl cAMP (1, 10, 100, and 500 µM), and forskolin (1, 5, 50, and 100 µM) were used to study the optimum concentration of these effectors. The effect of TPA (500 nM) was also studied from 1 to 12 h.

Immunoblots

Actively growing cells in the presence or absence of effectors were used for -pol detection. For rapid cell lysis, exponentially growing tissue culture cells were washed twice with ice-cold phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 2.7 µg/ml aprotinin, and 0.5 µg/ml each of leupeptin, pepstatin A, and chymostatin. The cells were lysed in a buffer containing 10 mM Tris-HCl, pH 7.5, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and the protease inhibitors mixture as described above. The lysate was centrifuged at 8,000 g for 5 min, and the supernatant fraction (20 µg of protein unless otherwise specified) was electrophoresed in a 12.5% SDS-polyacrylamide gel. Proteins in the gel were transferred electrophoretically to nitrocellulose membrane. Equal loading of samples in different lanes and transfer to nitrocellulose membrane was verified in all cases by staining the membrane with Ponceau S. -Pol was measured by incubating the membrane with mouse anti--pol monoclonal antibody 18S, and then with antibody to mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase. Immobilized horseradish peroxidase activity was detected by enhanced chemiluminescence.

Different amounts of pure recombinant rat -pol (2 to 20 ng) were also electrophoresed along with various unknown samples on a 12.5% SDS-polyacrylamide gel and blotted to a nitrocellulose membrane; -pol was detected as described. The amount of -pol was determined from the linear portion of a dose-response curve (Fig. 1) obtained by plotting concentration versus integrated optical density. The integrated densitometry signals of -pol bands from autoradiograms (proportional to chemiluminescence) were measured using Millipore BioImage Visage Software on a Sun Sparc Station II Workstation (BioImage Products, Ann Arbor, MI). We found that densitometric signals of recombinant -pol were 7-fold higher when added to whole cell extract than for the same amount of -pol run alone (without extract). The magnitude of this effect was reminiscent of that observed with crude cell extract in the activity gel assay for -pol enzymatic activity (Swack et al., 1985). Hence, the values for -pol level reported for different cell lines and tissues were calculated on the basis of standard curves derived after addition of purified recombinant -pol to each whole cell extract.


Figure 1: Standard curve for -pol quantification. Varying amounts of purified rat -pol, from 5 to 20 ng, were mixed with 20 µg E. coli whole cell extract and electrophoresed on 12.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and probed with mAb 18S. Integrated OD was measured as described and then plotted against -pol.




RESULTS

Monoclonal Antibody Characterization

Purified rat -pol was used as antigen to produce a panel of mouse monoclonal antibodies (mAbs). Sixteen hybridoma cell lines producing antibody were isolated, and the antibodies were purified; hybridomas were selected for their ability to produce probes for immunoblotting. For epitope mapping of the mAbs, controlled proteolysis of -pol was used to prepare 5 domain peptides, as summarized in Fig. 2. We also overexpressed and purified an 18-kDa N-terminal segment of -pol corresponding to residues 1-154. The final preparation of the 18-kDa peptide also contained a smaller peptide, designated as 16 kDa, sharing the same N-terminal sequence. These 7 peptides were transferred to membrane, which then was probed separately with each mAb for epitope localization (Fig. 2). Epitopes for 12 of the mAbs were not localized to a smaller region, whereas epitopes for 10S, 16S, 18S, or 22S were localized to a respective 10-20 amino acid segment (Fig. 2). The epitope for 35S is in the 140-220 segment of -pol (Fig. 2), and the epitope for 18S is between residues 140 and 154 (Fig. 3A). The immunoblotting signal produced by 18S could be verified by competition with a synthetic peptide corresponding to the region KYFEDFEKRIPREEM, as this peptide blocks 18S reactivity with intact -pol (Fig. 3B).


Figure 2: Epitope mapping of monoclonal antibodies. Epitope mapping was performed using an immunoblotting assay. Recombinant rat -pol and its various fragments generated by controlled proteolysis with trypsin were subjected to 15% SDS-PAGE. The proteins were electrotransferred to nitrocellulose membranes. Each blot was probed separately with a mAb. Epitopes were identified by comparing the reactivity of each mAb with rat -pol and its various fragments. The placing of mAb or mAbs shows their epitope position on -pol (A and B). 8- (1-80) and 31-kDa (87-335) fragments were produced by 1:1,000, trypsin:-pol digestion, whereas 27- (140-335), 10- (140-220), and 12-kDa (230-335) fragments were produced with trypsin at a trypsin:-pol ratio of 1:10 (w/w) at 25°C. 18 kDa (1-154) and a smaller 16-kDa fragment with the same N terminus were overexpressed and purified from E. coli (C). These peptides and -pol fragments obtained from selective digestion with trypsin were used to identify epitopes of monoclonal antibodies.




Figure 3: Epitope mapping and specificity of mAb 18S to various DNA polymerases. PanelA, immunoblot of overexpressed human or rat -pol along with its various domains (8, 10, 12, 27, and 31 kDa) produced by selective tryptic digestion, plus overexpressed fragments (16 and 18 kDa), DNA polymerase , the large fragment of E. coli DNA polymerase I, and human immunodeficiency virus type-1 reverse transcriptase. The immunoblot was probed with anti--pol 18S mAb. The migration positions of -pol and its various peptides are indicated on right-hand side. The positions of protein markers are shown on the left-hand side of the photograph. PanelB, competition of the 18S mAbs epitope with a synthetic peptide of -pol (corresponding to residues 140-154). All lanes (lanes 1-6) have an equal amount of pure -pol (100 ng). Lanes 1-6 were probed with mAb 18S which was preincubated for 2 h at 25°C with 0, 0.1, 1, 2, 5, and 10 µg of synthetic peptide, respectively.



Monoclonal antibody 18S did not cross-react with reference enzymes human DNA polymerase , human immunodeficiency virus type-1 reverse transcriptase, or with Klenow fragment (Fig. 3A), but it did react strongly with human -pol, as shown in Fig. 3A and summarized under ``Materials and Methods.'' Finally, none of the mAbs exhibited neutralizing activity against purified -pol on poly(dA)oligo(dT) as template-primer (data not shown). Once this panel of high-titer -pol specific mAbs was available, we characterized one of them (18S) as a probe for quantitative immunoblotting. Whole cell extracts from a variety of sources, including bovine testis (known to contain a high level of -pol; Hirose et al.(1989) and Nowak et al.(1989)), were surveyed by quantitative immunoblotting. Among the sources tested, the -pol level was higher in bovine testis than in the other sources.

Regulation of -Pol Expression in Vivo

Transient expression activity of the cloned human -pol promoter in transfected CHO cell can be altered through the protein kinase A signal transduction pathway (Englander and Wilson, 1992b), which is activated by MNNG treatment of cells (Kedar et al., 1991; Englander and Wilson et al., 1992a). With the availability of mAb 18S, we could examine the effect on -pol protein levels of altering protein kinase A by treating CHO cells with dibutyryl cAMP or forskolin. The -pol level in our wild type CHO cell line was found to be 7 ng/mg protein in the whole cell lysate. Treatment for 4 h with forskolin (1 µM), or with dibutyryl cAMP (100 µM), resulted in an increase to 18 and 15 ng/mg, respectively. Dexamethasone (500 nM) treatment caused only a modest increase in -pol level, to 10 ng/mg of whole cell protein. It is known that MMS or MNNG treatment of CHO cells increases -pol mRNA level (Fornace et al., 1989) and that MNNG treatment activates the cloned -pol promoter in transient expression experiments through an effect on the ATF/CREB transcriptional activator (Kedar et al., 1991; Englander and Wilson, 1992a). We examined -pol protein levels in CHO cells treated with either MMS or MNNG. CHO cells treated with either MMS or MNNG showed an increase in -pol protein level (Fig. 4, A and B); with MNNG, the highest level was observed 2.5-5 h after treatment (Fig. 4B).


Figure 4: Effect of MMS and MNNG treatment on -pol expression in CHO-K1 cells. PanelA, CHO-K1 cells were exposed for 4 h at varying concentrations of MMS as indicated at the top of each lane (lanes 2-5). Lane 1, CHO-K1 cells were grown in presence of 1% MeSO (vehicle); lane6, recombinant rat -pol. PanelB, CHO-K1 cells were grown in the absence (lane 1) or presence of 30 µM MNNG (lanes 2-6) for the time (h) shown at the top of each lane. Lane7, recombinant rat -pol; and lane8, CHO-K1 cells grown in presence of 30 µM MNNG for 5 h and probed with nonimmune IgG. PanelC, GC-1 cells (lanes1-3) and CHO-K1 (lanes4-6) cells were grown in the absence (lanes1 and 4) or presence of 10 µM (lanes2 and 5) or 30 µM MNNG (lanes3 and 6) for 4 h. Purified recombinant rat -pol (lane7) was used as a reference. Whole cell lysates of CHO-K1 (20 µg) and GC-1 (12 µg) were subjected to 12.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and immunoblotted with 18S. Levels of -pol in GC-1 cells were 7, 6, and 6 ng/mg whole cell protein in lanes 1-3, respectively, whereas in CHO-K1 cells levels were 7, 28, and 50 ng/mg whole cell protein in lanes 4-6, respectively.



Next, to determine whether this up-regulation in -pol level was linked to DNA alkylation, we studied the effect of MNNG treatment using a genetically altered CHO cell line termed GC-1. GC-1 cells, which were derived from our wild type CHO cell line, strongly overexpress the alkylation damage DNA repair enzyme O-methylguanine DNA methyltransferase and, therefore, are resistant to cell killing by MNNG (Dunn et al., 1991). These cells are expected to have much less MNNG-induced methylation damage to DNA, by virtue of removal of methyl groups from DNA by the overexpressed DNA repair methyltransferase. Our results with GC-1 cells are shown in Fig. 4C. In contrast to wild type CHO cells, GC-1 cells did not show any increase in -pol expression after MNNG treatment. These results suggest that the -pol response to MNNG treatment is, indeed, a DNA alkylation damage response.

Regulation of -pol by phorbol ester (TPA) treatment in CHO cells was examined as a function of time and TPA concentration. Maximum expression occurred 4 h after treatment with 500 nM TPA (Fig. 5); the effect of different TPA concentrations also depended upon the time of exposure. When cells were exposed to TPA in the presence of actinomycin D (5 µg/ml) expression of -pol did not increase, suggesting that the TPA induction of -pol required transcription (Fig. 5A). These results on up-regulation of the endogenous -pol gene by phorbol ester, and presumably the protein kinase C pathway, may be consistent with our earlier results with mouse 3T3 cells; overexpression of activated Harvey p21 strongly up-regulated the cloned -pol promoter (Kedar et al., 1990). Although the -pol promoter does not have a known protein kinase C responsive DNA element, it is possible that stimulation could occur through the ATF/CREB protein which is known to be a key transcriptional regulator of -pol gene expression (see ``Discussion'').


Figure 5: Effect of phorbol ester (TPA) on -pol expression in Chinese hamster ovary (CHO-K1) cells. PanelA, CHO-K1 cells were treated with 500 nM TPA (lanes2-11) for different time intervals from 1 to 24 h as indicated at the top of each lane. The level of -pol measured was: 7, 14, 20, 53, 42, 46, and 31 ng/mg whole cell protein in lanes 1-7, respectively. CHO-K1 cells were exposed first to actinomycin D for 15 min (5 µg/ml) with subsequent addition of 500 nM TPA for 8 h (lane9), for 4 h (lane 10), and for 1 h (lane11), and the levels of -pol in each lane was 7 ng/mg whole cell protein. Lane1 is without TPA. PanelB, CHO-K1 cells were grown in the absence (lane1) or presence of 10 nM (lanes2 and 6), 100 nM (lanes3 and 7), 500 nM (lanes 4 and 8), and 1 µM TPA (lanes 5 and 9) for 2 (lanes2-5) or 4 h (lanes6-9). Whole cell lysate (20 µg) was loaded in each lane, electrophoresed, and immunoblotted as described under ``Materials and Methods.'' The position of -pol is indicated by an arrow.



Effect of Simultaneous Treatment with TPA and MNNG

The results described above and findings recently reported by others on antagonism between the protein kinase A and protein kinase C signal transduction systems (Graves et al., 1993; Sevetson et al., 1993), prompted us to study the effect of treatment with combinations of TPA and MNNG on -pol protein level. In these experiments, CHO cells were treated with increasing concentrations of either TPA or MNNG, in the presence of different concentrations of the other agent. First, TPA had an abrogating effect on up-regulation of -pol by MNNG (): the up-regulation by MNNG in these experiments was as much as 8.5-fold and was seen at concentrations as low as 0.1 µM; TPA at each concentration tested blocked the up-regulation.

The effect of MNNG-induced DNA damage on the up-regulation by TPA was also examined (). TPA treatment up-regulated -pol by as much as 14-fold in these experiments, and this induction was either blocked or partially blocked at each concentration of MNNG (). These results, in a general sense, indicate that the mechanism of activation by these two agents is not identical, and the results are consistent with the possibility that the mediator of the MNNG effect and the mediator of the phorbol ester effect interact with one another to form a dead-end complex, or have an antagonistic effect on the same target protein in -pol gene expression (e.g. ATF/CREB).


DISCUSSION

We explored -pol expression in vivo using new mAbs as probes for immunoblotting. Of the 16 mAbs isolated, we localized the epitope for four (10S, 16S, 18S, and 22S) to 10-20 amino acid regions of -pol (Fig. 2). None had an epitope in the N-terminal domain (1-75), and none inhibited the -pol DNA polymerase activity (data not shown). 35S and 18S were found to be good probes for immunofluorescence staining and immunoblot analysis of whole cell extracts, respectively. 18S did not show cross-reactivity with the other polymerases tested here (Fig. 3A), in contrast to earlier mAbs reported by Recupero et al.(1992). 18S could be used for quantitative measurements of -pol and could also be used in combination with a synthetic epitope peptide to verify identity of the 39-kDa immunoblot signal by competition. In this immunoblot assay, the efficiency of 18S for detection of -pol was found to be enhanced by the presence of other proteins in the extract; hence, when pure -pol was added to a whole cell extract, more signal was detected than with assays of pure -pol alone. This difference in detection efficiency was taken into account in our quantification of absolute -pol levels in the whole cell lysate (see ``Materials and Methods'').

Mammalian cells respond to treatment with DNA-damaging agents via several intracellular signal transduction pathways that alter gene expression (Fornace et al., 1989; Deng and Nickoloff, 1994; Nelson and Kastan, 1994; Xanthoudakis et al., 1994). In one such pathway protein kinase C appears to be activated, resulting in phosphorylation of proteins through a cascade, eventually leading to induction of the so-called DNA damage-inducible genes (Buscher et al., 1988; Fornace et al., 1989; Wilson, 1990). In earlier studies, the protein kinase A pathway was implicated in DNA-damaging agent induction of -pol gene expression. We observed up-regulation of -pol mRNA in CHO cells after exposure to the DNA-alkylating agents MMS or MNNG (Fornace et al., 1989), and this up-regulation required transcription. Furthermore, induction by MNNG was observed with a cloned -pol promoter in transient expression experiments with CHO cells; this induction is mediated by the ATF/CREB transcriptional activator (Kedar et al., 1991; Narayan et al., 1995), and required the cellular protein kinase A signal transduction system (Englander and Wilson, 1992b).

In the current study, we selected protein kinase A signaling agents such as dibutyryl cAMP, forskolin, and the DNA damaging agent MNNG to study their effect on expression of -pol protein in CHO cells. An initial finding of interest was that treatment of cells with either MMS or MNNG led to an increase in -pol protein level. We had found earlier that the increase in -pol mRNA level was not accompanied by a significant increase in -pol activity level in CHO cells. This earlier work involved activity gel analysis to quantify -pol; although the analysis was conducted in a range of proportionality between activity gel signal and crude extract applied to the gel (Fornace et al., 1989), and the analysis showed only a very modest increase (about 1.5-fold) in -pol enzymatic activity level. The present results represent a more complete assessment of -pol protein level in the crude extract, and measurement by immunoblotting with mAb 18S is considered to be more quantitative than by activity gel analysis.

After observing -pol protein up-regulation, we examined the interesting question of whether the induction is secondary to DNA alkylation. To approach this issue, we studied the effect of intracellular overexpression of a DNA methyl group DNA repair enzyme, O-methylguanine DNA-methyltransferase, on the MNNG induction of -pol (Fig. 4B). Our results showed that the induction did not occur in an otherwise isogeneic cell line (GC-1) carrying the overexpressed DNA methyltransferase repair enzyme. The mere presence of overexpressed methyltransferase itself did not produce a change in steady-state level of -pol, in the absence of MNNG. Furthermore, there have been no reports that the methyltransferase itself alters transcriptional regulation. The results, taken together, indicate that this transcription-based stimulation of -pol expression is through a DNA damage response pathway, rather than through a direct effect of MNNG, on protein kinase A or on the transcriptional machinery, as has been proposed for the oxidative stress response involving AP-1 (Devary et al., 1991; Xanthoudakis et al., 1994).

The precise mechanism of the -pol promoter DNA damage response remains to be revealed, but clearly the response is mediated by the ATF/CREB transcriptional activator (Kedar et al., 1990; Englander and Wilson, 1992; Narayan et al., 1995). Evidence for this includes the finding that ATF/CREB protein purified from MNNG-treated cells is more active in stimulating in vitro transcription initiation than the corresponding activator protein from normal cells (Narayan et al., 1995). The increases in -pol protein level observed here when CHO cells are grown in the presence of dibutyryl cAMP or forskolin confirmed that the endogenous -pol promoter is capable of responding to activation of the protein kinase A signal transduction pathway.

The phorbol ester TPA is known in some cases to influence gene transcription by binding to and activating protein kinase C, which in turn regulates synthesis and post-transcriptional modification of transcriptional activator proteins, such as AP-1, among others. CHO cells, when treated with as little as 10 nM TPA for 4 h, showed a strong increase in -pol level, and this up-regulation required transcription because cells pretreated with actinomycin D did not show any TPA-mediated increase in -pol (Fig. 5A). Hence, these experiments, in a general sense, suggest that both this pathway and the protein kinase A pathway, which mediates the influence of DNA alkylation damage by MNNG, regulate the -pol gene. We note that involvement of the growth factor/protein kinase C/mitogen-activated protein kinase pathway had been suggested by earlier -pol promoter transient expression experiments with overexpression of activated Harvey p21 in 3T3 cells (Kedar et al., 1990).

It was interesting to observe that the up-regulation of -pol expression afforded by MNNG or TPA was not additive. Instead, these agents were antagonistic to each other at all concentrations tested. This observation suggests that mediators of these stimulatory effects interact at some point with the same component in the signal transduction or transcriptional apparatus. Protein kinase C pathway transcriptional activators, such as c-Jun, can dimerize with ATF/CREB and influence transcriptional activation by proteins binding at the ATF/CRE site (Benbrook and Jones, 1990), whereas in the case of some ATF/CREB family members, protein kinase C can stimulate phosphorylation of the ATF/CREB protein (Sakurai et al., 1991). AP-1 also has been reported to recognize and activate transcription through binding at both AP-1 and ATF elements (Buckbinder et al., 1989). Further studies will be required to understand how TPA treatment can induce or abrogate -pol promoter expression. However, taken together, these results point to the interesting possibility that the DNA damage response for -pol in mammalian cells can be regulated by agents that alter the activity of the protein kinase C signal transduction pathway. Similar observations on possible antagonism between the protein kinase A and C pathways have been reported for others genes (Graves et al., 1993; Sevetson et al., 1993).

In summary, we report new immunological probes for mammalian -pol, including a mAb (18S) that can be used to measure the -pol protein level in a crude cell extract. We demonstrated that DNA alkylation is required for the induction of -pol gene expression, secondary to MNNG treatment of CHO cells. Induction of the -pol level in CHO cells also was observed after phorbol ester treatment. When simultaneously applied to cells, MNNG and phorbol ester were antagonistic toward the up-regulation seen with either agent alone. The results may be interpreted as an example of antagonistic effects of stimulation of the protein kinase A and protein kinase C signal transduction pathways.

  
Table: Effect of TPA on the MNNG-induced up-regulation of -pol expression in CHO-K1 cells

CHO-K1 cells were exposed to different concentrations of MNNG for 2 h either with or without simultaneous exposure to TPA. The results shown are averages representative of two independent experiments. The -pol level in untreated CHO-K1 cells was 7 ng/mg of whole cell protein. Value in parentheses is the relative -pol level, ratio of -pol in treated cells/untreated cells.


  
Table: Effect of MNNG on the TPA-induced up-regulation of -pol expression in CHO-K1 cells

CHO-K1 cells were exposed to different concentrations of TPA for 4 h either with or without simultaneous exposure to MNNG. The results shown are averages representative of two independent experiments. The -pol level in untreated CHO-K1 cells was 7 ng/mg of whole cell protein. Value in parentheses is the relative -pol level, ratio of -pol in treated cells/untreated cells.



FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants ESO6839 and ESO6492 (to S. H. W.) and Robert A. Welch Foundation Grant H-1265. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Sealy Center for Molecular Science, 301 University Blvd., University of Texas Medical Branch, Galveston, TX 77555-1068. Tel.: 409-772-3367; Fax: 409-772-6334.

The abbreviations used are: -pol, DNA polymerase ; CHO, Chinese hamster ovary; MGMT, O-methylguanine DNA methyltransferase; MMS, methyl methanesulfonate; MNNG, N-methyl-N`-nitro-N-nitrosoguanidine; mAb, monoclonal antibody; protein kinase A, cAMP-dependent protein kinase; ELISA, enzyme-linked immunosorbent assay; ATF/CREB, activating transcription factor/cAMP-response element binding; PAGE, polyacrylamide gel electrophoresis; TPA, 12-O-tetradecanoylphorbol-13-acetate.

D. K. Srivastava and S. H. Wilson, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. Amalendra Kumar, Rajendra Prasad, Julie K. Horton, William A. Beard, and David A. Konkel for helpful discussions and critical reading of the manuscript, and also Kay Miller for typing the manuscript.


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