Carcinogenesis, Vol. 20, No. 6, 1049-1054,
June 1999
© 1999 Oxford University Press
Carcinogenesis |
DNA polymerase ß expression differences in selected human tumors and cell lines
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, PO Box 12233 and
1 Department of Functional Genetics, GlaxoWellcome Inc., Five Moore Drive, Research Triangle Park, NC 27709 and
2 Department of Medicine and Cell Biology, Vanderbilt University, School of Medicine, Nashville, TN 37232, USA
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Abstract |
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A long-standing question in cancer biology has been the extent to which DNA repair may be altered during the process of carcinogenesis. We have shown recently that DNA polymerase ß (ß-pol) provides a rate-determining function during in vitro repair of abasic sites by one of the mammalian DNA base excision repair pathways. Therefore, altered expression of ß-pol during carcinogenesis could alter base excision repair and, consequently, be critical to the integrity of the mammalian genome. We examined the expression of ß-pol in several cell lines and human adenocarcinomas using a quantitative immunoblotting method. In cell lines from normal breast or colon, the level of ß-pol was ~1 ng/mg cell extract, whereas in all of the breast and colon adenocarcinoma cell lines tested, a higher level of ß-pol was observed. In tissue samples, colon adenocarcinomas had a higher level of ß-pol than adjacent normal mucosa. Breast adenocarcinomas exhibited a wide range of ß-pol expression: one tumor had a much higher level of ß-pol (286 ng/mg cell extract) than adjacent normal breast tissue, whereas another tumor had the same level of ß-pol as adjacent normal tissue. Differences in ß-pol expression level, from normal to elevated, were also observed with prostate adenocarcinomas. All kidney adenocarcinomas tested had a slightly lower ß-pol level than adjacent normal tissue. This study reveals that the base excision repair enzyme DNA polymerase ß is up-regulated in some types of adenocarcinomas and cell lines, but not in others.
Abbreviations: BER, base excision repair; ß-pol, ß-polymerase; PBS, phosphate-buffered saline.
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Introduction |
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Endogenous metabolic events and exposure to external agents can lead to lesions in genomic DNA, most of which are faithfully repaired by the multitude of cellular DNA repair pathways. Thus, error-free repair, recombinational repair, mismatch repair, nucleotide excision repair and base excision repair (BER) act in concert to effectively ensure that most mutagenic and cytotoxic lesions are removed from DNA prior to their fixation. On occasion, however, an error occurs during repair or, in other cases, the repair systems fail to correct a DNA lesion, ultimately leading to genomic instability and/or cell death. There are many examples in model systems of a linkage between an alteration in DNA repair and genomic instability (1) and it had been proposed that alterations in DNA repair could be associated with a hypermutable condition known in many tumor cells (2–6). Recent studies have shown that variations in DNA mismatch repair genes are associated with hereditary non-polyposis colon cancers (7–9). Earlier studies had also shown that several of the inherited deficiencies in DNA repair are associated with a higher risk of cancer (1).
ß-Polymerase (ß-pol) is one of five well-characterized mammalian cellular DNA polymerases. Except in specialized cases (10), the enzyme has not been considered to have a role in semi-conservative DNA replication (11). Instead, ß-pol was proposed for gap-filling synthesis during DNA repair (12,13) and, in the past several years, studies have established a role for ß-pol in the short patch or simple (single nucleotide gap filling) BER pathway (14–20). There is also evidence that ß-pol functions in the long patch or alternative BER pathway (21). The expression level of ß-pol has been found to be independent of cell cycle stage (22), but the enzyme is regulated in a tissue-specific fashion (12).
Interestingly, in vitro studies of ß-pol demonstrate that it is more error prone than other cellular DNA polymerases. Relatively error prone DNA synthesis by ß-pol has been observed during the single nucleotide gap filling reaction (23–25), suggesting that ß-pol-dependent DNA repair synthesis in vivo could be error prone. In addition to providing DNA repair synthesis function during BER, ß-pol provides dRP lyase function for this repair pathway. dRPase is the rate-limiting step for BER in vitro in the presence of high concentrations of BER enzymes (26). Therefore, it appears that differences in the expression level of ß-pol could up-regulate or down-regulate the BER capacity of a cell.
The availability of a specific monoclonal antibody against ß-pol that can be used for quantitative immunoblotting (27) facilitates study of the expression level of this enzyme in different tumors and cancer cell lines. Our results show increased expression of ß-pol in some tumors and corresponding cell lines, as compared with their normal counterparts. The implications of these results are discussed.
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Materials and methods |
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Cell culture
Unless otherwise indicated, cell lines were obtained from the American Type Culture Collection (Rockville, MD). These cells were grown as per ATCC recommendations. C127 and its transfectants (7-4, CB2MX3 and BXL-40) were gifts from Dr G.N.Pavlakis (NCI/FCRDC, Frederick) and were maintained as described (28,29). The non-tumor breast epithelial cell lines 184 and B5 were provided by Dr M.Stampfer (University of California, Berkeley, CA) and were maintained in EGF-containing serum-free medium as described (30). The adriamycin-resistant MCF-7/ADR breast cancer cell line was provided by Dr K.Cowan (NCI, Bethesda, MD).
Blood sampling and isolation of lymphocytes
All blood samples were heparinized, transported on ice and processed within 3 h. The lymphocytes were separated from polymorphonuclear leukocytes and erythrocytes by layering 10 ml whole blood onto Histopaque gradients (Sigma, St Louis, MO) followed by centrifugation at 2000 r.p.m. for 30 min at room temperature. Lymphocytes were aspirated from the gradient–plasma interface and washed twice and resuspended in phosphate-buffered saline (PBS) (0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.4). For each sample, the number of cells in the suspension was adjusted to ~1x106 cells/0.5 ml.
Tissue sample procedures
The breast tumor tissue which showed a significantly higher level of ß-pol was from a 45-year-old patient with invasive stage II breast adenocarcinoma. A second breast tumor also was an invasive adenocarcinoma. The adjacent non-tumor specimen came from the same surgically resected breast tissue. Colorectal, prostate and kidney tumors and their adjacent normal tissue counterparts were obtained from Duke University Medical Center and the University of North Carolina. All specimens were frozen in liquid nitrogen immediately after excision and stored at –80°C. The pathology of all tissues was confirmed microscopically and all specimens were found to be free of necrosis and damage due to preservation or preparation (31).
Immunoblotting
For rapid cell lysis, exponentially growing tissue culture cells were washed twice with ice-cold PBS containing a protease inhibitor mixture with final concentrations of 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 RIPA buffer containing 150 mM NaCl, 10 mM Tris–HCl, pH 7.5, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA and the protease inhibitor mixture described above. The lysate was centrifuged at 8000 g for 5 min and the nuclear extracts from breast cells and tissues were prepared as described (32,33). Extracts were diluted 10-fold with PBS and used for protein estimation using an assay originally described by Bradford (34). Soluble protein samples were separated by SDS–PAGE and transferred to nitrocellulose membrane. Equal loading of samples and even transfer to the nitrocellulose membrane was verified in all cases by staining the membrane with Ponceau S. ß-pol was measured by incubating the membrane with mouse anti-ß-pol monoclonal antibody 18S (18S mAb) and then with antibody to mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase. Purified recombinant human/rat ß-pol was used as the positive control in quantitative immunoblotting as described (27). Immobilized horseradish peroxidase activity was detected by enhanced chemiluminescence (ECL).
Colorectal, prostate and kidney tissues were homogenized in 10 mM Tris–HCl, pH 7.5, 10 mM 2-mercaptoethanol, 10 mM EDTA, 0.25% Triton X-100, 1 mM PMSF and 1 µM pepstatin at 4°C and then mixed with an equal volume of SDS sample buffer (4% SDS, 10% 2-mercaptoethanol, and 0.004% bromphenol blue in 160 mM Tris–HCl, pH 6.8). Proteins were separated electrophoretically and transferred to Immobilon-P membrane at 45 V for 25 min in transfer buffer (20 mM Tris, 192 mM glycine, pH 8.2, containing 0.08% SDS and 20% methanol). The blots were incubated overnight in blocking buffer, 5% powdered non-fat milk in TBST (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20 and 0.02% sodium azide). The blots were then incubated for 6 h with anti-ß-pol affinity-purified mAb 18S diluted 1:1000 in blocking buffer; the blots were washed three times with TBST and incubated with 125I-labeled protein A in blocking buffer (1 µCi/ml) for 45–60 min. Blots were washed three times with TBST and then subjected to autoradiography. ß-pol-related bands were quantified by densitometry or by phosphoimagery.
Thus, ß-pol level was measured through both ECL and a 125I-labeled protein A method. The ECL method was used to calculate the absolute amount of ß-pol in different cell lines and tissues by the method described (27). ß-Pol levels determined in replicate experiments were similar (i.e. ±10% for the ECL method and ±25% for the 125I-labeled protein A method). In experiments that required a lower stringency of hybridization during immunoblotting, incubations with antibody were carried out in the absence of blocking agents.
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Level of ß-pol in reference samples
Monoclonal antibody 18S (mAb 18S) was raised against rat ß-pol and characterized earlier (27). Under appropriate conditions, this antibody recognizes human, hamster, mouse and bovine ß-pol, but does not cross-react with other proteins in cell extracts, including the other cellular DNA polymerases. We further examined this antibody for use as a probe to measure ß-pol in crude cell extracts by quantitative immunoblotting after SDS–PAGE (27). First, we used CHO-K1 cells to evaluate the ß-pol level after abrupt inhibition of expression of ß-pol mRNA. CHO-K1 cells were exposed to actinomycin D at a concentration sufficient to block mRNA synthesis (5 µg/ml). Cells were cultured for different time periods and extracts were prepared and examined for ß-pol level by immunoblotting (Figure 1A
). Only one significant immunoreactive polypeptide was detected in each whole cell extract (Figure 1A, lanes 1–4), illustrating the specificity of mAb 18S. The polypeptide detected exhibited gel migration similar to that of purified rat ß-pol used as reference. The amount of ß-pol in CHO-K1 cells did not change significantly over the 12 h period of actinomycin D treatment, indicating that acute down-regulation of ß-pol mRNA synthesis is an unlikely cause of any differences in ß-pol level observed in these cells. The 18S monoclonal antibody demonstrated a similar level of specificity as seen in Figure 1A, when it was used with other samples during the course of this study.
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ß-pol expression level was examined in cell extracts from several sources. In all samples, the apparent molecular mass of the ß-pol protein was ~39 kDa, which is in good agreement with the open reading frame in the predominant ß-pol mRNA from mammalian sources (35–38). The ß-pol level in the reference cell lines in logarithmic phase culture varied in the following order: NIH 3T3 cells > CHO-K1 cells > mouse CB2MX3 cells > mouse BXL-40 cells > mouse 7-4 cells > mouse C127 cells > HeLa cells (Figure 1B
). The absolute level of ß-pol per mg of cell protein is shown in Table I. Among all the reference samples analyzed, the ß-pol level was highest in bovine testis nuclear extract (120 ng/mg protein; Figure 1B). It is interesting to note that the level of ß-pol in HeLa and C127 cells was ~100-fold lower than the level in bovine testis nuclei.
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ß-pol expression level was also determined in normal human lymphocytes from different individuals. Peripheral blood lymphocytes were collected from five donors and cell extracts were prepared and analyzed (Figure 1C
, lanes 1–5). The extracts had a similar ß-pol level, suggesting that interindividual differences in lymphocyte ß-pol level are minimal.
Level of ß-pol in human breast and colon adenocarcinomas
The level of ß-pol was determined in cell lines from several human breast and colon adenocarcinomas during log phase growth. The level of ß-pol in two normal human breast cell lines (184 and B5) was 1 ng/mg cell extract protein (Table I). On average, the expression of ß-pol in breast adenocarcinoma cell lines was higher than in normal breast cell lines. The highest level of ß-pol was in BT-474 cells (28 ng/mg protein); other breast adenocarcinoma lines showed 4- to 13-fold higher ß-pol level than the normal breast cell lines (Figure 2A and Table I). Similarly, the expression of ß-pol in colon adenocarcinoma cell lines was higher than in normal colon cell lines (Figure 3A). The highest expression was observed in the HCT 116 cell line (24 ng/mg protein). Other colon adenocarcinoma lines showed >10-fold higher ß-pol level than the normal colon cell line CCD-18Co (Figure 3A and Table I).
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Using tissue samples, less consistent patterns were observed when tumor and normal samples were compared. In one breast adenocarcinoma, the ß-pol level was similar to that of the adjacent normal tissues (Table II
). However, expression of ß-pol in another breast adenocarcinoma was 286 ng/mg protein, as compared with 1 ng/mg protein from the adjacent normal tissue specimen (Table II). The ß-pol level was also analyzed in colon adenocarcinoma and adjacent mucosa isolated from four patients. In each case, the ß-pol level was higher in the tumor than in the sample of adjacent normal mucosa (Table II). The ß-pol level in tumor tissue samples ranged from 6- to 22-fold higher than in the adjacent normal mucosa.
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In order to confirm the specificity of the mAb 18S probe with tissue samples and cell, the following experiments were carried out. Using samples from the high ß-pol level breast adenocarcinoma (Figure 2B
) and the colon cell line with the highest ß-pol level (HCT 116; see Figure 3B), it was demonstrated that non-immune IgG does not produce a signal corresponding to the ß-pol protein (Figures 2B and 3B, lanes 1 and 2). In addition, preincubation of mAb 18S with a synthetic ß-pol epitope peptide, 142KYFEDFEKRIPREEM156, blocked the ß-pol specific signal (Figure 2B, lanes 6 and 7 and Figure 3B, lanes 6–8), both with the breast adenocarcinoma sample (Figure 2B, lanes 3–5) and the HCT 116 cell line sample (Figure 3B, lanes 3–5). Note that this experiment was conducted under less stringent blotting conditions (without using blocking agents during incubations with antibody) to demonstrate the specificity of the 18S mAb. The various minor bands observed, when 18S mAb was used (Figures 2B and 3B) were due to a non-specific secondary antibody reaction.
Expression of ß-pol in prostate and kidney tumors
The expression of ß-pol was also examined in prostate and kidney adenocarcinomas and the normal peripheral zone surrounding each tumor. In three of the four prostate tumors studied, the level of ß-pol was 3- to 11-fold higher than in the adjacent normal tissue (Table II). In contrast, the level of ß-pol in the four kidney tumors was less than in the adjacent normal tissue (Table II). The expression level in the normal tissue was dependent on the particular tissue; ß-pol level was higher in normal kidney than in normal prostate. Normal prostate tissue level of ß-pol was higher than the level observed in colorectal mucosa or breast tissue samples. In summary, the ß-pol level was highest in normal bovine testis nuclear extracts; normal human tissues had ß-pol levels in the order kidney > prostate > colorectal mucosa > breast (Table II).
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Discussion |
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In this study, we quantified ß-pol expression levels in vivo using immunoblotting with a ß-pol specific monoclonal antibody. The expression level was elevated in all of the colon adeno-carcinomas examined, in three of four prostate adenocarcinomas examined and was remarkably elevated in one breast adenocarcinoma. All of the breast and colon adenocarcinomas cell lines showed elevated ß-pol expression. These results represent the first report of increased ß-pol expression in human adenocarcinoma tissue and cell lines. Interestingly, the level of topoisomerase I, an enzyme involved in controlling the topological state of DNA, is also elevated in the same samples of colon (5- to 35-fold) and prostate tumors (2- to 11-fold) used here (31). The kidney tumors used here did not exhibit increased expression of topoisomerase I (31) and this correlates with the absence of elevated ß-pol expression in this study. Increased expression of both topoisomerase I and ß-pol has been demonstrated in cisplatin-resistant human kidney tumor cell lines (39). Although there may be no reason to expect a DNA repair role for topoisomerase or a DNA topology role for DNA polymerase ß, both enzymes can be involved in recognizing abasic sites in DNA. Thus, one plausible explanation for the up-regulation of both topoisomerase I and ß-pol is that the enzymes participate in the cellular response to DNA damage. For example, it has been suggested that topoisomerase I could play a role in DNA damage recognition during cell death (1,31,40) and ß-pol plays a key DNA repair role by participating in the recognition and repair of abasic sites by the BER pathway (19,20,41). Another possibility is that up-regulation of topoisomerase activity could lead to an increase in DNA strand breaks and that ß-pol is up-regulated in the mechanism repairing such breaks.
Wang et al. (2) and Dobashi et al. (5,6) reported ß-pol mRNA isoforms and polymorphisms of the ß-pol gene in colorectal and prostate cancers, respectively. In colorectal cancers, deletions and a point mutation were identified (2,42). The 87 bp deletions described by Wang et al. (2) probably represent an alternatively spliced form of ß-pol mRNA lacking exon 11 (43). However, 21, 42 and 217 bp deletion isoforms were also found in these colorectal tumors and none of these in our view can be readily explained by alternative splicing (2,43). The epitope for the ß-pol monoclonal antibody used here does not correspond to regions deleted in the mRNA isoforms reported by Wang et al. (2) and we did not find any significant ß-pol protein isoforms (other than the 39 kDa species) in colorectal tumors or other tissues studied. These results indicate that none of the ß-pol mRNA isoforms found previously in tumors lead to accumulation of a different size ß-pol protein at a level that could be detected in our immunoblotting studies.
In conclusion, our results indicate that the level of ß-pol is significantly elevated in some human adenocarcinomas and cell lines relative to the ß-pol level in normal tissues and cells. ß-pol provides two of the enzymatic steps in the simple BER pathway: gap filling DNA synthesis and dRP lyase (20–22,26). With regard to the function of the dRP lyase, up-regulation of ß-pol appears to have significance to the overall DNA repair capacity of a cell, since this is the rate-determining enzymatic activity in the BER pathway in vitro (26). It is interesting to consider the question of the mechanism by which a DNA repair enzyme such as ß-pol is up-regulated in some tumors. The process of carcinogenesis is known to involve increased cellular production of genotoxic agents, including, among others, reactive oxygen species, lipid peroxidation products and alkylation intermediates capable of reacting with DNA. Such endogenous genotoxic agents may alter gene expression patterns directly and may lead to a significant overall increase in genomic DNA lesions. An increase in the expression of DNA repair protein level may be a response to an increased number of DNA lesions. It is known that some cells can adapt to DNA alkylating agent exposure by increasing the expression of DNA polymerase ß mRNA, which is mediated through transcription factor CREB-1 binding to the ATF/CREB site in the ß-pol promoter (44–46). Based upon the results reported here, the precise cell type or differentiation stage may play a part in determining ß-pol expression level. As shown previously, ß-pol expression in normal tissue is regulated in a tissue-specific manner (12). As shown here, up-regulation of ß-pol during carcinogenesis occurs at variable levels and is in some cases tissue specific as well. It should be interesting to compare levels of DNA damage in the tumors studied here, as well as the question of whether the CREB-1 response system for the ß-pol promoter is operative in the kidneys as well as in the cell type studied.
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Acknowledgments |
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We thank Dr Miriam Sander for critical reading of the manuscript and Angela Woodcock for typing the manuscript.
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3 To whom correspondence should be addressed Email: wilson5{at}niehs.nih.gov
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