Decreased expression of glutathione S-transferase M1 in HPV16-transfected human cervical keratinocytes in culture
Chu Chen2 and
Wilas Nirunsuksiri1
Program in Cancer Biology, Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 981091024, USA
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Abstract
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Glutathione S-transferase (GST) M1 is a member of the GST µ family of cytosolic enzymes that have been hypothesized to catalyze the conjugation of glutathione to a large number of hydrophobic substances, including carcinogens such as polynuclear aromatic hydrocarbons present in tobacco smoke, leading to their excretion. Epidemiologic and experimental evidence suggests that the risk of cervical cancer is related to both human papillomavirus (HPV) infection and cigarette smoking. We compared the enzymatic activities and mRNA levels of GSTs in GSTM1-positive human cervical keratinocytes (HCKs) that had been transfected with HPV16 with those in the parental cells. The GSTM1 activity toward the substrate trans-stilbene oxide was 5- to 7-fold lower than in the parental cells. The relative mRNA level in HCK transfected with HPV16 E6/E7, as quantified by reverse transcriptasepolymerase chain reaction (RTPCR) with normalization against endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression, was 6% that of the parental cells. It was 16 and 82%, respectively, in cells that were transfected with HPV16 E6 alone or HPV16 E7 alone. When quantified by competitive RTPCR using an exogenous nuclease-resistant synthetic cyclophilin RNA transcript as control, the mRNA level in HCK transfected with HPV16 E6 was ~10-fold lower that that in the parental cells. It was ~5- to 7-fold lower in the HPV16 E7 or HPV16 E6/E7 cells. Our results suggest that viral infections, through the modulation of cellular xenobiotic-metabolizing enzymes, may play a role in the ability of cells to handle environmental carcinogens.
Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; HCK, human cervical keratinocyte; HPV, human papillomavirus; KSFM, keratinocyte serum-free medium; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RTPCR, reverse transcriptasepolymerase chain reaction; SDS, sodium dodecyl sulfate; SSC, sodium chloridesodium citrate buffer; TSO, trans-stilbene oxide.
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Introduction
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Although human papillomavirus (HPV) infection is now considered to be a cause of most or all squamous anogenital tumors, HPV infection is not sufficient for complete progression to malignancy. Several lines of evidence support a role of cigarette smoke in the etiology of anogenital cancers, suggesting that this exposure may be one cofactor that acts in conjunction with HPV (1).
Epidemiologic studies have observed that cigarette smoking is strongly related to the risk of squamous cancers of the anogenital tract (25). Although at one time there was concern that the relationship of smoking to cervical cancer was the result of uncontrolled confounding related to sexual factors, there are now reasons to believe the association is one of cause and effect: (i) for squamous tumors of the cervix, a recent meta analysis of 14 studies found the size of odds ratio associated with recent smoking, after control for number of sexual partners and other confounders, to be 1.8 (95% CI 1.71.9) (6); (ii) there is no association between cigarette smoking and the incidence of cervical adenocarcinoma (710), a tumor that also is strongly influenced by several aspects of sexual history and by the presence of some types of HPV infection; (iii) increased gene expression of several P450 enzymes (CYP1A1, CYP1A2, CYP2D6 and CYP2E1) that are potentially important in the activation of tobacco-specific carcinogens have been demonstrated in HPV16 immortalized cervical epithelial cell lines more than in parental cell lines (11); (iv) human ecto- or endocervical cells immortalized with HPV16 or -18 undergo malignant transformation after treatment with tobacco condensate (12,13); human foreskin keratinocytes immortalized with HPV18 undergo malignant transformation after treatment with nitrosomethylurea (14); (v) smoking-related DNA damage in cervical epithelial cells has recently been demonstrated (15); and (vi) the cervical mucus of cigarette smokers contains significantly higher concentrations of the potent carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone than does that of non-smokers (16).
Glutathione S-transferase (GST) M1 has been hypothesized to be involved in the detoxification of polynuclear aromatic hydrocarbons in tobacco smoke. The results of several (though not all) studies suggest that the GSTM1 null genotype or phenotype is associated with an increased risk of cancers of the lung (1723), bladder (2426) and colon (27). A summary of the epidemiologic studies of GSTM1 and cancer incidence was published recently (28). In a casecontrol study that we conducted recently (29), we observed little association between the GSTM1 null genotype and the risk of cervical cancer. Indeed, we found a modest negative association; only 51% of cases had the GSTM1 null genotype (age-adjusted odds ratio = 0.8, 95% CI = 0.61.2) as compared with 56% of the controls. The corresponding odds ratio for adenocarcinoma of the cervix was 0.8 (95% CI = 0.51.3). We sought to investigate the reason that the GSTM1 null genotype is not a susceptibility marker for cervical cancer. We hypothesized that, unlike other tissues in which smoking-related cancers develop, HPV infection down-regulates GSTM1 in cervical tissue. We tested the hypothesis by using established human cervical keratinocyte cell lines transfected with HPV16 E6/E7, HPV16 E6 or HPV16 E7 to evaluate the effect of HPV infection on GSTM1 enzyme activity and GSTM1 mRNA level.
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Materials and methods
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Cell culture of human foreskin keratinocytes and human cervical epithelial cells
For this study, we used monolayer cultures of human foreskin keratinocytes and normal human ectocervix cells established from surgical specimens, and cell lines that were derived from these cells through stable transfection of open reading frames of HPV16/18 E6, E7, or E6 and E7 using a murine retroviral vector LXSN. The establishment procedures and characteristics of these cells have been described previously (30). The transfected cell lines express appropriate HPV E6 or E7 proteins as judged by radioimmunoprecipitations. Infection with vector LXSN alone had little, if any, effect on overall growth or lifespan of the cells. Normal cells with or without the vector senesced between passages 10 and 11 (2225 population doublings). Transfection with HPV extends the lifespan of the cells: to passage 2023 (4552 population doublings) with HPV16 E6 alone; to passages 1516 (3436 population doublings) with HPV16 E7 alone. Transfection with HPV16 E6 and E7 gave rise to immortalized cells. We used passages 45 of the parental cells, passages 913 of HPV 16 E6 or HPV 16 E7 transfected cells, and passages 1418 of HPV 16 E6 and E7 transfected cells to conduct experiments. Earlier-passage normal cells were used because those cells with vector alone senesced soon after infection with retrovirus and were not available for these experiments.
Cryopreserved cell lines of human foreskin keratinocytes and cervical epithelial cells that have and have not been transfected with HPV16 E6, HPV16 E7, or HPV16 E6/E7 genes were thawed quickly in a 37°C water bath and cultured in Gibco BRL keratinocyte serum-free medium (KSFM; Life Technologies, Gaithersburg, MD) supplemented with bovine pituitary extract (25 µg/ml) and human epidermal growth factor (0.1 ng/ml) (KSFM). Cells from four confluent p100 Petri plates were combined after trypsinization at 37°C for 5 min with 0.05% trypsin0.53 mM ethylenediaminetetrachloroacetic acid·4Na, addition of pre-warmed trypsin-inactivating solution [2% fetal bovine serum in phosphate-buffered saline (PBS), pH 7.4], centrifugation at 1000 r.p.m. for 5 min in a Juan C412 centrifuge, and washing with PBS, and they were frozen at 70°C in a freezing medium with final concentration of 10% dimethyl sulfoxide, 15% fetal bovine serum in KSFM.
GSTM1 and GSTP1 genotyping and GSTM1/TSO and GST/CDNB enzymatic activity assays
GSTM1 and GSTP1 genotypes of the cultured cell lines were assessed as previously described (31,32). GST µ enzyme activities toward substrate trans-stilbene oxide (TSO) and total GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) among human keratinocyte cell lines that have or have not been immortalized with HPV16 or 18 were compared. The substrate [3H]TSO was either purchased from American Radiochemicals (St Louis, MO) or synthesized. The synthesis of substrate [3H]TSO was accomplished by reduction of 2-chloro-2-phenylacetophenone (desyl chloride) (Aldrich Chemical, St Louis, MO) with tritiated sodium borohydride (JT Baker, Phillipsburg, NJ) to chlorohydrin and subsequent alkaline epoxide formation according to the method of Gill et al. (33). The resulting TSO was separated by thin layer chromatography in hexane:water 10:1 and eluted off the thin layer plate with ethanol. The specificity of the final product was determined by UV spectrophotometry and liquid scintillation counting.
Frozen cell pellets were thawed and resuspended in PBS, sonicated twice with a Fisher Model 50 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA) at 2 W, 10 s each, in a cold room (48°C), and centrifuged at 13 000 r.p.m. in an Eppendorf 5415C microfuge for 30 min at 4°C. The cytosol was removed, aliquoted and the protein concentration was determined using Coomassie Protein Assay Reagent (Pierce, Rockford, MD). The 100 µl GST/TSO enzyme reaction mixture in PBS contained 10 nmol [3H]TSO (sp. act. 50 mCi/mmol) and cytosol containing 100 µg protein. The reaction was initiated with the addition of 10 µl 50 mM reduced glutathione and incubated for 30 min at 37°C in a water bath. Subsequently, the reaction was stopped and extracted with 200 µl hexanol. After centrifugation for 10 min in an Eppendorf microfuge at 7200 r.p.m., 10 µl of the aqueous phase was added to 4 ml Sigmafluor and counted in a Wallac 1409 liquid scintillation counter to determine the amount of conjugated [3H]TSO. Each result was determined in duplicate, from which was subtracted a heat-inactivated (100°C) non-enzymatic control to give the final result expressed as pmol product formed/mg protein/min.
The GST/CDNB assay was performed in the presence of 100 mM potassium phosphate buffer, pH 6.25, 625 µM CDNB and 1 µg cytosol, and initiated with 833 µM reduced glutathione and incubated at 30°C. The reaction was followed at 340 nm using a Roche Cobas Mira Plus Chemistry Analyzer and the initial rate of the reaction was determined by linear regression analysis. Each sample was analyzed in triplicate and the background was calculated by omission of the sample.
Quantitative measurement of GSTM1 expression by competitive RTPCR
Poly(A)+ RNA was isolated according to the protocol of Invitrogen (San Diego, CA). Heterologous competitor constructs that carry the primer binding sites used for the GSTM1 polymerase chain reaction (PCR) but yield a different size fragment when amplified using these primers were constructed. We used a previously described, unrelated rat cDNA cloned into pBluescript SK+ (Stratagene, San Diego, CA) for the competitor constructs (34). The GSTM1 forward primer 5'-GAT CCA CCA TGC CCA TGA TAC TGG GAT-3' was cloned into BamHI/EcoRV sites and the GSTM1 reverse primer 5'-CTC TGG ATT GTA GCA GAT CAT GCA AGC T-3' was cloned into HindIII/Psp5II sites upstream of the poly(A)+ site of the rat cDNA. The construct was sequenced, using applied Biosystems Taq Dye Primer cycle sequencing kit and the sequencing products were analyzed on an ABI 373A DNA sequencer, in order to verify the copy number and intactness of the primer binding sites. The EGCG package version 8 was utilized for determining sequence homology (35). PCR amplification of the rat construct using the GSTM1 forward and reverse primers yields a 450 bp fragment; PCR amplification of the human cervical keratinocytes (HCK) GSTM1 cDNA yields a 350 bp fragment. The competitor construct for the quantification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was created by deletion of a 135 bp NcoI/NsiI fragment from a rat cDNA clone (36). The primer binding sites were chosen in a region homologous to human GAPDH.
To perform competitive reverse transcriptasepolymerase chain reaction (RTPCR) (37,38) using heterologous competitor, ~400 ng of poly(A)+ RNA was reversely transcribed at 37°C for 1 h using M-MLV reverse transcriptase and oligo (dT)15 primers. Forward and reverse primers for GSTM1 and GAPDH were labeled with [
-32P]ATP. Competitive PCR was performed using a fixed amount of reversely transcribed cDNA, a varying amount of GSTM1 competitor, and GSTM1 specific forward and reverse primers. Parallel reactions were performed using GAPDH competitor and GAPDH-specific forward and reverse primers. Hot-start amplification reactions were performed in a Perkin Elmer (Norwalk, CT) thermocycler with the following program: 95°C for 2 min, and 25 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min, followed by a final elongation step at 72°C for 10 min. The PCR products were resolved on a 5% polyacrylamide gel, exposed to an X-ray film and analyzed with PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and quantified using image analysis software (ImageQuant Version 5.6; Molecular Dynamics). Comparison of GSTM1 mRNA among the sample was based on normalization using the GAPDH signals.
We also performed quantitative RTPCR using total RNA and a
GSTM1 clone to provide a competitor RNA template. Total RNA was isolated from human cervical keratinocyte (HCK) cell lines having the GSTM1+ genotype according to the method of Chomczynski and Sacchi (39). To prepare the competitive template (competitor), a 522 bp RTPCR product was amplified from total RNA extracted from GSTM1+ cervical keratinocyte cell line, using the Access RTPCR system (Promega, Madison, WI) and GSTM1-specific primers (5'-ACC ATG CCC ATG ATA CTG-3' and 5'-GTT GGG CTC AAA TAT ACG GTGG-3'). Reaction conditions were as follows: 48°C for 45 min, 94°C for 2 min, and 40 cycles of amplification with denaturing temperature 94°C for 30 s, annealing temperature 56°C for 1 min, and extension temperature 68°C for 2 min. The PCR product was subsequently cloned into the pPCR-SCRIPT Amp SK(+) cloning vector according to the manufacturer's recommendations (Stratagene), and the clone containing the sense orientation for T7 promoter was selected (pWN-3). The construct was then digested with NcoI, and self-ligated to obtain a derivative construct with 125 bp shorter than the original. Following linearization of the plasmid with NotI, a large quantity of RNA competitor template was synthesized in vitro using T7 polymerase according to the Ribomax kit (Promega).
A known amount of the
GSTM1 internal competitive template, ranging from 104109 molecules (40), was added to each RTPCR reaction containing a fixed amount of the RNA extracted from the control cell line (cervical keratinocyte with GSTM1+ genotype), and the cell lines transfected with HPV16 E6, HPV16 E7 and HPV16 E6/E7. Parallel reactions containing equal amount of total RNA, cyclophilin primer pair and cyclophilin competicon (Ambion, Austin, TX) were performed. Comparisons of the GSTM1 mRNA levels among the cell lines were based on equal cyclophilin levels.
Northern analysis
Ten micrograms of total RNA were fractionated on glyoxal gels (41), transferred onto a Genescreen Plus membrane (Du Pont NEN, Boston, MA) by capillary blotting, and hybridized to radiolabeled cDNA probes listed below: GSTM1 (the 522 bp insert of pWN-3), GSTP1 (ATCC, Rockville, WI) and human S26 ribosomal protein (HS26, a gift from Dr P.Fort, Montpellier, France). The hybridization was performed overnight at 65°C using fresh hybridization buffer containing 106 c.p.m. [
-32P]dCTP labeled probe/ml buffer. The membrane was washed with 2x sodium chloride-sodium citrate buffer (SSC) containing 0.1% sodium dodecyl sulfate (SDS) twice at 65°C for 30 min each, and with 0.1x SSC containing 0.1% SDS at 65°C three times for 30 min each. The washed northern blots were wrapped in Saran wrap and exposed to X-ray film overnight. After autoradiography, the blots were scanned using the PhosphorImager and quantified. Comparisons of the mRNA levels among the cell lines were based on equal HS26 hybridization signals.
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Results
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HPV16-positive, GSTM1-positive (by genotyping), HCK cell lines had greatly reduced GSTM1 enzymatic activity toward the model substrate TSO, when compared with that of GSTM1-positive cells without HPV transfection. Results from triplicate cell preparations and duplicate assays showed a 5-fold lower GST/TSO activity in cells stably transfected with HPV16 E6 or HPV16 E6/E7 than in the parental HCK cells (Figure 1
). Similar results were obtained in several replicate experiments. The low activities were seen in pre-crisis (passages 15 and under) as well as post-crisis (passage 50) (data not shown) cervical cell lines transfected with HPV, suggesting that the low activity of GST/TSO was not a consequence of long-term culture. We observed similar low levels of GST/TSO enzymatic activities in another GSTM1-positive HCK cell line, M31660, which had been transfected with HPV16. The low activity levels were seen both in early passage cells (passage 7) and late passage cells (passage 34) (data not shown).

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Fig. 1. GST/TSO activities (pmol/min/mg protein) of human cervical keratinocytes in culture. Results are averages of duplicate assays from triplicate cell preparations from one representative experiment. HCK, parental cells that have not been transfected with HPV (passage 5); E6, HCK parental cells transfected with HPV16 E6 (passage 13); E7, parental cells transfected with HPV 16 E7 (passage 11); E6/7, HCK parental cells transfected with both HPV16 E6 and E7 (passage 14).
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Steady state levels of GSTM1 mRNA in cervical keratinocyte cell lines expressing HPV16 E6, E7, E6/E7 or empty vector, were analyzed in two separate sets of experiments using different approaches of quantitative RTPCR. The analysis was based on quantitative RTPCR because the GSTM1 RNA levels in these cells were not high enough to be analyzed by northern blot analysis. In the first set, poly(A)+ RNA was extracted from these cells and reversely transcribed using an oligo-dT primer. Quantitative PCR was performed using varying amount of an internal competitive fragment carrying the same primers as those for GSTM1 mRNA. Cells carrying HPV16 E6/E7 or HPV16 E6 had GSTM1 mRNA levels that were 6 and 16%, respectively, that of the parental keratinocytes (HCK), based on normalization with the endogenous GAPDH mRNA level. Cells expressing HPV16 E7 alone, however, had GSTM1 mRNA that was ~82% that of the parental cell line (Table I
). In the second set of experiments, total cellular RNA was used to conduct competitive RTPCR to determine the level of GSTM1 expression of these cell lines. For these experiments, an exogenous nuclease-resistant synthetic cyclophilin RNA transcript was used as a control. Results obtained from the same three cell preparations of which GST/TSO activities were determined (Figure 2
) were similar to the results obtained in the first set of experiments. There was a 5- to 10-fold reduction in the GSTM1 mRNA levels in cells harboring HPV. These observations demonstrated that reduced GSTM1 activity in HCK cells transfected with HPV16 E6 or HPV16 E6/E7, was due to a decrease in the steady state level of GSTM1 mRNA.

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Fig. 2. Competitive standard RNA molecules were added to fixed amounts of total RNA (0.5 µg per reaction) as indicated. The mixture of RNAs were subjected to RTPCR and a fraction of the products were electrophoresed on a 2% Nusieve gel. Cellular RNA was isolated from cells of the same passages as those used for the GST/TSO assays described in Figure 1 . Top: GSTM1, endogenous GSTM1; cGSTM1, GSTM1 competitor; bottom: Cyclo, endogenous cyclophilin; cCyclo, cyclophilin competitor.
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Activities of other GSTs in the foreskin keratinocyte cell lines or the cervical keratinocyte cell lines appeared not to be affected by HPV16 or 18, judging by enzymatic activities toward substrate CDNB in cells that were or were not transfected (Figure 3
). We suspect that the CDNB activity might be largely that of GSTP1 since GSTP1 is expressed in cervical cells and utilizes CDNB as substrate. This observation was supported by our GSTP1 genotyping results as well. The only two foreskin keratinocyte cell lines that showed reduced GST/CDNB activities were found to carry one (FEAp54) and two (FEHp38) copies of GSTP1b mutant alleles (data not shown). Relative to the homozygous GSTP1 wild-type cell line M31660 (expressing HPV16 E6/E7), HCK, HPV16 E6/E7 Cxp15 and HPV16 E6/E7Cxp50 cell lines, although heterozygous for GSTP1, all showed high enzymatic activities toward CDNB suggesting that the cervical cell lines have large amounts of stable, active GST
enzyme that are not affected by HPV 16 infection.

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Fig. 3. GST/CDNB activities of human foreskin keratinocytes and human cervical keratinocytes in culture. GSTM1 genotypes of the cell lines are as indicated. FEAp54 and FEHp38 are heterozygous for the GSTP1 polymorphism at codon 105, while other cell lines carry the homozygous wild-type allele.
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Results of our northern blot analysis also indicated that the expression of GSTP1, unlike that of GSTM1, was not altered to any appreciable extent by HPV16. Representative results from duplicate experiments showing steady state levels of GSTP1 mRNA of the various cell lines studied are shown in Figure 4
. The blot was also probed with HS26 cDNA, a house-keeping gene with a long half-life, to show that an equal amount of total RNA from each cell line was used (Figure 4
). Intensities of hybridization signals obtained from GSTP1 and HS26 were quantified by PhosphorImager analysis and the ratio between their expression levels determined (data not shown). The results indicated that the levels of GSTP1 in transfected cells were similar to those of control cells.

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Fig. 4. A representative autoradiogram of a northern blot of total RNA extracted from cultures of a control HCK cell line, and those transfected with HPV16 E6 (E6) or HPV16 E7 (E7) or HPV16 E6/E7 (E6/7), is shown. Total RNA was isolated from the corresponding cell line at confluence and subjected to northern blot analysis (10 µg per lane). The same RNA was used in the quantitative competitive RTPCR experiments that gave rise to results shown in Figure 2 . The filter was sequentially hybridized with probe specific for GSTP1 and human S26 ribosomal protein (HS 26). Molecular weights of RNA standards are indicated on the left.
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Discussion
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We have demonstrated decreased GSTM1 activity toward the substrate TSO in cells that are transfected with HPV16 E6/E7 or HPV16 E6, and that such decreases were possibly associated with decreased GSTM1 mRNA levels. A marginal decrease in GST/TSO activity and GSTM1 mRNA level were seen in cells that were transfected with HPV16 E7 alone. Although the study was conducted with parental cells that were devoid of sham retroviral vector LXSN, the results we obtained were unlikely to be due to the effect of transfection. This is because: (i) normal cells and LXSN transfected cells exhibited similar life span and growth characteristics; (ii) cells that were transfected with HPV16 E6 gave different results from those that were transfected with HPV16 E7; and (iii) we observed different effects of HPV transfection on different GSTs. Unlike GSTM1, the enzymatic activity and the mRNA level of GSTP1 were similar in cells with or without HPV. Our study suggests that HPV infection may have a substantial effect on the activities of at least one xenobiotic-metabolizing enzyme, GSTM1, and provides a possible explanation for the observation of a lack of association between GSTM1 genotypes and the risk of cervical cancer in our epidemiologic study. It points to the importance of taking into account the potential effect of viral infection and integration has on the activity of xenobiotic-metabolizing enzymes when studying the role of their genetic polymorphisms as susceptibility markers for cancers. It also raises the question of how chronic viral infection may affect cellular defenses against carcinogens in general.
It is not known at the present time whether the decreased levels of GSTM1 mRNA level is due to decreased transcription or to mRNA instability. HPV16 E6 is known to inactivate the tumor suppresser gene p53, but whether the effect of HPV16 E6 on GSTM1 is via the p53 pathway also remains to be elucidated.
To our knowledge, this is the first report that a gene that is potentially important in the metabolism of tobacco-related carcinogens is down-regulated in HPV-transfected cells. Whether this in vitro finding is applicable to an in vivo situation is unknown. It is interesting to point out that during the preparation of this manuscript, two reports describing the effect of viral infection on the expression of GSTs appeared in the literature. The first article described the regulation of the GST
gene by simian virus SV40 T antigen in the rodent Fisher rat F1111 and human hepatoma HepG2 cell lines. In both cell lines, down-regulation was observed for the GST
gene, but not the GST µ gene (42). Down-regulation of GST
gene, but not the GST µ gene was also observed in adenovirus E1A-expressing F1111 or HepG2 cell lines. Furthermore, the down-regulation of GST
gene in these cell lines appeared not to require the complex formation of the large T antigen with p53, p300 or the pRb proteins. The second article showed markedly reduced
, µ, and
GST activities in hepatitis B virus-DNA positive hepatocellular carcinoma than in normal tissue (43). Taken together, these results suggest that viral infection can have a substantial impact on the ability of cells to handle environmental carcinogens by influencing the expression of genes that encode for carcinogen-metabolizing enzymes.
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Acknowledgments
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We thank Dr James K.McDougall and Dr Aloysius J.Klingelhutz for providing the cell lines and helpful comments, Dr P.Fort for providing the HS26 cDNA clone, Dr Jeannette Bigler for providing the rat cDNA clone, Mr Keith L.Munson and Ms JoAnn Prunty for providing technical support in the early phase of this study, and Ms Anita Randle for assistance in the preparation of this manuscript. This work was supported by grant RO1 ES06728 from the National Institute of Environmental Health Sciences and by institutional support of the Fred Hutchinson Cancer Research Center.
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Notes
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1 Present address: Dow AgroSciences, 9330 Zionsville Road, Indianapolis, IN 462681054, USA 
2 To whom correspondence should be addressed Email: cchen{at}fhcrc.org 
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References
|
---|
-
Winkelstein,W.Jr (1990) Smoking and cervical cancercurrent status: a review. Am. J. Epidemiol., 131, 945957.[ISI][Medline]
-
Holly,E.A., Whittemore,A.S., Aston,D.A., Ahn,D.K., Nickoloff,B.J. and Kristiansen,J.J. (1989) Anal cancer incidence: genital warts, anal fissure or fistula, hemorrhoids, and smoking. J. Natl. Cancer Inst., 81, 17261731.[Abstract]
-
Daniell,H.W. (1985) Re: Causes of anal carcinoma [letter]. JAMA, 254, 358.
-
Holmes,F., Borek,D., Owen Kummer,M., Hassanein,R., Fishback,J., Behbehani,A., Baker,A. and Holmes,G. (1988) Anal cancer in women. Gastroenterology, 95, 107111.[ISI][Medline]
-
Daling,J.R., Sherman,K.J., Hislop,T.G., Maden,C., Mandelson,M.T., Beckmann,A.M. and Weiss,N.S. (1992) Cigarette smoking and the risk of anogenital cancer. Am. J. Epidemiol., 135, 180189.[Abstract]
-
English,D.R., Holman,C.D.J., Milne,E. et al. (1995) The quantification of drug-caused morbidity and mortality in Australia. Section 4.6.14. Commonwealth Department of Human Services and Health, Canberra, Australia.
-
Brinton,L.A., Tashima,K.T., Lehman,H.F., Levine,R.S., Mallin,K., Savitz,D.A., Stolley,P.D. and Fraumeni,J.F.Jr (1987) Epidemiology of cervical cancer by cell type. Cancer Res., 47, 17061711.[Abstract]
-
Parazzini,F., La Vecchia,C., Negri,E., Fasoli,M. and Cecchetti,G. (1988) Risk factors for adenocarcinoma of the cervix: a case-control study. Br. J. Cancer, 57, 201204.[ISI][Medline]
-
Hopkins,M.P. and Morley,G.W. (1991) A comparison of adenocarcinoma and squamous cell carcinoma of the cervix. Obstet. Gynecol., 77, 912917.[Abstract]
-
Daling,J.R., Madeleine,M.M., McKnight,B. et al. (1996) The relationship of human papillomavirus-related cervical tumors to cigarette smoking, oral contraceptive use, and prior herpes simplex virus type 2 infection. Cancer Epidemiol. Biomarkers Prev., 5, 541548.[Abstract]
-
Farin,F.M., Bigler,L.G., Oda,D., McDougall,J.K. and Omiecinski,C.J. (1995) Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial cells immortalized by human papillomavirus type 16 E6/E7 genes. Carcinogenesis, 16, 13911401.[Abstract]
-
Nakao,Y., Yang,X., Yokoyama,M., Pater,M.M. and Pater,A. (1996) Malignant transformation of human ectocervical cells immortalized by HPV 18: in vitro model of carcinogenesis by cigarette smoke. Carcinogenesis, 17, 577583.[Abstract]
-
Yang,X., Jin,G., Nakao,Y., Rahimtula,M., Pater,M.M. and Pater,A. (1996) Malignant transformation of HPV 16-immortalized human endocervical cells by cigarette smoke condensate and characterization of multistage carcinogenesis. Int. J. Cancer, 65, 338344.[ISI][Medline]
-
Garrett,L.R., Perez-Reyes,N., Smith,P.P. and McDougall,J.K. (1993) Interaction of HPV-18 and nitrosomethylurea in the induction of squamous cell carcinoma. Carcinogenesis, 14, 329332.[Abstract]
-
Simons,A.M., van Herckenrode,C.M., Rodriguez,J.A., Maitland,N., Anderson,M., Phillips,D.H. and Coleman,D.V. (1995) Demonstration of smoking-related DNA damage in cervical epithelium and correlation with human papillomavirus type 16, using exfoliated cervical cells. Br. J. Cancer, 71, 246249.[ISI][Medline]
-
Prokopczyk,B., Cox,J.E., Hoffmann,D. and Waggoner,S.E. (1997) Identification of tobacco-specific carcinogen in the cervical mucus of smokers and nonsmokers. J. Natl Cancer Inst., 89, 868873.[Abstract/Free Full Text]
-
Seidegard,J., Pero,R.W., Miller,D.G. and Beattie,E.J. (1986) A glutathione transferase in human leukocytes as a marker for the susceptibility to lung cancer. Carcinogenesis, 7, 751753.[Abstract]
-
Zhong,S., Howie,A.F., Ketterer,B., Taylor,J., Hayes,J.D., Beckett,G.J., Wathen,C.G., Wolf,C.R. and Spurr,N.K. (1991) Glutathione S-transferase mu locus: use of genotyping and phenotyping assays to assess association with lung cancer susceptibility. Carcinogenesis, 12, 15331537.[Abstract]
-
Heckbert,S.R., Weiss,N.S., Hornung,S.K., Eaton,D.L. and Motulsky,A.G. (1992) Glutathione S-transferase and epoxide hydrolase activity in human leukocytes in relation to risk of lung cancer and other smoking-related cancers. J. Natl Cancer Inst., 84, 414422.[Abstract]
-
Hirvonen,A., Husgafvel-Pursiainen,K., Anttila,S. and Vainio,H. (1993) The GSTM1 null genotype as a potential risk modifier for squamous cell carcinoma of the lung. Carcinogenesis, 14, 14791481.[Abstract]
-
Nazar-Stewart,V., Motulsky,A.G., Eaton,D.L., White,E., Hornung,S.K., Leng,Z.T., Stapleton,P. and Weiss,N.S. (1993) The glutathione S-transferase mu polymorphism as a marker for susceptibility to lung carcinoma. Cancer Res., 53, 23132318.[Abstract]
-
Alexandrie,A.K., Sundberg,M.I., Seidegard,J., Tornling,G. and Rannug,A. (1994) Genetic susceptibility to lung cancer with special emphasis on CYP1A1 and GSTM1: a study on host factors in relation to age at onset, gender and histological cancer types. Carcinogenesis, 15, 17851790.[Abstract]
-
Anttila,S., Hirvonen,A. and Husgafvel-Pursiainen,K. (1994) Combined effect of CYP1A1 inducibility and GSTM1 polymorphism on histological type of lung cancer. Carcinogenesis, 15, 11331135.[Abstract]
-
Bell,D.A., Taylor,J.A., Paulson,D.F., Robertson,C.N., Mohler,J.L. and Lucier,G.W. (1993) Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer. J. Natl Cancer Inst., 85, 11591164.[Abstract]
-
Daly,A.K., Thomas,D.J., Cooper,J., Pearson,W.R., Neal,D.E. and Idle,J.R. (1993) Homozygous deletion of gene for glutathione S-transferase M1 in bladder cancer. Br. Med. J., 307, 481482.[ISI][Medline]
-
Lopez,M.F., Patton,W.F., Sawlivich,W.B., Erdjument-Bromage,H., Barry,P., Gmyrek,K., Hines,T., Tempst,P. and Skea,W.M. (1994) A glutathione S-transferase (GST) isozyme from broccoli with significant sequence homology to the mammalian theta-class of GSTs. Biochim. Biophys. Acta, 1205, 2938.[ISI][Medline]
-
Zhong,S., Wyllie,A.H., Barnes,D., Wolf,C.R. and Spurr,N.K. (1993) Relationship between the GSTM1 genetic polymorphism and susceptibility to bladder, breast and colon cancer. Carcinogenesis, 14, 18211824.[Abstract]
-
Rebbeck,T.R. (1997) Molecular epidemiology of the human glutathione S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility. Cancer Epidemiol. Biomarkers Prev., 6, 733743.[Abstract]
-
Chen,C., Madelaine,M.M., Weiss,N.S. and Daling,J.R. (1999) Glutathione-S-transferase M1 genotypes and the risk of squamous carcinoma of the cervix: a population-based case-control study. Am. J. Epidemiol., in press.
-
Klingelhutz,A.J., Barber,S.A., Smith,P.P., Dyer,K. and McDougall,J.K. (1994) Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells. Mol. Cell. Biol., 14, 961969.[Abstract]
-
Chen,C., Madeleine,M.M., Lubinski,C., Weiss,N.S., Tickman,E.W. and Daling,J.R. (1996) Glutathione S-transferase M1 genotypes and the risk of anal cancer: a population-based case-control study. Cancer Epidemiol. Biomarkers Prev., 5, 985991.[Abstract]
-
Harries,L.W., Stubbins,M.J., Forman,D., Howard,G.C.W. and Wolf,C.R. (1997) Identification of genetic polymorphisms at the glutathione S-transferases Pi locus and association with susceptibility to bladder, testicular and prostate cancer. Carcinogenesis, 18, 641644.[Abstract]
-
Gill,S.S., Ota,K. and Hammock,B.D. (1983) Radiometric assays for mammalian epoxide hydrolases and glutathione S-transferase. Anal. Biochem., 131, 273282.[ISI][Medline]
-
Bigler,J. and Eisenman,R.N. (1995) Novel location and function of a thyroid hormone response element. EMBO J., 14, 57105723.[Abstract]
-
Devereux,J., Haeberli,P. and Smithies,O. (1984) A comprehensive set of sequence analysis programs for the vax. Nucleic Acids Res., 12, 387395.[Abstract]
-
Tso,J.Y., Sun,X.H., Kao,T.H., Reece,K.S. and Wu,R. (1985) Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res., 13, 24852502.[Abstract]
-
Gilliland,G., Perrin,S., Blanchard,K. and Bunn,H.F. (1990) Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc. Natl Acad. Sci. USA, 87, 27252729.[Abstract]
-
Siebert,P.D. and Larrick,J.W. (1992) Competitive PCR. Nature, 359, 557558.[ISI][Medline]
-
Chomczynski,P. and Sacchi,N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162, 156159.[ISI][Medline]
-
O'Connell,J., Goode,T. and Shanahan,F. (1998) Quantitative measurement of mRNA expression by competitive RTPCR. Methods Mol. Biol., 92, 183193.[Medline]
-
Sambrook,J., Fritsch,E.F. and Maniatis,T. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY, p. 740.
-
Sompayrac,L. (1997) SV40 and adenovirus may act as cocarcinogens by downregulating glutathione S-transferase expression. Virology, 233, 130135.[ISI][Medline]
-
Zhou,T., Evans,A.A., London,W.T., Xia,X., Zou,H., Shen,F. and Clapper,M.L. (1997) Glutathione S-transferase expression in hepatitis B virus-associated human hepatocellular carcinogenesis. Cancer Res., 57, 27492753.[Abstract]
Received October 6, 1998;
revised November 19, 1998;
accepted December 4, 1998.