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Right arrow Epidemiology: Molecular Epidemiology
[Cancer Research 66, 6439-6448, June 15, 2006]
© 2006 American Association for Cancer Research

Epidemiology and Prevention

Haplotype-Environment Interactions That Regulate the Human Glutathione S-Transferase P1 Promoter

Stephane Cauchi1, Weiguo Han1, Shalini V. Kumar1 and Simon D. Spivack1,2,3

1 Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health; 2 Pulmonary and Critical Care Medicine, Albany Medical College; and 3 Environmental Health Sciences, University at Albany, School of Public Health, Albany, New York

Requests for reprints: Simon D. Spivack, Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, E624 Wadsworth's Laboratory, Empire State Plaza, Albany, NY. Phone: 518-473-0782; E-mail: spivack{at}wadsworth.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phase II detoxification of carcinogens is reported to mediate some of the anticarcinogenesis effects of candidate chemopreventive agents. We explored the interaction between sequence variation in the GSTP1 gene promoter and candidate chemopreventive exposure in regulating human GSTP1 expression. Polymorphisms along 1.8 kb of the GSTP1 promoter were identified in leukocytes [peripheral blood mononuclear cells (PBMC)] from 40 Caucasian subjects. Ten promoter polymorphisms (9 previously unreported) displayed strong linkage disequilibrium, yielding identification of three frequently observed haplotypes [HAP1 (43%), HAP2 (36%), and HAP3 (8%)]. Each haplotype was cloned into luciferase reporter constructs and transfected into normal human bronchial epithelial cells. Basal HAP3 reporter activity was significantly elevated (1.8-fold) but decreased to the same levels as HAP2 and HAP1 with increasing concentrations of sulforaphane, benzyl isothiocyanate (BITC), and epigallocatechin gallate (EGCG). To confirm native HAP3 functionality, we quantitated mRNA expression in uncultured PBMCs and in laser microdissected normal lung epithelial cells (MNLEC) from the same patients. Basal mRNA expression was higher in HAP3 individuals [1.8-fold (PBMC) and 4-fold (MNLEC) for HAP3 heterozygotes and 2.3-fold (PBMC), and 15-fold (MNLEC) for the HAP3 homozygote] than in the other genotypes. PBMC GSTP1 mRNA expression correlated to MNLEC expression (R2 = 0.77). After culture and in vitro exposure to sulforaphane, BITC, or EGCG, the elevated GSTP1 mRNA expression of PBMCs from HAP3 individuals decreased to common expression levels. Elevated HAP3 function was confirmed at the protein level in PBMCs (5-fold higher for HAP3 heterozygotes and 7.6-fold for the HAP3 homozygote). These data suggest a potentially protective GSTP1 promoter haplotype and unpredicted inhibitory chemopreventive agent-haplotype interactions. (Cancer Res 2006; 66(12): 6439-48)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Of the several thousand chemicals found in tobacco smoke, at least 50 are carcinogenic, including polycyclic aromatic hydrocarbons (PAH), aromatic amines, and nitroso-compounds (1). Susceptibility to tobacco smoke is plausibly attributable, in part, to each individual's capacity to activate and detoxify inhaled carcinogens. After phase I bioactivation, phase II enzymes protect a cell by detoxifying reactive metabolites into inactive compounds that are readily excreted (2).

Among the ubiquitously expressed phase II glutathione S-transferase (GST) superfamily (3), the family consists of a single gene, GSTP1, located on chromosome 11q13. This enzyme has multiple substrates, including epoxides of PAH, such as benzo(a)pyrene-diolepoxide, acrolein, and other unsaturated carbonyls, generated by lipid peroxides and by oxidative damage to DNA (4). GSTP1 is the major metabolic GST enzyme in nonmalignant human lung as assessed by mRNA expression (5) and by activity (6). It is also expressed in various tumors and cancer cell lines (4, 7).

GSTP1 polymorphisms may modify the association between cumulative exposure to active smoking and lung cancer. Ten single nucleotide polymorphisms (SNP) have been published in the GSTP1 coding region but only one in the promoter (8, 9). Thus far, genetic studies of the human GSTP1 promoter have been largely limited to the identification of essential responsive elements involved in basal gene expression. The first 100 bp 5' to the transcription start site have been annotated as the minimal promoter, containing nuclear factor-{kappa}B (NF-{kappa}B), Sp1, antioxidant-responsive element (ARE), and activator protein-1 (AP-1) sites (10–13).

GSTP1 expression differences may be involved in interindividual protection differences. Increased skin tumorigenesis was observed in mice lacking {pi} class GSTs after PAH exposure (14). Transient GSTP1 overexpression inhibits, and underexpression enhances, the cytotoxic effects of cigarette smoke extract in human lung fibroblast-derived cells (15). In humans, there are wide interindividual differences of GSTP1 expression in buccal and lung cells (6, 16).

A variety of phytochemical agents have been studied for their GSTP1-inducing potential, including cruciferous vegetable-derived sulforaphane (17), benzyl isothiocyanate (BITC; ref. 18), sodium butyrate (19), oltipraz (20), red grape–derived resveratrol (21), and tea-derived epigallocatechin gallate (EGCG; ref. 22). The oxidants hydrogen peroxide (H2O2; ref. 23) and cigarette smoke extract (24) have also been tested.

In this study, we report the presence and function of polymorphisms in a large portion of the human GSTP1 promoter region (–1,814 to +38) from patients from a lung cancer case-control tissue study. The most frequent haplotypes were reconstituted into reporter constructs, transfected into normal human bronchial epithelial (NHBE) cells, and evaluated for their activity on exposure to a panel of putative inducing agents, including candidate chemopreventive agents. Then, haplotype-specific cultured peripheral blood mononuclear cells (PBMC) were assessed for native mRNA and protein expression responses to the same panel of compounds. Finally, mRNA expression was measured in paired samples of PBMCs and microdissected normal lung epithelial cells (MNLEC) from the same patients.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subjects. Forty Caucasian subjects were analyzed in the present study (20 control and 20 newly diagnosed, untreated lung cancer patients). These patients were randomly selected from a New York State Department of Health institutional review board–approved and Albany Medical Center institutional review board–approved case-control study based in Albany, NY, with demographic, exposure, and clinical features similar to those described previously (20). Informed consent was explicit, and privacy was protected by stripping traceable identifiers from clinical samples. Patients were interviewed and phlebotomized preoperatively before clinically indicated diagnostic or therapeutic lung resection surgery.

Preparation of genomic DNA. Isolation of 40 PBMC samples was done in standard fashion using a Ficoll gradient technique. Genomic DNA was isolated using a standard isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's recommendations.

PCR amplification and sequencing. Because of the high GC content, the 5'-flanking region was amplified by six separate amplification reactions using primers listed in Table 1 in an overlapping fragment-restriction site ligation strategy. Promoter primers were designed (GCG, Madison, WI) against the GSTP1 genomic sequence (Genbank accession no. AY324387). Promoter PCR amplifications were carried out using the HotStarTaq DNA polymerase kit (Qiagen, Valencia, CA), in a final volume of 50 µL, in the presence of 200 ng genomic DNA template, 1.5 mmol/L MgCl2, 0.2 mmol/L of each of four deoxynucleotide triphosphates, 0.5 µmol/L of each primer, and 2.5 units HotStartTaq DNA polymerase (Qiagen). The PCR thermal profiles included an initial denaturation step at 95°C for 15 minutes and 30 cycles of denaturation at 94°C for 30 seconds, touchdown annealing from 65°C to 50°C (–0.5°C every two cycles) for 30 seconds, and extension at 72°C for 1 minute. A final extension step at 72°C for 7 minutes terminated the reaction. PCR products were then resolved by electrophoresis and purified with a gel extraction kit (Qiagen).


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  		Table 1. GSTP1 promoter primers for PCR and directed mutagenesis

 
Fragments were then subjected to direct cycle sequencing. It was done by first purifying the product using a Centri-Sep (Princeton Separations, Adelphia, NJ) column and then sequencing by a Perkin-Elmer Biosystems (Shelton, CT) ABI PRISM 377XL automated sequencer. Overall, the regions containing polymorphisms were amplified and sequenced at least two separate times.

Haplotype reconstitution. The THESIAS program (http://ecgene.net/genecanvas/) allowed the simultaneous estimation of the magnitude of polymorphism disequilibrium, the haplotype frequencies, and the effect of the haplotypes on the biological phenotype of interest. It is based on the maximum likelihood model described by Tregouet et al. and is linked to the SEM algorithm (25). The designations HAP1, HAP2, and HAP3 stand for the first, second, and third most frequent haplotypes, respectively, in our 40-sample set. HAP3-3 stands for the HAP3 homozygote haplotype combination (genotype HAP3-HAP3). HAP1/2-3 stands for HAP3 heterozygote haplotype combinations (genotypes HAP1-HAP3 and HAP2-HAP3). HAP1/2-HAP1/2 stands for the non-HAP3-containing haplotype combinations (genotypes HAP1-HAP1, HAP1-HAP2, and HAP2-HAP2).

Promoter-luciferase constructs. The 5'-flanking region of the GSTP1 gene was amplified from the sample carrying wild-type sequence by PCR described above, except that we substituted the restriction site–tagged primers GSTP1-FM4, GSTP1-FM5, GSTP1-RM5, and GSTP1-FM6 for the primers GSTP1-F4, GSTP1-F5, GSTP1-R5, and GSTP1-F6 to introduce a KpnI and/or a HindIII site immediately upstream of the forward and reverse primers, respectively (Table 1). For the cloning of the GSTP1 promoter, three overlapping PCR products were purified from an agarose gel and were digested with the following enzymes: (a) GSTP1-P5m was digested with KpnI and HindIII, (b) GSTP1-P4m was digested with KpnI and BssHII, and (c) GSTP1-P6m was digested with KpnI and BsmBI. The BsmBI restriction site is located in the overlapping sequence between GSTP1-P6m and GSTP1-P4m; the BssHII restriction site is located in the overlapping sequence between GSTP1-P5m and GSTP1-P4m. The three digested fragments were ligated successively by T4 DNA ligase and cloned into the KpnI/HindIII site of the promoter-less pGL3-basic firefly luciferase reporter vector (Promega, Madison, WI) to produce the GSTP1 wild-type (GSTP1-WT) construct. All constructs were sequenced before use to ensure a correct insertion sequence and direction.

Multisite-directed mutagenesis. The polymorphisms of interest were introduced into GSTP1-WT plasmid by oligonucleotide-directed PCR mutagenesis (QuikChange Multisite-Directed Mutagenesis kit, Stratagene, La Jolla, CA) using the primers listed in Table 1. The desired mutations were verified by sequencing of the whole promoter insert. To avoid any possibility that the base-change and amplification procedure would introduce a mutation into the noninsert portions of the pGL3-basic vector, we subcloned the mutant insert into a virgin pGL3-basic vector. The insert sequence was then verified by direct sequencing.

Primary lung cell epithelial culture. Commercially obtained NHBE cells are primary, nontransformed, and nonimmortal (BioWhittaker, Inc., Walkersville, MD). In this study, NHBE cells were maintained in BEGM (BioWhittaker) containing BEBM supplemented with 52 mg/mL bovine pituitary extract, 0.5 mg/mL hydrocortisone, 0.5 ng/mL human epidermal growth factor, 0.5 ng/mL L-epinephrine, 10 mg/mL transferrin, 5 mg/mL insulin, 0.1 ng/mL retinoic acid, 6.5 ng/mL triiodothyrinine, 50 mg/mL gentamicin, and 50 ng/mL amphotericin B at 37°C in a humidified 5% CO2 atmosphere. Transfections were done in triplicate in 24-well plates. Each well was plated with 1 x 105 cells on passage 4 24 hours before transfection. Reporter construct transfection was done using 0.2 mg GSTP1/Luc reporter gene plasmid with 0.2 mg reference PRL-TK vector (Promega) expressing Renilla luciferase constitutively; the latter serves as an internal control of transfection efficiency. The mixture was combined with 1 mL LipofectAMINE 2000 in Opti-MEM reduced serum medium (Invitrogen Life Technologies, Inc., Grand Island, NY).

Phytochemical agent exposure. After a 6-hour incubation, the medium was replaced with growth medium (BEGM) containing one of the following compounds: cigarette smoke extract (Murty Pharmaceuticals, Inc., Lexington, KY) dissolved in DMSO, resveratrol (Sigma-Aldrich, St. Louis, MO) dissolved in DMSO, oltipraz (LKT Laboratories, Inc., St. Paul, MN) dissolved in DMSO, EGCG (Sigma-Aldrich) dissolved in DMSO, sodium butyrate (Sigma-Aldrich) dissolved in deionized water, sulforaphane (LKT Laboratories) dissolved in deionized water, BITC (LKT Laboratories) dissolved in deionized water, and H2O2 (Sigma-Aldrich) dissolved in deionized water. For sulforaphane, H2O2, BITC, and sodium butyrate exposures, four different concentrations were tested: 0.05, 0.5, 5, and 40 µmol/L. When cell viability was assessed by the trypan blue exclusion test using a hemocytometer and light microscope (trypan blue vital stain, 0.4%; BioWhittaker), pilot studies suggested toxicity at high dose (≥5 µmol/L) for oltipraz, resveratrol, and EGCG. Therefore, for these three molecules, four concentrations were tested: 0.05, 0.1, 0.5, and 2 µmol/L. For cigarette smoke extract exposure, four concentrations were tested: 0.05, 0.5, 5, and 40 µg/mL. Control cells were exposed to either DMSO or deionized water vehicle only. In each condition, the final concentration of DMSO was adjusted to 0.1% (v/v).

After a 24-hour exposure, the cells were lysed by a passive lysis buffer (Promega). The activities of firefly and Renilla luciferases were determined by dual-luciferase reporter assay system (Promega). Firefly luciferase activity was then numerically normalized to Renilla luciferase activity.

PBMC culture. Twenty of 34 representative randomly selected PBMC samples were seeded with a concentration of 0.5 x 106 cells/mL and cultured in RPMI 1640 (2 mmol/L L-glutamine) supplemented with 10% fetal bovine serum, 100 µg/mL streptomycin, 100 units/mL penicillin, and 1% (v/v) phytohemagglutinin-M (Gibco-Invitrogen, Carlsbad, CA) for 24 hours. The cells were then exposed to either cigarette smoke extract, sulforaphane, EGCG, or BITC under the same conditions described above for reporter construct-transfected NHBE cells. Cigarette smoke extract-sulforaphane, cigarette smoke extract-EGCG, and cigarette smoke extract-BITC combinations were also tested. After a 24-hour exposure, mRNA was extracted and quantitated as described below.

Laser capture microdissection of lung tissue. Among the 20 individuals selected for PBMC culture, surgical lung specimens from a subset of 11 individuals was available for quantitative gene expression. Each sample of nonmalignant lung tissue from the surgical resection adjacent to the malignant tumor (n = 7) or benign lesion in question (n = 4) was flash frozen in liquid isopentane within 10 to 15 minutes of resection and stored at –80°C. Each block was then subjected to frozen microtome sectioning at –20°C, alcohol-based fixation and hematoxylin staining, and laser capture microdissection (Pixcell IIe, Arcturus, Mountain View, CA; ref. 26) within a single day. We did sampling of four areas of a frozen tissue section using 200 pulses per area (each pulse activated annealing of a 15-µm-diameter area of tissue, yielding about two to four cells), so that each cap (of a 500-µL tube containing an RNA extraction buffer) contained >1,000 nonmalignant, predominantly alveolar type I and II cells for downstream quantitative real-time reverse transcription-PCR (RT-PCR; ref. 27).

Quantitative real-time RT-PCR. The quantitative real-time RT-PCR approach used a previously published RNA-specific strategy that avoids the coamplification of contaminating gDNA-encoded pseudogenes homologous to the target transcript (GSTP1) and reference "housekeeping" internal transcript (36B4; refs. 16, 28). The RNeasy Total RNA Extraction kit (Qiagen) was used with minor adaptations of the manufacturer's directions for the PBMC and MNLEC sample sets. Reverse transcription was done using the protocol for SuperScript II reverse transcriptase RNase H– (Invitrogen), employing our universal tagged reverse transcription primer. For PCR, a single 25-µL reaction in a glass capillary PCR tube (Roche Applied Science, Indianapolis, IN) included the following: 5 µL of the 5x One-Step RT-PCR buffer (Qiagen), to yield 2.5 mmol/L MgCl2; 15.5 µL RNase/DNase-free H2O; a total of 1.6 µL deoxynucleotide triphosphate mixture (10 mmol/L each triphosphate nucleotide); 1.25 µL SYBR Green dye (1:10,000); 0.4 µL Platinum Taq (Invitrogen) DNA polymerase; 0.25 µL of 50 pmol/L/µL each oligonucleotide of the cDNA-specific primer pair; and 1 µL template cDNA from the reverse transcription reaction. Parallel uniplex reactions were done in separate capillary reaction tubes.

PCR thermocycling reaction conditions included one denaturing cycle at 95°C for 30 seconds and 50 cycles consisting of up-ramp (20°C/s) to melt at 95°C for 10 seconds, down-ramp (3.5°C/s) to anneal at 60°C, and up-ramp (3.5°C/s) to anneal at 72°C for 30 seconds. Melting analysis for one cycle was as follows: up-ramp (20°C/s) to melt at 95°C for 10 seconds, down-ramp (20°C/s) to anneal at 58°C, and slow up-ramp (0.1°C/s) for continuous acquisition to 95°C. As a criterion for inclusion of quantitative data, the PCR product had to both display a single peak with characteristic melt temperature on melting analysis and be corroborated by agarose ethidium bromide electrophoresis, displaying a single product at the appropriate size. All RT-PCRs were run in triplicate. Relative concentrations of target transcript were normalized to valid internal RNA controls in the quantitative real-time RT-PCR steps as described below.

Western immunoblotting. For cytosolic [GSTP1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] proteins, PBMCs were lysed in passive lysis buffer. Total protein level was quantified using the BCA protein assay reagent kit (Pierce, Rockford, IL). Each cell lysate sample (20 µg) was subjected to electrophoresis in 10% acrylamide NuPAGE Novex Bis-Tris denaturing gels (Invitrogen) as recommended by the manufacturer. Proteins were blotted onto 0.2-mm nitrocellulose membranes (Invitrogen). Blots were blocked and treated with antibodies according to the WesternBreeze chemiluminescent Western blot immunodetection kit (Invitrogen). Rabbit polyclonal antihuman GSTP1 (Oxford Biomedical Research, Inc., Oxford, MI) and GAPDH (Imgenex Corp., San Diego, CA) primary antibodies were diluted at 0.1 µg/mL in blocking solution for 1 hour at room temperature with the blotted membranes. The secondary anti-rabbit antibodies were supplied in the WesternBreeze kit and incubated with the membranes for 30 minutes at room temperature. Detection was accomplished with a ready-to-use CDP-Star chemiluminescent substrate for alkaline phosphatase kit, exposed on Kodak X-OMAT film (Sigma-Aldrich), and quantified by densitometry with a scanning laser densitometer (Molecular Dynamics, Sunnyvale, CA).

Statistical analyses. Two-way ANOVA was done to test the contributions of the haplotype and the chemical concentration factors to GSTP1 promoter activity, mRNA expression, and protein expression in cultured PBMCs (see above). Where there was a significant haplotype effect, we did the post hoc Tukey test for all pair-wise comparisons of the average responses to the different haplotypes. Where there was a significant concentration effect, we did the post hoc Dunnett's test to compare several treatment groups to a single control group. Mann-Whitney tests were done for native uncultured GSTP1 mRNA expression in PBMCs and MNLECs to test the haplotype combinations (genotypes) effect. For all of these tests, P < 0.05 was considered significant. Normality and variance equality hypotheses were assessed by the Kolmogorov-Smirnov and Levene tests, respectively; if P > 0.01, then the data were considered normally distributed and/or with equal variance, respectively. Spearman's (nonparametric) rank correlation test was done to correlate the 11 sets of uncultured or cultured PBMCs with MNLEC GSTP1 mRNA expression. All statistical analyses were done with the SigmaStat 2.03 software (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Detection of GSTP1 promoter polymorphisms. The five pairs of primers (P1-P5) used to amplify the five overlapped fragments are listed (Table 1). Polymorphisms in the GSTP1 promoter detected by direct sequencing included nine SNPs that were not reported previously along with a previously published ATAAA pentanucleotide repeat variation (ref. 9; Table 2 ). The TRANSFAC 7.0 public program (BIOBASE Biological Databases, Wolfenbüttel, Germany) was used in silico to search for putative responsive elements in the polymorphisms regions. That search yielded only one putative functional motif, a Sp1 site in the region of polymorphism 5 (G–566C) with no significant homology change between the wild-type and the mutated allele (homology score = 0.95 for G–566 and 0.93 for C–566). None of the polymorphisms was associated with higher or lower lung cancer risk in this small 40-sample set (data not shown). All genotype distributions followed the Hardy-Weinberg law.


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  		Table 2. Ten polymorphisms assembled into haplotypes

 
Linkage disequilibrium evaluation and haplotype reconstitution. We calculated the linkage disequilibrium (LD) and the haplotype frequencies using the THESIAS program (see Materials and Methods). The polymorphisms could be divided into three blocks in strong LD [1-2 (100%), 3-4 (100%), and 5-6-7-8-9-10 (>90%); Table 2]. We then calculated haplotype frequencies. The three most frequent haplotypes represented, in aggregate, 87% of all observed haplotypes. HAP1 was the most frequent (43%) followed by HAP2 (36%) and HAP3 (8%). None of these "common variant" haplotypes (>5% haplotype frequency) was found to be associated with higher or lower lung cancer risk in this small 40-sample set (data not shown).

Evaluation of the activity of the three most frequent promoter haplotypes. The three reconstituted haplotypes inserted into luciferase reporter constructs were transfected into NHBE cells from one donor and were functionally evaluated by exposure for 24 hours to each of the eight potential inducers: cigarette smoke extract, resveratrol, oltipraz, EGCG, sodium butyrate, sulforaphane, BITC, and H2O2 (Fig. 1 ). Statistical analyses measuring the effect of the haplotype and concentration factors revealed that HAP3 had a significantly higher activity than did the other haplotypes regardless of the culture conditions (1.8-fold higher than other genotypes for vehicle controls; P < 0.001). Three compounds, sulforaphane (0.05 µmol/L), EGCG (0.1 µmol/L), and BITC (0.5 µmol/L), each showed a significant inhibition effect at low concentration, decreasing the HAP3 promoter activity to the basal level (P < 0.05). The other haplotypes were not affected by any of the exposure conditions.


Figure 1
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  		Figure 1. Reporter luciferase activities of the three most frequent haplotypes after exposure to each of eight potential inducers. Reconstructed sequences identical to those of the three most frequent haplotypes were inserted into luciferase reporter constructs and transfected into NHBE cells from one donor. For every exposure, luciferase activity was significantly higher for HAP3 constructs than for the other haplotype constructs (P < 0.001). Sulforaphane, BITC, and EGCG each had a repressor effect at low concentration but only on HAP3. *, P < 0.05. Luc, firefly/Renilla luciferase activity; CSE, cigarette smoke extract; Sul, sulforaphane; Olt, oltipraz; Res, resveratrol; NaBu, sodium butyrate.

 
Quantitative mRNA expression in PBMCs and MNLECs. Quantitative native GSTP1 mRNA expression in the 40 uncultured PBMC samples was distributed normally (Kolmogorov-Smirnov P > 0.2) over a 16-fold range (Fig. 2A ). The genotype of 34 of 40 individuals could be defined by the three most frequent haplotypes (Fig. 2B). After reorganizing the GSTP1 mRNA expression data by genotype, we found a significantly higher PBMC expression for HAP3 than for the other genotypes [1.8-fold for HAP3 heterozygotes (P < 0.001) and 2.3-fold for the single HAP3 homozygote]. There was no significant smoking status difference (current versus former, pack years, and time since last cigarette) found between HAP1/2-HAP1/2 and HAP1/2-3.


Figure 2
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  		Figure 2. GSTP1 mRNA distribution across uncultured PBMCs. A, native GSTP1 mRNA expression distribution among the 40 uncultured PBMC samples. B, genotype of 34 samples could be defined by the three most frequent haplotypes. On this chart, the data have been displayed to show the expression distribution across the different haplotype combinations. Pooled together, HAP3-containing combinations are significantly associated with higher GSTP1 expression (P < 0.001). Values have been scaled to validated RNA-specific housekeeping reference transcript 36B4.

 
For assessment of the mRNA expression of PBMCs in culture, a set of 20 of 34 representative PBMC samples (5 each of HAP1-1, HAP1-2, and HAP2-2, 2 each of HAP1-3 and HAP2-3 heterozygotes, and the single HAP3-HAP3 homozygote available) was arbitrarily chosen. After culture for 24 hours, vehicle (DMSO) exposure revealed that the basal GSTP1 mRNA expression of the HAP3 heterozygotes and the one HAP3 homozygote was significantly higher by 2-fold (P < 0.001) and 15-fold, respectively, than that of the other genotypes (Fig. 3A ). Twenty-four-hour exposures to cigarette smoke extract (1 g/mL) and each of the single agents (0.05 mol/L sulforaphane, 0.1 µmol/L EGCG, or 0.5 µmol/L BITC) that have previously shown activity in the reporter construct experiments as well as to selected combinations (cigarette smoke extract-sulforaphane, cigarette smoke extract-EGCG, and cigarette smoke extract-BITC) revealed that exposure to low concentrations of sulforaphane, EGCG, or BITC decreased the higher expression of HAP3-containing individuals to levels identical to those of other genotypes (P < 0.001; Fig. 3A). No compound induced GSTP1 mRNA expression. Cigarette smoke extract exposure did not have any significant additive effects over that of the single compounds.


Figure 3
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  		Figure 3. Quantitative GSTP1 mRNA expression in PBMCs (n = 20) and MNLECs (n = 11). A, 20 of 34 representative PBMC samples were randomly chosen for expression studies of cultured and exposed blood cells. Quantitative GSTP1 mRNA expression was assessed by RNA-specific quantitative real-time RT-PCR. Exposures were cigarette smoke extract (1 µg/mL), sulforaphane (0.05 µmol/L), EGCG (0.1 µmol/L), and BITC (0.5 µmol/L). HAP3 heterozygotes and the HAP3 homozygote expressed 2-fold (P < 0.001) and 15-fold more GSTP1 mRNA in PBMCs, respectively, compared with expression by the other individuals. After exposure to sulforaphane, EGCG, or BITC, however, their mRNA levels decreased to identical basal levels. Combinations of cigarette smoke extract and selected agents did not show any additive effects beyond the single-compound effects. B, uncultured MNLEC specimens from a subgroup of 11 of the 20 individuals from (A) were used for comparison. Native GSTP1 mRNA expression was quantified as above. HAP3 heterozygotes and the HAP3 homozygote expressed 4-fold (P < 0.001) and 15-fold increased levels of GSTP1 mRNA in MNLECs, respectively, compared with expression by the individuals without HAP3-containing genotypes. C, logarithmic correlation between uncultured PBMC GSTP1 mRNA and MNLEC GSTP1 mRNA expression from these same 11 individuals was apparent (R2 = 0.77; P < 0.001). D, linear correlation between expression of GSTP1 mRNA in unexposed PBMCs in culture and GSTP1 mRNA in uncultured MNLECs from these 11 individuals was apparent (R2 = 0.77; P < 0.001).

 
Among the 20-sample subset selected for measurement of PBMC GSTP1 mRNA described above, 11 paired MNLEC samples were available from the same individuals: 2 each of HAP1-1, HAP1-2, and HAP2-2, 2 each of HAP1-3 and HAP2-3 heterozygotes, and the single HAP3-3 homozygote (Fig. 3B). HAP3 heterozygotes and the HAP3 homozygote expressed more native GSTP1 mRNA by 4-fold (P < 0.001) and 15-fold, respectively, than did the other genotypes. As a control, native GSTP1 mRNA expression was also measured in commercially obtained cultured NHBE cells (Cambrex, Walkersville, MD) derived from one anonymous commercial donor, genotyped as a combination of HAP1 and a rare haplotype. None of the tested chemical agents influenced the GSTP1 mRNA expression in these NHBE cells (data not shown).

GSTP1 mRNA expression from uncultured PBMCs and native MNLECs from the same patients was logarithmically correlated [y = 0.0713ln(x) + 0.3005; R2 = 0.77; n = 11; P < 0.001; Fig. 3C]. GSTP1 mRNA expression from 24-hour cultured PBMCs (DMSO) and MNLECs obtained from the same patients was linearly correlated (y = 0.8419x; R2 = 0.77; n = 11; P < 0.001; Fig. 3D). The GSTP1 mRNA expression from the two types of PBMCs, from uncultured PBMCs and from 24-hour cultured PBMCs (in DMSO), from the same patients was also logarithmically correlated [y = 0.0655ln(x) + 0.3234; R2 = 0.65; n = 20; P < 0.001; data not shown in Fig. 3].

GSTP1 protein expression in PBMCs. Western blot experiments were done to quantitate the GSTP1 protein expression in cultured PBMCs. Figure 4A shows representative GSTP1 and loading reference GAPDH Western blot gels from 24-hour cultured PBMCs exposed to DMSO, cigarette smoke extract (1 µg/mL), sulforaphane (0.05 µmol/L), EGCG (0.1 µmol/L), and BITC (0.5 µmol/L). Semiquantitative protein expression was measured and is displayed by genotype in Fig. 4B. For controls, HAP3 heterozygotes and the HAP3 homozygote expressed 5-fold (P < 0.001) and 7.6-fold more GSTP1 protein, respectively, than did the other genotypes. After exposure to sulforaphane, EGCG, or BITC, the elevated expression of HAP3-containing individuals decreased to levels identical to those of the other haplotypes (P < 0.001). Cigarette smoke extract did not have any significant effect on any genotype.


Figure 4
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  		Figure 4. GSTP1 protein expression in PBMCs (n = 20). A, representative GSTP1 and GAPDH Western blot gels from 24-hour cultured PBMCs. Lane 1, DMSO; lane 2, cigarette smoke extract (1 µg/mL); lane 3, sulforaphane (0.05 µmol/L); lane 4, EGCG (0.1 µmol/L); lane 5, BITC (0.5 µmol/L). B, quantitative protein expression displayed as a function of the genotype. HAP3 heterozygotes and the single HAP3 homozygote expressed 5-fold (P < 0.001) and 7.6-fold more GSTP1 protein, respectively, than did the other genotypes. Values are scaled to expression levels of the cytosolic housekeeping reference protein GAPDH.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To explore factors influencing the wide interindividual differences previously observed in expression of the carcinogen-metabolizing enzyme GSTP1 (6, 16) and to directly test the modulation potential of this phase II enzyme by chemopreventive agents, we investigated common variants of the human GSTP1 promoter in reporter constructs, peripheral blood cells, and microdissected normal epithelial lung cells.

Ten polymorphisms in the human GSTP1 promoter were found in 1.8 kb of the GSTP1 promoter region. Strong LD between several of these polymorphisms yielded three haplotypes that covered the majority of variation among our predominantly Euro-Caucasian subjects. Because we completely sequenced the region in all 40 individuals, this result reaffirms the feasibility of using tag-SNPs for efficient detection of the most common haplotypes on a larger scale in genetic epidemiology studies (29).

None of the SNPs was located in the previously reported minimal promoter region. However, when these haplotypes were reconstituted in luciferase reporter constructs that were transfected into normal human lung cells (i.e., NHBE), the basal GSTP1 promoter activity was found to be significantly higher for one haplotype (HAP3) than for the other two haplotypes. Unexpectedly, among eight different potential inducers, human exposure-relevant concentrations of three of them, sulforaphane, EGCG, and BITC, decreased the HAP3 promoter activity to a common basal level. The other agents had no effect. The repressive effect observed for these three agents is contrary to much of the data derived from whole animal, nonhuman cell studies (see below) and thus warranted further verification for the native promoter.

We therefore quantitated native GSTP1 mRNA expression using a previously published RNA-specific RT-PCR assay unconfounded by GSTP1 or internal housekeeping (36B4) pseudogenes encoded in any potential DNA contaminants (16, 28); the mRNA expression data were thus specific, precise, and reproducible. Among the uncultured PBMC samples from the 40 subjects, the HAP3 individuals again displayed a higher mRNA expression than did the other genotypes. When 20 representative PBMC samples were then cultured in the presence of sulforaphane, EGCG, or BITC at human exposure-relevant concentrations identical to those used in the reporter construct studies, the original elevated baseline expression for the HAP3 individuals once again decreased down to a common basal level. GSTP1 protein expression studies confirmed the mRNA findings in the same 20 representative PBMC samples after culture with the same phytochemical agents in the same concentrations.

The anticarcinogenic effects of the isothiocyanates, including sulforaphane and BITC, in animal models have been summarized (30, 31). For example, in rat liver cells, GSTP1 mRNA is induced by sulforaphane binding to the ARE in the minimal promoter (32). Northern and Western blot analyses showed that 24-hour exposure to BITC specifically enhanced the production of GSTP1 in rat liver cells (18, 33). Similar inductive effects were seen in mouse models in various tissues (34). However, the degree of anticarcinogenesis protective effects in animals may not be solely attributable to protection against carcinogen bioactivation and DNA adduct formation (31, 32).

In humans, sulforaphane protected cultured human lymphocytes from micronucleus induction by various mutagens (35), and in DU-145 prostate cancer cells, its use resulted in the inhibition of growth and tumorigenesis (36). The existing reported evidence for direct sulforaphane influence on human GSTP1, however, is sparse. The GSTP1 protein expression was increased by 3- to 5-fold in nonneoplastic human mammary MCF-10F cells treated with sulforaphane (37). However, no increase in GSTP1 expression was observed after treatment of human keratinocytes (HaCaT) with sulforaphane (38). Indeed, increased vegetable intake seems to decrease GSTP1 expression in human lymphocytes (17). Our data are consistent with these latter two studies.

Given that gene regulatory data are frequently discordant between rodent species and humans, the difficulty of cross-species inferences for behavior of the GSTP1 promoter could be predicted. Rat GSTP1 gene expression is dominantly regulated by GSTP1 enhancer I and II, two powerful enhancers, which are located ~2.5 and 2.2 kb upstream, respectively, from the transcriptional initiation site of this gene (39). These enhancer elements have not been found in humans and thus may contribute to interspecies difference of expression. The roles of Keap1, Nrf2, and the ARE in phase II transcriptional events have been extensively explored in certain models (40–42). Whereas BITC and sulforaphane are known to act through the Nrf2 binding to ARE, three ARE elements exist in the mouse GSTP1 promoter, which is thought similar to the human GSTP1 promoter (38, 43). Therefore, the details of interspecies and intertissue differences in the isothiocyanate-modulated GSTP1 transcriptional cascades remain unclear.

EGCG is the most abundant polyphenol in tea (44). Reductions in human lung cancer have been reported to be associated with green tea consumption (45, 46). Tea components increase GST activity in rat (47) and human (22) liver cells. EGCG increases AP-1 factor levels in normal human keratinocytes (48) but inhibits NF-{kappa}B activation in cancer cells (49). Here again, further studies are necessary to understand the direct human transcriptional regulation of GSTP1 by this tea polyphenol on a cell type– and species-specific basis (50). Clearly, high-dose effects of inducers in animal models are not directly comparable with the effects of submicromolar levels plausibly achievable in humans (51).

Because the mechanistic basis for differential haplotype-specific responses to the assayed agents was unclear, we sought putative responsive elements in silico in and around the individual polymorphisms that we observed. Because no polymorphism was found to occur in a CpG site, differential epigenetic regulatory effect of the haplotypes did not seem likely. The only readily apparent, consensus-identified element encompassing a SNP was a putative Sp1 site found in the G–566C region; however, no significant change between the two alternative alleles was associated with this site (homology score = 0.95 for G–566 and 0.93 for C–566). Clearly, additional experimental and in silico genetic studies will be necessary to determine which transcription factors or coactivators are able to bind to the variable regions detected in this group of subjects.

In the current study, MNLECs also revealed significantly higher GSTP1 mRNA expression in HAP3 individuals than in the other genotypes. A good correlation was apparent for GSTP1 mRNA expression in paired samples of PBMCs and MNLECs from the same individual. From a translational perspective, the potential surrogacy of blood genotypic or expression data for the lung could offer potential for the noninvasive, semiquantitative, or ordinal assessment of lung GSTP1 expression.

Given that the human GSTP1 promoter haplotype data were concordant at three different expression levels (GSTP1 promoter reporter, mRNA, and protein) and that they correlated with findings in microdissected lung cells, our findings seem to aid in the functional definition of these common three haplotypes. The observed repression by sulforaphane, BITC, and EGCG was observed only for HAP3; these agents were otherwise completely inactive for the other more common haplotypes in PBMCs and in NHBE cells with non-HAP3-containing genotypes. The consistency across chemopreventive agents, experimental conditions, and human cell types additionally argues against the idea that these haplotype-environment phenomena are attributable to artifactual model selection factors.

There are, clearly, several limits to the study. The size of the cohort examined was necessarily modest because of the detailed analysis inherent in the haplotype and functional assessment across several human cell types, experimental designs, and exposure panels. Corroboration of the major findings in a larger population sample is certainly warranted to enable subgroup and stratification analyses and meaningful case-control analyses. As a corollary, the uniformity of ethnicity clearly limits the generalizability of a particularly complex gene-environment interaction, because modifier alleles that vary by region-of-origin are likely (52). Finally, studies of downstream functional consequences for DNA adduction and mutation are necessary before definitive assignment of functionality to the GSTP1 promoter haplotypes can be made.

In summary, these data raise not only the possibility of specific GSTP1 haplotypes that could confer resistance to mutagenesis but also the possibility that haplotype-environment interactions exist that may render the development of phase II phytopreventive strategies potentially more complex than originally envisaged. Given that three of the studied phytochemical agents (sulforaphane, EGCG, and BITC) have been commonly perceived to be cancer protective, based on animal models that predominate in the literature, our current findings and future functional data warrant consideration in advance of human chemopreventive trials employing these agents.


   Acknowledgments
 
Grant support: NIH grants R21-104812 and R01-106186 (S.D. Spivack) and American Lung Association research fellowship grant (W. Han).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Gregory Hurteau (now at Ordway Research Institute, Albany, NY) for collaboration in designing the original RNA-specific RT-PCR strategy, the Wadsworth Center Molecular Genetics Core Facility for sequencing, the Biochemistry Core Facility for instrument support, and the research nurses at Albany Medical College for subject entry and human study coordination.

Received 12/14/05. Revised 3/ 1/06. Accepted 4/17/06.


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 Introduction
 Materials and Methods
 Results
 Discussion
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