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Carcinogenesis Advance Access originally published online on June 8, 2007
Carcinogenesis 2007 28(11):2268-2273; doi:10.1093/carcin/bgm135
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Differential effects of glutathione S-transferase pi (GSTP1) haplotypes on cell proliferation and apoptosis

Sarah L. Holley, Anthony A. Fryer, John W. Haycock1, Sarah E.W. Grubb1, Richard C. Strange and Paul R. Hoban*

Human Disease and Genomics Research Group, Institute of Science and Technology in Medicine, Keele University, University Hospital of North Staffordshire, Stoke-on-Trent, Staffordshire, ST4 7QB, UK
1 Department of Engineering Materials, Kroto Research Institute, Sheffield University, Sheffield, S3 7HQ, UK

* To whom correspondence should be addressed. Tel: +44 1782 555226; Fax: +44 1782 717079; Email: p.r.hoban{at}keele.ac.uk


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Expression of the glutathione S-transferase, GSTP1, is associated with phase 1 detoxification of the products of oxidative stress. Recently, GSTP1 expression has been implicated in the regulation of cell proliferation and apoptosis through direct interaction with the c-Jun N-terminal kinase, (JNK). GSTP1 is polymorphic and allelic variants have been associated with disease susceptibility and clinical outcome. However, the influence of GSTP1 alleles on proliferation and apoptosis has not been studied previously. To investigate this, we have examined the effects of inducible expression of wild-type GSTP1*A and mutant GSTP1*C haplotypes on cell proliferation and apoptosis in NIH3T3 fibroblasts. Cells expressing GSTP1*A displayed increased doubling times and a delayed G1–S phase transition compared with cells expressing GSTP1*C. Both GSTP1*A and GSTP1*C haplotypes protected cells from undergoing apoptosis when exposed to oxidative stress. However, analysis of JNK status revealed that only GSTP1*C expression led to a reduction in JNK activity compared with GSTP1*A-expressing cells and non-induced cells. We further examined the effect of GSTP1 alleles on colony-forming efficiency (CFE) in soft agar following exposure to oxidative stress and found that GSTP1*A-expressing clones had increased CFE compared with non-induced and GSTP1*C-expressing clones. Our data suggest that GSTP1 alleles have differential effects on proliferation and apoptosis; GSTP1*A reduces cellular proliferation and protects against apoptosis through a JNK-independent mechanism. In contrast, GSTP1*C does not influence cellular proliferation but protects cells from apoptosis through JNK-mediated mechanisms.

Abbreviations: CFE, colony-forming efficiency; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GST, glutathione S-transferase; IPTG, isopropyl-beta-D-thiogalactopyranoside; JNK, c-Jun N-terminal kinase


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The glutathione S-transferases (GST; EC 2.5.1.1 [EC] 8) are a supergene family of dimeric enzymes found in cells of nearly all life forms (1–3). Traditionally, the GST are considered to be phase II detoxifying enzymes that catalyse the conjugation of reduced glutathione with a wide variety of electrophilic substrates (3). It is now clear that the functions of GST are much broader than this classical concept of xenobiotic detoxification. For example, the pi class enzyme, GSTP1, also has leukotriene synthetase activity and, as a monomer, can inhibit the c-Jun N-terminal kinase (JNK) (1). Consequently, in addition to detoxification, GSTP1-1 appears to be involved in responses to cellular stress (exogenous and/or endogenous oxidative stress) and control of the cell cycle.

A number of recent reports support a role for GSTP1 in cell cycle control (1,4–6). A study by Ruscoe et al. (4) supports the idea that the GSTP1–JNK interaction has a role in cell proliferation. Suppression of GSTP1 led to elevated JNK activity, increased proliferation and reduced apoptosis in mouse embryonic fibroblasts from GSTP1 knockout mice (GSTP1–/–) mice. The doubling time of cells from was7.4 h shorter compared with wild-type mouse embryonic fibroblasts. The authors also demonstrated that re-expression of GSTP1 in GSTP1–/–cells through retroviral transfection increased cell doubling time.

A role for GSTP1 in regulating apoptosis has been formulated from studies which demonstrate that, under non-stressed conditions, GSTP1 monomer inhibits JNK through formation of a protein complex (5). Exposure to oxidative stress causes GSTP1 dissociation from JNK and subsequent formation of inactive covalent dimers. Under these conditions, full JNK activity is restored by JNK leading to activation of c-jun through phosphorylation at Ser-63 or Ser-73 residues and increased transcription of AP-1-responsive genes. This results in activation of signalling pathways for stress response, proliferation and apoptosis (4,6). In addition, it has been suggested that ROS-generating agents activate GSTP1 transcription via the JNK/Jun cascade and form a subtle regulatory loop dependent on the duration and magnitude of stress kinase activity. Through transcriptionally active JNK substrates, including c-Jun, new synthesis of GSTP1 occurs, which is expected to resume JNK inhibition. Indeed, suppression of GSTP1 leads to elevated activities of JNK (4). Thus, GSTP1 influences signalling pathways for apoptosis by modulating JNK kinase activity (5).

GSTP1 is polymorphic with two common functional variants described based on substitutions at amino acids 105 (Ile-Val) and 114 (Ala-Val). Thus, four haplotypes have been identified: the wild-type GSTP1*A (Ile105+Ala114) and three variant haplotypes, GSTP1*B (Val105+Ala114), GSTP1*C (Val105+Val114) and GSTP1*D (Ile105+Val114) (7–11). The Val105 variant, compared with Ile105, appears to confer a higher catalytic efficiency for polycyclic aromatic hydrocarbon diol epoxides but a lower efficiency for 1-chloro-2,4-dinitrobenzene (7–11). The crystal structure of GSTP1 Val105 reveals that it is probable to fit less bulky substrates but has broader substrate specificity than the GSTP1 Ile105 variant of the enzyme (12). In addition, heat stability of the proteins is different with half-lives of 19 min (GSTP1 Ile105) and 51 min (GSTP1 Val105) described. Amino acid 105 lies in close proximity to the active centre and it is therefore not surprising that this residue influences catalytic activity (12). The effect of the Ala114–Val114 substitution is unclear, though there is some evidence that it enhances the effect of the Ile105–Val105 substitution (8).

Many studies have examined the association of these polymorphic variants with susceptibility or outcome in a range of pathologies including cancer and inflammatory diseases. In asthma, for example, homozygosity of the GSTP1 Val105 allele is associated with reduced risk of airway hyperresponsiveness and improved lung function (1,13), whereas in head and neck cancer this genotype is associated with increased risk (14). Furthermore, in transplant patients exposed to high- and low-dose corticosteroids (15), we showed that, relative to the wild-type genotype, the Val105/Val105genotype was associated with reduced risk of cutaneous squamous cell carcinoma in patients on low-dose prednisolone but increased risk in those on high-dose prednisolone. To date, it is unknown whether these apparent contradictory findings reflect the role of GSTP1 in cell cycle control and/or detoxication.

While some studies have focussed on the role of GSTP1 on cell cycle regulation and others on the functional effect of GSTP1 polymorphism on detoxification of exogenous substrates, no study to date has examined the effect of polymorphism on the cell cycle-associated endpoints of proliferation and apoptosis. In this study, we have examined the effect of expression of GSTP1 haplotypes on these tightly regulated processes.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell culture
NIH3T3 fibroblasts were routinely maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) (BioSera, Sussex, UK), 2 mM L-glutamine, 2 mM antibiotic antimycotic solution (Invitrogen, Paisley, UK) at 37°C in 5% CO2. Cells were grown as monolayers in tissue culture flasks or plates (Appleton Woods, Birmingham, UK) as required. Cell monolayers were detached using 0.25% trypsin–ethylenediaminetetraacetic acid (Sigma, Dorset, UK). Induction of the LacSwitchTM expression system (Stratagene, Amsterdam, Netherlands) was achieved through growth in medium containing 10% FBS in the presence of 5 mM isopropyl-beta-D-thiogalactopyranoside (IPTG; Invitrogen) for the duration of each experiment. In experiments where cell synchronization was required, serum starvation was performed in medium containing 0.1% FBS for 72 h (in the absence of IPTG).

Vector construction and transfection
Using site-directed mutagenesis (GeneTailor; Invitrogen) on a human GSTP1*B cDNA clone (IMAGE), we constructed inducible expression systems containing GSTP1*A (Ile105–Ala114) and GSTP1*C (Val105Val114) haplotypes.

Induced expression of GSTP1 cDNAs was achieved using the LacSwitchexpression system (Stratagene). Full-length GSTP1 cDNAs were subcloned in the sense orientation into the EcoR V site of pOPRSVIMCS (Stratagene, UK). Recombinant pOPRSVI/GSTP1*A and pOPRSVI/GSTP1*C molecules were confirmed through direct sequencing of a full-length polymerase chain reaction product on an ABI Prism 310 Genetic Analyzer (PE Applied Biosystems, Warrington, UK). Briefly, NIH3T3 (7 x105) cells were seeded overnight in six-well plates and were then transfected with pCMVLacI plasmid (4 µg/ml) using standard calcium phosphate precipitation. Transfectants were grown for 3 days, split 1:3 and grown under hygromycin B (500 µg/ml; Invitrogen) selection for 14 days. Clones were picked through 0.33% noble agar (Difco, MI) and those expressing LacI repressor transcript (as determined by reverse transcriptase–polymerase chain reaction) were transfected with 4 µg/ml pOPRSVI/GSTP1*A or pOPRSVI/GSTP1*C as before. Seventy-two hours post-transfection cells were split 1:3 and grown under medium containing 500 µg/ml G418 (Geneticin; Invitrogen) and hygromycin B for 2-3 weeks. Single NIH3T3GSTP1*A and NIH3T3GSTP1*C clones were picked through 0.33% noble agar and each clone was plated separately. Stock cells were routinely grown under hygromycin B and G418 selection (500 µg/ml). NIH3T3GSTP1*A and NIH3T3GSTP1*C clones will be referred to as GSTP1*A and GSTP1*C throughout the remainder of this article. Experimental assays were performed in the absence of antibiotics. Three inducible clones for each haplotype were selected for further study.

Western blot analysis
Nuclear proteins were extracted from 1 x 106 cells. Pellets were resuspended in 50 mM Tris–HCl (pH 8), 150 mM NaCl, 1% Triton-X, 100 µg/ml phenylmethylsulfonylfluoride, 1 µg/ml aprotonin and 50 µg/ml leupeptin and a cocktail of phosphatase inhibitors (sodium fluoride, sodium orthovanadate, sodium pyrophosphate and ß-glycerophosphate) (Perbio Bioscience, Rockford, IL). Denatured protein lysates (20 µg) were subjected to 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analysis. Gels were electroblotted onto ProtranTM polyvinylidene fluoride paper (Amersham Pharmacia Biotech, Buckinghamshire, UK). Following blotting, filters were blocked with 5% skimmed milk powder in Tris-buffered saline containing 0.1% Tween-20 overnight. Western blots were probed with 1:1000 dilution of GSTP1 antibody (16), followed by three washes in Tris-buffered saline containing 0.1% Tween-20. Antibodies were detected at room temperature using appropriate horseradish peroxidase-conjugated antibody (Dako, Glostrup, Denmark) diluted 1:1000. Western blots were developed using chemiluminescence (Pierce West Pico Supersignal kit; Perbio Bioscience, Cheshire, UK).

Trypan blue exclusion cell counts
Cells (1 x 104) per well were plated on a 12-well plate and allowed to adhere overnight. Cells were then synchronized by serum starvation (0.1% FBS). After 72 h, the cells were reintroduced into 10% FBS in Dulbecco's Modified Eagle Medium with or without 5 mM IPTG. Three wells were harvested each day from 0 h to 96 h and cells were counted using a haemocytometer with viability determined by trypan blue exclusion.

Bromodeoxyuridine (BrdU) incorporation assay
DNA synthesis was assessed by a colourimetric enzyme-linked immunosorbent assay (ELISA) method according to the manufacturers recommendations (Roche, Sussex, UK). Cells (1 x 103) were seeded onto 96-well plates (1 plate for each time point) and left for 24 h. The cells were then synchronized in situ for 72 h. At each time point, one plate was removed and subjected to BrdU incorporation for 2 h, and processed following the manufacturers recommendations. The colourimetric change was measured on a Dynatech MR5000 microplate reader (Dynex Laboratories, UK).

Flow cytometric analysis of DNA content
Cells were grown under the desired conditions (with or without IPTG) and after the allocated time were harvested by trypsinization and centrifuged at 1200 r.p.m. for 5 min. The pellets were washed twice with 1xphosphate-buffered saline. Cells were then resuspended in 0.2% Triton-X and 50 µM PI(Sigma). Resuspended cells were incubated at room temperature for 15 min and then stored at 4°C until analysis. FACS analysis was performed on a Dako Cytomation MoFlo (Dako UK Ltd, Cambridgeshire, UK).

Detection of apoptosis by Hoechst 33342 staining
Cells were seeded into flasks in 5 ml of medium, left to adhere overnight and then induced with 5 mM IPTG for 72 h prior to addition of 100 µM H2O2. Cells were assayed for apoptotic response after 24 h. Cells were harvested and washed in phosphate-buffered saline before subsequent centrifugation. Twenty microlitres of cell suspension was mixed with 1 µl (1 mg/ml) Hoechst 33342 (Invitrogen) and analysed on a wet mount slide using a Leica DMR fluorescent microscope (Leica Corp., Deerfield, IL). The percentage of cells exhibiting apoptosis was determined from 200 cells in three separate fields, and the mean from three independent experiments was calculated.

Detection of apoptosis using externalized phosphatidyl serine by Annexin V–phycoerythrin flow cytometry
Cells were seeded into T25 cm2 flasks in 5 ml of medium, left to adhere overnight and then induced with 5 mM IPTG for 72 h prior to addition of 100 µm H2O2. Cells were assayed for apoptotic response after 24 h. Cells were prepared for Annexin V–phycoerythrin flow cytometry as recommended by the manufacturer (Guava Technologies, Hayward, CA) and as described previously (17). In brief, cells were harvested and washed as described by the manufacturer, after which Annexin V-PE and 5 µl 7-aminoactinomycin D was added to the cell suspension. The reactions were then incubated on ice for 20 min and the results acquired on a Guava PCA flow cytometer (Guava Technologies). Data were analysed using CytoSoft (v2.0) software and corrected for background apoptosis using values for non-induced and induced cells not treated with H2O2.

Colony-forming efficiency
Cells were seeded at 1 x 104 cells into noble agar (Difco). The resultant suspension was dispensed into gridded 0.09 cm2 dishes (Sarstedt, Leicester, UK), allowed to set and overlaid with 5 ml of cloning media (9:1 ratio of fresh Dulbecco's Modified Eagle Medium containing 20% fetal calf serum to conditioned media). Cells were cultured at 37°C for 2 weeks, following which the number of colonies was counted using a x4 objective lens on a microscope (Nikon, Surrey, UK). A single colony was defined as a group of 10 or more cells in close proximity.

JNK activity immunoassay
JNK activity was assessed using the JNK activity immunoassay kit from Merck (Darmstadt, Germany) as per the manufacturer's instructions. In brief, total protein was extracted from 2 x 106 cells (with and without H2O2 treatment) and the protein concentration determined. Total protein (200–400 µg) was then used in immunoprecipitation reactions. A JNK-specific antibody was used to immunoprecipitate JNK from the cell lysates. JNK activity was detected via a kinase reaction using c-Jun as a substrate. Phosphorylated c-Jun was analysed by Western blot using a c-Jun phospho-specific antibody. Differences in active c-Jun levels were determined by comparison with total c-Jun levels measured via scanning densitometry.

Cellular activation of JNK signalling ELISA
The SuperArray CASE kit was used for cell-based detection of JNK protein phosphorylation (SuperArray, Frederick, MD). Cells (between 50 and 80% confluency) were treated with H2O2 for 30 min before fixation in a 4% formaldehyde:phosphate-buffered saline solution. The ELISA was then performed as per the manufacturer's specifications. The colourimetric change on cell total and phosphorylated JNK protein expression was measured at 450 nm on a Dynatech MR5000 microplate reader (Dynex). Relative cell number was also determined using the absorbance of 595 nm. The relative extent of JNK protein phosphorylation was then determined by normalizing the phosphoprotein-specific antibody OD450:OD595 ratio to the pan-protein-specific antibody OD450:OD595 ratio for the same experimental conditions.

Statistical analysis
For both GSTP1*A and GSTP1*C transfectants, cell growth, DNA synthesis, apoptosis and colony formation experiments were performed on three clones. Experiments were performed independently three times and data presented as the mean ±SEM. Data were analysed using a one-tailed t-test or Mann–Whitney U-test with Stata software (version 8, Stata Corporation, TX). Results were judged to be statistically significant when the calculated P value was <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Inducible expression of GSTP1 haplotypes in NIH3T3 cells
GSTP1*A and GSTP1*C clones grown under non-inducing conditions did not express GSTP1 protein using Western blotting with an antibody specific to GSTP1. This confirms firstly that NIH3T3 cells have a low basal level of endogenous GSTP1 protein and secondly that LacI repression of the RSV promoter is efficient (Figure 1). We found that when grown under inducing conditions, GSTP1*A and GSTP1*C cells expressed a 23 kDa protein of the expected size (Figure 1).


Figure 1
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  		Fig. 1. Inducible expression of GSTP1in NIH3T3 transfectants. Western blots probed with GSTP1 antibody. Shown is a representative example from a single clone for each GSTP1 haplotype. Lane 1: GSTP1*Anon-induced, lane 2: GSTP1A*induced, lane 3: GSTP1*C non-induced and lane 4: GSTP1*C induced.

 
Effect of induced expression of GSTP1*A on cellular proliferation
To examine the influence of induced expression of the haplotypes on cell growth, we calculated doubling times for GSTP1*A and GSTP1*C cells using firstly trypan blue exclusion cell counts and secondly by measuring the rate of DNA synthesis using an ELISA method to measure BrdU incorporation in synchronously growing cells (Table I).


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  		Table I. Effects of induced expression of GSTP1 in NIH3T3 fibroblasts on cell doubling times

 
GSTP1*A cells showed differences in cell doubling times when grown under inducing conditions. Expression of GSTP1*A increased the cell doubling time by 3.85 ± 0.43 h (mean difference ± SEM, P = 0.058) and induction of GSTP1*C increased cell doubling times by 1.10 ± 0.60 h (P = 0.193), compared with non-induced cells (Table I).

Similar results were observed using BrdU ELISA analysis (Table I). Induction of GSTP1*A significantly increased the cell doubling time by 3.41 ± 0.59 h mean difference ± SEM P = 0.040). Induction of GSTP1*C decreased cell doubling times by 1.56 ± 0.22 h, compared with non-induced cells (P = 0.802). Thus, induced expression of GSTP1*A, but not GSTP1*C, in cells resulted in a significant increase in cell doubling times and thus slower cellular growth.

We next studied the effect of GSTP1alleles on transition through G1phase of the cell cycle. FACS analysis illustrated that, compared with the non-induced control, the proportion of cells expressing GSTP1*A that entered S phase was lower (at ~16 h after release from serum starvation) than cells not expressing this protein (Figure 2). This lag in G1–S progression, however, was not apparent in cells expressing GSTP1*C, which, as observed in the BrdU analysis, showed a similar proportion of cells moving through the cell cycle irrespective of whether GSTP1*C expression was induced or not (Figure 2). Time course analysis showed that by 18 h, the proportion of cells in the S phase from expressing GSTP1*A were similar to non-induced controls at this time point (data not shown), suggesting a delay of up to 2 h in GSTP1*A, but not GSTP1*C-expressing cells.


Figure 2
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  		Fig. 2. FACS analysis illustrating the effect of GSTP1 haplotype expression on cell cycle progression. Cells were starved for 48 h prior to the start of the experiment to ensure cell synchronization. Cells were then released into media containing 10% serum, harvested at 16 h (as indicated by initial experiments) and assessed for DNA content by flow cytometry. Shown is a representative example from a single clone.

 
The effect of GSTP1 haplotype expression on apoptosis
We next investigated the effect of expression of GSTP1 haplotypes on apoptosis. Cells were treated for 24 h with 100 µM H2O2 before Hoechst 33342 staining and assessment of condensed chromatin status via immunofluorescence. Upon induction of both GSTP1 haplotypes in NIH3T3 cells, apoptosis was significantly reduced when challenged with H2O2. Compared with non-induced cells, a 2.6-fold decrease in the proportion of cells staining for Hoechst 33342 when expressing GSTP1*A (P < 0.001) (Mann–Whitney U-test) and a 2.4-fold decrease when expressing GSTP1*C (P < 0.001) were found (Figure 3).

Similar results were observed using Annexin V FACS analysis. Compared with non-induced cells, a 2.1-fold decrease in the proportion of cells staining positive for Annexin V when expressing GSTP1*A (P = 0.050) and a 2-fold decrease when expressing GSTP1*C (P = 0.127) were observed (Figure 3).


Figure 3
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  		Fig. 3. The effect of GSTP1 haplotype expression on ROS-induced apoptosis. The ability of GSTP1 to protect cells from apoptosis was assessed by Hoechst 33342 staining (A), Annexin V FACS analysis (B) and CFE efficiency (C). The data in (A) and (B) depict the number of cells staining for each procedure when induced to express GSTP1. Data are shown as percentages of the non-induced sample, which has been ascribed a value of 100%, to allow direct comparison between the Hoechst 33342 and Annexin V FACS analysis. (C) CFE (over a 2-week period) was performed at 100 µM H2O2. Number of colonies are presented as a percentage of the sample not treated with H2O2.

 
The effect of GSTP1 haplotypes on cell viability following exposure to H2O2
We next examined the effect of GSTP1 haplotypes on cell survival, as measure using colony-forming efficiency (CFE), following exposure to H2O2. Cells were exposed to 0 or 100 µM H2O2 for 24 h before suspension in soft agar. Induction of GSTP1*A significantly increased CFE by 9.02 ± 2.40% at 100 µM (P = 0.020). Conversely, induction of GSTP1*C led to decreased CFE by 8.86 ± 5.76% at 100 µM (P = 0.052) (Figure 3C).

Influence of GSTP1 haplotypes on JNK pathway
We next explored the mechanism by which GSTP1 haplotype expression protects cells from apoptosis. Activity of JNK following exposure to H2O2 was examined in cells under non-induced and induced conditions (Figure 4). Densitometry of Western blots showed that GSTP1*C expression reduced the degree of H2O2-induced JNK activity by 69.0%, conversely GSTP1*A expression increased JNK expression by 34.6% (Figure 4A). Similar results were obtained using an ELISA to detect both total and phosphorylated JNK (Figure 4B). This showed that GSTP1*C expression significantly reduced the degree of H2O2-induced JNK phosphorylation by 71.4% (P < 0.001) and as observed previously GSTP1*A expression led to a 34.6% increase in phosphorylated JNK expression (P = 0.002).


Figure 4
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  		Fig. 4. JNK immunoassay. The effect of GSTP1 expression on H2O2 induction of JNK activity in GSTP1-transfected NIH3T3 cells. Analysis was carried out via immunokinase reaction using antibodies to JNK kinase with a c-Jun substrate (A) and by ELISA analysis of total and phosphorylated proteins (B). (A) Proteins were prepared 30 min after H2O2 exposure. Lane 1: GSTP1*A non-induced, lane 2: GSTP1*A induced, lane 3: GSTP1*A non-induced treated with H2O2, lane 4: GSTP1*A induced treated with H2O2, lane 5: GSTP1*C non-induced, lane 6: GSTP1*C induced, lane 7: GSTP1*C non-induced treated with H2O2 and lane 8: GSTP1*C induced treated with H2O2. (B) ELISA analysis was performed using antibodies to total and phosphorylated JNK. Samples are presented in graphical format as the percentage of phosphorylated protein in the sample. Values were corrected for background JNK expression.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
For the first time, we have investigated the function of GSTP1 haplotypes on cell growth. We used the normal diploid fibroblast cell line, NIH3T3, which has been used previously in the study of GSTP1 (5). Expression was examined through inducible regulation of an RSVpromoter using the Lac Switch system. In our hands, this proved to be a robust system. Thus, when grown under non-inducing conditions, no protein could be detected from either GSTP1*A or GSTP1*C transfected cell lines when performing Western blotting with an anti-human GSTP1 antibody which is known to cross-react with mouse GSTP1/2. However, on release of LacI repression, a 23 kDa protein encoded from GSTP1 was detected in both GSTP1*A or GSTP1*C cells.

In GSTP1*A transfected cells, induced expression of GSTP1*A significantly decreased the proportion of cells undergoing DNA synthesis (BrdU incorporation), resulting in an increased cellular doubling time. This is in accordance with previous reports demonstrating that expression of GSTP1 slows proliferation in rodent fibroblasts (4). Conversely, cells expressing the GSTP1*C haplotype demonstrated similar levels of DNA synthesis and doubling times compared with non-induced cells. FACS analysis illustrated these differences were due to a lag in G1–S phase progression when cells were induced to express GSTP1*A, but not when expressing the GSTP1*C variant. Previous data have suggested that cells not expressing GSTP1 had elevated levels of ERK1/ERK2 kinases. This was thought to be due to absence of GST-mediated regulatory control leading to elevated kinase activity and increased proliferation, though this could be a consequence rather than a cause of proliferation (4). In addition, Chen et al. (18) using an amplified differential gene expression array, also identified genes associated with GSTP1 expression that are involved in cellular proliferation (particularly within G1and S phases of the cell cycle), including p21-activated kinase 1, tousled-like kinase 2 and cullin 1. For example, they found that in the absence of GSTP1, cullin 1 expression was increased which could lead to an increase in cyclin D1 ubiquitination and thus slowing of the cell cycle as we observed following GSTP1*A induction (18). This view is supported by our preliminary data which suggest that induced GSTP1*A, but not GSTP1*C, reduces cyclin D1 expression in proliferating NIH3T3 cells (data not shown). Gate et al. (19) showed that GSTP1-null mice have higher circulating levels of white blood cells than wild-type mice, a finding that supports the observation that TLK199, a selective GSTP1 inhibitor, is associated with myeloproliferation. They proposed that this increased proliferation in GSTP1-null cells was associated with down-regulation of negative regulators of the Janus kinase–STAT pathway that can lead to activation of JNK, ERK and p38 mitogen-activated protein kinase pathways. The effect of GSTP1 expression on proliferation may also provide an explanation for the finding by Wolf et al. (20) that the lungs of GSTP–/– (null) mice have much larger lungs than wild-type animals (up to twice the size as a percentage of lung wet weight).

Using H2O2to induce apoptosis in our transfected NIH3T3 fibroblasts, we demonstrated that GSTP1 expression did indeed protect cells from apoptosis as described previously (21). No differences could be observed between the haplotypes, using our measurements of apoptosis. However, a differential effect was observed when analysing cell viability. More clones grew in soft agar following exposure to hydrogen peroxide in GSTP1*A haplotype-expressing cells than GSTP1*C-expressing cells. To understand the mechanism behind the observed protection from apoptosis by GSTP1, we examined JNK activity as previously reported by Adler et al. (5). We found JNK activity was reduced in cells treated with H2O2 and induced to express GSTP1*C as described by Yin et al. (21). Conversely, cells induced to express GSTP1*A showed elevated levels of JNK. Turella et al. (22) showed that the new generation of pharmacological GSTP1 inhibitors appear to act on the GSTP1–JNK interaction and trigger a ROS-independent activation of JNK-mediated pathway. They also identified a secondary ROS-dependent pathway, in the leukaemic cell line K562 but not CCRF-CEM T-lymphoblastic leukaemia cells, which involves p38 mitogen-activated protein kinase signal transduction. It is interesting to speculate that these differences may be due to differing GSTP1 haplotypes in these cell lines. Adler et al. (23) demonstrated that peptides covering amino acid residues 99–121 influence the binding of GSTP1 to JNK, suggesting that the Ile105–Val105 and Ala114–Val114 substitutions that define the GSTP1*A and GSTP1*C haplotypes may be crucial for JNK binding and potentially other protein–protein interactions. Accumulating data suggest that GSTP1 may be crucial in regulating the activation of multiple stress kinase cascades, including the ASK1, MEKK1, mitogen-activated protein kinase, ERK and IKK–NF{kappa}B signalling pathways. Among the substrates for these signalling cascades are p53, NF{kappa}B, c-Jun, ATF2 and c-Fos which dictate protection from, or promotion of cell death (24,25). Wu et al. (26) have recently shown that the mechanism of the GSTP1-mediated inhibition of JNK, p38 and ASK1 appears to be via forming ligand-binding interactions with tumour necrosis factor receptor-associated factor 2. Prx1, a peroxiredoxin family member, has also been shown to interact the GSTP1–JNK complex and suppresses release of JNK (27). The influence of GSTP1 haplotypes on these interactions is currently being investigated.

Our observation of differential effects of GSTP1 haplotypes on cell cycle control, in addition to detoxification reactions, may have significant implications for the interpretation of genetic association studies. It could provide an explanation why some studies suggest that the GSTP1*C allele is associated with risk, whereas in others, it appears protective, as exogenous environmental exposure may determine the balance between the relative importance of these two activities. For example, in our study on head and neck cancer patients (14), the ability to detoxify cigarette smoke-derived carcinogens may predominate implicating GSTP1*C as a risk factor, whereas in patients with asthma (13), the increased proliferative effect associated with this allele may be associated with increased lung growth and hence improved function.

Our present data confirm the role of GSTP1 in cell proliferation and apoptosis as described previously in the literature (4,21). However, for the first time, we have shown that GSTP1 haplotypes have differential effects on cellular proliferation and apoptosis, which may be translated into cell viability. Thus, expression of the GSTP1*A variant slows cell proliferation and protects from apoptosis probably through a JNK-independent pathway regulated by ERK signalling pathways whose targets are G1–S regulators. Expression of the mutant GSTP1*C variant, however, has no effect on cell proliferation and protects from apoptosis through a JNK-dependent mechanism. Understanding the normal function of GSTP1 haplotypes in vivo will help elucidate their role in tumourigenesis and cell growth.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
This work was funded by the International Association for Cancer Research, project AICR 04-021.


   Acknowledgments
 
Conflict of Interest statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

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Received April 2, 2007; revised May 23, 2007; accepted May 23, 2007.


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