Abstract
Glutathione S-transferase P1–1 (GSTπ) is an abundant and ubiquitously expressed protein in normal and malignant mammalian tissues and possesses catalytic and ligand binding properties. Our present data suggest that the protein contributes to the regulation of cell proliferation. Mouse embryo fibroblasts (MEFs) isolated from mice with a GSTP1–1 [glutathioneS-transferase P1–1 (isozyme in nonhepatic tissue)] null genotype (GSTπ−/−) doubled their population in 26.2 h versus 33.6 h for the wild type (GSTπ+/+). Retroviral transfection of GSTP1–1 into GSTπ−/− MEF cells slowed the doubling time to 30.4 h. Both early passage and immortalized MEF cells from GSTπ−/− animals expressed significantly elevated activity of extracellular signal-regulated kinases ERK1/ERK2, kinases linked to cell proliferation pathways. In vivo, GSTπ−/−mice had higher basal levels of circulating white blood cells compared with GSTπ+/+. Administration of a peptidomimetic inhibitor of GSTP1–1, TLK199, (γ-glutamyl-S-(benzyl)cysteinyl-R-phenyl glycine diethyl ester), stimulated both lymphocyte production and bone marrow progenitor (colony-forming unit-granulocyte macrophage) proliferation, but only in GSTπ+/+ and not in GSTπ−/− animals. Selection of a resistant clone of an HL60 tumor cell line through chronic exposure to TLK199 resulted in cells with elevated activities of c-Jun NH2 terminal kinase (JNK1) and ERK1/ERK2, and allowed the cells to proliferate under stress conditions that induced high levels of apoptosis in the wild type cells. The in vitro and in vivo data are consistent with the principle that GSTP1–1 influences cell proliferation.
In mammalian cells, glutathione (GSH) is the major source of available nucleophilic thiol equivalents. Thiol homeostasis is carefully maintained, and thiol:disulfide exchange reactions are important to the functional status of many proteins (Thomas and Sies, 1992). Catalytic conjugations of glutathione to low molecular weight acceptors frequently involve glutathione S-transferases (GSTs) (Boyland and Chasseaud, 1969). It is now clear that the GST gene family is extensive, with distinct isozymes expressing different functional properties. The family is characterized by a promiscuous substrate specificity with low “catalytic efficiency”, characteristics integral to the evolution of GSTs as detoxifiers of a broad spectrum of endogenous and environmental chemicals. GSTP1–1 is the most prevalent isozyme in nonhepatic tissues, and increased expression of GSTP1–1 has been extensively linked to drug resistance and the malignant phenotype of many solid tumors (Tew, 1994). Although GST catalysis will result in glutathione S-conjugate formation of drugs with an electrophilic center, there are numerous examples of GSTP1–1 overexpressing drug-resistant cell lines where the selecting drug is not a substrate and no conjugate is formed (Tew, 1994). In addition, a functional link between GSTP1–1 overexpression and the transformed phenotype remains elusive. To this end, our recent description of the protein:protein interactions between GSTP1–1 and c-Jun NH2-terminal kinase (JNK) (Adler et al., 1999) has provided a framework for contemplating a role for this protein in regulation of stress response, proliferation, and apoptosis (Davis et al., 2001). In addition, a novel GSTP1–1 inhibitor, TLK199 [γ-glutamyl-S-(benzyl)cysteinyl-R-phenyl glycine diethyl ester], was synthesized (Lyttle et al., 1994) with the goal of potentiating the efficacy of anticancer drugs in tumors with a GSTP1–1 overexpressing resistant phenotype (Morgan et al., 1996). The drug is a peptidomimetic of GSH, esterified to enhance cellular uptake and designed to bind to the “G-site” of GSTP1–1. The inhibition constant (Ki) for GSTP1–1 catalytic activity (chlorodinitrobenzene as substrate) is 400 nM. This demonstrates significant specificity for the π-family, since theKi for the GSTα and -μ families range from approximately 20 to 75 μM. Independent of catalysis inhibition, TLK199 also disrupts the protein:protein interaction site(s) between GSTP1–1 and JNK1 (Adler et al., 1999; Wang et al., 2001). Interference with the ligand binding properties of GSTP1–1 may prove to be a critical pharmacological property for the drug in mediating its myeloproliferative effects.
Many drugs produce reactive oxygen species (ROS) as direct or indirect by-products of their metabolism. These can change redox conditions and trigger cellular responses through a number of different pathways. The nature and extent of the ROS insult can determine the threshold of the cellular response, manifest as proliferation, stress response and damage repair, or apoptosis (Adler et al., 1999). How these optional pathways are regulated is not known. However, links between thiol active molecules such as GSTπ and stress-activated protein kinases such as JNK and thioredoxin and apoptosis signaling kinase have been established (Saitoh et al., 1998; Adler et al., 1999). In an unstressed cellular environment, JNK and apoptosis signaling kinase are kept in an inactive mode by the presence of these ligand-binding proteins. Under conditions of oxidative stress or in the presence of inhibitors, GSTP1–1 dissociates from JNK (Adler et al., 1999), activating the catalytic kinase activity and phosphorylating c-Jun. This process can activate kinase cascades involving the numerous sequential downstream kinases. These results are evidence that GSTP1–1 can have a nonenzymatic regulatory role in controlling cellular response to external stimuli (Davis et al., 2001).
There are indications that GSH and associated enzymes play a role in control of cellular immunity. For example, GSH levels in antigen-presenting cells determine whether a Th1 or Th2 pattern of response predominates (Peterson et al., 1998). In human immunodeficiency virus patients, GSH levels in antigen-presenting cells may be important in determining disease progression (Herzenberg et al., 1997). By extending the GST:JNK link, recent results have shown that JNK expression may affect immune response/myeloproliferation. T cells from a JNK1 knockout mouse hyperproliferated, exhibited decreased activation, and induced cell death and preferentially differentiated into Th2 cells (Dong et al., 1998). Despite the redundancy accorded to this system by the expression of JNK2, it appears reasonable to conclude that JNK1 signaling pathways may play a role in T-cell receptor initiated T-cell proliferation and differentiation.
In this present study, we provide evidence from pharmacologic and genetic approaches that GSTP1–1 contributes to control of cell proliferation through a noncatalytic, ligand binding activity with signaling kinases. These results imply that drug targeting of the GST isozyme can be used as a therapeutic approach to stimulate myeloproliferation.
Materials and Methods
Cell Lines.
Human myeloid leukemic cells (HL60) were chronically exposed to TLK199, and drug-resistant cells were selected by passage. Cells were initially exposed to 2.5 μM (1/10th IC50), and the drug concentration was increased in increments of 5 μM after 3 to 4 passages. Cells resistant to 50 μM TLK199 were cloned from single cells and selected for investigation.
Mouse Embryo Fibroblasts (MEFs).
The development of GSTπ−/− mouse was described earlier (Henderson et al., 1998). Removal of the murine GSTπ gene cluster completely abolished the coding sequences of P1, leaving 5 exons for the coding region of P2. We established MEF cell lines from GSTπ+/+ and GSTπ−/−mice. Timed pregnant mice were euthanized by cervical dislocation and the uterus aseptically removed for dissection of the embryos. Embryos were harvested at day 11. Tissue was finely chopped, rinsed, and the pieces seeded onto the culture surface in a medium containing serum. Cultures were kept at 37°C for 18 to 24 h. Once the tissue pieces adhered, the medium was replaced until a substantial outgrowth of cells was observed, at which point the cells were passaged.
When a culture was initiated, cells were maintained in minimal essential medium with 10% fetal bovine serum (Biosources International, Rockville, MD), 1% nonessential amino acids, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 4 mM glutamine. Identical passage numbers for each cell line were used to compare growth characteristics. Primary cell lines exhibited limited culture lifespans (usually between 20–40 doublings), before reaching their Hayflick limit. Immortalized cultures were established by selecting colonies of cells that continued to divide spontaneously after 35 population doublings.
Transfection of GSTP1–1.
The influence of GSTP1–1 on proliferative rate was considered by implementing retroviral gene transfer into MEF cells from GSTπ−/− mice. Briefly, stable lipofectamine-mediated HEK293 packaging cell transfectants were made with a full-length cDNA for GSTP1–1 in the retroviral expression vector pLPCX containing a puromycin resistance selection marker. Several clones were selected and grown in drug-free medium for 48 h. High viral titer supernatants were recovered from the packaging cells, filtered, and added to the MEF cells. Efficiency of transfection was assessed by cotransfection of a green fluorescent protein vector into parallel cultures and by immunostaining. The average transfection efficiency was 65% (proportion of cells), and the transfected cells expressed levels of GSTπ equivalent to approximately 50% of the wild type (WT) MEF cells. Whole cell extracts were used for verification of expression patterns for the transfected cDNAs in the appropriate cell lines. Blots were probed with antisera against actin (Amersham, Piscataway, NJ) to verify equivalent loading.
Cell Doubling Experiments.
A master mix was prepared containing the appropriate number of cells to aliquot 7 flasks of 100,000 cells per 10 ml of RPMI media. Cell doubling rates for GSTπ+/+ and GSTπ−/−cells were determined by counting cells every 24 h for 7 days using a Coulter counter (Beckman Coulter, Fullerton, CA). Final counts were averaged from at least five experiments.
Drugs, Antibodies, and Enzyme Assays.
The GSTP1–1 inhibitor TLK199 (Fig. 1) was obtained from Telik (San Francisco, CA). Antibodies to GST isozymes were obtained from Biotrin (Dublin, Ireland). Anti-active phospho-JNK antibody was obtained from Promega (Madison, WI), and the anti-JNK1 (clone C-17), the anti-JNK2 (clone N-18), the anti-ERK2 (clone C14-G), and the anti-p-ERK (clone E-4) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
The ERK kinase activity was determined by analysis of ELK-1 phosphorylation using a p44/42 MAP Kinase Assay kit from Cell Signaling (Beverly, MA). Briefly, MEF or HL60 cells were resuspended in ice-cold lysis buffer, sonicated at 4°C and centrifuged at 12,000 rpm for 15 min at 4°C; the resulting supernatants were used for the kinase assay using an immobilized phospho-p44/42 MAP kinase monoclonal antibody. Then the samples were centrifuged 5 min at 12,000 rpm at 4°C, the pellets were washed twice in lysis buffer and twice in the kinase buffer, and the resulting pellets were suspended in the kinase buffer containing 200 μM ATP and 2 μg of ELK-1 fusion protein. After 30 min of incubation at 30°C, the reaction was stopped by addition of 3× sodium dodecyl sulfate loading buffer, the samples were boiled and centrifuged, and the resulting supernatants were loaded on a 12% acrylamide gel. After transfer overnight on a polyvinylidene difluoride membrane, the amount of phospho-ELK-1 was determined by immunoblot using a phospho-ELK-1 antibody provided in the kit.
Protein Analysis.
Cells for UV irradiation were exposed to UV-C (60 J/m2) in a minimum amount of phosphate-buffered saline. Protein concentrations were determined using the Bradford reagent (Bio-Rad Laboratories, Hercules, CA). Whole cell extracts (100 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer overnight at 30 V onto a polyvinylidene difluoride membrane. Blots were blocked in 10% nonfat dried milk in TBS-T (50 mM Tris, pH 7.5, 2.3% NaCl, 0.2% Tween 20) and washed between steps in TBS-T. All subsequent antibody incubations were conducted in blocking solution. Proteins were detected using a chemiluminescence detection kit (NEN Life Science Products, Boston, MA). Band intensity was analyzed using NIH Image software (version 1.57).
Apoptosis.
Cells were exposed to either TLK199 (0–50 μM), UV-C (60 J/m2), or a combination of the two. Following an incubation period of 8 h or 24 h at 37°C and 5% CO2, cells were washed in phosphate-buffered saline, resuspended in ethidium bromide (75 μM), and subjected to fluorescence-activated cell sorter analysis using a Beckman FACSCAN (Beckman Coulter, Fullerton, CA). The percentage of apoptotic cells was calculated manually using the Mac Cycle AV program (Phoenix Flow System, San Diego, CA) and statistical significance determined by Student's unpaired t test.
Animal Studies.
Mice (129 GSTπ+/+and GSTπ−/−) were maintained in a barrier facility with ad libitum access to food and water. For all bone marrow experiments, mice of 6 to 12 weeks of age were sacrificed with CO2, the pelt clipped, and hind limbs exposed. Both femurs were removed and cells collected in Iscove's modified Dulbecco's culture media containing 2% fetal bovine serum. Red blood cells were lysed with 0.165 M NH4Cl. Cells (1 ml) were plated (5 × 104 cells/ml) in Methocult M3434 methylcellulose medium (Stemcell Technologies, Vancouver, BC) into 35-mm culture dishes, treated with TLK199 (0.2% dimethyl sulfoxide in H2O) or vehicle, and incubated at 37°C and 5% CO2. In vivo, mice (GSTπ+/+ and GSTπ−/−) were treated with TLK199 (50 or 75 mg/kg; 0.2% dimethyl sulfoxide in water) or vehicle control. Seventy-two hours postinjection, mice were sacrificed with CO2 and cervical dislocation and the bone marrow removed as described previously. Cells were plated (5 × 104 cells/ml) in Methocult M3434. Colony-forming units (CFUs) were scored (colony >50 cells) 7 to 10 days after plating and expressed as a percentage of control. Statistical significance was determined by Student's pairedt test.
Detection of Superoxide Anion Levels.
Superoxide anions were detected using the LumiMax Superoxide detection kit (Stratagene, La Jolla, CA). Briefly, cells were collected from bone marrow (129 GSTπ+/+ or GSTπ−/−mice) and resuspended in superoxide assay medium (5 × 106 cells/ml). Cells (100 μl) were added to 100 μl of reagent mix containing 200 μM luminol and 250 μM enhancer, ±- phorbol-12-myristate-13-acetate (PMA, 0.4 μM; Sigma, St. Louis, MO) and/or TLK199 (0–100 μM) and assayed for superoxide anions using a luminometer over 60 min. Statistical significance was determined by Student's unpaired t test.
Results
GSTπ Status and Cell Proliferation. MEF cultures from GSTπ−/− and GSTπ+/+(11-day-old embryos) demonstrated consistently distinct growth patterns. GSTπ−/− cells doubled faster than GSTπ+/+ cells (26.2 h versus 33.6 h, respectively; Table 1) and reached confluence at higher cell density. These values were calculated from at least five separate MEF cultures and were consistent in cultures established from 13- or 15-day-old embryos. Introduction of GSTP1–1 cDNA into the GSTπ−/− MEF cells through retroviral transfection extended the doubling time to 30.4 h. Transfection efficiency was estimated at ∼65% with ∼50% GSTπ protein levels, compared with the GSTπ+/+ cells (data not shown). These results are consistent with the rationale that GSTP1–1 plays a direct role in control of proliferation. Selection of immortalized cell cultures was achieved by continued growth of cultures beyond 40 passages. These cells had doubling times in the 12- to 14-h range, but there was no difference between GSTπ+/+ and GSTπ−/−cells (Table 1).
The basal (unstimulated) white blood cell counts in GSTπ−/− mice were found to be significantly higher than in GSTπ+/+ animals (Table2). In vivo treatment of GSTπ+/+ mice with TLK199 (75 mg/kg i.p.) resulted in a significant 2-fold increase in white blood cells 3 days after treatment, due to an increase in circulating lymphocytes (Table2). Analysis of blood from GSTπ−/− mice showed no increase in white blood cell count above control following TLK199 treatment. Whereas basal proliferation for the GSTπ−/− cells was the highest of all groups, this result implies that the target of TLK199 may be required for the pharmacological myeloproliferative effects to occur.
Direct effects of TLK199 on mouse bone marrow progenitor cells were demonstrated either by in vivo or ex vivo treatment. Drug treatment induced a proliferative response (approximately 2-fold above vehicle control) in cells (or animals) wild type for GSTπ (Table3). Bone marrow cells null for GSTπ did not respond to TLK199 treatment. TLK199 enhanced the number of cells from the granulocyte-macrophage (GM) lineage with an increased number of GM-CFUs detected either by in vivo or ex vivo experiments.
The Effect of TLK199 on Superoxide Production.
To consider a possible role for ROS in the myeloproliferative effects of TLK199, bone marrow cells from GSTπ+/+ and GSTπ−/− animals were treated with combinations of PMA and TLK199. As a single agent, TLK199 did not stimulate superoxide anion production above control levels. In contrast, TLK199 inhibited the PMA-initiated production of superoxide anions in a dose-dependent fashion (Fig.2, a and b). For example, 50 μM TLK199 caused a significant (P < 0.05) reduction in superoxide detection measured from 5 to 60 min, reducing levels at 5 min from 174,800 RFU to 48,029 RFU. The GSTπ phenotype influenced the production of superoxide anions in bone marrow cells following PMA stimulation (Fig. 2c). Bone marrow cells isolated from GSTπ−/− mice produced less superoxide anions compared with GSTπ+/+ (134,600 RFU, compared with 20,650 RFU).
GSTπ Status and Kinase Activity.
Regulatory kinase pathways, especially those involving JNK and ERK, are frequently linked with cell proliferative status (Pedram et al., 1998; Rausch and Marshall, 1999). Because of our earlier studies ascribing a role for GSTπ in regulating these kinases (Adler et al., 1999; Yin et al., 2000; Davis et al., 2001), we analyzed ERK activity under conditions where GSTπ expression was genetically or pharmacologically manipulated. Figure3 shows that, whereas protein levels of ERK1/ERK2 were similar in early passage GSTπ+/+and GST−/− MEF cells (lanes 3 and 4), enhanced kinase activity was reflected either by ERK phosphorylation (∼3.4-fold) or phosphorylation of the downstream substrate ELK-1 (∼2.3-fold). In immortalized MEF cultures (passage 105, lanes 5 and 6) quantitative kinase activities still remained, albeit at a reduced level (1.3-fold p-ERK; 2-fold p-ELK-1).
To study critical downstream pathways influenced by GSTP1–1 inhibition, a TLK199-resistant HL60 cell line was created by sequential selection through incrementally increasing concentrations of the drug (O'Brien et al., 1999). In the resultant clonal resistant cell line (HL60/TLK199) protein levels of ERK1/ERK2 were unchanged; however, catalytic kinase activity was increased ∼2.3-fold (Fig. 3). Whereas the growth rates of the wild type and resistant cells were not significantly different, the resistant cells are maintained in TLK199 and survive despite constant drug exposure. This drug-induced stress presumably contributes to the approximately 3-fold increased expression of JNK1 (Fig. 4), determined through immunoblotting (normalized to actin). Exposure to UV light (60 J/m2) increased JNK1 catalytic activity as measured by the levels of phosphorylated JNK1 (2.6-fold; Fig. 4). This increase may be attributable to the higher levels of JNK1 in the resistant cells.
The Effect of TLK199 on Apoptosis.
Wild type HL60 cells were induced to undergo apoptosis by 8- and 24-h treatments with 10 and 50 μM TLK199 (Fig. 5). Additive apoptotic effects were seen when HL60 cells were co-treated with 50 μM TLK199 and UV. In contrast, the pattern of apoptosis was significantly different in the HL60/TLK199 cell line. For these experiments, TLK199 was removed from the resistant cell line for one passage prior to additional exposure. Figure 5 shows that overall levels of induced apoptosis were significantly lower, even for single modality UV light exposure. Whereas the HL60/TLK199 cells did undergo apoptosis following combined UV and TLK199 treatment, a significantly lower level (15.1% ± 1.9, compared with 54.6% ± 6.5 for HL60 WT at 24 h) was apparent.
Discussion
It has been known for many years that GSTP1–1 is expressed at high levels in many solid tumors and in cells made resistant to a wide range of anticancer drugs. This is true even when the selecting drug is not a substrate for thioether product formation through GST catalysis (Tew, 1994). In addition, whereas GSTP1–1 is essentially absent from adult rat liver, high levels are expressed in preneoplasticfoci in liver (Imai et al., 1997). In light of our recent data implicating GSTπ in regulation of JNK and other kinases, such observations have prompted an assessment of the role that GSTπ may play in influencing mitogenic pathways with a mechanism unrelated to its catalytic properties. The present study has used two model systems to establish whether different expression levels of GSTP1–1 influence cell proliferation. The GSTπ−/− mouse was established previously (Henderson et al., 1998) using gene-targeting technology to delete the murine GSTπ gene cluster (P1 and P2). The initial phenotype described an increased sensitivity to two-step carcinogenesis induction by polycyclic aromatic hydrocarbons and 12-O-tetradecanoylphorbol-13-acetate, resulting in a 3-fold increase in the incidence of skin papillomas 20 weeks following exposure. Although implying a detoxification function, these data did not distinguish between catalytic or ligand binding functions for the protein.
The present data show that GSTπ−/− mice have higher levels of circulating white blood cells when compared with wild type animals, predominantly observed as an increase in circulating lymphocytes. These elevated counts do not seem to be in response to infection, since the animals are free of apparent pathology or common veterinary disease. Previous reports have linked GSH and associated enzymes with regulation of cellular immunity (Herzenberg et al., 1997;Kim et al., 1998). Thus, the animal phenotype would be consistent with the involvement of GSTP1–1 in control of myeloproliferation. Furthermore, the fact that only GSTπ+/+ mice showed a myeloproliferative response when treated with TLK199, the selective GSTπ inhibitor, provides additional support for the importance of GSTπ to the proliferative response. These data are consistent with the results showing that TLK199 caused an increase in GM-CFU only in bone marrow from GSTπ+/+ mice. This suggests that the presence of, and subsequent inhibitor binding to, GSTπ is critical to the proliferative effects of the drug, and confirm that manipulation of GSTπ is a prerequisite for the stimulation of myeloid progenitor proliferation. In a series of rodent studies, TLK199 impacted positively on the nadir of neutrophil counts and enhanced the recovery period following a myelotoxic dose of 5-fluorouracil. Both in terms of quantitative pharmacologic response and time to recovery, the TLK199 effect was essentially equivalent to granulyte-colony stimulating factor [unpublished data and Dr. Rey Gomez (Telik, San Francisco) personal communication].
Further support for a direct role for GSTπ in control of proliferation is afforded by the MEF cultures established from the GSTπ+/+ and GSTπ−/−mice. The GSTπ−/− cells have a faster doubling time than the GSTπ+/+. Retroviral-mediated transfection of GSTP1–1 into GSTπ−/− MEF cells partially abrogated the doubling time difference. The fact that proliferative rate was not slowed to the equivalent of the GSTπ+/+ cells may be explained by the transfection efficiency (∼65%), which would be reflected by an overall “averaging” of heterogeneity in the cell populations. Immortalization of the MEF cultures resulted in a significantly enhanced doubling time for both cultures. Preliminary indications suggest that changes in p53 expression and mutations may account for the enhanced rates, and that this may abrogate the difference between GSTπ+/+ and GSTπ−/− (data not shown).
Insight into how GSTP1–1 may participate in regulation of proliferation may be gained from our recent reports showing that the protein is a negative regulator of JNK catalytic activity (Adler et al., 1999) and also influences other downstream kinase pathways (Yin et al., 2000). The functional ligand binding properties of GSTP1–1 are exemplified by fluorescence resonance energy transfer data showing aKd of ∼200 nM for the interaction with purified full-length JNK1 (Wang et al., 2001). There is now significant evidence that kinase pathways, particularly involving ERK and JNK, have prominent roles in regulation of proliferative pathways in a wide range of cell types (Pedram et al., 1998; Ishikawa and Kitamura, 1999; Rausch and Marshall, 1999). The increase in ERK1/ERK2 activity in GSTπ−/− MEF would be consistent with the absence of GST-mediated regulatory control producing elevated kinase activity and increased proliferation rates. Although it could be argued that elevation in ERK1/ERK2 and JNK activities is a result rather than a cause of proliferation, the specific nature of the defined GST null phenotype must causally link these pathways in some mechanistic fashion. Other small molecule thiol active agents, such as the aminothiol WR2721, have also been found to cause myeloproliferation (List and Gerner, 2000), and it may be through a mechanism such as thiol modification in either GSTπ (inhibiting its negative regulation of JNK) or other proteins important to proliferation that these agents share a common mechanism.
We established another model system to take advantage of the adaptive changes brought about by the process of chronic exposure and selection in TLK199. HL60 cells are of leukemic myeloid lineage. The HL60/TLK199 cell line grows routinely in 50 μM of drug, a concentration that induces apoptosis in the parent HL60 cell line. Such a model is valuable in determining possible adaptive responses that may indicate the molecular targets of the drug. Indeed, some of the changes have been directly associated with detoxification and enhanced drug efflux (e.g., increased expression of γ-glutamylcysteine synthetase and multidrug resistance-associated protein) (O'Brien et al., 1999). The present data suggest that changes in stress kinase response pathways are also an adaptation to chronic TLK199 exposure. This adaptive response extends our earlier in vitro data showing that TLK199 can cause a dissociation of the GSTP1–1:JNK1 interaction (Adler et al., 1999). In effect, the value of the chronic drug exposure lies in the extrapolation of the in vitro observations into a cellular system. Although correlative in nature, the cells do provide a functional in vivo link with TLK199 treatment and JNK expression.
Apoptosis induced by UV was observed in HL60 WT, as well as in HL60 cells resistant to TLK199. However, the levels of induced apoptosis were significantly lower in the resistant cell line presenting higher ERK1/2 and JNK1 activities. These data are consistent with the numerous studies on the roles of MAP kinases in apoptosis (Cross et al., 2000). Indeed, whereas the ERK family is reported to have antiapoptotic properties, JNK family members have been described as proapoptotic proteins. Moreover, it was demonstrated that the induction of ERK activity could protect HL60 cells against apoptosis induced by anisomycin, a potent inducer of JNK activity (Stadheim and Kucera, 1998). Then, following the concept that the balance between pro- and anti-apoptotic factors determines the fate of a cell, the increased activity of ERK in HL60 cells resistant to TLK199 may partially protect these cells against apoptosis induced by UV, despite the increased JNK1 activity. The pharmacological inhibition of GSTπ by non-cytotoxic concentrations of TLK199 did not modify significantly the level of UV-induced apoptosis in either WT or resistant HL60 cells. In addition, in WT cells, a cytotoxic concentration of TLK 199 and UV had only an additive effect on apoptosis. For example, even where the differences in response appear most marked (8 h in sensitive cells), 52% apoptosis caused by the combination of 50 μM TLK199 and 60 J/m2 UV is not significantly different from the additive values of TLK199 and UV alone (17 + 21 = 38%;P > 0.05). Thus, whereas the forced overexpression of GSTπ protects NIH3T3 cells against apoptosis induced by H2O2 by modifying the activation of MAP kinase pathways (Yin et al., 2000), the pharmacological inhibition of this enzyme may appear to be insufficient to alter the level of cell death observed in our cell lines after UV treatment. This discrepancy could be explained by the fact that, although UV irradiation causes ROS, ultimately cytotoxic effects could involve other mechanisms unrelated to ROS generation.
Paradoxically, a number of studies have emphasized the role of low-level ROS in governing cell proliferation in response to growth factor stimulation (Smith et al., 2000). Extremely low concentrations of anticancer drugs, such as Adriamycin, produced oxidative stress that can actually stimulate tumor cell proliferation. Thus, in clonogenic survival assays, proliferative indices will frequently exceed 100% prior to achieving therapeutic cell kill. For TLK199, we considered the possibility that the altered kinase activities were the consequence of drug-induced ROS. However, in bone marrow cells mitogenically stimulated by PMA, TLK199 appeared to act as a scavenger of ROS, such as superoxide anions. In the GSTπ−/− marrow samples, PMA produced lower levels of superoxide, providing support to the principle that the proliferative effects were not a function of simple changes in ROS levels.
In summary, we provide evidence that GSTπ is involved directly in controlling cellular mitogenic pathways that influence proliferation. The reported role of GSTπ as a negative repressor of JNK (and other kinases) could significantly impact on kinase cascades that influence stress response, proliferation, or apoptosis pathways. Conceivably, the extent and duration of kinase stimulation may determine cellular fate (Davis et al., 2001). Evolutionary conservation and ubiquitous expression of GSTπ at variable levels in proliferative tissues reinforces the principle that this protein has an important role as a ligand-binding protein involved in kinase-mediated stress and proliferation pathways. Pharmacological manipulation of GSTP1–1 with a GSH peptidomimetic agent can be achieved with a concomitant impact upon mitogenic response. This approach may have a useful therapeutic application where a nongrowth factor-mediated stimulation of myeloproliferation can be achieved.
Footnotes
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↵1 Current address: AstraZeneca, UK.
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↵2 Current address: Food and Drug Administration, Alexandria, VA.
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This work was supported in part by the National Institutes of Health Grants CA06927 and RR05539; by the National Institutes of Health Grants CA53893 to K.D.T. and CA77389 to Z.R.; and by appropriation from the Commonwealth of Pennsylvania.
- Abbreviations:
- GSH
- glutathione
- GST
- glutathioneS-transferase
- GSTP1–1
- glutathioneS-transferase P1–1 (isozyme in nonhepatic tissue)
- JNK
- c-Jun NH2 terminal kinase
- TLK199
- γ-glutamyl-S-(benzyl)cysteinyl-R-phenyl glycine diethyl ester
- ROS
- reactive oxygen species
- MEF
- mouse embryo fibroblast
- WT
- wild type
- ERK
- extracellular signal-regulated kinase
- MAP
- mitogen-activated protein
- UV
- ultraviolet
- CFU
- colony-forming unit
- PMA
- phorbol-12-myristate-13-acetate
- GM
- granulocyte-macrophage
- Received January 16, 2001.
- Accepted March 15, 2001.
- The American Society for Pharmacology and Experimental Therapeutics