Eur J Biochem 271, 1690–1701 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04067.x Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells Rajendra Sharma1,*, David Brown1,*, Sanjay Awasthi2, Yusong Yang1, Abha Sharma1, Brad Patrick1, Manjit K Saini1, Sharda P Singh3, Piotr Zimniak3, Shivendra V Singh4 and Yogesh C Awasthi1 Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX, USA; Department of Chemistry and Biochemistry, University of Texas at Arlington, TX, USA; 3Department of Pharmacology and Toxicology and Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA; 4Department of Pharmacology, University of Pittsburgh Cancer Center, PA, USA 4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end products of lipid peroxidation, has been shown to induce apoptosis in a variety of cell lines It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation We show for the first time that incorporation of 4-HNE-metabolizing glutathione S-transferase (GST) isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and CCL-75, by either cDNA transfection or microinjection of active enzyme, leads to their transformation The dramatic phenotypic changes due to the incorporation of hGSTA4-4 include rounding of cells and anchorage-independent rapid proliferation of immortalized, rounded, and smaller cells Incorporation of the inactive mutant of hGSTA4-4 (Y212F) in cells by either microinjection or transfection does not cause transformation, suggesting that the activity of hGSTA4-4 toward 4-HNE is required for transformation This is further confirmed by the fact that mouse and Drosophila GST isozymes (mGSTA4-4 and DmGSTD1-1), which have high activity toward 4-HNE and subsequent depletion of 4-HNE, cause transformation whereas human GST isozymes hGSTP1-1 and hGSTA1-1, with minimal activity toward 4-HNE, not cause transformation In cells overexpressing active hGSTA4-4, expression of transforming growth factor b1, cyclin-dependent kinase 2, protein kinase C bII and extracellular signal regulated kinase is upregulated, whereas expression of p53 is downregulated These studies suggest that alterations in 4-HNE homeostasis can profoundly affect cell-cycle signaling events Oxidative stress causes generation of reactive oxygen species, which leads to the onset of lipid peroxidation [1] 4-Hydroxynonenal (4-HNE) is one of the end products of this process [2] In recent years there has been an increasing interest in the role of 4-HNE in signaling mechanisms [3–12] There are reports suggesting that 4-HNE can cause cell cycle arrest [2], apoptosis [3,6,7,12], differentiation [12] or proliferation [11,12] in different cell types in a concentration-dependent manner These seemingly opposite effects of 4-HNE on cell cycle signaling (e.g cell cycle arrest and apoptosis vs proliferation) are intriguing If 4-HNE does indeed differentially affect signal transduction in a concentration-dependent manner, the regulation of 4-HNE homeostasis may be important for cell cycle signaling It is inherently difficult to characterize the functional consequences of changes in intracellular 4-HNE concentration because 4-HNE is formed by lipid peroxidation, mostly an uncontrolled nonenzymatic process In this study, we circumvented this problem by regulating 4-HNE concentration through its metabolism, and investigated the effect of altered 4-HNE homeostasis on proliferation and cell cycle signaling in two different adherent cell lines To test the hypothesis that 4-HNE may be a determinant in cell cycle regulation, we stably transfected the human lens epithelial cell line (HLE B-3) with cDNA for human glutathione S-transferase (GST, EC 2.5.1.18) isozyme hGSTA4-4 This isozyme conjugates GSH to 4-HNE with high efficiency [13], and cells overexpressing it, or similar enzymes [14], have lower steady-state levels of 4-HNE [12] In accordance with accepted convention [15], we refer to the gene and the dimeric enzyme as hGSTA4 and hGSTA4-4, Correspondence to Y C Awasthi, 551 Basic Science Building, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX 77555-0647, USA Fax: + 409 772 6603, Tel.: + 409 772 2735, E-mail: ycawasth@utmb.edu Abbreviations: 4-HNE, 4-hydroxy-2-trans-nonenal; GST, glutathione S-transferase; HLE B-3, human lens epithelial cell; CCL-75, human lung fibroblast cell; JNK, c-Jun N-terminal kinase; OG-dextran, Oregon green 488-dextran; GFP, green fluorescent protein; eGFP, enhanced green fluorescent protein; GS-HNE, glutathione conjugate of 4-HNE Enzyme: glutathione S-transferase (GST; EC 2.5.1.18) *These authors contributed equally to this work (Received 22 January 2004, revised 24 February 2004, accepted March 2004) Keywords: 4-hydroxy-2-trans-nonenal; glutathione S-transferase; lipid peroxidation; oxidative stress; transformation Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur J Biochem 271) 1691 respectively Surprisingly, the clonal lines of HLE B-3/ hGSTA4 transfectants overexpressing enzymatically active hGSTA4-4 acquired a transformed phenotype with time We then examined whether an adherent cell line other than HLE B-3 would also be affected by hGSTA4 transfection and exhibit a similar transformed phenotype Furthermore, to correlate specifically the effects of hGSTA4 transfection with the increased metabolism and depletion of 4-HNE, we investigated the effect of transfection with mutant hGSTA4 devoid of GST activity towards 4-HNE Finally, we compared the effect of microinjection of different GST isozymes from several species into HLE B-3 cells to rule out nonspecific effects of GST overexpression of active or mutant hGSTA4-4 protein The results show that lowering intracellular levels of 4-HNE by incorporation of active hGSTA4-4, by either transfection or microinjection, led to phenotypic transformation of attached cells into rounded, smaller cells which acquired immortality and grew rapidly in an anchorage-independent manner Transfection of HLE B-3 cells with p-Target-hGSTA4 expression vector HLE B-3 cells (2 · 105) at passage no 18 were plated in 60 mm dishes in complete growth medium When the cells reached nearly 80% confluency, the medium was changed, and the cells were transfected 3–4 h later with lg plasmid using the Profection mammalian transfection kit (Promega) according to the manufacturer’s protocol After h, the cells were treated with 10% dimethyl sulfamethoxazole in minimal essential medium for 30 s After dimethyl sulfamethoxazole shock, the cells were allowed to recover in complete growth medium for 48 h Stable transfectants were selected in 200 lgỈmL)1 G418 by the dilution method in 96 well plates Wells containing single cells were marked, and growth in these wells was monitored daily Expression of hGSTA4-4 protein was ascertained by Western blot analysis Site-directed mutagenesis of hGSTA4-4 Experimental procedures Cell culture HLE B-3 cells were a gift from U P Andley (Department of Ophthalmology and Visual Sciences, Washington University at St Louis, MO, USA) The cells were received on passage no 14 and were maintained in minimal essential medium containing 20% fetal bovine serum and 50 lgỈmL)1 gentamicin at 37 °C in 5% CO2 Human lung fibroblast cell line, CCL-75, obtained from ATCC (Manassas, VA, USA) was maintained in minimal essential medium containing 10% fetal bovine serum, mM sodium pyruvate and 10 mM nonessential amino acids Antibodies Polyclonal antibodies were developed against recombinant hGSTA4-4 in chicken as described previously [16] All other antibodies were from commercial sources Preparation of recombinant hGSTA4-4 and other GST isozymes hGSTA4-4 was expressed in Escherichia coli and purified as described previously [16] The purity of the enzyme was confirmed by SDS/PAGE; a single band at 26 kDa was recognized by hGSTA4-4 antibodies on Western blots Activity of the purified enzyme using 1-chloro-2,4-dinitrobenzene and 4-HNE as substrates was measured as described previously [6] Methods for preparation of recombinant GST isozymes mGSTA4-4 [14], Drosophila melanogaster DmGSTD1-1 [17], hGSTA1-1 [18] and hGSTP1-1 [19] have been described previously Preparation of hGSTA4-4 eukaryotic expression constructs The hGSTA4 ORF was amplified by PCR from the bacterial expression vector pET-30a[+]/hGSTA4, and subcloned into the pTarget-T mammalian expression vector (Promega) The hGSTA4 insert was confirmed by sequencing The Y212F mutation was introduced in both the bacterial and the mammalian hGSTA4-4 expression vectors using the Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) with the mutagenic sense primer 5¢-CCTGATGAATTTTCGTGAGAACCGT (mutation underlined) and the complementary antisense primer In this paper, hGSTA4-4(Y212F) is referred to as muthGSTA4-4 Immunohistochemical localization studies Immunofluorescence studies on adherent HLE B-3 and CCL-75 cells (wild-type, empty-vector-transfected and mut-hGSTA4-transfected) were carried out by seeding · 104 cells on to coverslips Next day, the coverslips with attached cells were washed in NaCl/Pi (pH 7.0) three times (5 each) and fixed in 4% paraformaldehyde solution prepared in NaCl/Pi (pH 7.4) for 15 at room temperature The fixed cells were washed three times with NaCl/Pi, permeabilized in cold methanol ()20 °C) for 30 s, treated with sodium borohydride (0.5 mgỈmL)1) for 15 to reduce aldehyde groups, and washed three times with NaCl/Pi (5 each) The cells were then incubated with blocking buffer (1% BSA + 1% goat serum in NaCl/Pi) for h at room temperature in a humidified chamber, and incubated with primary antibodies against hGSTA4-4 developed in chicken (1 : 200 dilution prepared in 1% BSA in NaCl/Pi) overnight at °C Cells were washed three times in NaCl/Pi and then incubated with Alexa fluor 488 fluorescein isothiocyanate-conjugated anti-chicken secondary IgG (Molecular Probes; : 200, diluted in 1% BSA in NaCl/Pi) for h at room temperature in a humidified chamber Cells were washed three times with NaCl/Pi, mounted on slides with 50% glycerol in NaCl/Pi, and visualized under a fluorescence microscope (Nikon Eclipse 600) The cells treated with preimmune chicken IgY were used as negative controls Slides for suspension culture of hGSTA4-transfected and transformed HLE B-3 cells were prepared by centrifuging the cells on polylysine-coated slides in a cytospin at 28 g Ó FEBS 2004 1692 R Sharma et al (Eur J Biochem 271) In situ detection of apoptosis To detect cells undergoing apoptosis during the course of microinjection experiments, we performed immunolocalization of cleaved caspase-3 by using monoclonal antibodies against cleaved caspase-3 After cytospinning the cells at 28 g for min, the cells were fixed in 4% paraformaldehyde (15 min) and washed three times in NaCl/Pi The cells were permeabilized by incubation in 0.1% Triton X-100 for min, washed with NaCl/Pi, treated with blocking buffer for h at room temperature in a humidified chamber, and then incubated with cleaved caspase-3 IgG (1 : 100 dilution prepared in 1% BSA) overnight at °C Cells were washed three times in NaCl/ Pi and then incubated with mouse tetramethyl rhodamine isothiocyanate-conjugated secondary antibodies (1 : 500) for h After the cells had been washed and mounted as described above, the expression of cleaved caspase-3, a marker of apoptosis, was ascertained by observing the cells under a fluorescence microscope Determination of intracellular levels of malondialdehyde and 4-HNE Lipid peroxide levels as determined by malondialdehyde and 4-HNE concentrations in hGSTA4-transfected and control HLE B-3 cells were determined using the Biotech LPO-586TM kit (Oxis International, Portland, OR, USA) according to the manufacturer’s protocol as described previously [6] SDS/PAGE and Western blot analysis For checking the expression of hGSTA4-4 by Western blot analysis, cells (1 · 106) were lysed in 10 mM potassium phosphate buffer, pH 7.0, containing 1.4 mM 2-mercaptoethanol, sonicated on ice for 30 s, and centrifuged at 28 000 g for 30 Buffer-soluble proteins (25 lg) present in the supernatants were mixed with Laemmeli’s sample buffer [20] and loaded in the wells of gels containing 12% polyacrylamide Proteins resolved on SDS/polyacrylamide gels were transferred to nitrocellulose or poly(vinylidene difluoride) membranes, and the blots probed by using hGSTA4-4 antibodies developed in chicken as primary antibodies, and secondary antibodies as horseradish peroxide-conjugated anti-chicken IgG developed in goat Blots were developed by West Pico-chemiluminescence’s reagent (Pierce) To check the expression of p53, transforming growth factor b1, cyclin-dependent kinase and protein kinase C bII proteins in HLE B-3 cells, Western blot analyses were performed by preparing whole cell extracts in RIPA buffer [20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P40, mM EDTA, mM NaF, mM sodium vanadate, mM phenylmethanesulfonyl fluoride and protease inhibitor cocktail (Sigma Chemical Co)] For these analyses extracts containing 100 lg protein were used for each sample Cell growth analysis The growth kinetics of HLE B-3 cells and their transfectants was measured both by manual cell count using a hemocytometer (after trypsinization in the case of adherent cells) and by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide analysis as described previously [18] Assay of soft agar colony formation This was performed as described previously [21] Briefly, 000 cells per dish, mixed in 0.35% agarose/complete medium, were seeded on to 0.7% agarose/complete medium bottom layer The Petri dishes were incubated at 37 °C and a drop of medium was added every days Four weeks later, cells were stained with 0.5% crystal violet (Sigma) in 20% methanol for h, and colonies were counted under a microscope Microinjection of cells Protein sample preparation Immediately before injection, the recombinant hGSTA4-4 protein (wild-type or mutant) was dialyzed against injection buffer (114 mM KCl, 0.5 mM K2HPO4 and 5.5 mM KH2PO4, pH 7.4) for 10 min, and brought to a concentration of mgỈmL)1 with injection buffer and a mgỈmL)1 stock solution of Oregon Green 488-dextran (OG-dextran; 70 kDa; Molecular Probes), bringing the injection samples to an OG-dextran concentration of 0.4 mgỈmL)1, a concentration used in previous studies that had no effect on cell viability and proliferation [22] The samples were then centrifuged at 10 000 g for 10 to remove large aggregates All steps of the sample preparation were performed at °C, and the samples kept on ice until injected into cells Samples of recombinant mGSTA4-4, DmGSTD1-1, hGSTA1-1 and hGSTP1-1 used for microinjection were prepared in an identical manner DNA sample preparation Both wild-type- and muthGSTA4-4 expression vectors were brought to a concentration of 20 copies per fL with injection buffer when injected as individual samples We had previously determined that optimal expression occurred with this concentration of DNA [22] For the experiments in which mGSTA4-4 expression vectors were coinjected with the green fluorescent protein (GFP) expression vector, all coinjected vectors were brought to a concentration of 40 copies per fL with injection buffer Just before injection, coinjected samples were mixed : bringing the coinjected vectors to a concentration of 20 copies of each vector per fL Just before injection of the vectors into cells, all samples were dialyzed against injection buffer for 10 min, and then centrifuged at 10 000 g for 10 at room temperature Glass-needle-mediated microinjection of proteins and DNA expression vectors HLE B-3 and CCL-75 cells were maintained as monolayer cultures as described above For the experiments performed in this study, HLE B-3 and CCL-75 cells were used at passage 18 On the day before each experiment, · 104 cells were plated in 35 mm2 tissue culture dishes (Corning) containing 1.5 mL medium Before plating of the cells, circles were etched into each of the dishes to facilitate subsequent identification of injected cells Injection needles were pulled from borosilicate capillaries using a Flaming/Brown Micropipette Puller, model P-97 Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur J Biochem 271) 1693 (Sutter Instrument Co., Novato, CA, USA) with a range of outer tip diameters of 2.5–3 lm, as determined by scanning electron microscopy [23] Phase contrast microscopy was used to visualize the injection procedure using an Olympus Corp (Melville, NY, USA) IX70 inverted microscope equipped with a temperature-controlled stage kept at 37 °C The cells were injected with either  fL sample containing protein (cytoplasmic injections, with OG-dextran as the marker of the injected cells) or sample containing DNA (nuclear injections, with GFP as the marker of the injected cells), using the electronically interfaced Eppendorf Micromanipulator (model 5171) and Transjector (model 5246) as described previously [23] All injections were performed manually, with each injection sample being injected into 75 cells per dish per experiment All experiments were repeated two or more times Only cells within an etched boundary were injected, to allow easy localization of the injected cells Single-cell assay of post-injection viability and GSinduced cell rounding or apoptosis Fluorescence microscopy (IX70 inverted microscope) was used to identify injected cells The percentage post-cytoplasmic and postnuclear injection viabilities were determined for both HLE B-3 and CCL-75 cells by calculating: (number of fluorescent cells 24 h after injection/75) · 100 Viabilities were determined from cells coinjected with either muthGSTA4-4 protein and OG-dextran or the mut-hGSTA4-4 expression vector expressing the mutant form of hGSTA4 (Y212F) and the GFP expression vector, or with fluorescent markers alone At 24 h after the injection, any cells killed by the injection procedure were lifted off the dish leaving only the injected cells that survived the injection Such cells were flat and attached to the dish as shown in Fig Stratagene enhanced GFP (eGFP) and OG-dextran fluorescent markers of injected cells as well as mutant hGSTA4-4 protein had no effect on post-injection viabilities The mean viability after nuclear injection into HLE B-3 and CCL-75 cells ranged from 40% to 70% To determine the effect of wildtype and mutant forms of GST on injected cells, all surviving HLE B-3 and CCL-75 cells were scored at 24 and 48 h and 24, 48, and 72 h, respectively, as being either flat, round or apoptotic The mean percentage of the injected cells showing the above morphologies was calculated with data from three or more experiments for each injection sample at each time point Results E1, were selected Initially, hGSTA4-transfected cells grew normally in monolayers (Fig 1A, b) with a doubling time identical with that of empty-vector-transfected cells However, four weeks after transfection (two passages) during their clonal selection in medium containing G418, cells stopped proliferating and some began to enlarge (Fig 1A, c) Even though the growth medium was changed every 72 h, the cells remained in a quiescent state for the next four weeks Eight weeks after transfection, cells originating from clones C4, D7 and E1 started to transform their shape, as was apparent from the characteristic budding of round cells from giant cells A typical example of this transformation is shown in Fig 1A, d The transformed round cells becoming anchorage-independent (Fig 1A, e) continued to express higher levels of hGSTA4-4 (Fig 1B, a-p and a-f), and had lower levels of 4-HNE (Fig 1C) To date, these cells have undergone about 365 doublings in suspension cultures, with no cells becoming senescent, a property characteristic of cancer-derived cell lines, e.g human erythroleukemic (K562) and small cell lung cancer (H69) cell lines The HLE B-3/hGSTA4 anchorage-independent cells had a significantly shorter doubling time than wild-type-transfected and empty-vector-transfected HLE B-3 cells (20 ± 3.4 h vs 50 ± 4.3 h) hGSTA4-4 expression and 4-HNE levels in transfected cells The expression of hGSTA4-4 in stably transfected cells was confirmed by Western blots, which showed no detectable expression of hGSTA4-4 in the wild-type-transfected or empty-vector-transfected HLE B-3 cells, but a strong band in hGSTA4-transfected cells (Fig 1D) All three clones (C4, D7 and E1) continued to express high levels of enzymatically active hGSTA4-4 and showed similar effects of hGSTA4 transfection on their phenotype with a significant reduction in intracellular 4-HNE levels Most of the data presented here were obtained using the representative clone C4 Although there was detectable constitutive GST activity towards 4-HNE in WT- HLE B-3 cells, this activity was about sixfold higher in the transfected cells [1.5 vs 9.7 nmol 4-HNE consumedỈmin)1Ỉ(mg protein))1], indicating successful expression of enzymatically active hGSTA4-4 in transfected cells The 4-HNE level in clone C4 used for these studies was found to be 40 ± 8% of that observed in the wild-type-transfected or empty-vector-transfected HLE B-3 cells (Fig 1C) These results further confirm overexpression of active hGSTA4-4 in the transfected cells Effect of transfection of HLE B-3 cells with hGSTA4 Anchorage-independent growth The HLE B-3 cell line was originally developed after infection with adenovirus (Ad12-SV40) [24] and is referred to here as WT-HLE B-3 These cells have been reported to be relatively resistant to oxidative stress [25], grow in monolayers (Fig 1A, a) with a population doubling time of 48–52 h, and become senescent after 76 population doublings [24] Keeping this in view, we used WT-HLE B-3 cells with low passage numbers (passages 18–20) for these studies WT-HLE B-3 cells were transfected with a eukaryotic expression vector containing hGSTA4 cDNA, and three clones overexpressing hGSTA4-4, designated C4, D7 and The anchorage-independent growth of phenotypically transformed cells was confirmed by assay of soft agar colonies [26] Clone C4 cells grew into colonies within weeks of plating, while WT-HLE B-3 cells did not form detectable colonies (data not presented) The colonyforming ratio of clone C4 (HLE B-3) cells to WT-K562 cells used as positive control in these experiments was found to be : Taken together, these results confirm the phenotypic transformation of WT-HLE B-3 cells to anchorage-independent growth on stable transfection with hGSTA4 1694 R Sharma et al (Eur J Biochem 271) Ó FEBS 2004 Fig Phenotypic transformation and biochemical characterization of hGSA4-transfected cells (A) Phenotypic transformation of hGSA4-transfected cells (a) Control WT-HLE B-3 cells; (b) HLE B-3 cells weeks after hGSTA4 transfection; (c) growth arrest and enlargement of HLE B-3 cells weeks after transfection; (d) budding of rounded cells from giant cells weeks after transfection; (e) anchorage-independent growth of transformed rounded cells (B) Transfection of HLE B-3 with WT-hGSTA4 and Y212F mutant hGSTA4 (mut-hGSTA4) with no activity towards 4-HNE: (a-p) a typical phase contrast micrograph of transformed cells after transfection with WT-hGSAT4; (a-f) fluorescence micrograph showing expression of WT-hGSTA4-4 protein in transformed cells detected immunohistologically using hGSTA4-4 antibodies; (m-p) phase contrast micrograph of cells weeks after transfection with mut-GSTA4; (m-f) fluorescence micrograph showing expression of mut-hGSTA4-4 protein in transfected cells (C) 4-HNE levels in HLE B-3 cells (D) Expression of hGSTA4-4 protein in transfected cells as detected by Western blots: lane 1, cells transfected with hGSTA4 Y212F mutant; lane 2, WT-HLE B-3 cells; lane 3, cells transfected with hGSTA4; lane 4, positive control of hGSTA4-4 Details for transfection, immunofluorescence studies, Western blots and 4-HNE determination are given in Experimental procedures Effect of transfection with enzymatically inactive mutant hGSTA4 To establish whether the observed phenotypic changes were specifically due to depletion of 4-HNE because of high activity of hGSTA4-4 towards 4-HNE in the transfected cells or to some unknown effect of transfection, we prepared a mutant cDNA of hGSTA4-4 isozyme in which Tyr212 was replaced with phenylalanine Consistent with the previous studies [13], recombinant mutant hGSTA44(Y212F) had only  3% of the activity towards 4-HNE compared with WT-hGSTA4-4 [1.9 vs 72 lmol 4-HN min)1Ỉ(mg protein))1] There was no noticeable change in morphology of the cells tarnsfected with mutant hGSTA4 (Y212F) even after six passages (Fig 1B, m-p) Despite high expression of mutant protein as indicated by immunolocalization (Fig 1B, m-f) and Western blot studies (Fig 1D, lane 1) using hGSTA4-4 antibodies, there was no significant change in either their GST activity towards 4-HNE or the steady-state levels of 4-HNE compared with those of WTHLE B-3 cells (Fig 1C) These results strongly suggest that overexpression of enzymatically active hGSTA4-4 resulting in accelerated metabolism of 4-HNE and thereby lowering of the intracellular concentrations of 4-HNE leads to the observed phenotypic transformation and immortalization of WT-HLE B-3 cells Microinjection of the active hGSTA4-4 induces similar phenotypic changes We also studied the effects of direct microinjection of the active hGSTA4-4, its inactive mutant, and their expression vector counterparts into WT-HLE B-3 cells To monitor the microinjection of active or inactive hGSTA4-4 recombinant protein, the cells were coinjected with OG-dextran, a fluorescent marker, as detailed in the legend of Fig 2A The Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur J Biochem 271) 1695 Fig Microinjection of active WT-hGSTA4-4, inactive mutant hGSTA4-4 recombinant protein or the respective expression vector into HLE B-3 cells (A) (a-p) Phase contrast micrograph of a typical transformed HLE B-3 cell 24 h after cytosolic microinjection with WT-hGSTA4-4 protein; (a-f) fluorescence micrograph of same cell showing fluorescent marker, OG-dex, coinjected with WT-hGSTA4-4; (m-p) phase contrast micrograph of a typical HLE B-3 cell after microinjection with inactive mut-hGSTA4-4 protein; (m-p&f) combined phase contrast micrograph and fluorescence micrograph of same cell indicating delivery of OG-dex marker Bar denotes 30 lm (B) (a-p&f) Combined phase contrast micrograph and fluorescence micrograph of a typical transformed HLE B-3 cell 24 h after nuclear microinjection with WT-hGSTA4 and the marker eGFP cDNAs; (a-f) fluorescence micrograph of same HLE B-3 cell (fluorescence represents expression of eGFP); (m-p) phase contrast micrograph of typical HLE B-3 cells 24 h after microinjection with inactive mut-hGSTA4 and eGFP cDNAs; (m-f) fluorescent micrograph of same cells (C) A small fraction of microinjected cells undergo apoptosis (p) phase contrast micrograph of cell undergoing apoptosis; (f) fluorescence micrograph of same cell These cells could be distinguished from the transformed cells as they were not fully rounded and expression of fluorescent marker eGFP was minimal (D) Quantitation of transformed (unfilled bars), nontransformed (grey bars) and apoptotic cells (black bars) after microinjection with WT-hGSTA4 or mut-hGSTA4 expression vectors Details of microinjection and immunofluorescence experiments are given in Experimental rocedures cells were monitored from 12 to 48 h after injection After 24 h, cells injected with active protein began to round up and detach (Fig 2A, a-p and a-f), whereas those injected with OG-dextran and inactive protein remained flat and attached (Fig 2A, m-p and m-p & f) There was a significant increase in the percentage (52.5 ± 5%; mean ± SD) of the round cells in active protein-injected cells over the first 48 h after injection (data not presented) In contrast, in the cells injected with the mutant hGSTA4-4 protein, only ± 2.5% of the cells were rounded, which was similar to the level observed in OG-dextran mock-injected cells (data not presented) In the experiments for microinjecting WT-hGSTA4 and the inactive mut- hGSTA4 (Y212F) cDNA into HLEB-3 cells, the expression vector of eGFP was used as a marker for successful microinjection As shown in Fig 2B, a-p and a-f, microinjection of WT-hGSTA4 cDNA led to characteristic rounding and anchorage-independent growth within 24 h Cells microinjected with mut-hGSTA4 cDNA maintained their original phenotype and did not undergo any change (Fig 2B, m-p and m-f) A small but clearly noticeable number of cells underwent apoptosis after microinjection The apoptotic cells could be identified, as they showed activation of caspase-3 detected by staining the cells with antibodies to cleaved caspase-3 (data not shown) and loss of fluorescence due to extrusion of cytoplasm These cells could be easily distinguished from the rounded, transformed cells As shown in Fig 2C, these cells were not fully rounded and showed only minimal fluorescence of eGFP which was prominent in rounded, transformed cells The percentages of unchanged flat cells, transformed rounded cells, and apoptotic cells after 24 h and 48 h of microinjection of WT-hGSTA4 and mut-hGSTA4 cDNA in a typical experiment are given in Fig 2D Together, these results further indicate that overexpression of active hGSTA4-4 is required for phenotypic transformation Only GST isozymes that have high catalytic efficiency with 4-HNE have transforming activity To further establish that high hGSTA4-4 activity was required for its transforming activity, we microinjected four different GST isozymes into HLE B-3 cells For these experiments, two GST isozymes with high activity and two Ó FEBS 2004 1696 R Sharma et al (Eur J Biochem 271) Fig Cytoplasmic microinjection of recombinant mGSTA4-4 and DmGSTD1-1 in HLE B-3 cells Cells were microinjected with the respective protein as described in Experimental procedures and scored 24 h and 48 h after injection (A) (p) Phase contrast micrograph of a typical transformed cell 24 h after microinjection with mGSTA4-4; (f) fluorescence micrograph of same cell showing fluorescent marker OG-dex coinjected with mGSTA4-4 (B) (p) Phase contrast micrograph of a typical transformed cell 24 h after microinjection with DmGSTD1-1; (f) fluorescence micrograph of same cell showing fluorescent marker OG-dex coinjected with DmGSTD1-1; (C) Bar graph showing percentage of nontransformed (flat; black bars), transformed (rounded; light grey bars) and apoptotic (dark grey bars) cells 24 h and 48 h after microinjection with minimal activity toward 4-HNE were selected Mouse enzyme mGSTA4-4 [14] and Drosophila enzyme DmGSTD1-1 [17] are known to have high activities for 4-HNE (specific activities: 65 mg)1 and 32 mg)1, respectively) On the other hand, human enzymes hGSTA1-1 and hGSTP1-1 have minimal activity towards 4-HNE [27] These results show that mGSTA4-4 and DmGSTD1-1 (Fig 3) trigger transformation and hGSTA1-1 and hGSTP1-1 (Fig 4) not A phase contrast micrograph and fluorescent micrograph of a typical transformed cell 24 h after microinjection of mGSTA4-4 or DmGSTD1-1 are presented in Fig 3A and Fig 3B, respectively Results presented in Fig 3C indicate that most microinjected cells are transformed within 48 h In contrast, results presented in Fig show that cells microinjected with either hGSTA11 or hGSTP1-1 retain their original phenotype and not undergo transformation These results further support the idea that the ability of the GST isozymes to induce transformation is dependent on their ability to conjugate 4-HNE to GSH Furthermore, these results argue against the possibility of a nonspecific effect of hGSTA4-4 causing the transformation Effect of hGSTA4-4 overexpression in the CCL-75 cell line The effect of hGSTA4-4 overexpression was also examined in a human lung fibroblast cell line, CCL-75, a nonviral transformed adherent cell line with a finite lifetime of 50 ± 10 population doublings [28] In these experiments, when CCL-75 cells were microinjected with active and mutant hGSTA4-4 proteins in a manner similar to WTHLE B-3 cells, comparable results were observed (Fig 5A– D) Interestingly, cell rounding was observed in CCL-75 cells 48 h after the microinjection of active protein, a delay of nearly 24 h compared with WT-HLE B-3 cells The reasons for this time lag are not clear These results also show that direct injection of active hGSTA4-4 protein or its cDNA into attached cells causes a characteristic transformed phenotype and further suggest that overexpression of hGSTA4-4 leading to such transformation may be a generalized phenomenon Effect of hGSTA4-4 on key cell-cycle genes We also studied the effects of the hGSTA4-4 expression on some of the key genes involved in cell-cycle regulation and apoptosis In the hGSTA4-transfected and phenotypically transformed, anchorage-independent HLE B-3 cells, we found upregulation of transforming growth factor, cyclindependent kinase 2, protein kinase C bII, and extracellular regulatory stress kinase vs downregulation of p53 (Fig 6) These observations are consistent with the idea that lowering the intracellular concentrations of 4-HNE upregulated genes involved in promotion of proliferation and Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur J Biochem 271) 1697 Fig Cytoplasmic microinjection of recombinant hGSTA1-1 and hGSTP1-1 in HLE B-3 cells (A) Typical cells 48 h after microinjection of hGSTA1-1 (p) Phase contrast micrograph showing that cells maintained their original phenotype; (f) fluorescence micrograph of same cells showing OG-dex fluorescent marker (B) Typical cells 48 h after microinjection of hGSTP1-1 (p) Phase contrast micrograph and (f) fluorescence micrograph of same cells (C) Bar graph showing percentage of nontransformed (flat; black bars) and transformed (rounded; light grey bars) cells 24 h and 48 h after microinjection downregulated genes (such as p53) that control the cell cycle and are pro-apoptotic [29] Discussion Previous studies strongly suggest that intracellular 4-HNE can influence signaling mechanisms and, depending on its concentration, can promote apoptosis [3,6,7,12], differentiation [12], or proliferation [11,12] of cells GSTs in general, and hGSTA4-4 and hGST5.8 in particular, are the major 4-HNE-metabolizing enzymes in humans [16] Dramatic phenotypic transformation of attached cells on transfection with hGSTA4-4 into smaller rounded immortalized cells which grow rapidly in suspension is surprising but it seems to be consistent with numerous previous studies suggesting that 4-HNE is involved in cell-cycle signaling mechanisms Our results show that transfection or microinjection of cells with enzymatically active hGSTA4-4 causes the emergence of the transformed phenotype, whereas hGSTA4-4(Y212F), a mutant with decreased activity for 4-HNE [13], is unable to transform cells This result provides a reasonable basis for proposing the hypothesis that the observed transformation of HLE B-3 and CCL-75 cells is a consequence of its conjugation of 4-HNE, rather than being linked to other possible effects of hGSTA4-4, such as a hypothetical direct binding to signaling kinases, as has been described for Pi-class GSTs [30] To test further the hypothesis that 4-HNE conjugation is relevant to cellular transformation, we microinjected cells with two additional GSTs which are known to metabolize 4-HNE but are structurally distinct and phylogenetically distant from hGSTA4-4 The murine enzyme mGSTA4-4 has a catalytic efficiency for 4-HNE that is similar to that of hGSTA4-4 [14] However, antibodies against one enzyme not cross-react with the other [16], indicating that at least parts of the surface of the two proteins differ substantially from each other The second microinjected protein was DmGSTD1-1 from D melanogaster [31] This Delta-class insect GST also has a relatively high catalytic efficiency for 4-HNE conjugation [17] Insects and mammals diverged at least 600 million years ago [32], and hGSTA4-4 is only 22%/40% identical/similar to DmGSTD1-1 Thus, it is unlikely that DmGSTD1-1 could replace hGSTA4-4 in any putative regulatory protein– protein interactions in which hGSTA4-4 may be involved Our studies clearly show that microinjection of hGSTA4-4, mGSTA4-4, and DmGSTD1-1 triggers cell transformation whereas microinjection of hGSTA4-4(Y212F), hGSTA1-1, and hGSTP1-1 does not The three proteins able to transform cells are structurally dissimilar but are all efficient at conjugating 4-HNE, whereas those that lack 4-HNEconjugating activity also fail to transform cells, even if they are structurally almost identical with an active enzyme, as in the case of hGSTA4-4(Y212F) Together, these results point to the conjugative metabolism of 4-HNE as the common denominator and the causative principle in the transformation process, and suggest that the level of 4-HNE or its glutathione conjugate (GS-HNE) is the 1698 R Sharma et al (Eur J Biochem 271) Ó FEBS 2004 Fig Microinjection of WT-hGSTA4-4, mut-hGSTA4-4 recombinant protein or respective expression vector in CCL-75 cells (A) (a-p) Phase contrast micrograph of a typical transformed CCL-75 cell 48 h after cytoplasmic microinjection with WT-hGSTA4-4 recombinant protein and OG-dex marker; (a-f) fluorescence micrograph of same cell (fluorescence indicates delivery of the marker OG-dex); (b-p) phase contrast micrograph of a typical transformed CCL-75 cell 48 h after nuclear microinjection of expression vectors of WT-hGSTA4 and eGFP cDNAs; (b-f) fluorescence micrograph of same cell (fluorescence indicates expression of the marker eGFP); (c-p) phase contrast micrograph of a typical CCL-75 cell 48 h after cytoplasmic microinjection of mut-hGSTA4-4 protein and the marker OG-dex; (c-f) fluorescence micrograph of same cell (fluorescence indicates the marker OG-dex); (d-p) phase contrast micrograph of a typical CCL-75 cell 48 h after nuclear microinjection of mut-hGSTA4 and eGFP cDNAs; (d-f) fluorescence micrograph of same cell (fluorescence indicates expression of the marker eGFP) Details are provided in Experimental procedures Bar represents 30 lm (B–D) Bar graphs showing percentage of transformed (rounded; unfilled bars), unaffected (flat; dark grey bars) and apoptotic (black bars) cells after cytoplasmic microinjection of recombinant protein of active WT-hGSATA4-4 (B) or inactive mut-hGSTA4-4 (C) and nuclear microinjection of expression vectors (D) most likely effector of the cell transformation we observed This is consistent with a lower 4-HNE level in cells overexpressing hGSTA4-4 but not in cells overexpressing the mutant Y212F, which is ineffective in triggering transformation Although a hypothetical substrate other than 4-HNE, perhaps another Michael acceptor, cannot be ruled out at present, 4-HNE is the only currently known common physiological substrate of proteins as different as hGSTA44 and DmGSTD1-1 However, the correlation of 4-HNEconjugating activity with the ability to transform cells which holds for six different proteins [hGSTA4-4, hGSTA44(Y212F), mGSTA4-4, DmGSTD1-1, hGSTA1-1, and hGSTP1-1] indicates that a causal involvement of 4-HNE in the mechanism of hGSTA4-4-mediated transformation of HLE B-3 and CCL-75 cells provides the simplest explanation of all the available experimental data Binding of GSTs, particularly hGSTP1-1 with c-Jun N-terminal kinase (JNK), modulates stress-mediated signaling for apoptosis In monomeric form, hGSTP1-1 binds to JNK and inhibits its activation, but under conditions of stress such as exposure to UV or H2O2 treatment, it oligomerizes and dissociates from the JNK complex leading to abrogation of JNK inhibition [30] Such interactions of hGSTA4-4 with JNK or other key kinases may also be considered as the mechanistic basis for the observed transformation However, the inability of mutant hGSTA4-4(Y212F) to induce transformation argues against such a possibility because GSTP1-1 with a mutation in its active site is still able to prevent JNK activation An effector domain critical for its binding to JNK (residues 191–201) has been identified in hGSTP1-1 [33], and it seems unlikely that mutation of a single active-site residue (Y212F) would abrogate the binding of hGSTA4-4 to kinases Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur J Biochem 271) 1699 Fig Expression of genes known to be involved in cell-cycle regulation (A,B) HLE B-3 cells (1 · 106) transfected with wild-type, empty vector or hGSTA4 were lysed in RIPA buffer containing protease inhibitor cocktail, mM phenylmethanesulfonyl fluoride and mM sodium orthovanadate The cell extracts were centrifuged at 15 000 g at °C Supernatant containing 100 lg protein was loaded in each well and subjected to Western blot analysis using antibodies against proteins identified in the left hand margins of (A) and (B) Lanes 1, 2, and in both panels represent extracts of HLE B-3 cells transfected with wild-type, empty vector and hGSTA4, respectively (C) For comparison of expression of extracellular signal regulated kinase 1/2, cells (1 · 106) from clone C4 (A4-4) and empty-vector-transfected (VT) HLE B-3 cells were serum starved for 24 h in separate Petri dishes and then treated with serum-containing medium (10%) for different times The cells were centrifuged at 654 g (5 min), their extracts were prepared in RIPA buffer as described in Experimental procedures, and Western blot analyses were performed using antibodies against extracellular signal regulated kinase 1/2 Lane 1, extract of cells before serum stimulation; lanes 2–6, extracts of the cells after treatment with 10% serum for 2, 5, 10, 15 and 30 min, respectively Furthermore, GSTP1-1-mediated activation of JNK does not appear to be applicable to all cell types because, in a human lung fibroblast cell line, GSTP1-1 does not affect JNK activation [34] We show that neither hGSTP1-1 nor hGSTA1-1 cause transformation but mouse and Drosophila 4-HNE-metabolizing GST isozymes (mGSTA4-4 and DmGSTD1-1, respectively) show transforming activity comparable to that of hGSTA4-4 Thus, transformation does not appear to be due to interaction of hGSTA4-4 with signaling kinases but seems to be linked to its catalytic ability to conjugate 4-HNE to GSH GSTA4-4-catalyzed conjugation of 4-HNE to GSH results in the formation of GS-HNE An increase in the level of GS-HNE may also be a trigger of transformation Previous studies have shown that overexpression of 4-HNEmetabolizing GST isozymes leads to accelerated formation of GS-HNE in cells As confirmed by identification and quantification of intact GS-HNE in the medium of cells loaded with radioactive GS-HNE, most GS-HNE thus formed is transported out of the cells through ATPdependent transport processes [6,7] However, a significant proportion of intracellular GS-HNE can be metabolized to the corresponding alcohol, glutathionyl dihydroxynonene formed through NADPH-dependent reduction of GS-HNE catalyzed by aldose reductase [35] In addition, GS-HNE can also be converted into mercapturic acids, which can then be x-hydroxylated by CYP-450 [36] to yield more hydrophilic products The possibility of GS-HNE or its metabolites being involved in the mechanisms of the observed transformation phenomenon is not ruled out by the present studies Aldose reductase, which can reduce GS-HNE, has been shown to mediate mitogenic signaling in vascular smooth muscle cells [37], and its inhibitors have been shown to inhibit tumor necrosis factor-a-induced apoptosis and caspase-3 activation [38] Channeling of 4-HNE towards accelerated formation of GS-HNE in hGSTA4-4-overexpressing cells may abruptly change the overall ÔphysiologicÕ homeostasis of 4-HNE, GS-HNE, and its metabolites maintained by a co-ordinated action of 4-HNE-metabolizing enzymes including GSTs [16], aldose reductase [35], aldehyde dehydrogenase [39], and transporters of GS-HNE [6,7] According to this interpretation, it is not just the concentration of 4-HNE but also changes in the homeostasis of 4-HNE and its metabolites that provides the mechanistic basis for the transformation This possibility needs to be explored in future studies Our results show that some of the more prominent genes suggested to be involved in promoting proliferation are upregulated in hGSTA4-4-overexpressing HLE B-3 cells This, along with almost complete suppression of p53, may account for the observed threefold higher growth rate of the transformed cells Our studies are limited to evaluating the expression of only a few key genes known to be involved in cell-cycle events An assessment of the effect of transfection with hGSTA4-4 and 4-HNE depletion on global gene expression using cDNA microarrays is planned for the future Taken together with the results of previous studies showing that at higher concentrations, 4-HNE causes apoptosis [3,6,7,12] and differentiation [12,40], the present results suggest that the intracellular level of 4-HNE may be one of the determinants for leading cells towards pathways for transformation, differentiation, proliferation, or apoptosis The mechanisms through which 4-HNE affects signaling processes in a concentration-dependent manner are obscure and appear to be complex 4-HNE is a strong electrophile which reacts with nucleophilic groups of proteins [41,42], nucleic acids [43,44], and lipids [45] It interacts with thiols Ó FEBS 2004 1700 R Sharma et al (Eur J Biochem 271) and also with nucleophilic nitrogen atoms in proteins and phospholipids, perhaps with varying affinity It may be postulated that, at low concentrations, 4-HNE may selectively affect pathways favoring proliferation by interacting with nucleophilic groups that have high affinity for the compound On the other hand, at higher concentrations of 4-HNE, the effects of these interactions may be overwhelmed by reactions with low affinity groups of cellular nucleophiles to trigger the pathways favoring apoptosis This speculation lacks direct experimental evidence but is consistent with the previous studies showing that bI and bII isoforms of phosphoinositide-specific protein kinase C in several cell types are activated by submicromolar levels but inhibited by higher levels of 4-HNE [9] Further studies on the possible chemical interaction of 4-HNE with cellular nucleophiles including proteins, nucleic acids and lipids and possible correlation between these interactions and signaling cascades may provide clues to the mechanisms by which 4-HNE affects signaling events 10 11 12 Acknowledgements Supported in part by NIH grants EY 04396 (to Y C A.), CA77495 (to S A.), ES 07804 (to P Z.) and CA 76348 (to S V S.) 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CCL-75 cells ranged from 40% to 70% To determine the effect of wildtype and mutant forms of GST on injected cells, all surviving HLE B-3 and CCL-75 cells were scored at 24 and 48 h and 24, 48, and. .. fluorescence of eGFP which was prominent in rounded, transformed cells The percentages of unchanged flat cells, transformed rounded cells, and apoptotic cells after 24 h and 48 h of microinjection of WT-hGSTA4