Constitutive expression of ectopic c-Myc delays glucocorticoid-evoked apoptosis of human leukemic CEM-C7 cells.

Sensitivity to glucocorticoid (GC)-evoked apoptosis in lymphoid cell lines correlates closely with GC-mediated suppression of c-Myc expression. To establish a functional role for c-Myc in GC-mediated apoptosis, we have stably expressed MycER(TM), the human c-Myc protein fused to the modified ligand-binding domain of the murine estrogen receptor alpha, in GC-sensitive CEM-C7-14 cells. In CEM-C7-14 cells, MycER(TM) constitutively imparts c-Myc functions. Cells expressing MycER(TM) (C7-MycER(TM)) exhibited a marked reduction in cell death after 72 h in 100 nM dexamethasone (Dex), with 10-20-fold more viable cells when compared to the parental CEM-C7-14 clone. General GC responsiveness was not compromised, as evidenced by Dex-mediated suppression of endogenous c-Myc and cyclin D3, and induction of c-Jun and the glucocorticoid receptor. MycER(TM) also blunted Dex-mediated upregulation of p27(kipI) and suppression of the Myc target p53. In comparison to parental CEM-C7-14 cells, Dex-evoked DNA strand breaks were negligible and caspase activation was delayed, but the extent of G1 cell cycle arrest was similar in C7-MycER(TM) cells. Myc-ER(TM) did not result in permanent, complete resistance to GC however, and the GC-treated cells eventually died, indicative of redundant or interactive mechanisms in the GC-evoked lytic response of lymphoid cells. Our results emphasize the importance of c-Myc suppression in GC-evoked apoptosis of CEM-C7-14 cells.

Glucocorticoid mediated transcriptional repression of c-myc in apoptotic human leukemic CEM cells.

Suppression of c-myc has been implicated as a critical event in some glucocorticoid-evoked apoptotic systems. It is therefore of interest to understand the mechanism of glucocorticoid-regulation of the c-myc gene. In the present study, a detailed analysis of dexamethasone (Dex)-evoked regulation of the human c-myc gene in human leukemic CEM-C7 cells has been performed. Dex suppresses c-myc mRNA and immunoreactive protein expression in clone CEM-C7 and subclone CEM-C7-14 cells. Nuclear run-on assays suggested that the regulation occurred at the level of transcription initiation. The half-life of c-myc mRNA was approximately 30 min and its stability was not affected by Dex treatment. In addition, Dex suppressed luciferase gene expression driven by -2052 to +34 bp c-myc promoter in transfected CEM-C7-14 cells. This result further supports that c-myc gene is suppressed by Dex at the transcriptional level in apoptotic human leukemic cells.

Hormonal regulation of physiological cell turnover and apoptosis.

Physiological cell turnover plays an important role in maintaining normal tissue function and architecture. This is achieved by the dynamic balance of cellular regeneration and elimination, occurring periodically in tissues such as the uterus and mammary gland, or at constant rates in tissues such as the gastrointestinal tract and adipose tissue. Apoptosis has been identified as the prevalent mode of physiological cell loss in most tissues. Cell turnover is precisely regulated by the interplay of various endocrine and paracrine factors, which modulate tissue and cell-specific responses on proliferation and apoptosis, either directly, or by altering expression and function of key cell proliferative and/or death genes. Although recent studies have provided significant information on specific tissue systems, a clearly defined pathway that mediates cell turnover has not yet emerged for any tissue. Several similarities exist among the various tissues with regard to the intermediates that regulate tissue homeostatis, enabling a better understanding of the general mechanisms involved in the process. Here we review the mechanisms by which hormonal and cytokine factors mediate cell turnover in various tissues, emphasizing common themes and tissue-specific differences.

Structure-apoptotic potency evaluations of novel sterols using human leukemic cells.

Three oxidized analogs of cholesterol have been characterized for their ability to cause apoptotic cell death in CEM-C7-14 human leukemic cells. In addition to testing 15-ketocholestenol (K15), 15-ketocholestenol hydroxyethyl ether (CK15), and 7-ketocholesterol hydroxyethyl ether (CK7), an oxysterol of known apoptotic response, 25-hydroxycholesterol (25OHC), served as a standard for comparison. Growth studies based on dye exclusion by viable cells while using a sublethal concentration of oxysterols ranked their potency for cell kill as 25OHC > K15 > CK15 > CK7. Both the TUNEL assay (terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling), which quantifies the amount of DNA nicks caused by a toxic agent, and the MTT assay, which measures cell metabolism and thus reflects cell viability, substantiated the same rank order. An ELISA assay for evaluating release of DNA fragments into the cytosol after treatment gave a similar potency order. The oncogene c-myc mRNA was suppressed by all three oxysterols, with 25OHC and K15 being the most potent suppressors. Hoechst and Annexin V staining documented that these oxysterols kill cells by an apoptotic pathway as evidenced by condensation of nuclear chromatin and plasma membrane inversion, respectively. From these in vitro studies, we believe that 25OHC, K15, and possibly CK15 have the potential to be chemotherapeutic agents.

The delayed induction of c-jun in apoptotic human leukemic lymphoblasts is primarily transcriptional.

Because of their ability to induce lymphoid cell apoptosis, glucocorticoids have been used for decades to treat certain human leukemias and lymphomas. Studies presented in this paper complement our previous work demonstrating that sustained induction of the proto-oncogene c-jun plays a crucial role in the glucocorticoid-induced apoptotic pathway in CEM cells, human leukemic lymphoblasts. Results from measurements of c-jun mRNA half-life with RNase protection assays and of transcription by nuclear run-on assays indicate that, in the dexamethasone-sensitive cloned CEM-C7 cells, c-jun is induced at the transcriptional level. Consideration of time-course, however, suggested that this might be a secondary or possibly a delayed primary response. Use of cycloheximide to block protein synthesis strongly induced c-jun mRNA, suggesting that there had been relief from a labile protein repressor of transcription. Comparing the level of induction by cycloheximide with that of dexamethasone indicated that the two did not induce by an identical mechanism. The high induction by cycloheximide obscured simple interpretation of elevated c-jun mRNA levels after concomitant administration of cycloheximide and dexamethasone. This was resolved by nuclear run-on experiments, which showed that the dexamethasone induction of c-jun mRNA in this system does require protein synthesis.

Glucocorticoids, oxysterols, and cAMP with glucocorticoids each cause apoptosis of CEM cells and suppress c-myc.

In clones of the CEM human acute lymphoblastic leukemic cell line, glucocorticoids, oxysterols and activators of the cAMP pathway acting synergistically with glucocorticoids, each can cause apoptotic cell death. Morphologically and kinetically, these deaths resemble one another. The kinetics are striking: in each case, after addition of the lethal compound(s), an interval of approximately 24 h follows, during which cell growth continues unabated. During this "prodromal" period, removal of the apoptotic agent leaves the cells fully viable. We hypothesize that a sequence of biochemical events occurs during the prodrome which eventually results in the triggering of the full apoptotic response as evidenced by the activation of caspases and DNA fragmentation. At some point, the process is irreversible and proceeds relatively rapidly to cell death. Suppression of c-Myc seems a universal early event evoked by each of these lethal compounds or combinations, and we conclude that the negative regulation of this proto-oncogene is an important aspect of the critical pre-apoptotic events in these cells.

Resistance of human leukemic CEM-C1 cells is overcome by synergism between glucocorticoid and protein kinase A pathways: correlation with c-Myc suppression.

Glucocorticoids (GCs) induce apoptosis in lymphoid cells that contain functional GC receptors (GRs). However, GC resistance often is seen in cells with demonstrable GRs; one such line is CEM-C1. We have tested the hypothesis that positive interactions between GC and cyclic AMP (cAMP) regulate GC actions in CEM clones. Treatment of both GC-resistant CEM-C1 [resistant to 1 microM dexamethasone (Dex)] and the sensitive sister clone, CEM-C7 (approximately 65% cell death with 20 nM Dex, approximately 99% death with 1 microM Dex), with a < or = 20 microM concentration of the protein kinase A activator, forskolin, had no significant effect on cell viability. Cotreatment with Dex and forskolin resulted in a strong synergistic death response, with only approximately 10% CEM-C1 cells surviving treatment with 1 microM Dex and 20 microM forskolin. This death was blocked by the GR antagonist RU 38486. However, the extent of apoptosis did not correlate with the amount of GR protein or binding activity in either C7 or C1 cells. As reported previously, Dex-evoked cell death was associated with suppression of c-Myc in C7 cells. In CEM-C1 cells, Dex alone did not affect c-Myc; however, Dex plus forskolin suppressed c-Myc levels. To evaluate mechanisms of Dex-forskolin synergism, fresh subclones of CEM-C7 (clone 14) and CEM-C1 (clone 15) were isolated, to ensure purity of phenotype. In these, forskolin (with or without Dex) caused a similar increase in cAMP (approximately 300-fold) and phospho-cAMP-responsive element binding protein (approximately 4-5-fold) levels, whereas total cAMP-responsive element binding protein expression was not affected. GR transcription function, as tested from a GR-responsive 330-bp mouse mammary tumor virus promoter-luciferase reporter construct, was induced 8- and 4-fold by 1 microM Dex treatment of CEM-C7-14 and CEM-C1-15 cells, respectively. Forskolin (10 microM) significantly potentiated Dex response in CEM-C1-15 cells (13.5-fold) but had only a modest effect (1.5-fold) in CEM-C7-14 cells. These studies suggest that sensitization of CEM-C1 cells by cross-talk between GR and protein kinase A pathways may occur via cooperative effects on GR-mediated gene transcription.

Agonist-specific modulation of glucocorticoid receptor-mediated transcription by immunosuppressants.

Although the immunosuppressive drugs FK506, rapamycin and cyclosporin A have been reported to potentiate transcriptional activation mediated by a non-saturating concentration of the glucocorticoid receptor agonist dexamethasone, the precise mechanism(s) underlying these responses remains unclear. The murine L-929-derived LMCAT cell line stably transfected with the mouse mammary tumor virus promoter-chloramphenicol acetyl transferase reporter gene construct was utilized in the present study to further investigate the mechanism(s) underlying this dexamethasone potentiation as well as the possible agonist specificity of this potentiation. The present data demonstrate that pretreatment (2 h) of LMCAT cells with 10 microM FK506, rapamycin or cyclosporin A results in the potentiation of reporter gene transcription mediated not only by dexamethasone (approximately 12-fold), but also by hydrocortisone (approximately 6-fold) and triamcinolone acetonide (approximately 2.5-fold). In sharp contrast, the data show for the first time that pretreatment with any one of these immunosuppressive drugs suppresses (approximately 2-8-fold) the transcriptional responses mediated by corticosterone, deoxycorticosterone, and cortexolone. Pretreatment of intact LMCAT cells with FK506 increases the subsequent whole cell specific binding of [3H]dexamethasone, but does not increase specific cytoplasmic binding when the tritiated agonist is added directly to cytosolic extracts prepared from the pretreated cells. These data suggest that the FK506-mediated potentiation of the transcriptional responses induced by some agonists, like dexamethasone, may be related to the ability of this immunosuppressant to inhibit the membrane-associated multidrug resistance (MDR) P-glycoprotein, which actively extrudes some steroids from cells. Identical pretreatment with FK506 has no detectable effect on the subsequent whole cell specific binding of [3H]corticosterone, a steroid which is not effectively extruded by the MDR pump. Two additional MDR pump inhibitors, verapamil and quinidine, potentiate (30-fold) the dexamethasone-mediated transcriptional response as expected, but have no detectable effects on a corticosterone-mediated transcriptional response. Unlike immunosuppressive drugs, these ion channel blockers do not bind to receptor-associated immunophilins (FK506-binding proteins or cyclophilins). Collectively, these results suggest that immunosuppressants potentiate a dexamethasone-mediated transcriptional response in LMCAT cells by inhibiting efflux of this steroid. In contrast, these drugs appear to suppress a corticosterone-mediated transcriptional response by a different mechanism, perhaps one involving their binding to glucocorticoid receptor-associated immunophilins.

Trans-retinoic acid and glucocorticoids synergistically induce transcription from the mouse mammary tumor virus promoter in human embryonic kidney cells.

Human embryonic kidney (K293) cells transfected with a mouse mammary tumor virus (MMTV) promoter-luciferase reporter construct (pHH-Luc) were utilized to investigate the potential effects of trans-retinoic acid (tRA), either by itself or in combination with glucocorticoid (GC) hormones, on a well-characterized, GC-sensitive transcriptional response. tRA or the synthetic GC hormone dexamethasone induced transcription from the MMTV promoter in a dose-dependent manner, with 1 micromol tRA and 1 micromol dexamethasone alone causing a four- to six-fold and a 40-fold induction of basal transcription, respectively. Simultaneous treatment with 1 micromol dexamethasone and 1 micromol tRA resulted in a synergistic transcriptional response that was 120-fold higher than basal level and 2.5 times the predicted response, based on a simple additive effect of both agonists. tRA does not appear to mediate this synergistic transcriptional response by enhancing GC receptor (GR) binding capacity, affinity, or nuclear translocation. tRA was unable to potentiate GC-induced transcriptional activity from a minimal GC response element (GRE), and GC were unable to potentiate tRA-induced transcriptional activity from a minimal retinoic acid response element (RARE). These data rule out direct protein-protein interactions between GC and retinoid receptors as a mechanism for the observed synergism. tRA also synergized with aldosterone-induced, mineralocorticoid receptor (MR)-mediated, transcriptional activation of the MMTV promoter, resulting in a response that was 1.7 times the predicted additive response. The MMTV GRE located between -187 and -165 was required for GC-induced and synergistic activation of the MMTV promoter, whereas sequences located within -151 to +5 were sufficient for tRA-induced transcription from the MMTV promoter. Mutation of a consensus RARE half-site (CCAAGT) identified at position -65 to -60 within the MMTV-LTR did not affect either tRA-induced transcriptional activation or synergism with GC. We propose that the tRA-induced transcriptional response from the MMTV promoter, as well as synergism with GC, may be mediated by the activation or induction of a factor(s) that either directly binds to the MMTV promoter or indirectly stabilizes binding of another transcription factor to these sequences.

Stimulation of tissue plasminogen activator production by retinoic acid: synergistic effect on protein kinase C-mediated activation.

Trans retinoic acid (t-RA) stimulated the production of tissue plasminogen activator (tPA) in HeLa-S3 and human umbilical vein endothelial cells (huvecs) in a dose-dependent manner with maximal release (four to five times control) at 40 nmol/L and 40 mumol/L, respectively. In endothelial cells, the stimulation of tPA production by phorbol 12-myristate 13-acetate (PMA) was potentiated 1.9-fold by 10 mumol/L t-RA, or 1.8 times the additive effect. In HeLa cells, total tPA secretion with 10 nmol/L PMA was increased from 43 ng/mL to 96 ng/mL by 40 nmol/L t-RA, which was two times the additive effect. Higher concentrations of t-RA (400 nmol/L) depressed tPA secretion by itself and also suppressed PMA-induced tPA production by 50%. Histamine and thrombin also synergized with t-RA. t-RA (40 nmol/L) and 10 micrograms/mL histamine or 10 U/mL thrombin combined to induce tPA production 3.4 and 1.3 times the additive effect in HeLa cells. Cyclic adenosine monophosphate (cAMP) levels were not significantly affected by 10 nmol/L to 10 mumol/L t-RA. Nor did 10 nmol/L PMA and 40 nmol/L t-RA together affect cAMP levels, suggesting that t-RA-mediated potentiation of PMA-induced tPA production occurred via a mechanism that was independent of cAMP levels. Downregulation of protein kinase C (PKC) by pretreatment of huvecs with 100 nmol/L PMA completely blocked a secondary response to PMA, but did not have a significant effect on t-RA induction. Pretreatment with 10 mumol/L t-RA, on the other hand, did not significantly affect a secondary stimulus by 100 nmol/L PMA, but completely suppressed a secondary stimulation by 10 mumol/L t-RA alone. These studies suggest that the mechanism mediating t-RA stimulation of tPA production interacts with the PKC pathway, resulting in synergism.

Characterization of a novel glutathione S-transferase isoenzyme from mouse lung and liver having structural similarity to rat glutathione S-transferase 8-8.

In mouse lung, glutathione S-transferase (GST, EC 2.5.1.18) isoenzymes belonging to the three major known classes, Alpha, Mu and Pi, have been previously characterized, along with an isoenzyme (pI 5.7) that could not be identified with the Alpha, Mu or Pi classes of GSTs. In the present studies we have demonstrated that this isoenzyme is also expressed in liver. Its structural, kinetic, and immunological properties have been determined and compared with those of the three classes of GSTs. GST 5.7 has a subunit molecular mass of 23 kDa, which is intermediate between that of the previously characterized Alpha (25 kDa) and Pi (22.5 kDa) class GST subunits of mouse lung. Comparison of peptide maps of GST 5.7 with those representative of Alpha, Mu and Pi class GST isoenzymes of mouse lung showed that it had a distinct peptide fragmentation pattern. Kinetic and immunological properties of GST 5.7 were also distinct from other mouse GST isoenzymes belonging to the Alpha, Mu or Pi classes. N-Terminal amino-acid-sequence analysis of a 6 kDa fragment generated by CNBr digestion of mouse lung GST 5.7 revealed a 15-residue sequence that was distinct from sequences of known Alpha, Mu and Pi class mouse GSTs. The sequence, however, matched with the sequence of rat GST 8-8 between amino acid residues 106 and 120 with a 73% identity. The 6 kDa and 12 kDa fragments generated by CNBr digestion of mouse liver GST 5.7 also gave sequences which matched with those of rat GST 8-8 between positions 106 and 120 and 167 and 186, with a high degree of identity. These studies suggest that mouse GST 5.7 structurally corresponds to rat GST 8-8 and belongs to the Alpha class.

Purification and characterization of dinitrophenylglutathione ATPase of human erythrocytes and its expression in other tissues.

S-(2,4-dinitrophenyl)glutathione (Dnp-SG) ATPase of human erythrocytes has been purified to apparent homogeneity by affinity chromatography. In reduced denaturing gels, the subunit Mr value of Dnp-SG ATPase was found to be 38,000. Dinitrophenyl glutathione (Dnp-SG) stimulated the hydrolysis of ATP by the purified enzyme whereas oxidized glutathione (GSSG) did not, indicating that Dnp-SG and GSSG are transported from the erythrocytes by different transporters. Results of Western blot analysis using the antibodies against Dnp-SG ATPase subunits indicated that the enzyme was expressed in human liver, lung, placenta and pancreas.

Selective expression of the three classes of glutathione S-transferase isoenzymes in mouse tissues.

The suitability of mouse as an animal model for studying the glutathione S-transferase (GST)-mediated detoxification mechanisms has been studied by analyzing the expression of the alpha, mu, and pi classes of glutathione S-transferase isoenzymes in mouse brain, heart, kidney, spleen, liver, and muscle. Individual isoenzymes from each of these tissues have been purified, characterized, and classified into the three known classes of GST. These studies demonstrate that GST isoenzymes are variably expressed in different mouse tissues, suggesting that their expression is tissue specific. A major isoenzyme, belonging to the pi class, with a pI value in the range of 8.6-9.1 and an approximate subunit Mr value of 22,500 was detected in each tissue investigated in this study. A variable number of mu class isoenzymes with subunit Mr values of 26,500 were expressed in all mouse tissues studied, except spleen and muscle. Only liver and kidney showed the expression of an alpha class isoenzyme, each having a basic pI value and subunit Mr of approximately 25,000. Another minor acidic alpha class isoenzyme, also with a subunit Mr value of 25,000, was detected in liver, kidney, and brain. While multiple GST isoenzymes were detected in all other tissues studied, only spleen showed the presence of a single isoenzyme, which belonged to the pi class. These results reveal considerable differences in the GST isoenzyme composition of mouse tissues as compared to rat and human tissues. However, several apparent similarities in mouse and human tissues exist, suggesting that the mouse model can be used to analyze the GST-mediated detoxification mechanisms in humans.

Glutathione S-transferase and P-glycoprotein in multidrug resistant Chinese hamster cells.

Glutathione S-transferases (GSTs) have been reported to be elevated in some forms of hepatic carcinogenesis, in multidrug resistant (MDR) cells exhibiting elevated P-glycoprotein, and in cells resistant to alkylating agents independent of the MDR phenotype. The reported elevation of GST in association with the MDR phenotype and the overexpression of P-glycoprotein along with induction of GST in hepatic carcinogenesis suggest a correlation in the two mechanisms of cellular detoxification. To evaluate this hypothesis we examined the expression of GSTs in an MDR Chinese hamster fibroblast cell line overexpressing P-glycoprotein. We were unable to demonstrate concordant elevation of GST in these MDR cells. We conclude that GST expression is independent of P-glycoprotein expression in MDR Chinese hamster fibroblasts. The overexpression of GSTs in certain cells may provide an alternative mechanism for the development of drug resistance, either in association with or independent of P-glycoprotein overexpression, but is not essential for the MDR phenotype.

Primary and secondary structural analyses of glutathione S-transferase pi from human placenta.

The primary structure of glutathione S-transferase (GST) pi from a single human placenta was determined. The structure was established by chemical characterization of tryptic and cyanogen bromide peptides as well as automated sequence analysis of the intact enzyme. The structural analysis indicated that the protein is comprised of 209 amino acid residues and gave no evidence of post-translational modifications. The amino acid sequence differed from that of the deduced amino acid sequence determined by nucleotide sequence analysis of a cDNA clone (Kano, T., Sakai, M., and Muramatsu, M., 1987, Cancer Res. 47, 5626-5630) at position 104 which contained both valine and isoleucine whereas the deduced sequence from nucleotide sequence analysis identified only isoleucine at this position. These results demonstrated that in the one individual placenta studied at least two GST pi genes are coexpressed, probably as a result of allelomorphism. Computer assisted consensus sequence evaluation identified a hydrophobic region in GST pi (residues 155-181) that was predicted to be either a buried transmembrane helical region or a signal sequence region. The significance of this hydrophobic region was interpreted in relation to the mode of action of the enzyme especially in regard to the potential involvement of a histidine in the active site mechanism. A comparison of the chemical similarity of five known human GST complete enzyme structures, one of pi, one of mu, two of alpha, and one microsomal, gave evidence that all five enzymes have evolved by a divergent evolutionary process after gene duplication, with the microsomal enzyme representing the most divergent form.

Glutathione and glutathione S-transferases in a human plasma cell line resistant to melphalan.

We report the development of a melphalan-resistant HS-Sultan human plasma cell line. The melphalan-resistant [MEL(R)] cell line was 16.7-fold more resistant to melphalan in vitro than the parent cell line [MEL(S)]. The wild type and MEL(R) HS-Sultan cell lines formed localized plasmacytomas when injected into nude mice. A dose-response effect of melphalan against the drug-sensitive plasmacytomas was present in vivo. A dose of 10 mg/kg of melphalan, which caused a 90% regression of MEL(S) plasmacytomas, had no effect on the MEL(R) plasmacytomas in vivo. In contrast to previous reports, there was no increase in the levels of glutathione (GSH) in the MEL(S) and MEL(R) plasmacytomas, suggesting that the association of elevated glutathione levels and melphalan resistance may not be common to all drug-resistant lines. In the MEL(R) plasmacytomas, there was a 1.5-fold induction of a pi type glutathione S-transferase (GST) as evidenced by isoelectric focusing (IEF) and Western blotting. This GST isoenzyme was purified and, although immunochemically similar to the pi type isoenzymes induced in other drug-resistant cell lines, was noted to have different functional characteristics. These data suggest that, depending on cell type and the drug studied, functionally different GST isoenzymes may be induced and they could be of importance in the development of drug resistance.

Anionic glutathione S-transferases of human erythrocytes, placenta, and lung: evidence for structural differences.

Studies were undertaken to elucidate the structural interrelationships among glutathione S-transferase (GST) isozymes of human placenta, lung, and erythrocytes. Results of the high-performance liquid chromatography of the trypsin digests of the three isozymes indicate minor but significant differences in their elution profiles. Although a number of peptides generated by proteolysis were common for either 2 or 3 of the isozymes, significant differences were observed in elution profiles of other peptides. Qualitative as well as quantitative differences were also observed in the electrophoretic peptide maps of these isozymes. These studies suggest that there may be fine structural differences among the pi class GST isozymes of human tissues.

Differential expression of alpha, mu and pi classes of isozymes of glutathione S-transferase in bovine lens, cornea, and retina.

Isozyme characterization of glutathione S-transferase (GST) isolated from bovine ocular tissue was undertaken. Two isozymes of lens, GST 7.4 and GST 5.6, were isolated and found to be homodimers of a Mr 23,500 subunit. Amino acid sequence analysis of a 20-residue region of the amino terminus was identical for both isozymes and was identical to GST psi and GST mu of human liver. Antibodies raised against GST psi cross-reacted with both lens isozymes. Although lens GST 5.6 and GST 7.4 demonstrated chemical and immunological relatedness, they were distinctly different as evidenced by their pI and comparative peptide fingerprint. A corneal isozyme, GST 7.2, was also isolated and established to be a homodimer of Mr 24,500 subunits. Sequence analysis of the amino-terminal region indicated it to be about 67% identical with the GST pi isozyme of human placenta. Antibodies raised against GST pi cross-reacted with cornea GST 7.2. Another corneal isozyme, GST 8.7, was found to be homodimer of Mr 27,000 subunits. Sequence analysis revealed it to have a blocked amino-terminus. GST 8.7 immunologically cross-reacted with the antibodies raised against cationic isozymes of human liver indicating it to be of the alpha class. Two isozymes of retina, GST 6.8 and GST 6.3, were isolated and identified to be heterodimers of subunits of Mr 23,500 and 24,500. Amino-terminal sequence analysis gave identical results for both retina GST 6.8 and GST 6.3. The sequence analysis of the Mr 23,500 subunit was identical to that obtained for lens GSTs. Similarly, sequence analysis of the Mr 24,500 subunit was identical to that obtained for the cornea GST 7.2 isozyme. Both the retina isozymes cross-reacted with antibodies raised against human GST psi as well as GST pi. The results of these studies indicated that all three major classes of GST isozymes were expressed in bovine eye but the GST genes were differentially expressed in lens, cornea, and retina. In lens only the mu class of GST was expressed, whereas cornea expressed alpha and pi classes and retina expressed mu and pi classes of GST isozymes.

Glutathione S-transferase isoenzymes in human lung tumors.

In the present studies we have compared the levels of glutathione (GSH) and GSH-related enzymes in lung tumors and corresponding normal tissues obtained from the same individuals. We have also immunologically quantitated the relative amounts of glutathione S-transferase pi (or GST-P) type antigen in tumors and adjacent normal tissues from five patients. GST activities towards 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid were found to be elevated in tumors from two out of five patients (patients #1 and 4), whereas the activity towards these substrates was markedly suppressed in the tumor tissue from one of the patients (#5). Immunotitration and Western blot studies using antibodies raised against pi-type GST isoenzymes of human lung and placenta indicated induction of GST pi-type isoenzyme in tumors from patients #1 and 4 and suppression of this isoenzyme in tumor from patient #5. The tumors from patients #2 and 3 did not show any increase in GST activity or GST pi-type antigen. Except for the tumor from patient #5, the GSH content was higher in the tumors from other patients. GSH reductase activity was found to be elevated in tumors of all the patients examined in this study. These results indicate that GSH and GSH related enzymes are differentially altered in lung tumors and GSH levels and GST pi- or GST-P-type isoenzyme(s) are not uniformly elevated in all tumors.