Expression of glutathione S-transferases and cytochrome P450 in normal and tumor breast tissue

The level of expression of glutathione S-transferases (GSTs) and cytochrome P450s in breast tissue are potentially important determinants in both the susceptibility of this tissue to the mutagenic effects of chemical carcinogens and in the response of breast tumors to chemotherapy. In this study we have investigated the expression of these proteins in 41 tumor and surrounding normal breast tissue samples by measurement of substrate metabolism. Western blot analysis and immunohistochemistry. In addition, we have quantitated the concentration of alpha, mu and pi class GST subunits using radioimmunoassay. All three classes of GST were expressed in breast tissue. The pi and mu class enzymes preponderate. Both the polymorphic mu class GST as well as a further form, present in all individuals, were found in high concentration. The polymorphic mu class GST was expressed in approximately 50% of the samples, which is consistent with the frequency of this polymorphism in the population and therefore does not appear to be a factor in susceptibility to this disease. Interestingly, although levels of the alpha class GST were very low, in two tumor samples extremely high levels of the B1B1 subunit were detected. Immunohistochemical studies showed significant variability in the localization of the pi class of GST between normal epithelial cells, infiltrating plasma cells and tumor cells, and in some samples GST pi appeared to be almost absent from the tumor tissue. No direct, or inverse correlation was found between GST pi concentration determined by radioimmunoassay and estrogen receptor levels. However, when studied by immunohistochemistry estrogen receptor negative tumors did tend to have higher GST pi content. The only cytochrome P450 detectable by Western blot analysis was a member of the P450IIC gene family. This was apparently distinct from the P450IIC proteins expressed in the liver and was detected in normal and tumor tissues to a similar extent.     

Differential activity of human, rat, mouse and bacteria glutathione transferase isoenzymes towards 4-nitroquinoline 1-oxide

The conjugation capacity of 4-nitroquinoline 1-oxide (4-NQO) with GSH by a number of human, rat, mouse and bacteria glutathione transferases (GSTs) was investigated. Pi and mu classes GSTs exhibited maximum conjugation capacity. Alpha class glutathione transferases as well as bacteria glutathione transferases were found to be unable to conjugate GSH to 4-NQO. The Km values as well as the catalytic efficiency (Kcat/Km) for most of the GSTs investigated were also determined. Mouse liver GST MIII (class mu) was the most efficient of the various isoenzymes tested. Its Kcat/Km value was 162 times higher than that of mouse liver GST MI (class alpha). The relatively high catalytic efficiency exhibited by GST-alpha (class pi) is prevalently due to its low affinity for 4-NQO.     

Glutathione transferase isoenzymes in normal and neoplastic human kidney tissue.

Glutathione transferase (GST) activity in the cytosolic fractions of renal cortex tumour was found to be significantly lower (215 +/- 156 mU/mg) than that present in the corresponding non-tumour (466 +/- 278 mU/mg) tissues. Using the immunoblotting technique, glutathione transferase isoenzymes expression in both tumour and non-tumour kidney was investigated. Alpha and pi class glutathione transferases were the most abundant enzymes in non-tumour kidney and were expressed by all samples investigated. Immunofluorescence analysis indicated that the pi class enzymes are localized mainly in the distal convoluted tubules, whereas alpha class enzymes are localized in the proximal tubules. In the tumour moiety the alpha class GST appears to be absent or expressed at low level as compared with non-tumour samples. On the contrary, no significant differences in the expression of pi class GST were found in tumour as compared with non-tumour tissues. Mu class GST protein was detected in 12 of 26 samples tested. When present, mu class GST constitutes a few per cent of total GST protein. Immunofluorescence studies indicate that mu class GSTs are localized within the distal convoluted tubules. According to the electrophoretic mobility at least two different mu GST subunits (26.5 and 27.5 kd) were found. In one sample only the faster mu class GST subunit was present, two samples expressed both types of GST subunits, whereas nine samples expressed only the slower GST subunit. With the exception of one sample, a reduction of mu class GST expression was seen in tumour as compared with non-tumour tissues. The decrease of activity seen in the cytosolic fraction of tumour kidney must be ascribed mainly to a reduction or to a lack of expression of alpha class GST and to a lesser extent of mu class GST.     

Gene-specific oligonucleotide probes for alpha, mu, pi, and microsomal rat glutathione S-transferases: analysis of liver transferase expression and its modulation by hepatic enzyme inducers and platinum anticancer drugs.

Glutathione S-transferases (GSTs) play an important role in the detoxification of diverse electrophilic chemicals, including anticancer drugs. Gene-specific oligonucleotide probes were developed to monitor the expression of individual GST mRNAs in livers of adult male rats treated with drugs and other chemical modulators of GST expression. Northern blot analysis of total liver RNA using probes specific for individual GSTs belonging to classes alpha (GSTs Ya1, Ya2, Yc), mu (GSTs Yb1, Yb2, Yb3), pi (GST Yp), and GSTms demonstrated the expression in liver of all but Yp mRNA. Kidney GST expression was at least as high as that in liver for GSTs Ya1, Yc, and Yp, while it was substantially lower but still detectable for GSTs Ya2, Yb2, and GSTms. Several of the liver GST class alpha mRNAs, in particular Ya2, were inducible by pretreatment of rats with phenobarbital or isosafrole. In contrast, dexamethasone preferentially induced Yb1, Yb2, and Ya2, while two other inducers of liver drug metabolism, isoniazid and clofibrate, were less effective with respect to GST induction. GSTms mRNA was induced to a small extent or not at all by the agents tested. Treatment of adult male rats with the anticancer drug cisplatin increased liver expression of GST Yc mRNA and suppressed Ya1 mRNA levels with little or no major effect on several other GST mRNAs. Western blot analysis of liver cytosols prepared from the cisplatin-treated rats revealed corresponding changes in GST Yc and Ya protein levels. Comparable changes in liver GST Ya1 and Yc expression were effected by the cisplatin analogue iproplatin but not by carboplatin or transplatin. This pattern of response to these platinum drugs is comparable to that seen with respect to platinum drug-induced gonadal toxicity and modulation of liver cytochrome P450 expression, suggesting a common mechanistic basis for these diverse effects of platinum anticancer drugs on hepatic enzymes of drug metabolism. Together, these studies demonstrate the utility of oligonucleotide probes for phenotyping liver tissue for the expression of GST enzymes that can contribute to anticancer drug metabolism and resistance. They also raise the possibility of drug-drug interactions involving cisplatin and alkylating agent anticancer drugs that can be metabolized in liver by alpha-class GSTs.     

Studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The glutathione transferases (GST) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). The aim of the present investigation was to study the nature and function of the GST pi gene in relation to the mutagenicity of BPDE in different cell lines. The studies were performed on three cell lines commonly used in toxicological studies, i.e. rat hepatoma cells (H4IIE), human mammary carcinoma cells (MCF-7) and Chinese hamster lung fibroblasts (V79). Western blotting with antisera against GST pi revealed a high level of reaction with cytosol from V79 and H4IIE cells. Furthermore, cytosol from the V79 cells demonstrated low levels of GSTs belonging to the alpha and mu classes, suggesting that a considerable portion of the total capacity of these cells to conjugate chlorodinitrobenzene (CDNB) was provided by GST pi. The level of mRNA for GST pi, as measured by Northern blots, was high in V79 and H4IIE and undetectable in the MCF-7 cell line. Analysis of the DNA fragment patterns using a series of restriction enzymes, revealed that all three cell lines have the pi class gene, although with different band patterns. The findings with H4IIE and MCF-7 cells with respect to their expression of the GST pi gene and their ability to conjugate BPDE were in agreement with the mutagenic effects of BPDE, produced by metabolic activation of (-)-7 beta, 8 alpha-dihydroxybenzo[a]-pyrene in the cells. In contrast, V79 cells although expressing high levels of GST pi, showed no ability to conjugate BPDE or to inhibit the mutagenicity of this compound. Based on these results, we suggest that V79 Chinese hamster lung cells contain a GST pi with a different substrate specificity from those of the human and rat GST pi enzymes.     

Specificity of murine glutathione S-transferase isozymes in the glutathione conjugation of (-)-anti- and (+)-syn-stereoisomers of benzo[g]chrysene 11,12-diol 13,14-epoxide.

Specificities of murine glutathione (GSH) S-transferase (GST) isozymes mGSTA1-1, mGSTA2-2, mGSTA3-3 and mGSTA4-4 (alpha class), mGSTP1-1 (pi class) and mGSTM1-1 (mu class) for GSH conjugation of (-)-anti- and (+)-syn-stereoisomers of benzo[g]chrysene 11, 12-diol 13,14-epoxide (B[g]CDE), the activated metabolites of the environmental pollutant benzo[g]chrysene (B[g]C), have been determined. When GST activity was determined as a function of varying (-)-anti- or (+)-syn-B[g]CDE concentration (10-320 microM) at a fixed saturating concentration of GSH (2 mM), each isozyme obeyed Michaelis-Menten kinetics. mGSTA1-1 was significantly more efficient than other murine GSTs in the GSH conjugation of not only (-)-anti-stereoisomer but also (+)-syn-B[g]CDE. For example, the catalytic efficiency (k(cat)/K(m)) of mGSTA1-1 towards (-)-anti-B[g]CDE was approximately 2.3- to 16.6-fold higher compared with other murine GSTs. Likewise, mGSTA1-1 was approximately 2.7-, 6.7-, 4.4- and 12.4-fold more efficient than mGSTA2-2, mGSTA3-3, mGSTP1-1 and mGSTM1-1, respectively, in catalyzing the GSH conjugation of (+)-syn-B[g]CDE. Interestingly, mGSTA4-4, which also belongs to class alpha, was virtually inactive towards both stereoisomers of B[g]CDE. The results of the present study indicate that murine GSTs, especially alpha class isozymes, significantly differ in their ability to detoxify B[g]CDE stereoisomers and that mGSTA1-1 plays a major role in the detoxification of both (-)-anti- and (+)-syn-B[g]CDE, which among four B[g]CDE stereoisomers are formed from the carcinogen B[g]C as major DNA binding metabolites.     

Immunohistochemical localization of glutathione S-transferases alpha, mu, and pi in normal tissue and carcinomas from human colon.

Analysis of the heterogeneity of glutathione S-transferase (GST) isozyme expression was carried out by immunohistochemical evaluation of human colon biopsy tissue from 30 patients. Using polyclonal antibodies specific for the GST alpha, mu and pi families of isozymes, an increased expression of pi was found in 21/30 carcinoma specimens compared to their pair-matched controls. This isozyme was the most prevalent in all colon samples. GST mu was expressed at reduced levels in 20/30 carcinoma specimens when compared to normal. GST alpha showed no consistent change. Analysis of the immunostaining in different cell types showed that the highest intensity stain for all isozymes was in the columnar epithelial cells. These cells were primarily responsible for the proportional changes in GST pi and mu between carcinoma and normal tissues. In addition, goblet (crypt), endothelial and muscle cells stained positively. In the lamina propria, lymphocytes and phagocytes stained positively, while fibroblasts, plasma cells and leukocytes were negative. Endocrine cells were also negative. The differential expression of GST pi and mu, confirming biochemical data, supports the potential utility of GST pi as a carcinoma marker.     

Proto-oncogene ras GTPase-linked induction of glutathione-S-transferase by growth factors in PC12 cells

This report provides evidence linking activation of Ras GTPase by growth factors and induction of glutathione-S-transferase isozymes in PC12 cells. Ras GTPase was activated by EGF, NGF, insulin and phorbolester in PC12 cells. Activation of Ras GTPase was found to be associated with induction of the expression of GST mu and pi isoenzymes while there was no detectable induction of GST alpha expression. GST pi was found to be induced by all the Ras GTPase activating agents tested while activation of Ras by phorbolester and insulin induced expression of GST mu only. These results suggest a role of Ras, at least in part, in controlling the expression of GST and that there might be independent signalling pathways for the expression of different GST isoenzymes. GST activity was found to be very high (4-fold) in the lysate obtained from retinoic acid treated PC12 cells when compared with untreated cells. Induction of GST expression was found to be initiated within 30 min of retinoic acid treatment in PC12 cells reaching a maximum level at 4 h. However, immunoblot analysis showed that retinoic acid (RA), unlike mitogens/growth factors, weakly induced the expression of GST pi but not the expression of alpha, mu and microsomal GSTs. Overxpression of inhibitory polypeptides that block signals generated from Ras and Cdc42 was found to reverse the retinoic acid activation-dependent induction of GST expression in PC12 cells. These results provide evidence for the first time suggesting a novel role of Ras GTPase in the regulation of GST expression which might have a significant implication in developing drug resistance and/or growth of cancer cells.     

Ethanol interactions with a choline-deficient,ethionine-supplemented feeding regime potentiate pre-neoplastic cellular alterations in rat liver

To examine the effect of ethanol on hepatocarcinogenesis induced by a choline-deficient, ethionine-supplemented (CDE) diet, rats were fed either an ethanol-supplemented diet or ethanol-free, isocaloric diet for 2 months, followed by a CDE diet or control diet for up to 8 months. Changes to cellular composition and pattern of gene expression in the liver were determined at 0 and 3 days, and 1, 2 and 3 weeks after commencing the CDE diet, using histological/immunochemical techniques and northern analysis. Oval cells in the liver were identified morphologically and by expression of pi-glutathione S-transferase (pi-GST), alpha-fetoprotein (AFP) and the embryonic isoform of pyruvate kinase (M2-PK). Oval cell numbers and changes in the pattern of gene expression induced by the CDE diet were accelerated by pre-treatment with ethanol. At all stages, the proportion of oval cells in the test group exceeded that in controls. After 1 week, oval cells had spread sufficiently from the periportal region to be observed pericentrally in test animals and by 3 weeks, extensive formation of ductal structures was apparent, which were absent in controls. Additionally, M2-PK and AFP mRNA were detected earlier, and in greater abundance in animals pre-treated with ethanol. After 8 months of CDE treatment, one or two small hepatic foci (<10 hepatocytes), strongly positive for pi-GST, were detected in the liver of ethanol-pre-treated animals. These foci were absent in CDE-treated animals; however, animals pre-treated with ethanol followed by chronic CDE treatment showed increased size (>40 hepatocytes) and numbers of foci, correlating with the extent of liver damage and varying from 5 to 50% of the liver section. Our data suggest that ethanol pre-treatment potentiates the short-term effects of the CDE diet by enhancing oval cell proliferation, while chronic CDE administration enhances the appearance of pre-malignant hepatic foci that are observed with ethanol pre-treatment alone.     

Induction of rat hepatic and intestinal glutathione s-transferases and glutathione by dietary naturally-occurring anticarcinogens

Effects of dietary naturally occurring anticarcinogens; quercetin, flavone, ellagic acid, ferulic acid, tannic acid, curcumin, coumarin, alpha-angelicalactone, fumaric acid and Brussels sprouts on male Wistar rat hepatic and intestinal (i) glutathione S-transferases (GST) enzyme activity, (ii) GST isozyme levels and (iii) glutathione (GSH) content were investigated. GST enzyme activity was significantly increased by all anticarcinogens tested, except fumaric acid, at least at one of the five sites investigated: proximal, middle, distal small intestine, large intestine and liver. Only alpha-angelicalactone gave an enhanced GST enzyme activity at all five sites. Large intestinal GST enzyme activity was increased only by quercetin (175%) and alpha-angelicalactone (138%). Concomitant changes in GST isozyme levels occurred. Class alpha GSTs were induced in 50% of the cases, especially in liver and upper parts of the intestine by quercetin, flavone, coumarin and alpha-angelicalactone. GST class pi levels were enhanced only at one site by quercetin, coumarin and alpha-angelicalactone. GST class mu changed in 14% of the cases, most profoundly in proximal and middle small intestine by flavone, coumarin and alpha-angelicalactone. Tannic acid and fumaric acid gave a significant raise in class alpha GSTs at almost all sites, whereas overall GST enzyme activity hardly changed. GSH was increased at various sites in 14% of the cases by Brussels sprouts, quercetin, flavone and alpha-angelicalactone. These data demonstrate that most anticarcinogens, in particular flavone, coumarin and alpha-angelicalactone, enhance GST activity in liver and intestine, mainly by induction of class alpha and mu isozymes.     

Glutathione S-transferase isoenzymes in human tumours and tumour derived cell lines

An increasing body of evidence indicates that glutathione S-transferases play a role in the intrinsic and acquired resistance of tumours to anticancer drugs. In view of the wide use of tumour cell lines to understand the factors which confer either sensitivity or resistance to chemotherapeutic agents we have determined glutathione S-transferase (GST) activity and isozyme composition in nine human cell lines. These data have been compared with the values obtained in solid tumours. In most cases overall GST activity was higher in the tumours than in the cell lines. This was most pronounced for the breast tumour samples relative to MCF7 cell line. The pi class GST subunit was present at similar concentration in the cell lines and the tumours, and in most cases was the most abundant subunit present. The alpha and mu class GST were expressed in most of the cell lines but at much lower concentration than the pi class subunit. Also considerable variability particularly in the expression of the mu subunits was observed. This was also the case for the expression of these subunits in the solid tumour samples. The levels of these GSTs (when expressed) in the solid tumours was invariably higher than that observed in the cell lines. There are therefore several similarities but also some significant differences in GST expression in solid tumours and cell lines. Whether the differences are because expression is lost during the generation of the cell lines or whether it reflects the individuality of human tumours remains to be clearly established.     

Differential induction of glutathione transferase isoenzymes of mice stomach by diallyl sulfide, a naturally occurring anticarcinogen

Diallyl sulfide (DAS), an organosulfur compound identified as the flavor component in garlic, has been shown to inhibit chemically induced neoplasia of forestomach and lung in mice. Even though the exact mechanism(s) of anti-neoplastic activity of DAS is not known, several independent studies suggest that this effect may, at least in part, be due to the elevation of glutathione-S-transferase (GST) activity. To gain further insight into the mechanism(s) of anti-carcinogenic activity of DAS, we have determined effect of orally administered DAS (25, 50 and 75 mumol) on levels of alpha, mu and pi class GSTs and glutathione (GSH) peroxidase and GSH reductase activities of female A/J mice stomach. Western blotting revealed presence of alpha, mu and pi class GSTs in mice stomach. A significant increase in all the three classes of GSTs was observed in the stomach of mice treated with DAS. Maximum increase in GST alpha and pi was evident by treating the animals with 75 mumol DAS whereas maximum induction of GST mu occurred after treating mice with 50 mumol DAS. GSH peroxidase activity towards t-butyl-hydroperoxide increased in a dose-dependent fashion in the mice stomach treated with DAS. Even though this activity towards hydrogen peroxide was similar in mice treated with 50 or 75 mumol DAS, these values were significantly higher than that of the control. GSH reductase was also elevated in the stomach of mice treated with 75 mumol DAS. These results suggest that DAS may exert anti-neoplastic effect by modulating GSH dependent detoxification enzymes.     

Decreased expression of the glutathione S-transferases alpha and pi genes in human renal cell carcinoma

Using the polymerase chain reaction (PCR), a human kidney glutathione S-transferase (GST) alpha cDNA clone (GST alpha 12 K) was synthesized; it is identical to a known liver GST alpha cDNA clone except for one base change (G----A), indicating that an alpha class gene expressed in human kidney is similar to one expressed in human liver. Comparisons were made in the expression of GST alpha and GST pi between renal cell carcinoma and adjacent non-neoplastic tissue. Messenger RNA expression in 30 cases was determined by Northern blotting, and GST protein from nine of these cases was analyzed by HPLC. The GST alpha gene products were expressed at near-zero levels. The GST pi gene product was the predominant GST in tumors, but was decreased in absolute amount compared with control tissue, the tumor/control ratios for the GST pi gene obtained by Northern blots and HPLC analysis being 0.50 +/- 0.07 and 0.36 +/- 0.07 respectively. The resulting pattern in renal cell carcinoma therefore shows a predominance of GST pi. Since it is assumed that renal cell carcinoma derives from the proximal tubular epithelial cells which are high in GST alpha, this implies a dedifferentation in the GST expression pattern.     

Antineoplastic drug sensitivity of human MCF-7 breast cancer cells stably transfected with a human alpha class glutathione S-transferase gene

Studies have suggested that the alpha class glutathione S-transferase (GST) may protect cells from the chemotherapeutic drugs chlorambucil and melphalan. In order to further define the function of human alpha class GST, a complementary DNA which encodes it was ligated into an expression vector under the direction of the human metallothionein-IIA promoter and stably transfected into human MCF-7 breast cancer cells in conjunction with the G418-selectable plasmid pSV2neo. Clonal cell lines were identified which expressed increased levels of GST enzyme activity (2.2- to 5.6-fold). The transfected cell lines also had increased peroxidase activity using cumene hydroperoxide as the substrate (1.9- to 3.8-fold) which is consistent with the intrinsic peroxidase activity of alpha class GSTs. Southern blot analysis indicated that genomic DNA from these cells contained a fragment indistinguishable from the transfected alpha class GST complementary DNA (850 base pairs); Northern blot analysis of total cellular RNA indicated that these cells contained appropriately sized alpha class GST RNA (980 nucleotides); and Western blot analysis indicated that, while MCF-7 cells contained no detectable alpha class GST protein, the transfected cells contained markedly elevated levels of alpha class GST but no detectable mu or pi class GST. These alpha class GST transfected cells had increased resistance to ethacrynic acid (2.1- to 3.0-fold). However, the transfected cells failed to show any increased resistance measured at the drug dosage which inhibited 50% of the colony formation to the chemotherapeutic drugs chlorambucil, melphalan, Adriamycin, or cisplatin under conditions of either continuous or 1-h drug exposure. Neither was there any change in sensitivity to the cytotoxins benzo(a)pyrene, benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (anti), or 1-chloro-2,4-dinitrobenzene. These studies indicate that expression of this human alpha class GST by itself in MCF-7 human breast cancer cells does not confer resistance to the chemotherapeutic drugs tested under the conditions used in these studies.     

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma

Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.     

Differential expression of glutathione-s-transferase isozymes in murine and human skin tumors

Glutathione S-transferases (GSTs) are a family of widely distributed multifunctional enzymes present in a number of organs and species. GSTs participate in the detoxification of a wide variety of xenobiotics and their metabolites. Mammalian GSTs have been grouped into three major classes alpha, mu and pi. The differential expression of specific GST isozymes has been reported as a phenotypic marker for the preneoplastic lesions and neoplastic tissues in liver and other extracutaneous organs of rat and other species including humans. However, the expression of GST isozymes in skin cancer is not known. In the present study, therefore, employing Western blot analysis, we assessed the GST isozyme expression in experimentally-induced murine skin tumors and clinically obtained human skin basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs). As compared to normal murine skin showing high levels of GST-pi, significantly lower expression of GST-pi was observed in murine skin papillomas and carcinomas induced chemically in SENCAR mice or by chronic ultraviolet B radiation in SKH-1 hairless mice. Similar results were also observed for GST-mu isozyme. On the other hand, compared to normal murine skin where the expression of GST-alpha isozyme was not detectable, significant levels of this GST isozyme were observed in all the murine skin papillomas and carcinomas. In case of human skin BCCs and SCCs, variable GST isozyme patterns were evident, however in each case, GST-pi was found to be significantly underexpressed as compared to that in skin from healthy subjects. Our results suggest that altered phenotypic expression of GST isozymes may be a useful marker for skin cancer.     

Low glutathione and glutathione S-transferase levels in Barrett's esophagus as compared to normal esophageal epithelium.

Patients with Barrett's esophagus, wherein squamous epithelium has been replaced by columnar epithelium, have an increased risk for developing esophageal adenocarcinoma as compared to the general population. Glutathione S-transferase (GST), a family of detoxification enzymes consisting of class alpha, mu, pi, and theta isoforms, is involved in detoxification of carcinogens and low levels of these enzymes correlated with high cancer risk. We have now compared GST enzyme activity, GST isoenzyme composition and glutathione (GSH) content of Barrett's mucosa with that of adjacent normal squamous epithelium. Biopsy specimens of 98 patients with Barrett's esophagus were taken from both Barrett's and adjacent normal squamous epithelium. GST enzyme activity towards 1-chloro-2,4-dinitrobenzene was measured, and GST isoenzyme levels were determined by densitometrical analyses of western blots after immunodetection with monoclonal antibodies. Total GSH content was determined by high-performance liquid chromatography after conjugation with monobromobimane. Wilcoxon's signed rank test and Spearman correlation analyses were used for statistical evaluation. As compared with adjacent normal squamous epithelium, GST enzyme activity in Barrett's epithelium was reduced by 35%, and GST mu, GST pi and GSH levels were reduced by 24%, 30%, and 63%, respectively. However, the minor GST alpha and GST theta levels were higher in Barrett's epithelium (by 625% and 33%, respectively). High levels of GSH and GSTs in general are correlated with protection against cellular or cytogenetic damage. The observed reduction in GSTs and GSH in Barrett's epithelium may therefore contribute to the increased cancer risk in this tissue.     

Expression of glutathione-s-transferase isotypes in relation to Doxorubicin sensitivity and prognosis in patients with renal-carcinoma

Specimens were obtained from the tumour and adjacent normal kidney in 29 patients with renal carcinoma. Expression of the alpha, pi and mu isoforms of gluthatione-S-transferase (GST) was estimated in formalin fixed paraffin embedded sections of the normal and neoplastic kidney following exposure to the appropriate polyclonal antibodies. binding of which was recognised by the indirect peroxidase technique. Expression of alpha, pi and mu GST in each tumour specimen was compared with the in vitro sensitivity of tumour cells to doxorubicin determined by drug inhibition of [Se-75] selenomethionine uptake by the appropriate tumour cells in culture. Expression of pi GST was associated with tumour cell resistance to doxorubicin. The level of alpha, pi and mu GST was not related to patient survival.     

Enhanced expression of glutathione S-transferases in colorectal carcinoma compared to non-neoplastic mucosa

Ten paired samples of primary human colorectal carcinoma and adjacent non-neoplastic mucosa were analysed for total glutathione S-transferase (GST) activities as determined by 1-chloro-2,4-dinitrobenzene assays. These tissues were also investigated for the expression of acidic (pi), basic (alpha) and neutral (mu) GSTs using Western blotting procedures and immunohistochemical staining. For each of the paired samples examined the total GST activity was higher in tumour than in adjacent non-neoplastic mucosa. Western blotting, using an antibody against acidic GST also showed strong immunoreactivity in all the samples with more intense reactions in tumour compared to mucosa in nine out of the ten paired samples. Low levels of basic GST were also expressed in all samples of tumour and mucosa. Neutral GST was not detectable in two samples of tumour and corresponding mucosa, but low levels of expression were demonstrated in the remaining eight. Immunohistochemical staining for acidic GST showed a dark brown reaction in all tumour cells; in non-neoplastic mucosa there was positive immunoreactivity for epithelial cells situated deep within the crypts and a negative reaction for surface epithelial cells. Immunohistochemical staining for basic GST was negative except for one sample of tumour and two of mucosa. Neutral GST was expressed only in two samples of tumour and two samples of mucosa. We therefore conclude that there is enhanced expression of GSTs, acidic GST being the predominant form, in tumour compared to normal mucosa, in keeping with a role for GSTs in colonic carcinogenesis and acquired or innate drug resistance.