Expression of anionic glutathione-S-transferase and P-glycoprotein genes in human tissues and tumors

The development of multidrug resistance in MCF-7 human breast cancer cells and the acquisition of broad resistance to xenobiotics in rat hyperplastic nodules are both associated with increased P-glycoprotein (mdr) gene expression as well as changes in activities of intracellular detoxication enzymes; among these changes is a significant increase in the activity of the anionic isozyme of glutathione-S-transferase (GST). We have isolated a cDNA encoding the human anionic glutathione-S-transferase, GST pi-1, from a cDNA library constructed from multidrug-resistant MCF-7 cells. The deduced amino acid sequence of GST pi-1 shows that while the human anionic GST displays 85% nucleotide and amino acid sequence homology to the rat anionic isozyme, it is markedly less related to human basic GST isozymes. We have examined the expression of GST pi and P-glycoprotein in 170 specimens of human tissues and tumors. P-Glycoprotein RNA expression was positive in eight of 23 lymphomas and two of 12 colon tumors; however, many other normal and malignant tissues, including lung, bladder, and breast tumors, had low or undetectable levels of P-glycoprotein RNA expression. In contrast, GST pi was readily detected in a wide variety of normal and malignant tissues. The level of GST pi mRNA expression in normal tissues was heterogeneous, with lowest levels found in liver and the highest levels found in lung, esophagus, and placenta. GST pi was also variably expressed in human tumors, with the lowest relative levels occurring in lymphoma and breast cancer and the highest levels found in lung cancer and head and neck tumors. In addition, comparison of paired specimens from the same patient indicated that GST pi expression was increased in many tumors relative to matched normal tissue.     

Characterization of glutathione S-transferase expression in lymphocytes from chronic lymphocytic leukemia patients

Chronic lymphocytic leukemia (CLL) is a disease state which frequently responds to alkylating agent chemotherapy but ultimately becomes refractory through acquired resistance mechanisms. In the present study, we have examined the expression of glutathione S-transferases (GST) in both CLL and normal control lymphocytes, as these enzymes have been implicated in mechanisms of natural and acquired resistance. Lymphocyte GST was purified from samples by high-pressure liquid affinity chromatography, and subunits were identified by two-dimensional gel electrophoresis and immunoblotting by using polyclonal antibodies specific for individual subunits. Analysis of CLL lymphocyte GST activity using the general substrate 1-chloro-2,4-dinitrobenzene showed a statistically significant 2-fold increase in cells from chlorambucil-resistant patients over those from untreated patients and normal individuals. Furthermore, chlorambucil therapy was seen to cause a 1.3- to 1.5-fold elevation of enzyme activity in three previously drug-naive patients. Analysis of GST isozyme subunits indicated that 95% of the CLL patients examined were positive for the pi isozyme, and this appeared quantitatively to be the major isozyme present. The alpha and mu isozymes were also expressed in 63 and 53% of the patients, respectively. Examination of control lymphocytes, as well as separated B- and T-cell subpopulations, yielded similar results. The present study indicates that a high degree of interindividual variation occurs and that the pattern of CLL lymphocyte GST expression differs from that of other tumor tissues. While there were no obvious correlations between the disease state or stage and isozymes expressed, the quantitative increase in GST activity in chlorambucil-resistant CLL patients may be of relevance to the overall resistant phenotype.     

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.     

Expression of alpha, mu and pi class glutathione S-transferases in oval and ductal cells in liver of rats placed on a choline-deficient, ethionine-supplemented diet.

Expression of the alpha, mu and pi class glutathione S-transferases (GSTs) in hepatocytes, oval cells and ductal cells derived from the livers of rats placed on a choline-deficient, ethionine-supplemented (CDE) diet for 5 weeks was investigated. An overall decrease in the expression of alpha and mu class GSTs and an over-expression of pi class GST was observed in the liver after CDE treatment as indicated by Northern blotting analysis. Massive disruption of the liver with oval cell infiltration in the sinusoids throughout the lobule occurred after 5 weeks CDE treatment. 'Duct-like' structures consisting of oval-like cells (ductal cells) with rounder nuclei and more cytoplasm than oval cells within the sinusoids were also apparent. Immunocytochemical analysis revealed that the altered expression of GST in the whole liver is attributed to a differential expression of alpha, mu and pi class GSTs in the different cell types in the liver, including hepatocytes, oval cells around the portal region and among the sinusoids, and oval-like cells (ductal cells) in the 'duct-like' structures. In vitro studies using purified oval-ductal cells and hepatocyte populations confirmed the differential expression of GSTs in the varying cell populations in situ. The expression of the alpha and mu class GSTs in hepatocytes does not appear to be altered by the CDE diet. Heterogeneity in distribution of pi class GST was observed in the hepatocyte population, some hepatocytes were stained strongly while no staining was observed in others. Oval and ductal cells represent two distinct populations displaying different expression of GSTs. Pi class GST was detected in the majority of oval and ductal cells. Alpha class GST was detected in < 5% of the oval cell population and was found in > 50% of the ductal cell population. In contrast, mu class GST was absent in ductal cells and was present in 24% of oval cells around the portal region. This supports the view that ductal cells are not of bile ductal origin since mu GST is present in normal bile duct epithelial cells. Furthermore the change in expression of GSTs in the liver after CDE treatment is attributed to the large increase in oval and ductal cell populations.     

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.     

Glutathione S-transferase and glutathione peroxidase expression in normal and tumour human tissues

Glutathione S-transferases play a central role in drug detoxification and have been implicated in the sensitivity of tumour cells to anticancer drugs. In this study, glutathione S-transferase (GST) isozyme expression in normal and tumour tissue from human lung, colon, stomach, breast, kidney and liver tissue has been quantified using sensitive and subunit specific radioimmunoassays (RIA), together with Western blot analysis and measurement of substrate metabolism. Glutathione S-transferase pi was the predominant GST in the majority of the tumours examined. The concentration of this enzyme was increased significantly in tumour tissue relative to normal lung, colon, and stomach tissue. A strong correlation was observed (r = 0.77, P less than 0.01) between GST activity and GST pi levels in those tumour samples. The concentrations of the alpha class GST, the predominant isoenzymes in normal stomach, kidney and liver, decreased dramatically in tumour tissue from these organs. Western blot analysis revealed the presence of novel polypeptides that cross-reacted with antisera raised against alpha and mu class GST. Our data demonstrates that although GST pi is the predominant GST isoenzyme in many tumours, significant levels of the other GST subunits are also present and collectively can represent a significant proportion of the GST content. Therefore the properties of all the GST isoenzymes need consideration when assessing the role of these proteins in drug resistance. Selenium-dependent glutathione peroxidase, an enzyme activity also implicated in the mode of action of certain antitumour agents, was also studied and shown to be the predominant glutathione-dependent peroxidase in all tumours except the hepatoma.     

Influence of glutathione S-transferases on cellular glutathione determination by flow cytometry using monochlorobimane

Intracellular glutathione (GSH) levels for seven mammalian cell lines (four human tumors, two rodent, one monkey) were determined by flow cytometry following staining with monochlorobimane (MBCl), and the results were compared with GSH levels measured by the Tietze assay. The mean fluorescence intensity for all but the two rodent lines did not correlate with GSH levels determined biochemically. Good agreement between the two assays was observed for the rodent lines following depletion of GSH by buthionine sulfoximine, but the level of GSH depletion achieved in the human and monkey lines was always underestimated by MBCl/flow cytometry. These discrepancies were not resolved by increasing stain concentration or staining time. Total glutathione S-transferase (GST) activity and GST isozyme profiles were determined for each of the cell lines. Western analysis with antibodies raised against rat Ya, Yb1, and Yc and human pi isozymes revealed that the rodent cell lines expressed abundant alpha (Ya, Yc subunits) and mu (Yb1 subunits) class isozymes. In contrast, GST-pi was the predominant isozyme detected in the human tumor cell lines and Cos-7 monkey cells. Michaelis-Menten analysis with purified GSTs from rat liver as well as purified human placental (pi) GST revealed that the conjugation of MBCl and GSH catalyzed by the alpha (1-1 and 2-2) and mu (3-3 and 3-4) class GST isozymes was approximately 10 and 80 times more efficient than was conjugation by the GST pi form, respectively. These data indicate that the GST-catalyzed conjugation of GSH and MBCl is isozyme dependent and that MBCl is a relatively poor substrate for the pi isozyme. As a consequence of this isozyme rate differential, the MBCl/flow cytometry technique for GSH quantitation must be applied cautiously, particularly with human tumor cells, many of which have been shown to have high GST-pi activity. Application to other cell types should also be made after careful characterization of GSH levels and GST isozyme composition and only after comparison with other independent assays of GSH concentration.     

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.     

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.     

Glutathione S-transferases and cytochrome P450 detoxifying enzyme distribution in human cerebral glioma

Malignant astrocytomas are frequently resistant to cytotoxic chemotherapy. A possible mechanism of chemoresistance is drug inactivation within malignant astrocytes by detoxifying enzymes (glutathione transferases (GST) and cytochrome P450's). The aim of this study was to assess whether there was differential expression of these detoxifying enzymes in the central nervous system and any relationship to histological grade (WHO) of the tumours.Immunostaining was performed in 30 consecutive glioma samples, using class specific polyclonal antibodies to subtypes of GST (pi, alpha, mu) and to human cytochrome P450 reductase.GST immunostaining was evident in astrocytes and endothelium but not neurones or oligodendrocytes in normal brain. Immunostaining for GST increased in intensity from well differentiated tumours to glioblastoma. Staining was least evident in surrounding normal brain, strong in reactive astrocytes and astrocytic tumour cells and very intense in gemistocytic and giant tumour cells. Small anaplastic tumour cells had very little GST staining. Where endothelial proliferation was evident, GST staining in endothelial cells was increased. Pi was always the predominant subclass, although GST alpha and mu were also expressed in some tumours. Cytochrome P450 reductase immunostaining was present in normal neurones and malignant astrocytes.Gemistocytic astrocytic tumour cells stained intensely. Further work is necessary to see if there is any correlation between immunostaining intensity survival or response to chemotherapy.     

Glutathione, glutathione S-transferases, and related redox enzymes in Adriamycin-resistant cell lines with a multidrug resistant phenotype

Friend erythroleukemia cells (FLC) selected by exposure to Adriamycin (doxorubicin) express an approximate 2.5-fold (ARN1) or 13-fold (ARN2) resistance to the drug with various degrees of cross-resistance to other anthracyclines, vinca alkaloids, and epipodophyllotoxins. Because the redox cycling of the quinone moiety of Adriamycin is known to produce oxidative stress, however, an analysis of glutathione (GSH) and related enzyme systems was undertaken in the wild-type and selected resistant cells. In ARN1 and ARN2, superoxide dismutase (SOD) and catalase activities were slightly decreased, intracellular GSH and GSH reductase were essentially unchanged, and total GSH peroxidase, glutathione S-transferase (GST), and DT-diaphorase activities were slightly elevated. In each case there was no stoichiometric relationship between degree of resistance and level of activity. GST isozymes were purified from each cell line by HPLC GSH affinity column chromatography. Two-dimensional gel electrophoresis and western blot immunoreactivity against a battery of GST isozyme polyclonal antibodies determined that both the resistant and sensitive cells expressed isozymes of the alpha, pi, and mu classes (alternative murine nomenclature: M1, M2, M3). Of significance, both ARN1 and ARN2 cell lines expressed a unique alpha subunit which was absent from the parent FLC cell line. This isozyme presumably accounted for the increased GSH peroxidase activity (cumene hydroperoxide as substrate) found in ARN1 and ARN2 and may play a role in the small incremental resistance to melphalan found for both resistant lines. Expression of the isozyme was not stoichiometric with respect to degree of resistance. The presence of this isozyme may contribute to the resistant phenotype or may be the consequence of a more general cellular response to oxidative stress.     

Glutathione S-transferase isoenzymes and glutathione peroxidase activity in normal and tumour samples from human lung

An increasing body of evidence suggests that glutathione-dependent enzymes are an important factor in determining the sensitivity of tumours to cytotoxic drugs. Ten randomized normal and tumour samples from individuals with lung cancer were analysed for glutathione S-transferase isoenzyme (GST) content and glutathione peroxidase (Gpx) activity. The normal tissue samples exhibited a 5.1- and 7.0-fold variation in GST and Gpx activity respectively. High levels of the pi class, acidic Yf, GST subunit were found in all the samples, with little variation between individuals. The concentration of alpha and mu class subunits was 5- to 10-fold lower and were subject to significant individual variability. The mu class subunit identified had a faster mobility on SDS-PAGE than the hepatic GST mu standard and did not appear subject to the genetic polymorphism associated with certain members of this gene family. This suggests the presence of a novel pulmonary protein which may correspond to the rat Yn Yn protein. The normal to tumour ratio for GST activity varied significantly between the samples and tended to follow the relative expression of the mu class subunit, and to a lesser extent the alpha class GST subunit. The pi subunit was present in the normal and tumour cells in very similar concentration. The expression of the mu class GST appeared to follow the differences in GST enzymic activity and although the numbers were small appeared to segregate according to tumour type. Gpx activity was also elevated in certain tumours. Of particular interest were the two adenocarcinomas which had a 20- to 30-fold higher tumour Gpx activity.     

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.     

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.     

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.     

Increased activities of thymidine kinase isozymes in human colon polyp and carcinoma

Thymidine kinase (TK), the enzyme in the pyrimidine salvagepathway, and its isozymes were examined in 15 specimens of normalmucosa, 14 polyps and 14 carcinomas in human colon. The averageTK activities in colon polyps and carcinomas were about 1.6and 2.9 times that in normal colon mucosa, respectively. Thecolon TK isozymes were separated into 2 types by DEAE-cellulosecolumn chromatography. The activity of the TK isozyme elutedwith 0.1 M NaCI in buffer was 1.6-fold higher in colon polypand 1.5-fold higher in colon carcinoma than that in normal colonmucosa. In colon carcinoma, but not colon polyp, the activityof the other isozyme eluted with buffer alone was increasedto 5.1-fold that in normal tissues. As the activity of the latterisozyme was not affected by deoxycytidine triphosphate, it maybe involved in DNA replication. The results suggest that increasesin the activities in the former and latter TK isozymes may indicatetumorigenesis and carcinogenesis, respectively, in the humancolon.     

Expression of glutathione-s-transferase (gst) subtypes in resected lung-tumors - relationship to histological type and proliferation indexes

Samples from 19 resected lung tumours have been analysed for the levels of GST alpha, mu and pi mRNA and protein. No significant survival disadvantage was found for patients with tumours containing any of the GST subtypes at the levels of either protein or mRNA. Both GST mu and GST pi mRNAs were expressed at significantly higher levels in squamous carcinomas than adenocarcinomas but GST alpha mRNA was not. The significant increases in expression of mu and pi GST in squamous relative to adenocarcinomas of the lung is not related to differences in the proliferation rates between these cancers.     

The spontaneous and glutathione S-transferase-mediated reaction of chlorambucil with glutathione

Incubation of 100 microM chlorambucil (CMB) with 1 mM glutathione (GSH) in 0.1 M potassium phosphate buffer yielded a mixture of GSH conjugates and hydrolytic products that were separable by HPLC. The appearance in HPLC analysis of four of these products was GSH-dependent. 35S-Label from 35S-GSH eluted with all four chemical species that also gave UV spectra characteristic of CMB-containing compounds. Mass spectral analysis of three of these compounds gave molecular weights consistent with the following adducts: adduct 2: diglutathionyl CMB; adduct 3: monohydroxy monochloroglutathionyl CMB; adduct 4: monochloro monoglutathionyl CMB. Purified GST alpha and partially pure GST pi and mu class protein prepared from adult male mouse liver cytosol significantly increased the formation of the monoglutathionyl CMB adduct. At 5 min incubations, this adduct was quantitatively most important and was increased 4.4-fold by the addition of GST alpha isozymes. At 1 hr incubations, all four adducts were measurable, although enzymes primarily affected the formation of adduct 4. At 1 hr, addition of GST alpha, pi, or mu protein increased the production of monochloro monoglutathionyl CMB 2-fold, 1.5-fold, and 1.7-fold, respectively, demonstrating that CMB is indeed a substrate for GST isozymes in vitro.     

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.