Heterogeneity of glutathione S-transferase enzyme and gene expression in ovarian carcinoma.

Expression of the three major cytosolic classes of glutathione S-transferases (GST; Pi, Alpha and Mu) was examined by 2D gel analysis and Western blotting of biopsies from 26 patients diagnosed with ovarian carcinoma. In contrast to other tissues, at least one 'constitutive' subunit from each of the three major cytosolic GST classes was expressed. In most cases, pi appeared to be the major form present, although levels of alpha and mu subunit expression were approximately equal to pi in some patients. There was no detectable effect of prior chemotherapy on enzyme activity. Mean transferase activity for primary carcinoma was 79.9 +/- 11.9 (mean +/- SEM; nmol min-1 mg-1), with three pair-matched normal tissues showing minor decreases in transferase activity. One sample, in which a 32% increase in tumour enzyme activity was noted, was from a patient with primary disease and was associated with marked overexpression of a relatively basic form of alpha which was absent from the matching normal tissue, but present in 20% of all tumours examined. RFLP analysis of genomic tumour DNA using a human mu class cDNA probe indicated that at least two of the three mu forms (the 'constitutive' form and one other) observed in ovarian tissue were allelic variants, as a one-to-one correlation was observed between the presence of two Hind III fragments at 13.1 and 2.2 kb and expression of a second, more basic, variable form. This latter form was positively identified as the mu class subunit mu based on Southern analysis and was seen to be present in 40% of the samples examined. However, in the absence of mu expression, at least one other mu class subunit probably corresponding to GST psi, was seen to be present. Thus, at least in ovarian tissues, absence of the mu subunit does not necessarily imply a lack of ability to metabolize mu substrates, as psi has similar catalytic activity. A third mu subunit, probably corresponding to GST phi based on its relatively acidic pI, was also noted in 72% of samples examined, but has unknown substrate specificity. Increased expression of both alpha and mu forms may be of relevance to disease diagnosis and drug response.

Effects of chlorinated hydrocarbons on cellular volume regulation in slices of rat liver.

The effects of carbon tetrachloride and 1,2-dichloroethane (1,2DCE) on the recovery of slices of rat liver from cellular swelling in vitro were studied. Slices took up water during pre-incubation at 1 degrees C, then cellular volume and ultrastructure were rapidly restored during subsequent incubation at 38 degrees C. Ouabain (2 mm) inhibited water extrusion by less than 50%, while inducing formation of peri-canalicular vesicles, apparently derived from the Golgi apparatus. Neither CCl(4) nor 1,2DCE (up to 10 mm) affected the initial extrusion of water at 38 degrees C in the absence of ouabain, but renewed swelling occurred after 60 min with either agent; this was associated with loss of membrane selectivity and some histological damage. By contrast, 1,2DCE inhibited water extrusion in the presence of ouabain after less intensive exposure, for example with 5 mm-1,2DCE for 60 min or 10 mm for 15-30 min. With ouabain present, 1,2DCE (10 mm) caused marked swelling of the endoplasmic reticulum, reduced the peri-canalicular vesicles seen with ouabain alone and reduced the formation of canalicular microvilli. Both CCl(4) and 1,2DCE inhibited the ATP-dependent accumulation of Cl(-) by isolated vesicles of the Golgi apparatus, The delayed swelling of hepatocytes at high concentrations of 1,2DCE and CCl(4) in the absence of ouabain is probably a non-specific consequence of membrane damage. By contrast, 1,2DCE specifically inhibits the ouabain-resistant extrusion of water, possibly by interfering with a postulated mechanism for the exocytotic expulsion of water.

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.

Involvement of the cellular vacuolar system with the cytotoxicity of bleomycin-like agents.

The role of acidic cellular organelles in regulating the toxicity of selected antitumor drugs was studied with L1210 cells using modifiers of vesicular pH or function. A 1 hr exposure to a non-toxic concentration of the acidotropic weak base ammonium chloride increased the lethality of bleomycin A2 (BLM A2), demethyl BLM A2, peplomycin, and talisomycin S10b to L1210 cells grown in culture. Enhanced BLM lethality was also seen with the lysosomal disruptive agents verapamil and diltiazem. The increased lethality with verapamil and BLM A2 was schedule dependent, being seen only when cells were exposed to the drugs simultaneously or to BLM A2 first and then verapamil. Non-toxic concentrations of the monovalent cationic ionophore monensin also increased the cytotoxicity of BLM A2 and talisomycin S10b but not doxorubicin. This enhanced cytotoxicity seen with monensin occurred without an increase in either cell associated BLM A2 or single-strand DNA damage as measured by alkaline elution. We propose that acidic cellular organelles or their contents participate in controlling the cytotoxicity of the BLM class of antitumor agents.

Initial single-strand DNA damage and cellular pharmacokinetics of bleomycin A2.

The cellular association and fate of high specific activity [3H]bleomycin A2 (BLM A2) were examined in three previously untreated cultured cell lines. Human head and neck A-253 carcinoma cells were 10-fold more sensitive to a 1-hr exposure to BLM A2 than either murine leukemic L1210 or human ovarian SK-OV cells. Both murine and human cells displayed rapid drug association with steady-state drug levels being reached within 15-30 min. At steady state, the T1/2 of drug dissociation was slow (between 65 and 155 min), unaltered by 100-fold excess of unlabeled BLM A2, and unrelated to cellular sensitivity to BLM. Approximately 15% of the total cellular drug was found in the nuclei at steady state. In intact cells, BLM hydrolase activity appeared latent; significant BLM hydrolase activity was detected using broken cell homogenates with all cell types, but no extensive drug metabolism was evident in intact cells. Murine L1210 cells differed from both human cell lines in that they had only 50% of the steady-state drug levels, had lower nuclear drug content, and had markedly less initial single-strand DNA damage. Human SK-OV cells had 2.4-fold greater initial single-strand DNA damage despite similar nuclear content and a much lower rate of DNA repair. Thus, cellular or nuclear factors, in addition to BLM A2 content, affect initial single-strand DNA damage. Collectively, our data support the proposition that lesions other than single-strand DNA breaks contribute to the cytotoxicity of BLM.

Characteristics of bleomycin-resistant phenotypes of human cell sublines and circumvention of bleomycin resistance by liblomycin.

Three bleomycin (BLM)-resistant sublines were isolated from a human head and neck squamous cell carcinoma cell line (A-253); these sublines (C-10, D-10, and G-11) were 4-, 9-, and 21-fold resistant to BLM A2, respectively. These sublines were selectively resistant to other members of the BLM class, namely BLM B2, peplomycin, talisomycin S10b, and bleomycinic acid; none of the sublines displayed cross-resistance to vincristine, doxorubicin, cis-diamminedichloroplatinum or melphalan; only one subline (G-11) was cross-resistant to X-irradiation. None of the BLM-resistant cell lines demonstrated resistance to the novel BLM analogue liblomycin, which contains a lipophilic terminal amine. The cell cycle distributions of the clonally derived BLM-resistant cell populations were similar to the distribution of the parental cell population. In vitro BLM hydrolase activity in homogenates of D-10 and G-11 BLM-resistant cell lines was two- to threefold higher than that in homogenates of A-253 or C-10 cells. Nonetheless, no deamido BLM A2 was found associated with any cell type or in the culture medium and more than 80% of the radioactivity in all cells appeared as unmetabolized BLM A2 by high pressure liquid chromatography. Thus, the appearance of large quantities of the deamido BLM metabolite was not a prominent feature of acquired resistance to BLM in these human tumor cells. The cellular accumulation of radiolabeled BLM A2 by C-10 and G-11 cells during a 1-h incubation with [3H]BLM A2 was 1/2 that seen with A-253 and D-10 cells. C-10 cells maintained a lower nuclear content of radioactivity than A-253, G-11, or D-10 cells. Initial single strand DNA damage, based upon alkaline elution analysis, also was lower in C-10 cells compared to A-253 cells. D-10 cells, in contrast, exhibited high initial genomic DNA damage but demonstrated a greater repair rate than either A-253 or C-10 cells. Thus, multiple BLM-resistant phenotypes can be obtained from a population of human squamous carcinoma cells, and modification of the terminal amine in the BLM molecule can produce compounds capable of circumventing all of these BLM-resistant phenotypes. Liblomycin, which appears to be a nonclassical BLM, may be a useful therapeutic agent with a spectrum of activity distinct from other members of the BLM class.

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.