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Several lines of evidence implicate glutathione (GSH) depletion and/or GSH transferase inhibition in the sensitizing action of nitroimidazoles to alkylating agents. To characterize this interaction, scid mice bearing subcutaneously implanted HT-29 colon tumor (0.75 to 1.25 cm diameter) were treated with SR2508 (2 g/kg, i.p.). At intervals following treatment, samples of blood, liver, spleen, kidney and central non-necrotic tumor core and tumor periphery were obtained and analyzed for SR2508 content by high-pressure liquid chromatography. Tissues were assayed spectrophotometrically for GSH and GSH transferase. SR2508 plasma pharmacokinetics in this model were similar to those described previously (t 1/2 beta = 5.83 hr). The volume of distribution of 0.32 L/kg suggests minimal tissue binding. In tumor periphery and core samples SR2508 levels peaked at 1 hr, and declined exponentially in parallel with plasma. During the terminal phase core SR2508 levels were 10-fold and tumor periphery levels 4.3-fold those of concurrent plasma concentrations. Consistent with these data, tumor GSH levels in both periphery and core fell below 30% of control at 4 hr, and remained depressed > 12 hr. Delayed recovery of GSH content of tumor tissue may explain in part the selectivity of SR2508 for tumor (oxic or hypoxic). GSH transferase activity in tumor was inhibited both at the center and periphery to 75 and 71% of control, respectively, and it appeared that recovery occurred more slowly in the hypoxic core. The mild degree of inhibition observed does not support an important role for inhibition of GSH transferase in sensitization by SR2508 in this tumor. The pronounced selective depletion of GSH in tumor supports the further development of SR2508 in the reversal of alkylating agent resistance.

Subpopulations of HT 29 human colon carcinoma cells (HT/M and HT/S) were selected for resistance to the glutathione S-transferase (GST) inhibitor ethacrynic acid (EA). Both clones displayed a 2-fold resistance to the selection agent and required its constant presence for the maintenance of the resistant phenotype. Purification and characterization of GST isoforms showed similar profiles in the wild-type (WT) and EA-resistant clones, with microheterogeneous forms of the pi isoenzyme detected in each case. Metabolism of EA in vitro in the presence of GSH and the isolated GST from each cell line was characterized by a biphasic disappearance of the parent drug; the initial rate at which each of these enzymes metabolized EA was similar. These enzymes also displayed similar Km values for 1-chloro-2,4-dinitrobenzene. However, the amount of GST isolated per total cellular protein was 3.0-fold in HT/M and 1.6-fold in HT/S relative to WT in the continuous presence of EA. Under these conditions GST activity was increased by 2.3-fold in HT/M and 3.2-fold in HT/S as were GSH levels (2.7- and 4.1-fold for HT/M and HT/S respectively). When EA was removed, enzyme activity and GSH concentrations decreased to values similar to those of the WT. Slot-blot and Southern analyses of the DNA gave no evidence of GST-pi-gene amplification or rearrangement. However, RNA analyses by both slot-blot and Northern studies indicate a 2.5-3.5-fold elevation in the GST pi transcript in the EA-resistant population. Results from these studies indicate that: (1) maintenance of the EA-resistant phenotype requires constant presence of the agent; (2) the 2-fold resistance to EA can be quantitatively related to a 2-3-fold increase in GST activity and amount which appears to be the result of a 2.5-3.5-fold elevation in GST transcript; (3) EA, a Michael-reaction acceptor, can induce GST at the transcriptional level.

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.

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.

A novel method for the rapid purification of glutathione S-transferases (GST) from tissue and cell culture samples is reported. A high-performance glutathione affinity column was used and produced results comparable to those obtained with classical agarose affinity columns. Experiments with purified rat liver GST standards resulted in 87% recovery of total activity. Sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) of the affinity-purified GST was identical to the GST standard and revealed three major protein bands, corresponding to the Ya, Yb, and Yc subunits. A fourth protein band (relative molecular mass 25,000), migrating slightly faster than the Ya subunit, was present in both the standard and eluted GST samples. This polypeptide was tentatively identified as the Yk subunit. Successful purification from rat liver and Walker 256 rat carcinoma cell cytosols was also performed. Recovery of total GST enzymatic activity from Walker cell and rat liver cytosol was 49 and 58%, respectively. SDS-PAGE of these samples indicated a high degree of purity. This methodology requires less than 1 h and can be performed using small quantities of tissue. These features make this technique applicable to analysis of a broad range of biological applications including human biopsy material for GST content.