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.

Determinants of drug response in a cisplatin-resistant human lung cancer cell line.

To elucidate the mechanism(s) of cisplatin resistance, we have characterized a human non-small cell lung cancer cell line (PC-9/CDDP) selected from the wild type (PC-9) for acquired resistance to cisplatin. PC-9/CDDP demonstrated 28-fold resistance to cisplatin, with cross resistance to other chemotherapeutic drugs including chlorambucil (X 6.3), melphalan (X 3.

Glutathione-S-transferase pi as a determinant of drug resistance in transfectant cell lines.

A series of glutathione S-transferase pi (GST-pi) transfectant cell lines have been constructed in activated c-H-ras-transformed NIH-3T3 cells (pT22-3) by using a pKOneo plasmid and an expression vector containing cDNA for GST-pi with a beta-actin gene promoter. From the wild type pT22-3 cells, two clones were selected and designated RGN1 and RGN2. The degree of overexpression of GST-pi was estimated by Northern and Southern blot analysis to be incrementally higher in RGN2 compared with RGN1.

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.

Cytotoxicity and DNA damaging effects of a new nitrosourea, fotemustine, diethyl- 1-(3-(2-chloroethyl)-3-nitrosoureido) ethylphosphonate-S10036.

Fotemustine is a new chloroethylnitrosourea which has recently entered a Phase II clinical trial. Using standard cytotoxicity analyses, Fotemustine was shown to be preferentially active in two Mer- cell lines, human colon BE and human lung A427. Comparative cell kill in the Mer+ counterparts HT29 and A549 (respectively) was significantly lower.

Identification of a glutathione S-transferase associated with microsomes of tumor cells resistant to nitrogen mustards.

Walker 256 rat mammary carcinoma cells resistant to chlorambucil (WR) exhibited an approximate 4-fold increase in glutathione S-transferase (GST) activity using 1-chloro-2,4-dinitrobenzene as compared to the sensitive parent cell line (WS). WR cells maintained without biannual exposure to chlorambucil (WRr) reverted to the sensitive phenotype and possessed GST levels equivalent to WS. Mitochondria, microsomes and cytosol were isolated from WS, WR and WRr cell lines and analyzed for their GST composition.

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.

Purification of glutathione S-transferases from rat liver and Walker 256 mammary carcinoma cells by high-performance liquid chromatography and a glutathione affinity column.

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.

Estramustine binds a MAP-1-like protein to inhibit microtubule assembly in vitro and disrupt microtubule organization in DU 145 cells.

The twofold purpose of the study was (a) to determine if a MAP-1-like protein was expressed in human prostatic DU 145 cells and (b) to demonstrate whether a novel antimicrotubule drug, estramustine, binds the MAP-1-like protein to disrupt microtubules. SDS-PAGE and Western blots showed that a 330-kD protein was associated with microtubules isolated in an assembly buffer containing 10 microM taxol and 10 mM adenylylimidodiphosphate. After purification to homogeneity on an A5m agarose column, the 330-kD protein was found to promote 6 S tubulin assembly.

Ethacrynic acid and piriprost as enhancers of cytotoxicity in drug resistant and sensitive cell lines.

Ethacrynic acid and piriprost (6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1) have been shown to potentiate the cytotoxic activity of chlorambucil in rat and human tumor cell lines. Walker 256 rat breast carcinoma cells (WS), with acquired resistance to nitrogen mustards (WR), and two human colon carcinoma cell lines, HT 29 and BE, were sensitized to chlorambucil when either ethacrynic acid or piriprost was administered at the same time as the alkylating agent. Both as single agents and in combination with chlorambucil, there was inhibition of glutathione S-transferase activity as measured with 1-chloro-2,4-dinitrobenzene as a substrate.

Estramustine binds MAP-2 to inhibit microtubule assembly in vitro.

We have investigated the ability of estramustine to bind to rat brain microtubule-associated proteins (MAPs) and purified MAP-2 in vitro. [3H]estramustine's relative affinity for tubulin and MAPs was assessed by gel filtration chromatography, immunoprecipitation and binding assays. Scatchard analysis demonstrated a specific affinity of the drug for MAP-2.

Immunofluorescent studies of the anti-microtubule effects of the anti-cancer drug estramustine.

Immunofluorescent studies in human prostatic carcinoma cells (DU 145) and cultured squirrel fish epithelial cells (a non-cancer cell) revealed that estramustine, a conjugate of estradiol and nor-nitrogen mustard, possessed microtubule disassembly properties. Sixty microM estramustine produced disassembly at both the proximal and distal ends of microtubules, producing short pieces of less than 2 microM which were "wavy" and oriented in a random manner. With increased time of drug exposure these short microtubules disappeared, to be accompanied by a gradual disassembly of a small population of longer microtubules (greater than 7-8 microM).

Glutathione S-transferases in human prostate.

A number of human prostatic tissue biopsies have been analyzed for glutathione S-transferase activity, using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. Samples from nine patients (age range 61-90) with benign prostatic hypertrophy who had received no prior chemotherapy had a mean glutathione S-transferase activity of 137 +/- 44 nmol/min per mg with a range of 97-237. A qualitative comparison of the glutathione S-transferase of normal prostate and benign prostatic hypertrophy samples was carried out.

Glutathione S-transferases in nitrogen mustard-resistant and -sensitive cell lines.

Tumor cell resistance to alkylating agents was studied by examining Walker 256 rat mammary carcinoma cells differentially sensitive to nitrogen mustards. A resistant subpopulation (WR) was selected by exposure to chlorambucil. WR cells showed approximately a 15-fold resistance to the cytotoxic effects of nitrogen mustards and elevated glutathione S-transferase (GST) activity when compared to the sensitive parent cell line (WS).

C-banding of two Walker 256 rat carcinoma cell lines sensitive and resistant to bifunctional mustards.

Walker 256 rat carcinosarcoma cell lines sensitive (WS) or resistant (WR) to bifunctional nitrogen mustards have modal chromosome numbers of 60 and 55 respectively. Karyotype analysis revealed that these cell lines have retained the major marker chromosomes present in the original in vivo Walker tumours. One new marker chromosome, a metacentric, was found in the WR cell line.

Hormone-independent, non-alkylating mechanism of cytotoxicity for estramustine.

Over two decades, experience with estramustine has provided limited data which support an estrogenic mechanism of action and no data which indicate the nitrogen mustard involvement in the cytotoxic properties of the drug. Consideration of the carbamate-ester portion of estramustine supports the pharmacokinetic evidence that estramustine has a long half life since enzymatic hydrolysis of the carbamate is an uncommon event. Using a variety of immunocytochemical and cellular morphology procedures, estramustine per se has been found to express anti-cytoskeletal properties through non-covalent binding to microtubule associated proteins (MAP's).

The effect of a novel taurine nitrosourea, 1-(2-chloroethyl)-3-[2-(dimethylaminosulfonyl)ethyl]-1-nitrosour ea (TCNU) on cytotoxicity, DNA crosslinking and glutathione reductase in lung carcinoma cell lines.

A novel nitrosourea, 1-(2-chloroethyl)-3-[2-(dimethylaminosulfonyl)ethyl]-1-nitrosourea (TCNU) has been investigated with respect to cytotoxic mechanisms in rat and human cell lines which either possess (Mer+) or lack (Mer-) 0(6)-alkylguanine transferase activity. TCNU produced significantly greater cytotoxicity in the Mer- cells (Walker 256 rat breast carcinoma resistant to nitrogen mustards; human lung carcinoma A427) than in the Mer+ cells (Walker 256 wild-type; human lung carcinoma A594). This correlated with results generated by alkaline elution studies which showed that TCNU caused DNA interstrand crosslinks in A427 but not in A549 cells.

Differences in the nuclear matrix phosphoproteins of a wild-type and nitrogen mustard-resistant rat breast carcinoma cell line.

Using polyclonal antibodies raised against a rat liver nuclear envelope protein, lamin protein A, the nuclear matrix proteins of a Walker 256 rat mammary carcinoma wild-type (WS) and a selected cell line with acquired resistance to nitrogen mustards (WR) were found to possess antigenic determinants which were recognized by the antibodies. In one-dimensional immunoblotting analysis, the nuclear matrix protein fractions of both cell lines revealed a common band at Mr 75,000; however only the WS nuclear matrix protein fraction contained a broad band at approximately Mr 70,000. Two-dimensional gel blotting studies of these proteins showed that this Mr 70,000 WS protein had a pI of approximately 7.

Selection of nitrogen mustard resistance in a rat tumor cell line results in loss of guanine-O6-alkyl transferase activity.

Cell killing, DNA-interstrand crosslinks, and DNA-protein crosslinks were assayed in nitrogen mustard-resistant Walker 256 carcinoma (WR) cells and the parent cell line (WS) after treatment with 5-[3-(2-chloroethyl)-1-triazenyl]imidazo-4-carboxamide (MCTIC). The WR cells, which also express collateral sensitivity to chloroethylnitrosoureas, were approximately twice as sensitive to the cytotoxic effects of MCTIC as were WS cells. Following treatment with 100 microM MCTIC, there was a rapid accumulation of both DNA-interstrand and DNA-protein crosslinks in the WR cell line, which reached a maximum at 6 and 12 hr, respectively.

Relationship of glutathione depletion and inhibition of glutathione-S-transferase activity to the antimitotic properties of estramustine.

A depletion of intracellular glutathione (GSH) is accompanied by a subsequent inhibition of GSH-S-transferase activity in a DU145 human prostatic carcinoma cell line treated with cytotoxic concentrations of estramustine. When GSH depletion reached approximately 50% of normal (approximately 2 hours after 10 microM estramustine), the mitotic index of logarithmically dividing cultures began to increase. A linear increase from 3.

A comparative analysis of drug-induced DNA effects in a nitrogen mustard resistant cell line expressing sensitivity to nitrosoureas.

In the Walker 256 rat mammary carcinoma cell line, WR, resistance to nitrogen mustards (NM) is accompanied by collateral sensitivity to chloroethylnitrosoureas (CENUs). DNA-interstrand cross-links, DNA-protein cross-links, and sister chromatid exchange (SCE) induction were assayed in WR and the parent cell line (WS) after treatment with nitrogen mustard (HN2), phosphoramide mustard (PM), chlorozotocin (CLZ) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU). Treatment of cells with HN2 caused extensive levels of cross-links, approximately 50% of which were DNA-interstrand, equal in both WR and WS, whereas PM caused no detectable cross-links in either cell line.

Dansylated estramustine, a fluorescent probe for studies of estramustine uptake and identification of intracellular targets.

Fluorescence-microscopic studies with dansylated estramustine (DnsEM) has permitted investigation of the mechanism of estramustine (EM) uptake in live human prostatic tumor cells (DU 145). DnsEM appeared to enter cells rapidly at the peripheral cell margins. A progressive increase in fluorescence was observed until the perinuclear material and cytoplasm were labeled brightly and the nucleoplasm was labeled faintly.