Biomedical subjects

A J Townsend

Publications and source records attributed to A J Townsend.

At least 37 records · Page 2Linked to original sources

PubMed1998

Multidrug resistance protein and glutathione S-transferase P1-1 act in synergy to confer protection from 4-nitroquinoline 1-oxide toxicity.

Model cell lines developed from MCF7 breast carcinoma cells were used to examine the roles of glutathione S-transferase P1-1 (GSTP1-1) and multidrug resistance protein (MRP) in the protection of cells from 4-nitroquinoline 1-oxide (4NQO) toxicities. Increased expression of GSTP1-1 alone in MCF7 cells results in limited protection from the formation of 4NQO-derived covalent adducts of nucleic acids but affords no protection from 4NQO-mediated cytotoxicity. Increased expression of MRP alone conferred modest protection while co-expression of GSTP1-1 with MRP produced high-level protection from both 4NQO-derived adduct formation and 4NQO cytotoxicity. This synergistic resistance to 4NQO toxicities (both nucleic acid adduct formation and cytotoxicity) is associated with a GSTP1-1-dependent increase in 4NQO-glutathione (QO-SG) conjugate formation and a MRP-dependent increase in QO-SG efflux. These data indicate that MRP is an important export transporter for the glutathione conjugate of the carcinogen, 4NQO. Moreover, this MRP-dependent efflux activity is necessary to achieve the full protection from 4NQO toxicity-protection that is potentiated by GSTP1-1-mediated QO-SG formation.

4-Nitroquinoline-1-oxide↗

PubMed1998

Overexpression of stably transfected human glutathione S-transferase P1-1 protects against DNA damage by benzo[a]pyrene diol-epoxide in human T47D cells.

The (+)-anti enantiomer of benzo[a]pyrene-7,8-dihydrodiol-9, 10-epoxide (BPDE) is a potent mutagenic and carcinogenic metabolite of benzo[a]pyrene (BP), and a major fraction is conjugated with glutathione in vivo. The chemopreventive role of glutathione S-transferases (GSTs) in protecting against covalent modification of DNA and other cellular macromolecules by BPDE was modeled in human T47D and MCF-7 cell lines previously stably transfected with human GSTpi1 (hGSTP1). Cells were exposed to [3H]BPDE (30-600 nM). Dose-response experiments indicated that the high level of expression of hGSTP1-1 in the T47Dpi cell line (4411 +/- 183 milliunits/mg of cytosolic protein, using 1-Cl-2,4-dinitrobenzene as substrate), resulted in 70-90% reduction in the covalent 3H-adduct formation in DNA or RNA isolated from the GSTP1-transfected T47Dpi cell line. The lower level of hGSTP1-1 expression in the transfected MCF-7 cell line (91 milliunits/mg) provided only marginal protection against [3H]BPDE adduct formation and did not affect sensitivity to BPDE-induced cytotoxicity. Protection against BPDE-induced cytotoxicity was observed only in the T47Dpi cell line, which had an IC50 value 5.8-fold greater than that of the T47Dneo control cell line. Measurement of glutathione conjugates of BPDE indicated that the total conjugation was 5-fold higher in the GSTpi-transfected T47D line, most of which was exported into the culture medium over the 20-min exposure period. These results indicate that hGSTP1-1 protects effectively against DNA and RNA modification by BPDE, but moderate to high level expression may be required for strong protection against BPDE-induced genotoxicity and cytotoxicity.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed1997

Coding region-specific destabilization of mRNA transcripts attenuates expression from retroviral vectors containing class 1 aldehyde dehydrogenase cDNAs.

Class 1 aldehyde dehydrogenases (ALDH-1) function as drug resistance gene products by catalyzing the irreversible conversion of aldophosphamide, an active metabolite of cyclophosphamide, to an inert compound. Because the dose-limiting toxicity of cyclophosphamide is myelosuppression, retrovirus-mediated transfer of ALDH-1 to bone marrow cells has been proposed as a protective strategy. Here we show that expression of ALDH-1 vectors was problematic due to low levels of ALDH-1 mRNA accumulation. A number of vectors containing several different ALDH-1 cDNAs were introduced into a variety of different cell lines either by transfection or transduction. Detectable ALDH-1 protein and enzyme activity was only seen in one transfected cell clone. Cells transduced with ALDH-1 retroviral vectors had no detectable protein expression and very low levels of ALDH-1 mRNA. Analogous vectors containing other drug resistance cDNAs led to much higher levels of steady-state mRNA. The mRNA half-life from ALDH-1 vectors was less than 2 hr suggesting that vector-derived mRNAs were destabilized by ALDH-1 coding sequences. These results suggest that methods which increase the stability of ALDH-1 mRNAs will be important for increased drug resistance in retrovirally transduced hematopoietic cells.

Aldehyde Dehydrogenase↗

PubMed1997

Relation of glutathione S-transferase alpha and mu isoforms to response to therapy in human breast cancer.

Glutathione S-transferase (GST) represents a multifunctional enzyme family consisting of four known cytosolic isoforms (alpha, mu, pi, and Phi) that detoxify a variety of xenobiotic chemicals and may confer resistance to both chemotherapeutic drugs and carcinogens in various experimental models. GST-pi has already been extensively studied in clinical specimens, including breast cancer. We studied the immuno-histochemical distribution and relative immunopositivity of GST-alpha and GST-mu, based on a grading system for immunointensity, in samples of 51 neoplastic and 46 normal breast samples and 12 lymph node metastases from patients treated with intensive chemotherapy and bone marrow transplant. In normal breast tissue, GST-alpha localized predominantly to the cytoplasm of scattered cells lining the luminal aspects of the ducts. Occasional cells showed both cytoplasmic and nuclear GST-alpha immunoreactivity. GST-mu was stained in myoepithelial cells preferentially as well as in occasional ductal cells (including apocrine epithelium), vascular smooth muscle, and plasma cells. GST-alpha and GST-mu were detected in 22 of 51 (43%) and 24 of 48 (50%) invasive cancers, respectively. In paired samples of normal and malignant tissue from the same patient, GST-alpha immunostaining in cancers was significantly less intense compared to that of normal breast tissue in 13 of 41 (32%) cases. No such trend was found for GST-mu in paired samples. Neither GST-alpha nor GST-mu immunopositivity in tumor or nonneoplastic breast was found to correlate with relapse-free or overall survival in this clinical context; however, the apparent decreased expression of GST-alpha in malignant versus normal breast epithelial cells could have important implications in breast carcinogenesis.

Adult↗

PubMed1996

De novo expression of transfected human class 1 aldehyde dehydrogenase (ALDH) causes resistance to oxazaphosphorine anti-cancer alkylating agents in hamster V79 cell lines. Elevated class 1 ALDH activity is closely correlated with reduction in DNA interstrand cross-linking and lethality.

Human class 1 aldehyde dehydrogenase (hALDH-1) can oxidize aldophosphamide, a key aldehyde intermediate in the activation pathway of cyclophosphamide and other oxazaphosphorine (OAP) anti-cancer alkylating agents. Overexpression of class 1 ALDH (ALDH-1) has been observed in cells selected for survival in the presence of OAPs. We used transfection to induce de novo expression of human ALDH-1 in V79/SD1 Chinese hamster cells to clearly quantitate the role of hALDH-1 expression in OAP resistance. Messenger RNA levels correlated well with hALDH-1 protein levels and enzyme activities (1.5-13.6 milliunits/mg with propionaldehyde/NAD+ substrate, compared to < 1 milliunit/mg in controls) in individual clonal transfectant lines, and slot blot analysis confirmed the presence of the transfected cDNA. Expressed ALDH activity was closely correlated (r = 0.99) with resistance to mafosfamide, up to 21-fold relative to controls. Transfectants were cross-resistant to other OAPs but not to phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. Resistance was completely reversed by pretreatment with 25 microM diethylaminobenzaldehyde, a potent ALDH inhibitor. Alkaline elution studies showed that expression of ALDH-1 reduced the number of DNA cross-links commensurate with mafosfamide resistance, and this reduction in cross-links was fully reversed by the inhibitor. Thus, overexpression of human class 1 ALDH alone is sufficient to confer OAP-specific drug resistance.

Aldehyde Dehydrogenase↗

PubMed1996

Protection by transfected rat or human class 3 aldehyde dehydrogenase against the cytotoxic effects of oxazaphosphorine alkylating agents in hamster V79 cell lines. Demonstration of aldophosphamide metabolism by the human cytosolic class 3 isozyme.

Expression of class 3 aldehyde dehydrogenase (ALDH-3) has been associated with acquired or inherent resistance to oxazaphosphorine (OAP) antineoplastic alkylating agents (eg. cyclophosphamide). We previously demonstrated that expression of transfected rat ALDH-3 can confer OAP-specific resistance in human MCF-7 cells (Bunting, K. D., Lindahl, R., and Townsend, A. J. (1994) J. Biol. Chem. 269, 23197-23203). However, the aldophosphamide intermediate inactivated by human class 1 ALDH (hALDH-1) has not proven to be a good substrate for the purified hALDH-3. We have examined the ability of transfected human or rat ALDH-3 to confer OAP resistance in V79/SDl cells. Clones expressing elevated human (386-5938 milliunits/mg) or rat (4-597 milliunits/mg, benzaldehyde/NADP+ substrate) ALDH-3 activity were 1.3- to 12-fold resistant to mafosfamide relative to control cells (<1 milliunit/mg). Resistance was correlated with hALDH-3 activity, and was reversed by pretreatment with the ALDH inhibitor diethylaminobenzaldehyde. Transfectants were cross-resistant to 4-hydroperoxycyclophosphamide and 4-hydroperoxyifosfamide but not to phosphoramide mustard, ifosfamide mustard, melphalan, or acrolein. DNA interstrand cross-links were reduced commensurately with the fold resistance to mafosfamide in the highest activity clone. A key finding was the detection of a metabolite, most likely carboxyphosphamide, that is formed only by cytosols from cells expressing either class 3 or class 1 ALDH.

Aldehyde Dehydrogenase↗

PubMed1994

Oxazaphosphorine-specific resistance in human MCF-7 breast carcinoma cell lines expressing transfected rat class 3 aldehyde dehydrogenase.

Overexpression of either class 1 or class 3 aldehyde dehydrogenase (ALDH) has been found in cell lines selected for resistance to the oxazaphosphorine (OAP) alkylating anticancer agent cyclophosphamide (CPA). Direct oxidation of the CPA metabolic intermediate aldophosphamide (ALDO) is catalyzed efficiently in vitro by the class 1 ALDH isozyme, but the involvement of the class 3 isozyme in OAP resistance is problematic since in vitro studies do not show efficient oxidation of ALDO. Cell lines were established that express stably transfected rat class 3 ALDH to model the potential role of this isozyme in OAP resistance. Clonogenic survival assay data indicated that even modest expression of rat class 3 ALDH was associated with resistance (2-4-fold) to the CPA analog mafosfamide and that the fold resistance was directly proportional to the class 3 ALDH activity expressed in clonal transfectants. Pretreatment of the highest activity cell line (3A1-31A) with 75 microM diethylaminobenzaldehyde, an ALDH substrate and inhibitor of benzaldehyde oxidation, effectively reversed the 3.8-fold resistance in this line; drug sensitivity was unaffected by diethylaminobenzaldehyde in the control transfected cell line. The resistance conferred by ALDH to mafosfamide is OAP-specific since the 3A1-31A line is also resistant to 4-hydroperoxycyclophosphamide (2.9-fold) and 4-hydroperoxyifosfamide (3.2-fold) but not to the non-oxazaphosphorine drugs phosphoramide mustard and melphalan, which cannot be detoxified by aldehyde dehydrogenase enzymes.

Aldehyde Dehydrogenase↗

PubMed1994

Protection by transfected glutathione S-transferase isozymes against carcinogen-induced alkylation of cellular macromolecules in human MCF-7 cells.

Increased expression of glutathione S-transferase (GST) isozymes has been correlated with development of resistance both to cytotoxic anticancer agents and to genotoxic carcinogens. While most anticancer agents are poor GST substrates, the model alkylating carcinogen 4-nitroquinoline-1-oxide (NQO) is a good substrate for human pi class GST (hGSTP1-1) and murine GST mu-1 (mGSTM1-1), but not human GST alpha-2 (hGSTA2-2). We investigated whether expression of these GST isozymes in stably transfected clonal cell lines could protect against the genotoxic and cytotoxic effects of NQO. Compared to parental MCF-7 or pSV2neotransfected control cell lines, covalent labeling of total cellular macromolecules by [3H]NQO (0.1-1.0 mM) was reduced by 70% and 92% in hGSTP1-1- and mGSTM1-1-transfected cell lines, respectively, but was not affected in the hGSTA2-2 expressing line. The observed protection was closely correlated with the relative specific activity of each cell line for conjugation of NQO by the transfected GST isozymes and this protection was reversible by pretreatment of cells with the GST inhibitor ethacrynic acid. Similar results were obtained when covalent labeling of total cellular nucleic acid or DNA was measured. However, clonogenic survival assays indicated that the sensitivity of these cell lines to the cytotoxic effects of NQO was similar for the control and GST-transfected MCF-7 cell lines. Thus, while expression of hGSTP1-1 and mGSTM1-1 (but not hGSTA2-2) was highly protective against alkylation of cellular macromolecules by NQO, this protection was not effective against cytotoxicity induced by NQO as measured by clonogenic assay. These results indicate that expression of GST isozymes can protect differentially against the acute genotoxic and potentially mutagenic effects, as compared to the cytotoxic effects, of electrophiles that are detoxified by glutathione conjugation.

4-Nitroquinoline-1-oxide↗

PubMed1994

Coordinate induction of glutathione S-transferase alpha, mu, and pi expression in murine liver after a single administration of oltipraz.

The antischistosomal agent oltipraz displays a unique ability to inhibit chemically induced carcinogenesis in a variety of animal models. Its apparent lack of carcinogen specificity and low toxicity make it an attractive candidate for further development as a chemopreventive agent. The mechanism by which oltipraz affords cellular protection is thought to involve the modulation of phase II detoxication enzymes. The present study examines the regulation of each class of glutathione S-transferase (EC 2.5.1.18) in mice after a single oral administration of oltipraz. Glutathione S-transferase activity in the liver increased in a dose-dependent manner after drug exposure. Oltipraz administration (1 g/kg, by gavage) elevated glutathione S-transferase activity to a maximum (4.5-fold) on day 4 after treatment. Western blot analyses demonstrated the induction of all three classes of glutathione S-transferase (alpha, mu, and pi) by oltipraz. Our murine studies suggest that the chemopreventive activity of oltipraz may be due in part to its ability to elevate glutathione S-transferase-mu activity. Consistent with this possibility, associations between the glutathione S-transferase-mu-null phenotype and increased risk for lung, larynx, and bladder cancer have been recently demonstrated in humans. Coordinate elevations in enzymatic activity were preceded by significant elevations in glutathione S-transferase alpha, mu, and pi RNA on day 2 after treatment. Although nuclear run-on assays confirmed the transcriptional induction of all three classes, the maintenance of elevations in enzymatic activity after RNA levels returned to base-line suggests that additional mechanisms are required to regulate glutathione S-transferase expression. Preclinical findings are presented that characterize the response of each class of glutathione S-transferase to oltipraz exposure and support the use of these enzymes as intermediate markers of the chemopreventive activity of oltipraz.

Animals↗

PubMed1994

Mechanisms of resistance to ansamycin antibiotics in human breast cancer cell lines.

We recently reported that multidrug-resistant, P-170 glycoprotein-positive, Adriamycin-selected, human breast tumor (MCF7/ADRR) cells were resistant to the benzoquinonoid ansamycin antibiotics geldanamycin (GL) and herbimycin A (HA) and that significantly fewer hydroxyl radicals were formed in resistant cells. We have carried out additional studies to define the mechanisms of cytotoxicity of and resistance to GL and HA, by directly examining the interactions of these drugs with P-170 glycoprotein using photoaffinity labeling. We found that both GL and HA inhibited binding of azidopine to P-170 glycoprotein in a dose-dependent manner. We have developed a 10-fold GL-resistant cell line (MCF7/GLR) by continuous drug exposure. Our studies indicated no significant differences in free radical formation between wild-type MCF7 cells and MCF7/GLR cells. Uptake and efflux studies indicated a small decrease in the GL accumulation but no difference in the efflux of GL in these cells. Verapamil had no effect on cellular accumulation of GL in wild-type MCF7 cells or MCF7/GLR cells. Verapamil significantly increased the accumulation of GL in MCF7/ADRR cells and enhanced GL cytotoxicity 12-fold, suggesting that GL interacted with the P-170 glycoprotein. Using reverse transcription-polymerase chain reaction, we found no expression of the mdr1 gene; however, expression of the multidrug resistance-associated protein was about 2-fold higher in MCF7/GLR cells. Taken together, these studies indicate that the mechanisms of GL resistance are multifactorial. Although decreased free radical formation may not play a significant role in low levels of GL resistance, e.g., in MCF7/GLR cells, both overexpression of mdr1 and decreased free radical formation contribute to GL resistance in highly resistant cells such as MCF7/ADRR cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Glutathione transferase GST pi in breast tumors evaluated by three techniques.

The glutathione transferases are involved in intracellular detoxification reactions. One of these, GST pi, is elevated in some breast cancer cells, particularly cells selected for resistance to anticancer agents. We evaluated GST pi expression in 60 human breast tumors by three techniques, immunohistochemistry. Northern hybridization, and Western blot analysis. There was a significant positive correlation between the three methods, with complete concordance seen in 64% of the tumors. There was strong, inverse relationship between GST pi expression and steroid receptor status with all of the techniques utilized. In addition, there was a trend toward higher GST pi expression in poorly differentiated tumors, but no correlation was found between tumor GST pi content and DNA ploidy or %S-phase. GST pi expression was also detected in adjacent benign breast tissue as well as infiltrating lymphocytes; this expression may contribute to GST pi measurements using either Northern hybridization or Western blot analysis. These results suggest that immunohistochemistry is the method of choice for measuring GST pi in breast tumors.

Blotting, Northern↗

PubMed1992

Expression of human mu or alpha class glutathione S-transferases in stably transfected human MCF-7 breast cancer cells: effect on cellular sensitivity to cytotoxic agents.

Increased expression of certain glutathione S-transferase (GST) isoenzymes has frequently been associated with the development of resistance to alkylating agents and other classes of antineoplastic drugs in drug-selected cell lines. The question arises whether this phenomenon is causal or is a stress-induced response associated with drug resistance in these cell lines. We have constructed mammalian expression vectors containing the human GST mu and GST alpha 2 (Ha2) cDNAs and stably transfected them into the human breast cancer cell line MCF-7. Whereas the parental and pSV2neo-transfected cell lines display low GST activity, three individual transfected clones were identified in each group that expressed either GST mu or GST alpha 2. The range of GST activities was similar to those observed in cells selected for anticancer drug resistance. The GST mu specific activities were 56, 150, and 340 mlU/mg, compared with 10 mlU/mg of endogenous GST mu in control lines. Specific activities in GST alpha 2-transfected clones were 17, 28, and 52 mlU/mg, compared with no detectable alpha class GST in control lines. These clonal lines and the parental and pSV2neo-transfected control lines were tested for sensitivity to antineoplastic agents and other cytotoxic compounds. The clones with the highest activity in each group were 1.7-fold (GST alpha 2) to 2.1-fold (GST mu) resistant to the toxic effects of ethacrynic acid, a known substrate for GSTs. However, the GST-transfected cell lines were not resistant to doxorubicin, L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, cisplatin, chlorambucil, or the GST substrates 1-chloro-2,4-dinitrobenzene or tert-butyl hydroperoxide. Thus, although L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, chlorambucil, tert-butyl hydroperoxide, and 1-chloro-2,4-dinitrobenzene are known to be metabolized by glutathione-dependent GST-catalyzed reactions, there was no protection against any of these agents in MCF-7 cell lines overexpressing GST mu or GST alpha 2. We conclude that, at the levels of GST obtained in this transfection model system, overexpression of GST mu or GST alpha 2 is not by itself sufficient to confer resistance to these anticancer agents. These studies do not exclude the possibility that GST may be a marker of drug resistance or that other gene products not expressed in MCF-7 cells might cooperate with GST to confer drug resistance.

Antineoplastic Agents↗

PubMed1991

Monoclonal antibodies to glutathione S-transferase pi-immunohistochemical analysis of human tissues and cancers.

Mouse monoclonal antibodies (MAb) have been generated against the anionic isozyme of human glutathione S-transferase (GST pi). MAb AGST I can inhibit 50-70% of GST pi enzymatic activity and reacts with a 3-dimensional epitope which includes a putative glutathione binding site on GST pi. A sandwich enzyme-immunoassay established using MAb AGST I and a polyclonal antibody displayed a sensitivity of 0.5 ng/ml. Immunohistochemical analysis of human tissues demonstrated marked increases in GST pi levels in cancers of the brain, cervix, endometrium, colon, rectum and testis and in fibro- and chondrosarcomas.

Antibodies, Monoclonal↗

PubMed1991

Increased glutathione peroxidase activity in a human sarcoma cell line with inherent doxorubicin resistance.

Several mechanisms of drug resistance have been defined using cell lines selected for resistance in vitro. However, the relevance of these to tumor cell resistance in vivo remains unclear. We established tumor cell lines from biopsies of human sarcomas before and after doxorubicin therapy. One pretreatment sarcoma line, STSAR90, was 6-fold less sensitive to doxorubicin than was a normal fibroblast line, AG1522. The sensitivities of six other sarcoma lines were similar to that of AG1522. STSAR90 cells did not overexpress P-glycoprotein mRNA, by Northern analysis with the pCHP1 complementary DNA fragment. Photoaffinity labeling with the vinblastine analogue N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine did not show increased P-glycoprotein concentrations. Accumulation of [3H]daunomycin was not decreased in STSAR90 compared with a less resistant sarcoma line, STSAR11, nor was the doxorubicin sensitivity of STSAR90 increased by coincubation with verapamil. Glutathione levels were twice as high in STSAR90 as in STSAR11, and glutathione peroxidase activity was 3.5- to 6-fold higher. This was due mostly to an increase in selenium-dependent peroxidase activity. After exposure to doxorubicin, STSAR90 cells formed only half as much measurable hydroxyl radical as STSAR11, as detected by electron spin resonance spectrometry. Doxorubicin sensitivity was increased in STSAR90 cells when intracellular glutathione levels were reduced by buthionine sulfoximine. These results indicate that multidrug resistance due to P-glycoprotein-mediated drug efflux is not the only mechanism of doxorubicin resistance that occurs in sarcomas and that glutathione peroxidase-dependent detoxification of doxorubicin-induced oxygen radicals may contribute to clinical doxorubicin resistance.

Adolescent↗

PubMed1991

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.

Antineoplastic Agents↗

PubMed1991

Selenium-dependent glutathione peroxidase expression is inversely related to estrogen receptor content of human breast cancer cells.

The absence of estrogen receptors (ER) in human breast tumors has been associated with a poorer prognosis compared to patients with ER positive breast cancer. Previous studies from our laboratory have shown that a multidrug resistant human breast cancer cell line selected for resistance to Adriamycin (ADR) exhibited markedly increased expression of both the pi class glutathione S-transferase (GST-pi) and the selenium-dependent glutathione peroxidase. These studies also revealed that the ER status was inversely related to the expression of GST-pi in six human breast cancer cell lines and primary tumor specimens. In the present study, we have examined the relationship between ER status and several biological properties of these cells, including their levels of glutathione peroxidase (GSH-Px) and catalase expression, their capacity to generate toxic hydroxyl radicals (degrees OH) by redox cycling of ADR, and their sensitivities to the cytotoxic effects of ADR and the oxidant, H2O2. Our results show that expression of GSH-Px, but not catalase, is inversely related to the ER status in these cell lines. Formation of the degree OH induced by treatment of cells with ADR was inversely proportional to the GSH-Px activity in these cell lines, and thus directly related to the ER status. Sensitivity of these cells to ADR or to H2O2, however, was not consistently related to ER status, GSH-Px, or catalase activity, or to ADR induced degree OH radical formation. These results indicate that these parameters are not predictive of cellular susceptibility to oxidative damage in these cell lines under the conditions studied.

Blotting, Northern↗

PubMed1990

An immunohistochemical study of pi class glutathione S-transferase expression in normal human tissue.

Glutathione S-transferases (GSTs), a family of isoenzymes that play an important role in protecting cells from cytotoxic and carcinogenic agents, can be separated by biochemical and immunologic characteristics into three distinct classes named alpha, mu, and pi. Previous studies have indicated that there is marked heterogeneity in the expression of different GST isoenzymes in different normal and malignant tissues. To better understand the regulation of the human pi class glutathione S-transferase isoenzyme (GST-pi), the tissue distribution of this protein wa studied by an immunohistochemical technique using an anti-GST-pi polyclonal antibody in normal paraffin-embedded human tissues. These studies indicate that there is a broad distribution of GST-pi in normal human tissues and establish a precise localization within the different organs studied. GST-pi was expressed predominantly in normal epithelial cells of the urinary, digestive, and respiratory tracts, suggesting a possible role for GST-pi in detoxication and elimination of toxic substances. Previous studies have indicated that GST-pi and the putative drug efflux pump P-glycoprotein are both overexpressed in multidrug-resistant human breast cancer cells and in xenobiotic resistant preneoplastic rat hyperplastic liver nodules. Results from this study indicate that there are also similarities between the normal tissue distribution GST-pi and that previously reported for mammalian P-glycoprotein, particularly in secretory epithelia. This finding suggests that these two gene products, which have been implicated in the development of resistance to cytotoxic drugs, may be coregulated in normal and malignant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Biochemical correlates of the antitumor and antimitochondrial properties of gossypol enantiomers.

Racemic gossypol has been shown to have antitumor properties that may be due to its ability to uncouple tumor mitochondria or to its inhibitory effects on a variety of nonmitochondrial enzymes. We have studied the antimitochondrial and enzyme-inhibiting properties of gossypol in human carcinoma cell lines of breast (MCF-7, T47-D), ovarian (OVCAR-3) colon (HCT-8), and pancreatic (MiaPaCa) origin by comparing the effects of its purified (+)- and (-)-enantiomers. (-)-Gossypol shows up to 10-fold greater antiproliferative activity than (+)-gossypol in the cancer cell lines and in normal hematopoietic stem cells grown in vitro, with IC50 values ranging from 1.5 to 4.0 microM for the cancer cells and from 10 to 20 microM for the human marrow stem cells. As well, multidrug-resistant MCF/Adr cells appear more resistant to (-)-gossypol than their parental cell line. Electron microscopy indicates that the earliest ultrastructural change in tumor cells exposed to a cytotoxic (10 microM) concentration of (-)-gossypol is the selective destruction of their mitochondria. Consistent with this observation, 31P magnetic resonance spectroscopy detects pronounced changes in tumor cell high energy phosphate metabolism within 24 hr of (-)-gossypol treatment, manifest by 1.6- to greater than 50-fold differential reductions in the intracellular ratios of ATP/Pi, relative to (+)-gossypol-treated cell lines; the magnitude of these antimitochondrial effects correlates with the antiproliferative activity of (-)-gossypol. Northern blot RNA analyses suggest that treatment with a 5-10 microM dose of (-)-gossypol induces a transient increase in the expression of heat shock gene products, particularly hsp-70 transcripts. The mean 5-fold increase in (-)-gossypol-induced hsp-70 mRNA appears coincident with a comparable heat-stimulated increase in transcript levels, as compared with control or (+)-gossypol-treated cells. The enzyme-inhibiting properties of gossypol enantiomers were compared in cell-free assays measuring glutathione-S-transferase-alpha, -mu, and pi activities, calmodulin stimulation of cyclic nucleotide phosphodiesterase, and protein kinase C activity. Both enantiomers are near equivalent antagonists of calmodulin stimulation and protein kinase C activity, exceeding the potency of known inhibitors such as phenothiazines by as much as 50-fold. In contrast, (-)-gossypol is a 3-fold more potent inhibitor of glutathione-S-transferase-alpha and -pi isozyme activity, resulting in IC50 values of 1.6 and 7.0 microM, respectively, for these two isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)

Antineoplastic Agents↗