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.

Resistance to mitoxantrone in multidrug-resistant MCF7 breast cancer cells: evaluation of mitoxantrone transport and the role of multidrug resistance protein family proteins.

We examined the role of multidrug resistance protein (MRP) 1 (ABCC1) in the emergence of mitoxantrone (MX) cross-resistance in a MCF7 breast cancer cell line selected for resistance to etoposide. The resistant cell line, MCF7/VP, expresses high levels of MRP1, whereas the parental cell line, MCF7/WT, does not. MCF7/VP cells are 6-10-fold cross-resistant to MX when compared with MCF7/WT cells. Drug transport studies in intact MCF7/VP cells revealed that MX resistance is associated with reduced MX accumulation due to enhanced MX efflux. MX efflux is ATP dependent and inhibited by sulfinpyrazone and cyclosporin A. Inhibition of MX efflux with these agents sensitizes cells to MX cytotoxicity and partially reverses MX resistance in MCF7/VP cells. Whereas resistance is partially attributable to increased MX efflux in MRP1-expressing MCF7/VP cells, we found no evidence for glutathione or other conjugates of MX in these cells. Moreover, glutathione depletion with buthionine sulfoximine had no effect on MX transport or sensitivity in MCF7/VP cells. MRP1 substrates are generally amphiphilic anions such as glutathione conjugates or require the presence of physiological levels of glutathione for MRP1-mediated transport. Therefore we conclude that MRP1 overexpression is unlikely to be responsible for increased MX efflux and resistance in MCF7/VP cells. In considering the potential involvement of other MRP family isoforms, a 3-fold increase in the expression of MRP5 was observed in MCF7/VP cells. However, stable expression of a transduced MRP5 expression vector in MCF7/WT cells failed to confer MX resistance. Because other transporters known to be associated with MX resistance, including P-glycoprotein and BCRP/MXR (ABCG2), are not expressed in MCF7/VP cells, we conclude that increased MX efflux and resistance in MCF7/VP cells is attributable to a novel transport mechanism or that MX represents a novel class of cationic, glutathione-independent MRP1 substrates.

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.

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.

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.

Apoptosis in RAW 264.7 cells exposed to 4-hydroxy-2-nonenal: dependence on cytochrome C release but not p53 accumulation.

The toxic reactive aldehyde lipid peroxidation byproduct 4-hydroxy-2-nonenal (HNE) is thought to be a major contributor to oxidant stress-mediated cell injury. HNE induced apoptosis in RAW 264.7 murine macrophage cells in a dose-dependent manner within 6-8 h after exposure. Expression of the antiapoptotic protein Bcl-2 in stably transfected RAW 264.7 cells prevented HNE-induced internucleosomal DNA fragmentation and apoptosis, and these cells resume growth after a temporary (24-48 h) growth delay. While parental RAW 264.7 cells released mitochondrial cytochrome c within 3 h after HNE exposure, expression of Bcl-2 prevented cytochrome c release. In control cells, p53 protein levels peaked at 6-9 h after HNE exposure and then declined, while in Bcl-2 expressing cells, p53 levels were maximal at 6-9 h and remained elevated up to 96 h. Expression of SV40 large T-antigen, which forms a stable complex with p53 protein, via stable transfection-blocked transactivation of the p53-regulated gene p21(WAF1/CIP1), but did not affect induction of apoptosis by HNE, suggesting that p53 function is not important in HNE-induced apoptosis. These results suggest that cytochrome c release, but not p53 accumulation, plays an essential role in HNE-induced apoptosis in RAW 264.7 cells.

Dependence of aldehyde dehydrogenase-mediated oxazaphosphorine resistance on soluble thiols: importance of thiol interactions with the secondary metabolite acrolein.

Acrolein is a highly reactive and cytotoxic by-product released during activation of oxazaphosphorine (OAP) anticancer alkylating agents. Previously, we demonstrated that transfected human aldehyde dehydrogenase (ALDH, EC 1.2.1.3) isozymes (class 1 or 3) protect V79/SD1 cells from mafosfamide (MAF) cytotoxicity, but protection from 4-hydroperoxy-cyclophosphamide (4-hpCPA) was weaker. Acrolein, an ALDH inhibitor, may be detoxified by conjugation with the nucleophilic thiol 2-mercaptoethanesulfonate (MESNA), which is released from MAF but not from 4-hpCPA. We examined the effect of acrolein or acrolein/thiol conjugates on ALDH activity in vitro. We found that both ALDH isozymes were inhibited by acrolein, with IC50 values of 35 and 144 microM for ALDH-1 or ALDH-3, respectively. Both isozymes were partially protected by NAD+ cofactor, being at least five-fold more sensitive to acrolein if added before cofactor. In contrast, thiol conjugates of acrolein did not inhibit ALDH-3 activity, but were substrates only for ALDH-1. Further, acrolein was shown to be oxidized by ALDH-3, but not by ALDH-1. The effect of acrolein on ALDH-mediated resistance to OAP agents in intact cells was also examined. In control cells (without ALDH expression), acrolein and 4-hpCPA rapidly depleted intracellular GSH levels, whereas the effect of MAF was much less. Depletion of GSH by preincubation of V79/SD1 cells with a low concentration of acrolein (2 microM) before MAF exposure caused a two-fold reduction in ALDH-mediated resistance. Conversely, protection from 4-hpCPA cytotoxicity was enhanced by the addition of thiols (GSH, 2-mercaptoethanesulfonate, or N-acetylcysteine) during the drug exposure. These results suggest 1) that thiol content is an important determinant of the OAP resistance conferred by ALDH isoenzymes; and 2) a new mechanism whereby thiol modulation could increase the therapeutic index of OAP chemotherapy.

Combined expression of multidrug resistance protein (MRP) and glutathione S-transferase P1-1 (GSTP1-1) in MCF7 cells and high level resistance to the cytotoxicities of ethacrynic acid but not oxazaphosphorines or cisplatin.

We tested the hypothesis that combined increased expression of human glutathione S-transferase P1-1 (GSTP1-1), an enzyme that catalyzes the conjugation with glutathione of several toxic electrophiles, and the glutathione-conjugate efflux pump, multidrug resistance protein (MRP), confers high level resistance to the cytotoxicities of anticancer and other drugs. To accomplish this, we developed MCF7 breast carcinoma cell derivatives that express high levels of GSTP1-1 and MRP, alone and in combination. Parental MCF7 cells, which express no GSTP1-1 and negligible MRP, served as control cells. We found that either MRP or GSTP1-1 alone conferred significant resistance to ethacrynic acid cytotoxicity. Moreover, combined expression of GSTP1-1 and MRP conferred a high level of resistance to ethacrynic acid that was greater than resistance conferred by either protein alone. Increased MRP was also associated with modest resistance to the oxazaphosphorine compounds mafosfamide, 4-hydroxycyclophosphamide, and 4-hydroperoxycyclophosphamide. However, coordinated expression of GSTP1-1 with MRP failed to augment this modest resistance. Similarly, GSTP1-1 had no effect on the sensitivities to cisplatin of MCF7 cells regardless of MRP expression. These results establish that coordinated expression of MRP and GSTP1-1 can confer high level resistance to the cytotoxicities of some drugs, including ethacrynic acid, but that such resistance is variable and does not apply to all toxic drugs that can potentially form glutathione conjugates in either spontaneous or GSTP1-1-catalyzed reactions.

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.

Prenatal toxicity and lack of carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) following transplacental exposure.

Accumulating evidence from human and experimental animal studies indicates that consumption of heterocyclic amines (HA), derived from cooked meat and fish, may be associated with an increased incidence of cancer. Experiments were initiated to assess the role of one of these compounds, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), as a potential transplacental carcinogen, as well as to evaluate whether in utero exposure to IQ results in the induction of fetal cytochrome P4501A1 (Cyp1a1), P4501B1 (Cyp1b1), and/or glutathione S-transferase (GST). Inducible, or responsive, backcrossed fetuses resulting from a cross between congenic C57BL/6 (Ah(d)Ah(d)) nonresponsive female mice and C57BL/6 (Ah(b)Ah(b)) responsive male mice were transplacentally exposed to olive oil or 6.25, 12.5, or 25 mg/kg of IQ on day 17 of gestation. No macroscopically or microscopically visible liver, lung, or colon tumors were found in the transplacentally treated offspring by one year after birth. Ethoxyresorufin O-deethylase (EROD) and 1-chloro-2,4-dinitrobenzene assays were performed to evaluate whether transplacental exposure to IQ results in the induction of fetal Cyp1a1 and GST, respectively, in lung and liver tissues. Results showed levels of EROD and GST activity in tissues of IQ-treated mice to be very close, if not identical, to those of mice treated with olive oil. Similarly, ribonuclease protection assay data showed that the levels of Cyp1a1 and Cyp1b1 RNA in tissues of IQ-treated mice were not significantly different from those of oil-treated controls. Previous studies have shown that the developing organism expresses very low levels of Cyp1a2. Thus, in utero exposure to IQ does not lead to induction of Cyp1a1, Cyp1a2, or Cyp1b1 in the fetal compartment, thereby maintaining the low levels of these activating enzymes in the developing organism. Taken together, these data imply that, at least under the conditions employed for these experiments, IQ may not play an important role in transplacentally induced tumorigenesis.

Symposium overview: Characterization of xenobiotic metabolizing enzyme function using heterologous expression systems.

Genetically modified cell lines can be very useful models for assessing the toxicologic effects of modulation of expression of individual gene products in comparison to their isogenic parental control cell lines. This symposium begins with an overview of general issues related to development and utilization of model systems created by transfection of cell lines to induce elevated expression of metabolic enzymes of toxicologic relevance. Selected studies that illustrate the heterologous expression rationale and various approaches to transgenic-cell model construction are represented. Results to date with cells engineered to express specific transfected genes are discussed, with emphasis on the effects of expression of selected phase I or phase II enzymes on cellular sensitivity to several toxic end-points. The individual sections highlight the utility of these model cell lines for examining the role of enzyme catalysis and function in metabolism of biologically active xenobiotic or endobiotic compounds of interest in toxicology. Both activating and detoxifying enzymes are discussed, with principal emphasis on the latter. This symposium includes talks on transfected cells that express aldehyde dehydrogenases, superoxide dismutase, UDP-glycosyltransferases, glutathione transferases, and cytochrome P450 isozymes. In addition to the general toxicologic utility and advantages of these genetically engineered cell lines, this overview emphasizes their particular contributions to the insights obtained to date with the specific model cell lines.

A trypanosomiasis survey of wild animals in the Luangwa Valley, Zambia.

Between 1971 and 1974 546 wild animals of 34 species were examined for trypanosomes; 79 infections (9 mixed) were diagnosed and 29 stocks were cryopreserved. Of 14 stocks of the subgenus Trypanozoon tested by the blood incubation infectivity test three (two from Kobus ellipsiprymnus and one from Phacochoerus aethiopicus) retained their infectivity to rodents. There are indications that the number of animals harbouring trypanosomes is far in excess of those showing detectable parasitaemias even when using a combination of diagnostic methods. The standard microhaematocrit method involving microscopic examination of the buffy layer from a microhaematocrit tube between a slide and coverslip, and the inoculation of experimental animals produced the most satisfactory results.

Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells.

Toxic medium chain length alkanals, alkenals, and 4-hydroxyalkenals that are generated during lipid peroxidation are potential substrates for aldehyde dehydrogenase (ALDH) isoforms. We have developed transgenic cell lines to examine the potential for either human ALDH1A1 or ALDH3A1 to protect against damage mediated by these toxic aldehydes. Using crude cytosols from stably transfected cell lines, these aldehydes were confirmed to be excellent substrates for ALDH3A1, but were poorly oxidized by ALDH1A1. Expression of ALDH3A1 by stable transfection in V79 cells conferred a high level of protection against growth inhibition by the medium-chain length aldehyde substrates with highest substrate activity, including hexanal, trans-2-hexenal, trans-2-octenal, trans-2-nonenal, and 4-hydroxy-2-nonenal (HNE). This was reflected in a parallel ability of ALDH3A1 to prevent depletion of glutathione by these aldehydes. Expression of hALDH3 completely blocked the potent induction of apoptosis by HNE in both V79 cells and in a RAW 264.7 murine macrophage cell line, consistent with the observed total prevention of HNE-protein adduct formation. Structure-activity studies indicated that the rank order of potency for the contributions of HNE functional groups to toxicity was aldehyde >/=C2=C3 double bond>>C4-hydroxyl group. Oxidation of the aldehyde moiety of HNE to a carboxyl by ALDH3A1 expressed in stably transfected cell lines drastically reduced its potency for growth inhibition and apoptosis induction. In contrast, ALDH1A1 expression provided only moderate protection against trans-2-nonenal (t2NE), and none against the other six-nine carbon aldehydes. Neither ALDH1A1 nor ALDH3A1 conferred any protection against acrolein, acetaldehyde, or chloroacetaldehyde. A small degree of protection against malondialdehyde was afforded by ALDH1A1, but not ALDH3A1. Paradoxically, cells expressing ALDH3A1 were 1.5-fold more sensitive to benzaldehyde toxicity than control V79 cells. These studies demonstrate that expression of class 3 ALDH, but not class 1 ALDH, can be an important determinant of cellular resistance to toxicity mediated by aldehydes of intermediate chain length that are produced during lipid peroxidation.

Chemoprotective functions of glutathione S-transferases in cell lines induced to express specific isozymes by stable transfection.

The authors have shown that expression of mGSTM1-1 or hGSTP1-1 in MCF-7 cells protects against DNA alkylation by 4-nitroquinoline-1-oxide (NQO) in an isozyme-specific manner and is commensurate with relative specific activity. Expression of GSTs also conferred protection against both DNA strand breaks and sister-chromatid exchange induced by NQO. Interestingly, GST expression did not protect against NQO cytotoxicity in transfected MCF-7 cell lines, although resistance to NQO cytotoxicity was observed in a T47D pi transfectant line, expressing much higher specific activity of the transfected hGSTP1-1. However, high level expression of hGSTP1-1 or mGSTM1-1 in V79 transfectants did not confer resistance to cytotoxicity, indicating that expression of GST alone is not sufficient. The authors have also shown protection against AFB1 in cell lines expressing transfected rat CYP2B1 (V79MZr2B1) and transfected mGST-Yc (mGSTA3-3). Protection was observed against both alkylation of DNA (3-fold) by [3H]AFB1 and against AFB1 cytotoxicity (7-fold). Similarly, V79MZr1A1 cells that express CYP1A1 and either transfected human or murine GSTP1-1 (< 5000 mIU/mg, CDNB) exhibited > 70% decrease in covalent labeling of total nucleic acids by [3H]BPDE. However, no protection against the cytotoxicity of BPDE was conferred by expression of hGSTP1-1. Overall, these results indicate that in some (NQO or BPDE), but not all (AFB1) cases, protection by GST expression against DNA damage is more effective than protection against cytotoxicity. In addition, there is evidence to indicate that additional factor(s) other than high GST isozyme expression level and good substrate efficacy affect the degree of protection against cytotoxicity of reactive electrophiles. This includes the differential protection against NQO cytotoxicity in T47D pi, but not V79 Xh pi-33 cells and also the recent studies which showed that expression of the MRP GS-X conjugate efflux transporter confers synergistic protection against NQO cytotoxicity when co-expressed with transfected human GSTP1-1 in MCF-7 cells. Thus, protective efficacy conferred by GST expression can vary with different cellular targets and/or experimental end-points, as well as with variations in relative specific activity or in different cellular phenotypic contexts.

Sequence-specific effects of ara-5-aza-CTP and ara-CTP on DNA synthesis by purified human DNA polymerases in vitro: visualization of chain elongation on a defined template.

1-beta-D-arabinofuranosyl-5-aza-cytosine (ara-5-aza-Cyd) is an analog of 1-beta-D-arabinofuranosylcytosine (ara-C), which resembles ara-C in anabolic metabolism, incorporation into DNA, and inhibition of DNA replication. Human T-lymphoblastic cells (Molt-4) incorporate three- to fivefold more ara-5-aza-Cyd than ara-C into DNA during 5-8 hr exposure. Although ara-5-aza-Cyd and its triphosphate metabolite are unstable in aqueous solution, the aza-analog was much more stable in solution when incorporated into native DNA isolated from Molt-4 cells. By using gapped duplex DNA as a substrate for purified human DNA polymerases alpha and beta, inhibition of [3H]-dCTP incorporation by ara-5-aza-CTP and ara-CTP was competitive, with Ki values for alpha of 11 and 1.5 microM, respectively. Ki values for polymerase beta were 39 and 7.6 microM, respectively. A DNA elongation assay was adapted from DNA sequencing technology, using singly primed bacteriophage M13mp19 or M13mp9 (+)-DNA. Elongation of 5'-[32P]-labeled primer by polymerase alpha is slowed considerably by incorporation of one ara-CMP and to a lesser extent after incorporation of one ara-5-aza-CMP. Neither analog significantly affected elongation by polymerase beta after a single incorporation. However, neither polymerase alone could appreciably extend the growing chain if two consecutive ara-5-aza-CMP or ara-CMP analogs were incorporated. Thus, if similar mechanisms are operant in intact cells, the greater incorporation of ara-5-aza-Cyd than ara-C into DNA may be due to a more facile elongation of the nascent DNA strand by polymerase alpha after incorporation of a single analog. The effect in vitro of incorporation of either analog on DNA chain elongation is widely variable, depending on the identity of the polymerase involved and the sequence of the DNA template being copied.

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.

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.

Elevation of pi class glutathione S-transferase activity in human breast cancer cells by transfection of the GST pi gene and its effect on sensitivity to toxins.

Increased expression of the glutathione S-transferase (GST; E.C.2.5.1.18) pi class isozyme is associated with both malignant transformation and drug resistance, as well as with decreased estrogen receptor content in breast cancer. In order to further characterize the role of this enzyme in drug resistance, we cloned the cDNA encoding the human isozyme GST pi and developed two eukaryotic expression vectors using this cDNA and either the human metallothionein IIa or cytomegalovirus immediate-early promoters. These GST pi expression vectors were cotransfected with pSV2neo into drug-sensitive MCF-7 human breast cancer cells, which have low amounts of GST activity and which do not express GST pi. The transfected cells were selected for G418 resistance and individual clones were screened for GST activity. Three clones that demonstrated increased GST activity were selected for further study. Immunoprecipitation studies demonstrated that the increase in GST activity in these clones was due to expression of GST pi. Although the total GST activity of the positive clones was increased as much as 15-fold over that in wild-type MCF-7 cells, there was no change in glutathione peroxidase activity, as measured using cumene hydroperoxide as a substrate. Immunoblot studies revealed that the increased GST enzyme produced in the transfected cells was identical in size to endogenous GST pi. Southern blot analysis demonstrated the incorporation of the GST pi expression vector into the genome of the positive clones and Northern blot analysis showed that the transfected genes made a hybrid GST pi RNA that was slightly larger than the endogenous GST pi RNA. Primer extension studies demonstrated that this increase in length corresponded to the added length of the 5' leader sequence of the expression vector. The effect of increased GST pi activity on the sensitivity of the transfected clones to several cytotoxic agents was assessed by colony-forming assay. The transfected clones were slightly more resistant (1.3-4.1-fold) to benzo(a)pyrene and its toxic metabolite benzo(a)pyrene-(anti)-7,8-dihydrodiol-9,10-epoxide, as well as to ethacrynic acid (3.1-to 4.4-fold). Although increased GST pi expression is found in MCF-7 cells selected for doxorubicin resistance, the transfected clones were not consistently more resistant to doxorubicin than control cells. In addition, the transfected cells were not resistant to either melphalan or (cis)-platinum, even though conjugation with glutathione is known to play a role in the detoxification of both of these drugs.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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.