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GST-specific activities in cytusnlic preparations of nonlransduccd NIH 3T3 cells. NIH 3T3 cells transfected with MDRI and growing in 1280 ng/nil colchicine (3T3MDRI2m¡. and NIH 3T3 cells transduced with the pHaMASV.GST-rr ecolropic retrovirus and growing in increasing (M), MO. and 12X0 ng/ml) concentrations of colchi cine (GST-M), GST-MO, and GST-I2W).
Source publication
In the experiments, we examined the ability of a retroviral vector, pHaMASV, to encode two potential chemoprotective genes on separate transcription units. We previously described the pHaMSV vector, which includes the human MDR1 gene as a selectable marker and chemoprotective gene, plus an internal SV40 promoter for expressing a second heterologous...
Contexts in source publication
Context 1
... Activities and Gene Expression in Transduced Cells. To determine whether the GST ITgene was transduced along with MDRI, we measured total GST activity in control and transduced NIH 3T3 cells as well as in the 3T3.MDR line. The results are shown in Fig. 3 and Table 1. The population of A/D/?/-GST-n--transduced cells had about 2.5-fold higher total GST activity (assayed with CDNB) than either nontrans- duced NIH 3T3 cells or cells transfected with MDRI and independently selected for resistance to 1280 ng/ml of colchicine (P < 0.02). Further more, the level of GST increased progressively ...
Context 2
... cells had about 2.5-fold higher total GST activity (assayed with CDNB) than either nontrans- duced NIH 3T3 cells or cells transfected with MDRI and independently selected for resistance to 1280 ng/ml of colchicine (P < 0.02). Further more, the level of GST increased progressively with selection in higher concentrations of colchicine (Fig. 3). Whereas the total GST activity increased in concert with the degree of colchicine resistance, the activities of other glutathione-dependent enzymes and of glutathione itself were unchanged in response to selection in colchicine, even at the highest level of colchicine resistance (Table 1). The level of glucose-6-phosphate ...
Citations
... Given that anti-cancer chemotherapy usually is applied as a combination of multiple agents, simultaneous transfer of CTX-R genes, represents a logical strategy for myeloprotection, and particularly combinations including MDR1 have been studied. Combination partners have been, glutathione S-transferase (GST), MGMT or mutDHFR to allow for additional protection against alkylators such as nitrosoureas, temozolomide (MGMT), chlorambucil, melphalan (GST), as well as folate antagonists [13,14,20,42,43]. Similarly, CDD has been combined with GST [44] and mutDHFR [45][46][47] to add nitrogen mustards and folate antagonists to the resistance spectrum. ...
Background:
Hematologic toxicity represents a major side effect of cytotoxic chemotherapy frequently preventing adequately dosed chemotherapy application and impeding therapeutic success. Transgenic (over)expression of chemotherapy resistance (CTX-R) genes in hematopoietic stem- and progenitor cells represents a potential strategy to overcome this problem. To apply this concept in the context of acute myeloid leukemia and myelodysplasia, we have investigated the overexpression of the multidrug resistance 1 (MDR1) and the cytidine deaminase (CDD) gene conferring resistance to anthracyclines and cytarabine (Ara-C), the two most important drugs in the treatment of these diseases.
Methods:
State-of-the-art, third generation, self-inactivating (SIN) lentiviral vectors were utilized to overexpress a human CDD-cDNA and a codon-optimized human MDR1-cDNA corrected for cryptic splice sites from a spleen focus forming virus derived internal promoter. Studies were performed in myeloid 32D cells as well as primary lineage marker negative (lin(-)) murine bone marrow cells and flow cytometric analysis of suspension cultures and clonogenic analysis of vector transduced cells following cytotoxic drug challenge were utilized as read outs.
Results:
Efficient chemoprotection of CDD and MDR1 transduced hematopoietic 32D as well as primary lin(-) cells was proven in the context of Ara-C and anthracycline application. Both, CTX-R transduced 32D as well as primary hematopoietic cells displayed marked resistance at concentrations 5-20 times the LD50 of non-transduced control cells. Moreover, simultaneous CDD/MDR1 gene transfer resulted in similar protection levels even when combined Ara-C anthracycline treatment was applied. Furthermore, significant enrichment of transduced cells was observed upon cytotoxic drug administration.
Conclusions:
Our data demonstrate efficient chemoprotection as well as enrichment of transduced cells in hematopoietic cell lines as well as primary murine hematopoietic progenitor cells following Ara-C and/or anthracycline application, arguing for the efficacy as well as feasibility of our approach and warranting further evaluation of this concept.
... GSTP1 is expressed in alveoli, alveolar macrophages, and respiratory bronchioles in the peripheral lung (6). It has been suggested that overexpression of GST increases resistance to oxidative stress (36) and to various drugs including carcinogenic (10) and chemotherapeutic agents (8). Because GSTP1 has catalytic activity against base propenals (17), it may also have a major role in the protection of DNA and contribute to cellular viability. ...
Cigarette smoking is thought to be a major risk factor in various lung diseases including lung cancer and emphysema. However, the direct effect of cigarette smoke on the viability of lung-derived cells has not been fully elucidated. In this study, we investigated the viability of human lung fibroblast-derived (HFL1) cells to different concentrations of cigarette smoke extract (CSE). CSE induced apoptosis at lower concentrations (10-25%) and necrosis at higher concentrations (50-100%). We also examined the effects of glutathione S-transferase P1 (GSTP1), one of the xenobiotic metabolizing and antioxidant enzymes in the lung, against the cytotoxicity of CSE. Our results indicated that the level of HFL1 cell death was decreased by transfection with a GSTP1 expression vector and was increased by GSTP1 antisense vector transfection. Therefore, transient overexpression and underexpression of GSTP1 appeared to inhibit and enhance the cytotoxic effects of CSE on HFL1 cells, suggesting that GSTP1 may have protective effects against cigarette smoke in the airway cells.
... 17 Using a more selective approach by retroviral transfer of MDR1 alone or MDR1 and GSTP1 together to a single cell line, GSTP1 was found to confer no additional doxorubicin resistance over Pgp alone. 18 Conversely, doxorubicin cytotoxicity was enhanced by transfection of cells with an antisense vector against GSTP1. 19 The interaction between GST expression, conjugation and MRP1-mediated efflux of drug conjugates may be important in determination of drug resistance. ...
... 12 However, information relating to MRP1 expression has been lacking in previous studies. [15][16][17][18][19] Our study has used a representative model to investigate the relative roles of GST enzymes, Pgp and MRP1 in acquired doxorubicin resistance. GST, Pgp and MRP1 expression and function have been studied in a drug-sensitive human laryngeal carcinoma cell line (HEp2) and a subclone made resistant by prolonged culture in doxorubicin (HEp2A), together with HEp2 subclones stably transfected with GSTP1. ...
While P-glycoprotein (Pgp) and multidrug resistance-associated protein 1 (MRP1) are known to be important in acquired doxorubicin resistance, the role of glutathione S-transferases (GST) remains unclear. Our study assessed roles of these 3 factors in a human drug-sensitive carcinoma cell line (HEp2), a subclone made resistant by prolonged incubation in doxorubicin (HEp2A), and HEp2 cells stably transfected with human GSTP1. Drug-resistant HEp2A cells showed greater total GST activity, GSTP class enzyme expression, Pgp expression, MRP1 transcript expression, drug efflux and at least 13-fold greater resistance to doxorubicin than the parent HEp2 cell line. GSTM class enzyme expression was similar in both cell types, while GSTA class enzymes were not detected. In the resistant HEp2A cells, cytotoxicity was markedly enhanced by the Pgp/MRP inhibitor verapamil at low doxorubicin concentrations. The GST inhibitor curcumin also enhanced cytotoxicity in HEp2A cells when the Pgp/MRP efflux barrier had been reversed by verapamil or overcome by high doxorubicin concentrations. In addition, curcumin had a chemosensitising effect at low doxorubicin concentrations in HEp2 cells. Stable transfection of HEp2 cells with human GSTP1 increases doxorubicin resistance 3-fold over control cells. Our study indicates involvement of GSTP enzymes as well as efflux mechanisms in the acquired doxorubicin-resistance phenotype.
... NIH 3T3 fibroblasts transduced with rat glutathione-S-transferase Yc displayed resistance to chlorambucil and mechlorethamine, while their sensitivity to methotrexate, which is not a substrate of glutathione-S-transferase, remained unchanged [40]. A novel retroviral vector in which glutathione-S-transferase π and MDR1 are driven from separate promoters conferred a broad range of chemoresistance to NIH 3T3 cells [41]. ...
Chemoresistance genes, initially considered to be a major impediment to the successful treatment of cancer, may become useful tools for gene therapy of cancer and of genetically determined disorders. Various target cells are rendered resistant to anticancer drugs by transfer of chemoresistance genes encoding P-glycoprotein, the multidrug resistance-associated protein-transporter, dihydrofolate reductase, glutathione-S-transferase, O6-alkylguanine DNA alkyltransferase, or aldehyde reductase. These genes can be used for selection in vivo because of the pharmacology and pharmacokinetics of their substrates. In contrast, several other selectable marker genes conferring resistance to substrates like neomycin or hygromycin can only be utilized in tissue culture. Possible applications for chemoresistance genes include protection of bone marrow and other organs from adverse effects caused by the toxicity of chemotherapy. Strategies have also been developed to introduce and overexpress nonselectable genes in target cells by cotransduction with chemoresistance genes. Thereby expression of both transgenes can be increased following selection with drugs. Moreover, treatment with chemotherapeutic agents should restore transgene expression when or if expression levels decrease after several weeks or months. This approach may improve the efficacy of somatic gene therapy of hematopoietic disorders which is hampered by low or unstable gene expression in progenitor cells. In this article we review preclinical studies in tissue culture and animal models, and ongoing clinical trials on transfer of chemoresistance genes to hematopoietic precursor cells of cancer patients.
Administering chemotherapy is a major frontline approach in treating many cancers. Now a large number of agents used alone, but more often in combination, exert cytotoxic effects on tumor cells when delivered systemically. The general effectiveness of antitumor drugs is a function of dose (intended versus actual), scheduling, and the inherent relative resistance of tumor versus normal cells to some or all components of the chemotherapeutic cocktail. Although alterations in the dose and particularly the drug delivery schedule can favorably effect the balance between acceptable tumor kill and the side effects of therapy, the dose-limiting toxicities of anticancer drugs remain a major problem.
Our better understanding of chemotherapy resistance has led to the proposal that gene therapy can protect hematopoietic stem cells from cytotoxic drugs toxicity. Transfer of drug resistance genes into hematopoietic cells may allow the administration of higher doses of chemotherapy and, thus, increase regression or even cure for chemosensitive tumors. In addition, chemoresistance genes can be used to allow in vivo selection of transduced cells after the administration of cytotoxic drugs. Preclinical studies using MDR1 (multidrug resistance 1), mDHFR (mutants of dihydrofolate reductase) and MGMT (methylguanine DNA methyltransferase) genes have already proven the feasibility of this approach. Our group has studied the potential value of MRP (multidrug resistance-associated protein) to protect hematopoietic cells. Phase 1 clinical trials are currently in progress. However the low stem-cell transduction efficiency limits the clinical applications.
Using a retrovirus, foreign genes can be introduced into mammalian cells. The purpose of this study is to produce a retrovirus that can make the infected cells express two genes; the human multidrug resistance gene (MDR1) and the HLA-B7 gene, which is one of the major human histocompatlbility complex (MHC) class I genes. For the expression of these genes, the internal ribosome entry site (IRES) was used, which was derived from the encephalomyocarditis (EMC) virus. In order to produce retroviruses, a retroviral vector was transfected into a packaging cell line and the transfected cells were treated with vincristine, which is an anti-cancer drug and a substrate for the MDR1 gene product. This study revealed that two genes were incorporated into chromosomes of selected cells and expressed in the same cells. The production of the retrovirus was confirmed by the reverse transcription (RT)-PCR of the viral RNA. The retrovirus that was produced infected mouse fibroblast cells as well as the human U937. This study showed that packaging cells produced the retroviruses, which can infect the target cells. Once the conditions for the high infectivity of retrovirus into human cells are optimized, this virus will be used to infect hematopoietic stem cells to co-express MDR1 and HLA-B7 genes, and develop the lymphocytes that can be used for the immnogene therapy.
The mechanism of cisplatin resistance in ovarian cancer in complex. It is related to the protein concerned with many kinds of enzymes: a) The ascension of Glutathione (GSH) and Glutathione S transferase (GST), both of which can accelerate degeneration of the anticancer drugs and can bring about rapid reduction of medicamentous accumulation in the target site. Moreover, GST may also quicken excretion of the anti-cancer drugs. b) Ascension of the lung resistance-related protein (LRP), which may prevent the drugs taking the cell nucleus as its target from entering the cell nucleus through the nuclear pore, cause the medicine in the cytoplasm to enter the transport pouch can be excreted out of the cells by exocytosis. c) Analysis of reduction of the situ isomerase II a (topoisomerase II a Topo II a): Topo II can mediate breakage of the DNA chain, thus causing the apoptosis. d) Ascension of the metallothionein (MTN), which may eliminate the free radical and the superoxide free radical, with a deintoxication of the heavy metals, esp. for the platinums. In addition, the mechanism of cisplatin-resistance is possibly related with the ascension of the multidrug resistance-associated protein (MRP) too, and the P-GP protein with function of the medicamentous drug pump. In brief, the mechanism of drug resistance is complicated. A variety of mechanisms of the drug resistance might simultaneously exist in one drug resistance cell. To seek an index which can objectively and all-roundly reflect the resistance has become the pending issue for workers of the gynecological oncology.
BACKGROUND
To clarify the role of glutathione (GSH) in the chemotherapy resistance of ovarian carcinoma, the authors examined the expression of glutathione S-transferase-π (GST-π) and the concentration of glutathione in tumors before and after chemotherapy in the same patients.METHODS
The cohort for this study comprised 20 patients with ovarian carcinoma who had residual disease after primary surgery. These patients received two to three courses of postoperative chemotherapy, then underwent surgery for a second time. Chemotherapy consisted of 50 mg/m2 cisplatin, 40 mg/m2 doxorubicin, and 400 mg/m2 cyclophosphamide. The expression of GST-π in tumors was determined by immunohistochemical staining and Western blot analysis. GSH concentration was measured by an enzymatic assay.RESULTSOf the 20 patients, 10 responded to chemotherapy and 10 did not. Immunohistochemical staining for GST-π was positive in 3 tumors among the 10 responders and in 7 tumors among the 10 nonresponders, but Western blot analysis detected GST-π expression in all tumors. Among the responders, GST-π after chemotherapy increased in one patient, was unchanged in two patients, and decreased in seven patients. Among nonresponders, GST-π increased in six patients, was unchanged in one patient, and decreased in three patients. The ratio of GST-π density in tumors after chemotherapy to GST-π density before chemotherapy was significantly higher in nonresponders than in responders (2.0 ± 1.1 vs. 0.6 ± 0.4). The concentration of GSH in tumors was widely distributed, but it was found that the ratio of GSH concentration in each tumor after chemotherapy to GSH concentration before chemotherapy was significantly higher for nonresponders than for responders (3.0 ± 1.3 vs. 0.6 ± 0.3).CONCLUSIONS
Increased levels of GST-π expression after chemotherapy are linked to drug resistance in patients with ovarian carcinoma. Cancer 1997; 79:521-7. © 1997 American Cancer Society.
