Menadione-resistant Chinese hamster ovary cells have an increased capacity for glutathione synthesis

Vallis, Katherine A.; Reglinski, John; Garner, Matthew R.; Bridgeman, M.M.E.; Wolf, Claude

doi:10.1038/bjc.1997.477

“…In menadione-resistant Chinese hamster ovary cells an increase in glutathione levels to approx. 2-fold higher than the starting level occurs 24 h after depletion by menadione treatment [53]. The control cell line showed a more marked glutathione depletion than the menadione-resistant cell line, and its glutathione levels did not exceed the starting concentrations during recovery.…”

Section: Discussionmentioning

confidence: 73%

“…The enhanced ability to resynthesize glutathione following DEM treatment in LN73 cells was evident by an overshoot in glutathione concentrations by 2-fold over the starting levels 5 h after treatment. This overproduction of glutathione following its depletion was substantially less marked in HL9 cells, but it is of interest that previous studies have demonstrated increased glutathione levels following its depletion both in i o, following cyclophosphamide treatment of mice [52], and in cell lines [53]. In menadione-resistant Chinese hamster ovary cells an increase in glutathione levels to approx.…”

Section: Discussionmentioning

confidence: 93%

Overexpression of the regulatory subunit of γ-glutamylcysteine synthetase in HeLa cells increases γ-glutamylcysteine synthetase activity and confers drug resistance

Tipnis

¹

,

Blake

²

,

Shepherd

³

et al. 1999

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gamma-Glutamylcysteine synthetase (GCS) is reported to catalyse the rate-limiting step in glutathione biosynthesis, and is a heterodimer composed of a catalytic subunit [heavy subunit (GCSh) of Mr 73000] and a regulatory subunit [light subunit (GCSl) of Mr 31000]. In the present study, we have demonstrated for the first time a potential role for GCSl in resistance towards doxorubicin and cadmium chloride. Addition of recombinant GCSl to HeLa cell extracts in vitro was found to result in an increase in GCS activity of between 2- and 3-fold. Transient transfections of COS-1 cells with the GCSl cDNA cause an increase in GCS activity of approx. 2-fold, and a small but significant (P=0.008) increase in glutathione levels from 126.9+/-4. 2 nmol/mg protein to 178.8+/-19.1 nmol/mg protein. We proceeded to make a HeLa cell line (LN73), which stably overexpresses GCSl. These cells overexpress GCSl approx. 20-fold above basal levels. LN73 was found to have a 2-fold increase in GCS activity (437.3+/-85.2 pmol/min per mg) relative to the control cell line, HL9 (213.4+/-71. 8 pmol/min per mg). In contrast with the transient transfections in COS-1 cells, stable overexpression of GCSl was found not to be associated with an increase in glutathione content. However, when the LN73 and HL9 cells were treated with the glutathione-depleting agent, diethylmaleate, the LN73 cells were found to have an enhanced ability to regenerate glutathione, compared with HL9 cells. The cell lines were treated with various anti-cancer drugs, and their cytotoxicity was examined. No obvious differences in toxicity were observed between the different cell lines following treatment with cisplatin and melphalan. The redox-cycling agent doxorubicin, however, was found to be more toxic (approx. 2-fold) to the HL9 cells than the LN73 cells. When the cells were treated with the carcinogenic transition-metal compound, cadmium chloride, LN73 cells were found to be approx. 3-fold more resistant than HL9 cells.

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“…This overproduction of glutathione following its depletion was substantially less marked in HL9 cells, but it is of interest that previous studies have demonstrated increased glutathione levels following its depletion both in i o, following cyclophosphamide treatment of mice [52], and in cell lines [53]. This overproduction of glutathione following its depletion was substantially less marked in HL9 cells, but it is of interest that previous studies have demonstrated increased glutathione levels following its depletion both in i o, following cyclophosphamide treatment of mice [52], and in cell lines [53].…”

Section: Discussionmentioning

confidence: 99%

Overexpression of the regulatory subunit of γ-glutamylcysteine synthetase in HeLa cells increases γ-glutamylcysteine synthetase activity and confers drug resistance

Tipnis

¹

,

Blake

²

,

Shepherd

³

et al. 1999

Biochemical Journal

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gamma-Glutamylcysteine synthetase (GCS) is reported to catalyse the rate-limiting step in glutathione biosynthesis, and is a heterodimer composed of a catalytic subunit [heavy subunit (GCSh) of Mr 73000] and a regulatory subunit [light subunit (GCSl) of Mr 31000]. In the present study, we have demonstrated for the first time a potential role for GCSl in resistance towards doxorubicin and cadmium chloride. Addition of recombinant GCSl to HeLa cell extracts in vitro was found to result in an increase in GCS activity of between 2- and 3-fold. Transient transfections of COS-1 cells with the GCSl cDNA cause an increase in GCS activity of approx. 2-fold, and a small but significant (P=0.008) increase in glutathione levels from 126.9+/-4. 2 nmol/mg protein to 178.8+/-19.1 nmol/mg protein. We proceeded to make a HeLa cell line (LN73), which stably overexpresses GCSl. These cells overexpress GCSl approx. 20-fold above basal levels. LN73 was found to have a 2-fold increase in GCS activity (437.3+/-85.2 pmol/min per mg) relative to the control cell line, HL9 (213.4+/-71. 8 pmol/min per mg). In contrast with the transient transfections in COS-1 cells, stable overexpression of GCSl was found not to be associated with an increase in glutathione content. However, when the LN73 and HL9 cells were treated with the glutathione-depleting agent, diethylmaleate, the LN73 cells were found to have an enhanced ability to regenerate glutathione, compared with HL9 cells. The cell lines were treated with various anti-cancer drugs, and their cytotoxicity was examined. No obvious differences in toxicity were observed between the different cell lines following treatment with cisplatin and melphalan. The redox-cycling agent doxorubicin, however, was found to be more toxic (approx. 2-fold) to the HL9 cells than the LN73 cells. When the cells were treated with the carcinogenic transition-metal compound, cadmium chloride, LN73 cells were found to be approx. 3-fold more resistant than HL9 cells.

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“…The role of glutathione (GSH) level in menadione-induced cytotoxicity has been demonstrated in many studies. Intracellular GSH level was found to be two-fold greater in menadione-resistant cells (MRc40) than in the parental cells (CHO-K1) [17]. In addition, depletion of GSH by an inhibitor of glutathione synthesis (buthionine sulfoximine) has been found to enhance menadione cytotoxicity in menadione-resistant K300 cells [18].…”

Section: Resultsmentioning

confidence: 97%

Dihydroisotanshinone I Protects Against Menadione-Induced Toxicity in a Primary Culture of Rat Hepatocytes

Ip

¹

,

Yang²,

Sun³

et al. 2002

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Dihydroisotanshinone I is a phenanthrenequinone derivative isolated from the roots of Salvia trijuga Diels. The present study demonstrated the hepatoprotective effect of dihydroisotanshinone I against menadione-induced cytotoxicity in a primary culture of rat hepatocytes. Pretreating the cells with dihydroisotanshinone I at concentrations ranging from 2.5 microM to 20 microM for 24 hours caused dose-dependent protection against hepatotoxicity induced by menadione. Intracellular glutathione level and activity of DT-diaphorase have been suggested to play important roles in menadione-induced cytotoxicity. However, treating the hepatocytes with 20 microM dihydroisotanshinone I for 24 hours did not cause a significant change in glutathione level and DT-diaphorase activity. On the contrary, adding dihydroisotanshinone I to freshly isolated hepatocytes at concentrations between 50 nM to 200 nM inhibited NADH-induced superoxide production dose-dependently as indicated by the decrease of lucigenin-amplified chemiluminescence. In addition, dihydroisotanshinone I at concentrations ranging from 5 microM to 20 microM inhibited tert-butyl hydroperoxide-induced lipid peroxidation dose-dependently in isolated hepatocytes as indicated by the level of malondialdehyde. These results suggest that the protective action of dihydroisotanshinone I against menadione-induced hepatotoxicity is attributed to its antioxidant properties including the free radical scavenging activity and inhibition of lipid peroxidation. Abbreviations. DTD:DT-diaphorase GSH:glutathione LDH:lactate dehydrogenase MDA:malondialdehyde MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide TBHP: tert-butyl hydroperoxide

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Menadione-resistant Chinese hamster ovary cells have an increased capacity for glutathione synthesis

Cited by 7 publications

References 30 publications

Overexpression of the regulatory subunit of γ-glutamylcysteine synthetase in HeLa cells increases γ-glutamylcysteine synthetase activity and confers drug resistance

Overexpression of the regulatory subunit of γ-glutamylcysteine synthetase in HeLa cells increases γ-glutamylcysteine synthetase activity and confers drug resistance

Overexpression of the regulatory subunit of γ-glutamylcysteine synthetase in HeLa cells increases γ-glutamylcysteine synthetase activity and confers drug resistance

Dihydroisotanshinone I Protects Against Menadione-Induced Toxicity in a Primary Culture of Rat Hepatocytes

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