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

gamma-Glutamylcysteine synthetase (GCS) is reported to catalyse the rate-li miting step in glutathione biosynthesis, and is a heterodimer composed of a catalytic subunit [heavy subunit (GCS(h)) of M-r 73 000] and a regulatory subunit [light subunit (GCS(l)) of M-r 31 000]. In the present study, we ha ve demonstrated for the first time a potential role for GCS(l) in resistanc e towards doxorubicin and cadmium chloride. Addition of recombinant GCS(l) to HeLa cell extracts in vitro was found to result in an increase in GCS ac tivity of between 2- and 3-fold. Transient transfections of COS-1 cells wit h the GCS(l) 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 GCS(l). These c ells overexpress GCS(l) 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 overexpre ssion of GCS(l) was found not to be associated with an increase in glutathi one content. However, when the LN73 and HL9 cells were treated with the glu tathione-depleting agent, diethylmaleate, the LN73 cells were found to have an enhanced ability to regenerate glutathione, compared with HL9 cells. Th e cell lines were treated with various anti-cancer drugs, and their cytotox icity was examined. No obvious differences in toxicity were observed betwee n 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.