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Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines.
Cancer Res. 1995 Oct 01; 55(19):4367-74.CR

Abstract

We have shown previously that tumor cell resistance to cisplatin is associated with elevated intracellular levels of glutathione, which is accomplished at least in part by increased expression of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS). To investigate the mechanism by which gamma-GCS expression is elevated, we have examined four related human ovarian cancer cell lines with increasing cisplatin resistance. Relative amounts of steady-state gamma-GCS mRNA in CP70, C30, and C200 were 4.8, 6.0, and 10.6, respectively, compared to the parental A2780 cell line, and a proportional increase in the transcriptional rate but not RNA stability was demonstrated. In contrast, no increase in mRNA for the gamma-GCS light subunit was found. To determine the mechanism of upregulation of this mRNA, we have cloned the promoter of the gene that encodes the heavy subunit of gamma-GCS. This region contains AP-1, NF-kappa B, XRE, AP-2, EpRE, CAAT, and TATA box elements upstream of the transcription initiation site and two MREs between this site and the start codon for the protein. Using gel mobility shift assays, we have found nuclear extract binding activity to the AP-1 response element to be closely associated with the level of gamma-GCS gene expression. A supershift assay showed that the AP-1 DNA-binding complexes are predominantly formed by dimers of JUN family members. Consistent with this finding, the expression of c-JUN was found to be elevated in the resistant cells. In contrast to AP-1 binding, AP-2 and NF-kappa B binding were inversely related to resistance. Furthermore, we have examined a partial revertant of the C200-resistant cells, which shows lower glutathione levels, and found decreased gamma-GCS expression associated with decreased AP-1 binding activity.

Authors+Show Affiliations

Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

7671249

Citation

Yao, K S., et al. "Evidence for Altered Regulation of Gamma-glutamylcysteine Synthetase Gene Expression Among Cisplatin-sensitive and Cisplatin-resistant Human Ovarian Cancer Cell Lines." Cancer Research, vol. 55, no. 19, 1995, pp. 4367-74.
Yao KS, Godwin AK, Johnson SW, et al. Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines. Cancer Res. 1995;55(19):4367-74.
Yao, K. S., Godwin, A. K., Johnson, S. W., Ozols, R. F., O'Dwyer, P. J., & Hamilton, T. C. (1995). Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines. Cancer Research, 55(19), 4367-74.
Yao KS, et al. Evidence for Altered Regulation of Gamma-glutamylcysteine Synthetase Gene Expression Among Cisplatin-sensitive and Cisplatin-resistant Human Ovarian Cancer Cell Lines. Cancer Res. 1995 Oct 1;55(19):4367-74. PubMed PMID: 7671249.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Evidence for altered regulation of gamma-glutamylcysteine synthetase gene expression among cisplatin-sensitive and cisplatin-resistant human ovarian cancer cell lines. AU - Yao,K S, AU - Godwin,A K, AU - Johnson,S W, AU - Ozols,R F, AU - O'Dwyer,P J, AU - Hamilton,T C, PY - 1995/10/1/pubmed PY - 1995/10/1/medline PY - 1995/10/1/entrez SP - 4367 EP - 74 JF - Cancer research JO - Cancer Res. VL - 55 IS - 19 N2 - We have shown previously that tumor cell resistance to cisplatin is associated with elevated intracellular levels of glutathione, which is accomplished at least in part by increased expression of the heavy subunit of gamma-glutamylcysteine synthetase (gamma-GCS). To investigate the mechanism by which gamma-GCS expression is elevated, we have examined four related human ovarian cancer cell lines with increasing cisplatin resistance. Relative amounts of steady-state gamma-GCS mRNA in CP70, C30, and C200 were 4.8, 6.0, and 10.6, respectively, compared to the parental A2780 cell line, and a proportional increase in the transcriptional rate but not RNA stability was demonstrated. In contrast, no increase in mRNA for the gamma-GCS light subunit was found. To determine the mechanism of upregulation of this mRNA, we have cloned the promoter of the gene that encodes the heavy subunit of gamma-GCS. This region contains AP-1, NF-kappa B, XRE, AP-2, EpRE, CAAT, and TATA box elements upstream of the transcription initiation site and two MREs between this site and the start codon for the protein. Using gel mobility shift assays, we have found nuclear extract binding activity to the AP-1 response element to be closely associated with the level of gamma-GCS gene expression. A supershift assay showed that the AP-1 DNA-binding complexes are predominantly formed by dimers of JUN family members. Consistent with this finding, the expression of c-JUN was found to be elevated in the resistant cells. In contrast to AP-1 binding, AP-2 and NF-kappa B binding were inversely related to resistance. Furthermore, we have examined a partial revertant of the C200-resistant cells, which shows lower glutathione levels, and found decreased gamma-GCS expression associated with decreased AP-1 binding activity. SN - 0008-5472 UR - https://www.unboundmedicine.com/medline/citation/7671249/Evidence_for_altered_regulation_of_gamma_glutamylcysteine_synthetase_gene_expression_among_cisplatin_sensitive_and_cisplatin_resistant_human_ovarian_cancer_cell_lines_ L2 - http://cancerres.aacrjournals.org/cgi/pmidlookup?view=long&pmid=7671249 DB - PRIME DP - Unbound Medicine ER -