Modulation of glutathione peroxidase expression by selenium: effect on human MCF-7 breast cancer cell transfectants expressing a cellular glutathione peroxidase cDNA and doxorubicin-resistant MCF-7 cells.
We have studied the effect of selenium on the expression of a cellular glutathione peroxidase, GSHPx-1, in transfected MCF-7 cells and in doxorubicin-resistant (Adrr) MCF-7 cells. A GSHPx-1 cDNA with a Rous Sarcoma virus promoter was transfected into a human mammary carcinoma cell line, MCF-7, which has very low endogenous cytosolic glutathione (GSH) peroxidase activity and no detectable message. The transfectant with the highest GSH peroxidase activity among the isolates, MCF-7H6, was characterized. Adrr MCF-7 cells, a subline of MCF-7 cells, also has elevated GSH peroxidase activity. GSH peroxidase expressed by MCF-7H6 and Adrr MCF-7 cells is similar to the endogenous GSHPx-1 based on molecular weight, immunoreactivity, and metabolic labeling with 75Se. MCF-7H6 and Adrr MCF-7 cells grown in Se-deficient media had 2.6 +/- 2.4 (mean +/- S.D.) and 4.2 +/- 3.6 units/mg protein of GSH peroxidase specific activity, respectively. Se supplementation increased GSH peroxidase activity in a concentration- and time-dependent fashion. Enzymatic activity reached a level of 164 +/- 62 in MCF-7H6 cells and 114 +/- 27 in Adrr MCF-7 cells within 5 days of growth in media supplemented with 30 nM Se. Northern analysis revealed that Se-deficient MCF-7H6 cells expressed 2.1 +/- 0.4-fold less GSHPx-1 mRNA than their Se-sufficient counterparts. Similarly, Se-deficient Adrr MCF-7 cells expressed 3.3 +/- 1.8-fold less GSHPx-1 mRNA than their Se-supplemented counterparts after the quantity of mRNA was normalized with beta-actin. These studies suggest that modulation of GSH peroxidase activity by Se in both MCF-7H6 transfectants expressing pRSV-GSHPx-1 and Adrr MCF-7 cells expressing endogenous GSHPx-1 occurs largely at the translational level, and to a lesser degree at the level of mRNA, possibly by stabilizing GSHPx-1 mRNA since the transfected cDNA in MCF-7H6 cells has only 5 nucleotides 5' to the AUG initiation codon.
Chu
F F
FF
Department of Medical Oncology and Therapeutics Research, City of Hope National Medical Center, Duarte, CA 91010.
Esworthy
R S
RS
Akman
S
S
Doroshow
J H
JH
D001358:Avian Sarcoma Viruses / Q000235:genetics; D001483:Base Sequence; D015152:Blotting, Northern; D001943:Breast Neoplasms; D003001:Cloning, Molecular; D004247:DNA / Q000187:drug effects / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D015723:Gene Library; D005822:Genetic Vectors; D005979:Glutathione Peroxidase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D020887:Selenious Acid; D012643:Selenium / Q000494:pharmacology*; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology*
D015345:Oligonucleotide Probes; D004317:Doxorubicin; D004247:DNA; D005979:Glutathione Peroxidase; D020887:Selenious Acid; D012643:Selenium