Glutathione-S-transferase pi as a determinant of drug resistance in transfectant cell lines.
A series of glutathione S-transferase pi (GST-pi) transfectant cell lines have been constructed in activated c-H-ras-transformed NIH-3T3 cells (pT22-3) by using a pKOneo plasmid and an expression vector containing cDNA for GST-pi with a beta-actin gene promoter. From the wild type pT22-3 cells, two clones were selected and designated RGN1 and RGN2. The degree of overexpression of GST-pi was estimated by Northern and Southern blot analysis to be incrementally higher in RGN2 compared with RGN1. Translation of mRNA was estimated by Western blot analysis using isozyme-specific polyclonal antibodies and confirmed the relative GST-pi levels. Each cell line, including the wild type, expressed alpha and mu class isozymes to the same degree and had similar but negligible expression of the mdr 1 gene. Sensitivity to various anticancer drugs and radiation was estimated by a series of cytotoxicity assays. The data confirmed that GST-pi provided a degree of protection against the toxicity of ethacrynic acid and adriamycin, but sensitivity to alkylating agents such as chlorambucil, melphalan, and cis-platinum was not influenced by GST-pi. Similarly, the response to ionizing radiation was similar for each line. Since the levels of intracellular GSH were also not significantly different, the availability of co-substrate was not a factor in determining response. In creating the GST-pi transfectants, these data establish that while increased isozyme levels can play a role in determining sensitivity to some agents, the protective effect is selective.
Pharmacology Division, National Cancer Center Research Institute, Tokyo, Japan.
K
K
Nakagawa
N
N
Saijo
S
S
Tsuchida
M
M
Sakai
Y
Y
Tsunokawa
J
J
Yokota
M
M
Muramatsu
K
K
Sato
M
M
Terada
K D
KD
Tew
D000199:Actins / Q000235:genetics; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003001:Cloning, Molecular; D004351:Drug Resistance / Q000235:genetics*; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D051379:Mice; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
D000199:Actins; D000970:Antineoplastic Agents; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D005982:Glutathione Transferase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)