Abstract
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.
Highlights
Study we report the development of GST-?r transfectant cell lines and characterize some of their properties with respect to drug response
It is apparent that the transfectant cells overexpress the r isozyme only and that other parameters which may be linked to GST activity are not affected by the transfection process
The levels of the mouse basic GST isozymes remain unaltered in the transfectants, and there is no significant change in intracellular GSH
Summary
From the wild type pT22-3 cells, two clones were selected and designated RGNl and RGNS. The degree of overexpression of GST-u was estimated by Northern and Southern blot analysis to be incrementally higher in RGN2 compared with RGNl. Translation of mRNA was estimated by Western blot analysis using isozymespecific polyclonal antibodies and confirmed the relative GST-x levels. Each cell line, including the wild type, expressed a and CCclass isozymes to the same degree and had similar but negligible expression of the mdr 1 gene. The data confirmed that GST-r 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-*. In creating the GST-u transfectants, these data establish that while increased isozyme levels can play a role in determining sensitivity to some agents, the protective effect is selective
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