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A novel class of unstable 6-thioguanine-resistant cells from dog and human kidneys
M S Turker1, R J Monnat, K Fukuchi
1Department of Pathology, University of Washington, Seattle 98195.
Abstract:
Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion. In medium containing a relatively high concentration (10 micrograms/ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10(-4) to 10(-5). A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in thioguanine medium, consistent with a reduction in hypoxanthine phosphoribosyltransferase activity. When these thioguanine-resistant primary clones were subsequently grown in the absence of thioguanine and assayed for the thioguanine-resistant phenotype and hypoxanthine phosphoribosyltransferase activity, it was found that most were now thioguanine-sensitive and yielded cell-free extracts with substantial amounts of hypoxanthine phosphoribosyltransferase activity. In contrast, thioguanine-resistant human clones grown continuously in the presence of thioguanine yielded cell-free extracts with little or no detectable hypoxanthine phosphoribosyltransferase activity. Southern blot analysis demonstrated no structural alterations in the hypoxanthine phosphoribosyltransferase gene in thioguanine-resistant primary human kidney clones. These results suggest that a novel mechanism(s) for thioguanine resistance and the control of hypoxanthine phosphoribosyltransferase expression may occur in dog and human kidney cells.
Insights
Novel thioguanine resistance mechanisms were identified in dog and human kidney cells. These cells showed reduced hypoxanthine phosphoribosyltransferase activity, suggesting new pathways for drug resistance and gene expression control.
Area of Science:
- Cell Biology
- Genetics
- Pharmacology
Background:
- Thioguanine resistance is a critical factor in cancer chemotherapy.
- Understanding resistance mechanisms in kidney cells is crucial for treatment efficacy.
- Hypoxanthine phosphoribosyltransferase (HPRT) is a key enzyme in purine metabolism.
Purpose of the Study:
- To investigate the mechanisms of thioguanine resistance in primary dog and human kidney clones.
- To analyze the activity of hypoxanthine phosphoribosyltransferase (HPRT) in resistant cells.
- To explore novel pathways controlling HPRT expression and thioguanine resistance.
Main Methods:
- Enzymatic digestion of kidney tissues to obtain single cell suspensions.
- Culture of primary clones in medium containing thioguanine.
- Assay of hypoxanthine uptake and HPRT activity in cell-free extracts.
- Southern blot analysis to detect structural alterations in the HPRT gene.
Main Results:
- Thioguanine-resistant clones emerged at frequencies of 10(-4) to 10(-5).
- Resistant clones showed reduced hypoxanthine uptake and HPRT activity.
- Most resistant clones lost resistance and regained HPRT activity when thioguanine was removed.
- Continuously exposed human clones showed persistent low HPRT activity without HPRT gene mutations.
Conclusions:
- Dog and human kidney cells exhibit novel mechanisms of thioguanine resistance.
- These mechanisms involve the regulation of hypoxanthine phosphoribosyltransferase expression.
- Further research is needed to elucidate these new pathways for potential therapeutic targeting.