Selection of N-(phosphonacetyl)-L-aspartate resistant Chinese hamster mutants in the presence of the uridine uptake inhibitor dipyridamole.
In mammalian cells selected in culture for resistance to PALA the CAD gene is amplified and these cells are a widely used model system to study gene amplification. Selection of resistant mutants is routinely performed in medium supplemented with dialyzed serum, because the cytotoxic effect of PALA is reversed by uridine, which is contained in serum. We have shown that in Chinese hamster cells dipyridamole reduced uridine uptake to less than 5% with limited effect on cell survival. Moreover, in medium supplemented with complete serum and 10 microM dipyridamole the toxicity of PALA was similar to that obtained in medium containing dialyzed serum. We then used 10 microM dipyridamole to inhibit uridine uptake during selection of PALA resistant colonies and found that both the frequency and the type of mutants were as those obtained in the presence of dialyzed serum. In particular, in the five mutants tested, the mechanism of resistance to PALA was amplification of the CAD gene.
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Dipartimento di Genetica e Microbiologia Adriano Buzzati Traverso, Università di Pavia, Italy.
Mucciolo
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Bertoni
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Giulotto
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D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000096:biosynthesis; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D003412:Cricetulus; D004080:Dihydroorotase / Q000096:biosynthesis; D004176:Dipyridamole / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005784:Gene Amplification; D007700:Kinetics; D009097:Multienzyme Complexes / Q000096:biosynthesis; D016296:Mutagenesis; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D014529:Uridine / Q000378:metabolism* / Q000494:pharmacology
C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D001224:Aspartic Acid; D004176:Dipyridamole; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D010746:Phosphonoacetic Acid; D014529:Uridine