Pyrazofurin-resistant hepatoma cells deficient in adenosine kinase

A pyrazofurin-resistant subline of the Morris rat hepatoma 3924A has been isolated in vitro. These cells, designated subline 3924A/1P39-3, were 100-fold resistant to pyrazofurin, and showed cross-resistance towards 6-methylmercaptopurine riboside and 6-chloropurine riboside, but not towards 6-azauridine or arabinosyladenine. Measurement of cellular enzyme activities indicated that the level of adenosine kinase in the mutant was decreased to 2% of the value in the parental cell line, but activities of uridine kinase, adenosine deaminase, deoxyadenosine kinase and orotidine 5′-monophosphate decarboxylase showed no significant change. Despite the extensive deletion of adenosine kinase, the sensitivity of the mutant to adenosine toxicity was not greatly different from that of the parental 3924A cell line. Incubation of the cells for 20 hr in presence of 300 μM adenosine caused an increase of 43% in the adenine nucleotide pool of the 1P39-3 cells, as compared with an increase of 93% in the parental 3924A cells. In both lines 300 μM adenosine caused significant depletion in uridine nucleotides and phosphoribosylpyrophosphate pools. Nevertheless, uridine (1 mM) did not reverse the adenosine toxicity in either case. These cells have provided direct evidence that the antipyrimidine agent, pyrazofurin, is activated by adenosine kinase in rat hepatoma cells. The results also show that loss of adenosine kinase does not affect adenosine toxicity in these cells, suggesting that the toxicity may be attributable to free adenosine rather than overproduction of adenine nucleotides.