Purine Nucleotide Pyrophosphorylases in 8-Azaguanine-Sensitive and -Resistant P388 Leukemias

The mouse lymphocytic neoplasm P388 was grown in vitro and three separate, Stahle sublines with resistance to 8-azaguanine were selected and cloned. These sublines were resistant, respectively, to 5, 20, and 2000 × the concentration of 8-azaguanine that inhibited the in vitro growth of the parental line. This resistance has been maintained with continued growth in vitro in the absence of 8-azaguanine. The resistant sublines are capable of progressive growth in mice and retain resistance in vivo. Because many tumor and bacterial cells show a loss of nucleotide pyrophosphorylase concomitant with the development of resistance to purine analogues, these three sublines of P388 were studied for possible gradations of such a loss of inosinic-guanylic acid pyrophosphorylase activity. The enzyme assays were done in vitro with the use of soluble protein fractions from the cells, 5-phosphoribosyl-l-pyrophosphate, and radioactive substrates including adenine, guanine hypoxanthine, 8-azaguanine, and 6-mercaptopurine. All three resistant sublines had similar 99 percent loss of the inosinic-guanylic acid pyrophosphorylase activity of the parental line, while they were unchanged with respect to adenylic acid pyrophosphorylase.