A decrease in the intracellular guanosine 5'-triphosphate concentration is necessary for granulocytic differentiation of HL-60 cells, but growth cessation and differentiation are not associated with a change in the activation state of Ras, the transforming principle of HL-60 cells.

We found that when human promyelocytic leukemic cells (HL-60 cells) were induced to differentiate along the granulocytic lineage by two diverse mechanisms (starvation for an essential amino acid or treatment with DMSO), there was a marked decrease in the intracellular guanosine 5'-triphosphate (GTP) concentration with no change in the guanosine 5'-diphosphate (GDP) concentration. Differentiation was prevented by guanine or guanosine in a dose-dependent manner. We showed that: (a) guanine had to be converted to a nucleotide because it did not prevent differentiation of hypoxanthine-guanine phosphoribosyltransferase-deficient HL-60 cells; (b) the effect of guanine correlated with a return of the cytosolic GTP:GDP ratio to normal; and (c) other purine bases were not effective. We hypothesized that the decreased GTP:GDP ratio in differentiating HL-60 cells might decrease the relative amount of GTP bound to Ras, a key regulatory GTP-binding protein important to cell growth and differentiation. Consistent with data showing that HL-60 cells harbor an activating N-Ras mutation, we found that the percentage of Ras molecules in the GTP-bound state was high in proliferating HL-60 cells (27 +/- 3%) compared with other cultured mammalian cells (< 1%); however, we found no change in the activation state of Ras when cells ceased to proliferate and differentiated in response to DMSO, amino acid deprivation, or inhibitors of guanylate synthesis. We conclude that: (a) a decrease in the intracellular GTP concentration is necessary for HL-60 cells to undergo granulocytic differentiation; and (b) although a high degree of Ras activation contributes to the malignant phenotype of the cell, there is no change in the activation state of Ras during granulocytic differentiation.