Arabinosylguanine-induced apoptosis of T-lymphoblastic cells: Incorporation into DNA is a necessary step

9-beta-D-Arabinosylguanine (ara-G) is a recently introduced and effective t reatment for T-cell acute lymphoblastic leukemia, but how ara-G and ara-G t riphosphate (ara-GTP) kill cells is not known. We hypothesized that, in cyc ling T-lymphoblastoid cells, ara-G may act directly by incorporation into D NA, which may lead to apoptosis. Hence, blocking the incorporation of ara-G monophosphate (ara-GMP) into DNA may prevent apoptosis. To test this hypot hesis, we performed experiments in a T-lymphoblastic leukemia cell line (CC RF-CEM) after synchronization with a double aphidicolin block. Intracellula r accumulation of ara-GTP was neither cell cycle dependent nor affected by aphidicolin (53 +/- 5 mu M/h without aphidicolin, 50 +/- 5 mu M/h with aphi dicolin). Cells at the G(1)-S boundary accumulated 75 +/- 7 mu M ara-GTP wi th minimal incorporation into DNA (5 +/- 2 pmol ara-GMP/mg DNA) and had lit tle biochemical or morphological evidence of apoptosis. In marked contrast, cells in S phase had significantly more ara-G incorporated into DNA (24 +/ - 4 pmol ara-GMP/mg DNA), although the cytosolic concentration of ara-GTP ( 85 +/- 7 mu M) was similar to that in the G(1)-enriched population. In the S-phase cells, there was a corresponding increase in apoptosis (measured as high molecular weight DNA fragmentation and morphological changes), and th e incorporation of ara-GTP into DNA resulted in a >95% inhibition of DNA sy nthesis. There was a direct linear relationship between the number of cells in S phase and both the total number of ara-GMP molecules in DNA and the i nhibition of DNA synthesis. Blocking of ara-GTP incorporation into S-phase DNA abolished biochemical and morphological features of apoptosis, even in the presence of cytotoxic level of intracellular ara-GTP. Taken together, t hese data demonstrate that the incorporation of ara-GTP into DNA is the cri tical event that mediates the induction of apoptosis in CCRF-CEM cells.