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Arabinosylguanine-induced Apoptosis of T-Lymphoblastic Cells

Incorporation into DNA Is a Necessary Step¹

Departments of Clinical Investigation and Leukemia [V.G.], The University of Texas M. D. Anderson Cancer Center, and The Graduate School of Biomedical Sciences, Houston, Texas 77030

9-ß-D-Arabinosylguanine (ara-G) is a recently introduced and effective treatment for T-cell acute lymphoblastic leukemia, but how ara-G and ara-G triphosphate (ara-GTP) kill cells is not known. We hypothesized that, in cycling T-lymphoblastoid cells, ara-G may act directly by incorporation into DNA, which may lead to apoptosis. Hence, blocking the incorporation of ara-G monophosphate (ara-GMP) into DNA may prevent apoptosis. To test this hypothesis, we performed experiments in a T-lymphoblastic leukemia cell line (CCRF-CEM) after synchronization with a double aphidicolin block. Intracellular accumulation of ara-GTP was neither cell cycle dependent nor affected by aphidicolin (53 ± 5 µM/h without aphidicolin, 50 ± 5 µM/h with aphidicolin). Cells at the G₁-S boundary accumulated 75 ± 7 µM ara-GTP with minimal incorporation into DNA (5 ± 2 pmol ara-GMP/mg DNA) and had little 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 µM) was similar to that in the G₁-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 the incorporation of ara-GTP into DNA resulted in a >95% inhibition of DNA synthesis. 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 inhibition 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, these data demonstrate that the incorporation of ara-GTP into DNA is the critical event that mediates the induction of apoptosis in CCRF-CEM cells.

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