Abstract
9-β-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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 4937-4943 |
| Number of pages | 7 |
| Journal | Cancer Research |
| Volume | 59 |
| Issue number | 19 |
| State | Published - Oct 1 1999 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Cancer Research
- Oncology
Cite this
Arabinosylguanine-induced apoptosis of T-lymphoblastic cells : Incorporation into DNA is a necessary step. / Rodriguez, Carlos O.; Gandhi, Varsha.
In: Cancer Research, Vol. 59, No. 19, 01.10.1999, p. 4937-4943.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Arabinosylguanine-induced apoptosis of T-lymphoblastic cells
T2 - Incorporation into DNA is a necessary step
AU - Rodriguez, Carlos O.
AU - Gandhi, Varsha
PY - 1999/10/1
Y1 - 1999/10/1
N2 - 9-β-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.
AB - 9-β-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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UR - http://www.scopus.com/inward/citedby.url?scp=0345363112&partnerID=8YFLogxK
M3 - Article
C2 - 10519407
AN - SCOPUS:0345363112
VL - 59
SP - 4937
EP - 4943
JO - Journal of Cancer Research
JF - Journal of Cancer Research
SN - 0099-7013
IS - 19
ER -