Intracellular metabolism of Ara-C and resulting DNA fragmentation and apoptosis of human AML HL-60 cells possessing disparate levels of Bcl-2 protein.
We examined the effects of high intracellular levels of Bcl-2 on the metabolism and DNA incorporation of high-dose Ara-C (HIDAC) as well as on Ara-C-induced DNA strand breaks and apoptosis of human AML HL-60 cells. HL-60/Bcl-2 and HL-60/neo cells were created by retrovirally transfecting the human AML HL-60 cells with the pZip-bcl-2 and pZip-neo plasmids, respectively. As compared to HL-60/neo, HL-60/Bcl-2 cells contained significantly higher (approximately 10-fold) p26Bcl-2, but equivalent levels of Bax and undetectable levels of Bcl-xL. HIDAC (10 or 100 microM for 4 h) produced the kilobase size and internucleosomal DNA fragmentation associated with apoptosis in HL-60/neo but not in HL-60/Bcl-2 cells. Significantly greater loss of survival (by MTT assay) and flowcytometric and morphologically recognizable apoptosis were observed in HL-60/neo cells. HIDAC did not affect Bcl-2 levels in either cell type. The intracellular accumulation of Ara-CTP relative to dCTP, Ara-C DNA incorporation and Ara-C-induced early DNA damage in the form of strand breaks (detected by alkaline elution assay) were not significantly different between HL-60/Bcl-2 and HL-60/neo cells. In addition, HIDAC treatment caused similar DNA synthesis inhibition in the two cell types. These results indicate that high intracellular levels of Bcl-2 operate distally to inhibit the final apototic cell death pathway by preventing the conversion of HIDAC-induced early DNA damage into lethal DNA fragmentation associated with apoptosis.
Bullock
G
G
Medical University of South Carolina, Charleston, USA.
Ray
S
S
Reed
J C
JC
Krajewski
S
S
Ibrado
A M
AM
Huang
Y
Y
Bhalla
K
K
D000964:Antimetabolites, Antineoplastic / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D003561:Cytarabine / Q000378:metabolism / Q000494:pharmacology*; D004249:DNA Damage / Q000187:drug effects*; D004273:DNA, Neoplasm / Q000187:drug effects* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D018922:HL-60 Cells; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics* / Q000378:metabolism
D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D019253:Proto-Oncogene Proteins c-bcl-2; D003561:Cytarabine