Apoptosis induced by an alkylated purine, 6-dimethylaminopurine, and changes in gene expression in human lymphoma U937 cells.
Apoptosis induced by an alkylated purine, 6-dimethylaminopurine (6-DMAP), was investigated to explore the p53-independent pathway in a human lymphoma U937 cell line. Here, it was discovered that the apoptosis was induced by 6-DMAP in a dose- and time-dependent manner and treatment for 16 h at a concentration of 5 mM induced apparent DNA fragmentation, phosphatidylserine externalization and lowering of the mitochondrial membrane potential, which are typical markers of apoptosis. Western blotting revealed reduced expression in Bcl-XL, increased expression in Bax and release of cytochrome c. These were associated with activation of caspase-3. The 6-DMAP-induced apoptosis was preceded by an increase in the intracellular calcium ion concentration ([Ca2+]i), indicating the involvement of intracellular Ca2+ ions in apoptosis. Two independent sets of cDNA microarray systems were used to examine changes in gene expression. Of the 3,893 and 886 genes analyzed, 32 and 13 genes were identified as down-regulated in cells treated with 6-DMAP for 3 h. No up-regulated gene was found. Real-time PCR revealed a significant decrease in mRNA of proliferating cell nuclear antigen, insulin-induced gene 1, serine proteinase inhibitor 2 and v-myc. Pathway analysis also revealed the interaction of genes on down-regulation. These findings suggest that intracellular calcium ions and mitochondrial caspase-dependent pathways play major roles in 6-DMAP-induced apoptosis, and protein kinase inhibition by the agent causes massive down-regulation of all genes relating to cell proliferation and progression of the cell cycle to induce apoptosis.
Department of Radiological Sciences, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.
Masao
M
Nakashima
Zheng-Guo
ZG
Cui
Yoshiaki
Y
Tabuchi
Ichiro
I
Takasaki
Qing-Li
QL
Zhao
Takashi
T
Kondo
Da-Yong
DY
Yu
Miki
M
Shoji
Takashi
T
Honda
Hiroshi
H
Kitagawa
Yasuo
Y
Kojima
Takashi
T
Nakano
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008954:Models, Biological; D020411:Oligonucleotide Array Sequence Analysis; D015398:Signal Transduction / Q000187:drug effects; D020298:U937 Cells; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism
C494811:BCL2L1 protein, human; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; C001020:N(6),N(6)-dimethyladenine; D045304:Cytochromes c; D053148:Caspase 3; D000225:Adenine; D002118:Calcium