Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells

Shikibu Muranaka, Hirofumi Fujita, Takuzo Fujiwara, Tetsuya Ogino, Eisuke F. Sato, Jitsuo Akiyama, Isuke Imada, Masayasu Inoue, Kozo Utsumi

Research output: Contribution to journalArticlepeer-review

29 Citations (Scopus)

Abstract

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91phox, p22phox, p67phox, p47phox, and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22phox. Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22phox with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.

Original languageEnglish
Pages (from-to)1367-1376
Number of pages10
JournalAntioxidants and Redox Signaling
Volume7
Issue number9-10
DOIs
Publication statusPublished - Sep 2005
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Molecular Biology
  • Clinical Biochemistry
  • Cell Biology

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