Abstract
Recent reports have implicated a possible role of reactive oxygen species (ROS) in the induction and mediation of apoptosis and DNA damage. Oxidative DNA base modification induced by cupric nitrilotriacetate (Cu-NTA) and the following apoptosis were observed in human promyelocytic leukemia HL- 60 cells. We measured the level of ROS in the cells by using a fluorescence probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA), and the amount of a modified DNA base, 8-hydroxydeoxyguanosine (8-OHdG) by HPLC-ECD. It was found that Cu-NTA exposure significantly enhanced ROS and 8-OHdG formation in the cells. Meanwhile, we observed both DNA fragmentation and morphological changes characteristic of apoptosis, which was also determined quantitatively by flow cytometry and showed dose- and time-dependent manners. Furthermore, several antioxidants such as dimethyl sulfoxide (DMSO), superoxide dismutase (SOD), and catalase were used to detect whether the apoptosis could be blocked. Only DMSO protected against this form of cell death. To elucidate molecular events in the apoptosis, expressions of Bcl-2 protein family members, such as Bcl-2, Bcl-X and Bax, and heat shock protein 70 (HSP-70) were measured by western blotting using specific antibodies. The levels of Bax and Bcl-X(S) remained largely unchanged, but the Bcl-2 and Bcl-X(L) expression showed down-regulation. After 24 h incubation in the presence of copper, the levels of Bcl-2 and Bcl-X(L) reduced about 33.8% and 51.1% compared with untreated cells, respectively. Furthermore, after 16 h incubation, the level of HSP-70 expression was about 3.4-fold greater than that in untreated cells, suggesting that HSP-70 is important in increasing resistance to oxidative stress induced by Cu-NTA. But overexpression of HSP- 70 failed to protect HL-60 cells from apoptosis induced by Cu-NTA. We inferred that Cu-NTA may induce oxidative DNA damage through free radical injuries, which may turn on the apoptosis in HL-60 cells.
| Original language | English |
|---|---|
| Pages (from-to) | 568-575 |
| Number of pages | 8 |
| Journal | Free Radical Biology and Medicine |
| Volume | 25 |
| Issue number | 4-5 |
| DOIs | |
| Publication status | Published - Sep 1998 |
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Keywords
- Apoptosis
- Copper
- DNA damage
- Free radicals
- HL-60 cells
- Reactive oxygen species
ASJC Scopus subject areas
- Medicine(all)
- Toxicology
- Clinical Biochemistry
Cite this
Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells. / Ma, Yuxiang; Cao, Liu; Kawabata, Teruyuki; Yoshino, Tadashi; Yang, Burton B.; Okada, Shigeru.
In: Free Radical Biology and Medicine, Vol. 25, No. 4-5, 09.1998, p. 568-575.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells
AU - Ma, Yuxiang
AU - Cao, Liu
AU - Kawabata, Teruyuki
AU - Yoshino, Tadashi
AU - Yang, Burton B.
AU - Okada, Shigeru
PY - 1998/9
Y1 - 1998/9
N2 - Recent reports have implicated a possible role of reactive oxygen species (ROS) in the induction and mediation of apoptosis and DNA damage. Oxidative DNA base modification induced by cupric nitrilotriacetate (Cu-NTA) and the following apoptosis were observed in human promyelocytic leukemia HL- 60 cells. We measured the level of ROS in the cells by using a fluorescence probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA), and the amount of a modified DNA base, 8-hydroxydeoxyguanosine (8-OHdG) by HPLC-ECD. It was found that Cu-NTA exposure significantly enhanced ROS and 8-OHdG formation in the cells. Meanwhile, we observed both DNA fragmentation and morphological changes characteristic of apoptosis, which was also determined quantitatively by flow cytometry and showed dose- and time-dependent manners. Furthermore, several antioxidants such as dimethyl sulfoxide (DMSO), superoxide dismutase (SOD), and catalase were used to detect whether the apoptosis could be blocked. Only DMSO protected against this form of cell death. To elucidate molecular events in the apoptosis, expressions of Bcl-2 protein family members, such as Bcl-2, Bcl-X and Bax, and heat shock protein 70 (HSP-70) were measured by western blotting using specific antibodies. The levels of Bax and Bcl-X(S) remained largely unchanged, but the Bcl-2 and Bcl-X(L) expression showed down-regulation. After 24 h incubation in the presence of copper, the levels of Bcl-2 and Bcl-X(L) reduced about 33.8% and 51.1% compared with untreated cells, respectively. Furthermore, after 16 h incubation, the level of HSP-70 expression was about 3.4-fold greater than that in untreated cells, suggesting that HSP-70 is important in increasing resistance to oxidative stress induced by Cu-NTA. But overexpression of HSP- 70 failed to protect HL-60 cells from apoptosis induced by Cu-NTA. We inferred that Cu-NTA may induce oxidative DNA damage through free radical injuries, which may turn on the apoptosis in HL-60 cells.
AB - Recent reports have implicated a possible role of reactive oxygen species (ROS) in the induction and mediation of apoptosis and DNA damage. Oxidative DNA base modification induced by cupric nitrilotriacetate (Cu-NTA) and the following apoptosis were observed in human promyelocytic leukemia HL- 60 cells. We measured the level of ROS in the cells by using a fluorescence probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA), and the amount of a modified DNA base, 8-hydroxydeoxyguanosine (8-OHdG) by HPLC-ECD. It was found that Cu-NTA exposure significantly enhanced ROS and 8-OHdG formation in the cells. Meanwhile, we observed both DNA fragmentation and morphological changes characteristic of apoptosis, which was also determined quantitatively by flow cytometry and showed dose- and time-dependent manners. Furthermore, several antioxidants such as dimethyl sulfoxide (DMSO), superoxide dismutase (SOD), and catalase were used to detect whether the apoptosis could be blocked. Only DMSO protected against this form of cell death. To elucidate molecular events in the apoptosis, expressions of Bcl-2 protein family members, such as Bcl-2, Bcl-X and Bax, and heat shock protein 70 (HSP-70) were measured by western blotting using specific antibodies. The levels of Bax and Bcl-X(S) remained largely unchanged, but the Bcl-2 and Bcl-X(L) expression showed down-regulation. After 24 h incubation in the presence of copper, the levels of Bcl-2 and Bcl-X(L) reduced about 33.8% and 51.1% compared with untreated cells, respectively. Furthermore, after 16 h incubation, the level of HSP-70 expression was about 3.4-fold greater than that in untreated cells, suggesting that HSP-70 is important in increasing resistance to oxidative stress induced by Cu-NTA. But overexpression of HSP- 70 failed to protect HL-60 cells from apoptosis induced by Cu-NTA. We inferred that Cu-NTA may induce oxidative DNA damage through free radical injuries, which may turn on the apoptosis in HL-60 cells.
KW - Apoptosis
KW - Copper
KW - DNA damage
KW - Free radicals
KW - HL-60 cells
KW - Reactive oxygen species
UR - http://www.scopus.com/inward/record.url?scp=18144448088&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=18144448088&partnerID=8YFLogxK
U2 - 10.1016/S0891-5849(98)00088-4
DO - 10.1016/S0891-5849(98)00088-4
M3 - Article
C2 - 9741594
AN - SCOPUS:18144448088
VL - 25
SP - 568
EP - 575
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
SN - 0891-5849
IS - 4-5
ER -