Wortmannin-enhanced X-ray-induced apoptosis of human T-cell leukemia MOLT-4 cells possibly through the JNK/SAPK pathway.
We demonstrated that enhancement of X-ray-induced apoptosis/rapid cell death by wortmannin accompanied by increased activation of JNK/SAPK in human leukemia MOLT-4 cells. Rapid cell death/apoptosis was determined either by the dye exclusion test or by the appearance of Annexin V-positive cells and cleaved PARP fragments. Enhancement was observed only at higher concentrations of wortmannin, i.e. 1 microM or more. At these high concentrations, both DNA-PK and ATM were inhibited. X-ray-induced phosphorylation of Ser 15 of p53/TP53, accumulation of both p53/TP53 and p21/WAF1/CDKN1A, and phosphorylation of XRCC4 were all suppressed. The enhancement of apoptosis/rapid cell death by wortmannin was prevented by addition of caspase inhibitors, Z-VAD-FMK or Ac-DEVD-CHO, or by transfection and overexpression of mouse Bcl2, which is known as an anti-apoptosis protein. The requirement for a high concentration of wortmannin, i.e. 1 microM or more, indicates that inhibition of both DNA-PK and ATM was necessary for the enhanced apoptosis/rapid cell death. Phosphorylation of AKT/PKB was completely suppressed at a much lower concentration, i.e. 0.1 microM wortmannin, where no enhancement of X-ray-induced apoptosis/rapid cell death was observed. On the other hand, X-ray-induced phosphorylation of JNK and its kinase activity as well as apoptosis/rapid cell death were all significantly enhanced only at high concentrations of wortmannin, i.e. 1 microM or more. Furthermore, the extent of enhancement of both JNK phosphorylation and of apoptosis/rapid cell death by wortmannin was less in Rh1a cells, which are ceramide- and radiation-resistant variant cells compared to the parental MOLT-4 cells. Therefore, activation of the JNK pathway was considered important for the enhancement of X-ray-induced apoptosis/rapid cell death of MOLT-4 cells by wortmannin, because of the requirement for a higher concentration of wortmannin than that required for inhibition of AKT phosphorylation. The suppression of the AKT-dependent pathway by wortmannin may have some underlying role in activating the JNK pathway toward the enhancement of cell death in the current system.
Tomita
Masanori
M
Department of Radiation Oncology, Graduate School of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
Suzuki
Norio
N
Matsumoto
Yoshihisa
Y
Enomoto
Atsushi
A
Yin
Hong Lan
HL
Hosoi
Yoshio
Y
Hirano
Kazuya
K
Sakai
Kazuo
K
D000730:Androstadienes / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects*; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases; D015458:Leukemia, T-Cell / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology*; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D011836:Radiation Tolerance / Q000187:drug effects*; D014407:Tumor Cells, Cultured; D000077191:Wortmannin; D014965:X-Rays
D000730:Androstadienes; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D000077191:Wortmannin