Kakeya H, Onose R, Osada H
Antibiotics Laboratory, The Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Cancer Res. 1998 Nov 1;58(21):4888-94.
A novel anticancer drug, cytotrienin A, isolated from Streptomyces sp., induces apoptosis (or programmed cell death) in human promyelocytic leukemia HL-60 cells within 4 h. To elucidate the mechanism of this process, we performed an in-gel kinase assay using myelin basic protein (MBP) as a substrate and found the activation of kinase with an apparent molecular mass of 36 kDa (p36 MBP kinase). The dose of cytotrienin A required to activate p36 MBP kinase was consistent with that required to induce apoptotic DNA fragmentation in HL-60 cells. This p36 MBP kinase was activated with kinetics distinct from the activation of JNK (c-Jun N-terminal kinase)/stress-activated protein kinase and p38 MAPK (mitogen-activated protein kinase). Importantly, the p36 MBP kinase was immunologically different from MAPK superfamily molecules such as ERK1, JNK isoforms, and p38 MAPK. In addition, the p36 MBP kinase activation and apoptotic DNA fragmentation were inhibited by antioxidants such as N-acetylcysteine and reduced-form glutathione. The p36 MBP kinase activation was also observed during hydrogen peroxide (H2O2) and okadaic acid-induced apoptosis. Although a specific inhibitor of caspase-3-like proteases (Ac-DEVD-CHO) or a specific inhibitor of caspase-1-like proteases (Ac-YVAD-CHO) did not block the cytotrienin A-, H2O2-, or okadaic acid-induced apoptosis, a broad specificity inhibitor of caspases (Z-Asp-CH2-DCB) strongly inhibited the apoptosis of HL-60 cells. Surprisingly, Z-Asp-CH2-DCB inhibited the activation of p36 MBP kinase induced by cytotrienin A or H2O2, but did not inhibit the activation of JNK/stress-activated protein kinase and p38 MAPK. Taken together, these results indicate that p36 MBP kinase activation is downstream of the activation of Z-Asp-CH2-DCB-sensitive caspases, and reactive oxygen species could be included in the apoptotic events. Moreover, according to the Western blotting using the antibodies against MST1/Krs2 or MST2/Krs1, it is suggested that the p36 MBP kinase is an active proteolytic product of MST1/Krs2 and MST2/Krs1, which are originally cloned by virtue of its homology to the budding yeast Ste20 kinase. Thus, the p36 MBP kinase might be a common component of the diverse signaling pathways leading to apoptosis, and controlling this p36 MBP kinase pathway might be a novel strategy for cancer chemotherapy.
一种从链霉菌中分离出的新型抗癌药物——细胞三烯素A，能在4小时内诱导人早幼粒细胞白血病HL-60细胞发生凋亡（或程序性细胞死亡）。为阐明这一过程的机制，我们以髓鞘碱性蛋白（MBP）为底物进行了凝胶内激酶分析，发现一种表观分子量为36 kDa的激酶（p36 MBP激酶）被激活。激活p36 MBP激酶所需的细胞三烯素A剂量与诱导HL-60细胞凋亡性DNA片段化所需的剂量一致。这种p36 MBP激酶的激活动力学与JNK（c-Jun N端激酶）/应激激活蛋白激酶和p38 MAPK（丝裂原激活蛋白激酶）的激活不同。重要的是，p36 MBP激酶在免疫学上与ERK1、JNK亚型和p38 MAPK等MAPK超家族分子不同。此外，N-乙酰半胱氨酸和还原型谷胱甘肽等抗氧化剂可抑制p36 MBP激酶的激活和凋亡性DNA片段化。在过氧化氢（H2O2）和冈田酸诱导的凋亡过程中也观察到了p36 MBP激酶的激活。虽然caspase-3样蛋白酶的特异性抑制剂（Ac-DEVD-CHO）或caspase-1样蛋白酶的特异性抑制剂（Ac-YVAD-CHO）不能阻断细胞三烯素A、H2O2或冈田酸诱导产生的凋亡，但一种caspase的广谱特异性抑制剂（Z-Asp-CH2-DCB）能强烈抑制HL-60细胞的凋亡。令人惊讶的是，Z-Asp-CH2-DCB能抑制细胞三烯素A或H2O2诱导的p36 MBP激酶的激活，但不抑制JNK/应激激活蛋白激酶和p38 MAPK的激活。综上所述，这些结果表明p36 MBP激酶的激活位于Z-Asp-CH2-DCB敏感的caspase激活的下游，活性氧可能参与了凋亡事件。此外，根据使用针对MST1/Krs2或MST2/Krs1的抗体进行的蛋白质印迹分析，提示p36 MBP激酶是MST1/Krs2和MST2/Krs1的活性蛋白水解产物，它们最初是因其与芽殖酵母Ste20激酶的同源性而被克隆的。因此，p36 MBP激酶可能是导致凋亡的多种信号通路的共同组成部分，控制这条p36 MBP激酶通路可能是癌症化疗的一种新策略。
