Mishima Yuji, Matsumoto-Mishima Yuko, Terui Yasuhito, Katsuyama Misa, Yamada Muneo, Mori Masaki, Ishizaka Yukihito, Ikeda Kazuma, Watanabe Jun-ichiro, Mizunuma Nobuyuki, Hayasawa Hirotoshi, Hatake Kiyohiko
Biochemical Research Laboratory, Morinaga Milk Industry Co. Ltd., Kanagawa, Japan.
J Natl Cancer Inst. 2002 Jul 3;94(13):1020-8. doi: 10.1093/jnci/94.13.1020.
Attachment of leukemic cells to vascular endothelial cells induces the vascular endothelial cells to release endothelial cell-derived interleukin 8 (endothelial IL-8), which then induces leukemic cells to undergo apoptosis. NB4, a human promyelocytic leukemic cell line that expresses high levels of cell-surface CD13/aminopeptidase N, does not undergo endothelial IL-8-induced apoptosis. Consequently, we investigated the relationship between cell-surface aminopeptidase activity and endothelial IL-8 induction of apoptosis in various leukemic cell lines.
CD13/aminopeptidase N activity and IL-8-induced apoptosis were examined in leukemic cell lines. Endothelial IL-8-induced apoptosis was examined further in NB4 cells, K562 cells (human chronic myelogenous leukemic cells expressing low levels of CD13/aminopeptidase N), CD13/aminopeptidase N-transfected K562 (K562/CD13) cells that overexpress aminopeptidase, and mock-transfected K562 cells (vector only). These cells were also cocultured with a vascular endothelial cell layer to investigate the association between aminopeptidase activity and apoptosis in this system. All statistical tests were two-sided.
Endothelial IL-8 induced apoptosis in K562 cells but not in K562/CD13 cells. A combination of an aminopeptidase inhibitor (such as bestatin) and endothelial IL-8 induced apoptosis in NB4 cells and K562/CD13 cells (2.88-fold difference [95% confidence interval [CI] = 1.82-fold to 3.94-fold], P =.004 for bestatin-treated NB4 cells and 4.31-fold difference [95% CI = 3.52-fold to 5.10-fold], P<.001 for bestatin-treated K562/CD13 cells). When aminopeptidase activity in NB4 cells was modulated by aminopeptidase inhibitors, a statistically significant correlation was found between aminopeptidase activity and the proportion of apoptotic cells induced by endothelial IL-8 (r = -.837, P<.001 by Pearson's correlation coefficient; r = -.697, P =.013 by Spearman's correlation analysis by ranks). K562/CD13 cells cocultured with vascular endothelial cells did not undergo apoptosis, but the addition of bestatin resulted in the induction of apoptosis in K562/CD13 cells (2.70-fold difference [95% CI = 1.77-fold to 3.63-fold], P<.001). Bestatin treatment increased the level of IL-8 mRNA in and the amount of IL-8 secreted by vascular endothelial cells.
High levels of cell-surface CD13/aminopeptidase N appear to allow leukemic cells to resist endothelial IL-8-induced apoptosis. The combination of endothelial IL-8 and bestatin induce leukemic cells expressing high levels of CD13/aminopeptidase N to undergo apoptosis. Bestatin may be useful for treating patients with leukemia.
白血病细胞与血管内皮细胞的黏附会诱导血管内皮细胞释放内皮细胞衍生的白细胞介素8（内皮IL-8），进而诱导白血病细胞发生凋亡。NB4是一种人早幼粒细胞白血病细胞系，其高水平表达细胞表面CD13/氨肽酶N，不会发生内皮IL-8诱导的凋亡。因此，我们研究了各种白血病细胞系中细胞表面氨肽酶活性与内皮IL-8诱导凋亡之间的关系。
检测白血病细胞系中的CD13/氨肽酶N活性和IL-8诱导的凋亡。在NB4细胞、K562细胞（表达低水平CD13/氨肽酶N的人慢性髓性白血病细胞）、过表达氨肽酶的CD13/氨肽酶N转染的K562（K562/CD13）细胞以及mock转染的K562细胞（仅载体）中进一步检测内皮IL-8诱导的凋亡。这些细胞也与血管内皮细胞层共培养，以研究该系统中氨肽酶活性与凋亡之间的关联。所有统计检验均为双侧检验。
内皮IL-8诱导K562细胞凋亡，但不诱导K562/CD13细胞凋亡。氨肽酶抑制剂（如苯丁抑制素）与内皮IL-8联合使用可诱导NB4细胞和K562/CD13细胞凋亡（苯丁抑制素处理的NB4细胞差异为2.88倍[95%置信区间[CI]=1.82倍至3.94倍]，P=0.004；苯丁抑制素处理的K562/CD13细胞差异为4.31倍[95%CI=3.52倍至5.10倍]，P<0.001）。当用氨肽酶抑制剂调节NB4细胞中的氨肽酶活性时，发现氨肽酶活性与内皮IL-8诱导的凋亡细胞比例之间存在统计学显著相关性（Pearson相关系数r=-0.837，P<0.001；Spearman秩相关分析r=-0.697，P=0.013）。与血管内皮细胞共培养的K562/CD13细胞未发生凋亡，但加入苯丁抑制素可诱导K562/CD13细胞凋亡（差异为2.70倍[95%CI=1.77倍至3.63倍]，P<0.001）。苯丁抑制素处理可增加血管内皮细胞中IL-8 mRNA水平和IL-8分泌量。
高水平的细胞表面CD13/氨肽酶N似乎使白血病细胞能够抵抗内皮IL-8诱导的凋亡。内皮IL-8与苯丁抑制素联合可诱导高水平表达CD13/氨肽酶N 的白血病细胞发生凋亡。苯丁抑制素可能对白血病患者的治疗有用。
