Egle A, Villunger A, Kos M, Böck G, Gruber J, Auer B, Greil R
Laboratory of Molecular Cytology, Department of Internal Medicine, Innsbruck University Hospital, Austria.
Eur J Immunol. 1996 Dec;26(12):3119-26. doi: 10.1002/eji.1830261244.
The Apo-1/Fas (CD95) antigen is known to be involved in the process of T cell-mediated target cell killing and has recently been shown to be expressed on myeloma cell lines and native malignant plasma cells. Several cytokines have been reported to interfere with spontaneous and even Apo-1/Fas-induced apoptosis, but no attempt has been made yet to investigate these interactions and the possible underlying mechanisms in myeloma cells. Since in myeloma patients Interferon (IFN)-alpha2 displays a profound therapeutic effect in vivo, which is usually attributed to its growth inhibitory and/or immunomodulatory capacity, we set out to study the potential interference of IFN-alpha2 with Apo-1/Fas-induced apoptosis. Contrary to expectations, IFN-alpha2 reduced the degree of apoptosis caused by the treatment of five Apo-1/Fas-sensitive myeloma cell lines with a Fas monoclonal antibody (mAb). Simultaneous application of IFN-alpha2 and Fas mAb was superior to the prolonged (i.e. >8 h) preincubation with the cytokine as far as inhibition of Apo-1/Fas-induced apoptosis was concerned. This effect of IFN-alpha2 was neither explained by a down-regulation of the Apo-1/Fas receptor nor caused by modulation of the expression levels of c-myc, bcl-2-, bcl-xL, bax- or p53 genes. IFN-alpha2 did not alter the Apo-1/Fas-induced activity of Mitogen-activated protein kinase (MAPK) 1 and did not inhibit the Apo-1/Fas-mediated proteolytic cleavage of ADP-ribosyltransferase, a substrate of Interleukin-beta1 converting enzyme (ICE) and homologues. However, activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) mimicked the effects of IFN-alpha2. Furthermore, the bis-indolylmaleimide GF 109203X, a specific inhibitor of PKC, inhibited the effect of PMA as well as that of IFN-alpha2 on Apo-1/Fas-induced apoptosis. These results point to a PKC-dependent mechanism of transient interaction between the intracellular signaling along the IFN-alpha2 and the Apo-1/Fas pathway (downstream of MAPK signaling as well as of ICE homologues), which becomes exhausted by prolonged stimulation with the cytokine. According to our data IFN-alpha2, applied continuously and in high doses resembling the therapeutic situation in vivo, inhibits myeloma growth. However, based on the observed inhibitory effect of IFN-alpha2 on Apo-1/Fas-induced apoptosis, a partial inhibition of the natural immune surveillance on myeloma cells by endogenous IFN-alpha2 present in the bone marrow microenvironment of this malignancy should be investigated.
已知Apo-1/Fas（CD95）抗原参与T细胞介导的靶细胞杀伤过程，最近研究表明其在骨髓瘤细胞系和天然恶性浆细胞上表达。据报道，几种细胞因子可干扰自发的甚至是Apo-1/Fas诱导的细胞凋亡，但尚未有人尝试研究骨髓瘤细胞中的这些相互作用及其潜在机制。由于在骨髓瘤患者中，α2干扰素（IFN-α2）在体内具有显著的治疗效果，通常归因于其生长抑制和/或免疫调节能力，因此我们着手研究IFN-α2对Apo-1/Fas诱导的细胞凋亡的潜在干扰作用。与预期相反，IFN-α2降低了用Fas单克隆抗体（mAb）处理五种Apo-1/Fas敏感骨髓瘤细胞系所导致的凋亡程度。就抑制Apo-1/Fas诱导的细胞凋亡而言，同时应用IFN-α2和Fas mAb优于用该细胞因子进行延长（即>8小时）的预孵育。IFN-α2的这种作用既不是由于Apo-1/Fas受体的下调，也不是由c-myc、bcl-2、bcl-xL、bax或p53基因表达水平的调节所引起。IFN-α2没有改变Apo-1/Fas诱导的丝裂原活化蛋白激酶（MAPK）1的活性，也没有抑制Apo-1/Fas介导的ADP-核糖基转移酶的蛋白水解切割，ADP-核糖基转移酶是白细胞介素-β1转化酶（ICE）及其同源物的底物。然而，佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）激活蛋白激酶C（PKC）可模拟IFN-α2的作用。此外，PKC的特异性抑制剂双吲哚基马来酰亚胺GF 109203X可抑制PMA以及IFN-α2对Apo-1/Fas诱导的细胞凋亡的作用。这些结果表明，在IFN-α2和Apo-1/Fas途径（MAPK信号传导以及ICE同源物的下游）的细胞内信号传导之间存在一种依赖PKC的瞬时相互作用机制，这种机制会因细胞因子的长期刺激而耗尽。根据我们的数据，持续高剂量应用类似于体内治疗情况的IFN-α2可抑制骨髓瘤生长。然而，基于观察到的IFN-α2对Apo-1/Fas诱导的细胞凋亡的抑制作用，应研究这种恶性肿瘤骨髓微环境中存在的内源性IFN-α2对骨髓瘤细胞天然免疫监视的部分抑制作用。
