Growth inhibition of a human myeloma cell line by all-trans retinoic acid is not mediated through downregulation of interleukin-6 receptors but through upregulation of p21WAF1
Growth inhibition of a human myeloma cell line by all-trans retinoic acid is not mediated through downregulation of interleukin-6 receptors but through upregulation of p21WAF1
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DOI：
10.1182/blood.v94.1.251.413k42_251_259
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发表时间：
1999-07-01
期刊：
影响因子：
20.3
通讯作者：
Hankewych, M
中科院分区：
文献类型：
--
作者：
Chen, YH;Lavelle, D;Hankewych, M
全反式维甲酸(ATRA)此前已被证明可抑制OPM-2人骨髓瘤细胞的生长。这种生长抑制可能是由于IL-6Rα(IL-6Rα)转录下调，而IL-6Rβ(Gp130)未受影响。为了形式化地验证这一假设，构建了一个用于组成性表达IL-6Rα的表达载体，并将其用于转染OPM-2细胞。克隆了6个稳定的转染体。抗IL-6Rα抗体免疫荧光和I-125-IL-6结合免疫荧光检测IL-6Rα的表达。在6个转基因克隆中，有5个细胞的IL-6Rα比亲本细胞高1.5~6倍，而与配体结合的亲和力没有变化。ATRA降低亲本OPM-2细胞中IL-6Rα的表达，增强其在这五种转染体中的表达。然而，这些转染体的克隆生长仍然受到ATRA的强烈抑制。进一步的分析，比较亲本的OPM-2细胞和代表性的转基因克隆C5，发现IL-6在OPM-2和C5克隆中都能引起gp130的快速酪氨酸磷酸化。全反式维甲酸可显著降低IL-6诱导的OPM-2细胞中gp130的磷酸化，反映了细胞内IL-6Rα的减少。相反，全反式维甲酸对IL-6诱导的C5细胞中gp130的磷酸化没有影响，表明表达的IL-6Rα是有功能的。与OPM-2细胞相似，C5细胞对地塞米松的生长抑制敏感，而外源性IL-6可完全逆转地塞米松的抑制作用，提示IL-6受体后信号转导未受影响。在OPM-2和C5细胞中，ATRA进一步被证明上调p21(WAF1)的表达，并导致视网膜母细胞瘤蛋白(PRB)的去磷酸化。外源性IL-6也不能逆转ATRA的上述作用。因此，ATRA的生长抑制活性不是通过细胞IL-6Rα下调介导的，而可能是直接上调p21(WAF1)并随后导致pRb去磷酸化的结果。(C)1999年由美国血液病学会主办。
All-trans retinoic acid (ATRA) has previously been shown to inhibit the growth of OPM-2 human myeloma cells. The growth inhibition was postulated to result from a transcriptional downregulation of interleukin-6 receptor alpha (IL-6R alpha) with IL-6R beta (gp130) unaffected. To formally test this hypothesis, an expression vector designed for constitutive IL-6R alpha expression was constructed and used for transfection of OPM-2 cells. Six stable transfectants were cloned. The expression of IL-6R alpha was shown by immunofluorescence with anti-IL-6R alpha antibody and I-125-IL-6 binding. In five of six transfectant clones, cellular IL-6R alpha was 1.5- to 6-fold higher than the parental cells, with the ligand binding affinity unchanged. While ATRA reduced IL-6R alpha expression in the parental OPM-2 cells, it enhanced its expression in these five transfectants. The clonogenic growth of these transfectants, however, remained strongly inhibited by ATRA. Further analysis, comparing the parental OPM-2 cells and a representative transfectant, clone C5, showed that IL-6 caused rapid tyrosine phosphorylation of gp130 in both OPM-2 and C5 clones. Pretreatment with ATRA greatly reduced IL-6-induced gp130 phosphorylation in OPM-2 cells, reflecting a reduction in cellular IL-6R alpha. In contrast, IL-6-induced gp130 phosphorylation was not reduced by ATRA pretreatment in C5 cells, indicating that the expressed IL-6R alpha was functional. Similar to OPM-2 cells, C5 cells were sensitive to growth inhibition by dexamethasone, which was entirely reversed by exogenous IL-6, suggesting that the IL-6 postreceptor signal transduction remained intact. ATRA was further shown to upregulate p21(WAF1) expression and cause dephosphorylation of the retinoblastoma protein (pRB) in both OPM-2 and C5 cells. Exogenous IL-6 also failed to reverse these effects of ATRA. Thus, the growth inhibitory activity of ATRA is not mediated through cellular IL-6R alpha downregulation and is likely to result from a direct upregulation of p21(WAF1) and consequent dephosphorylation of pRB. (C) 1999 by The American Society of Hematology.