Institute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan; Division of Hematology and Oncology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
J Cell Physiol. 2014 Nov;229(11):1639-46. doi: 10.1002/jcp.24609.
Thymosin β4 (Tβ4 ) is a multifunctional protein already used clinically to treat various diseases; however, the promoting effect of this protein on tumor malignancy should not be neglected. Here, we assessed whether Tβ4 alteration influences normal intestinal epithelial cells because Tβ4 is deemed a novel target for treating colorectal cancer (CRC). For this purpose, we examined the consequences of shRNA-mediated knockdown of Tβ4 in IEC-6 normal rat small intestinal cells and found that inhibiting Tβ4 expression significantly suppressed their growth and induced apoptosis in some cells. Flow cytometric analysis further revealed a marked decrease of G0/G1 population but a drastic increase of polyploid ones in these cells. The increase of polyploidy likely resulted from DNA re-replication because not only the de novo DNA synthesis was greatly increased but also the expression levels of Cdc6 (a replication-licensing factor), cyclin A, and phosphorylated-checkpoint kinase 1 were all dramatically elevated. Moreover, marked reductions in both RNA and protein levels of Emi1 (early mitotic inhibitor 1) were also detected in Tβ4 -downregulated IEC-6 cells which might be accounted by the downregulation of E2F1, a transcription factor capable of inducing Emi1 expression, mediated by glycogen synthase-3β (GSK-3β). To our best knowledge, this is the first report showing that inhibiting Tβ4 expression triggers DNA re-replication in normal intestinal epithelial cells, suggesting that this G-actin sequester may play a crucial role in maintaining genome stability in these cells. More importantly, clinical oncologists should take this novel activity into consideration when design CRC therapy based on targeting Tβ4 .
胸腺素 β4（Tβ4）是一种多功能蛋白，已临床用于治疗多种疾病；然而，不应忽视该蛋白促进肿瘤恶性的作用。在这里，我们评估了 Tβ4 改变是否会影响正常肠上皮细胞，因为 Tβ4 被认为是治疗结直肠癌（CRC）的新靶标。为此，我们研究了 shRNA 介导的 Tβ4 敲低对 IEC-6 正常大鼠小肠细胞的影响，发现抑制 Tβ4 表达可显著抑制其生长并诱导部分细胞凋亡。流式细胞术分析进一步显示，这些细胞中的 G0/G1 期细胞明显减少，多倍体细胞急剧增加。多倍体的增加可能是由于 DNA 再复制所致，因为不仅新合成的 DNA 大大增加，而且 Cdc6（复制许可因子）、细胞周期蛋白 A 和磷酸化检查点激酶 1 的表达水平均明显升高。此外，还检测到 Tβ4 下调的 IEC-6 细胞中 Emi1（早期有丝分裂抑制剂 1）的 RNA 和蛋白水平均明显降低，这可能是由糖原合酶-3β（GSK-3β）介导的转录因子 E2F1 的下调引起的，E2F1 能够诱导 Emi1 的表达。据我们所知，这是第一项表明抑制 Tβ4 表达会在正常肠上皮细胞中触发 DNA 再复制的报告，这表明这种 G 肌动蛋白结合蛋白可能在维持这些细胞的基因组稳定性方面发挥关键作用。更重要的是，临床肿瘤学家在基于 Tβ4 靶向设计 CRC 治疗时应考虑到这一新的活性。
