Kamal Anupama, Datta K
Biochemistry Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
Apoptosis. 2006 May;11(5):861-74. doi: 10.1007/s10495-006-5396-4.
We have earlier reported that overexpression of HABP1 in fibroblast cells causes perturbed cell growth, extensive vacuolation and restricted entry to the S-phase, finally leading to apoptosis (Biochem Biophys Res Commun 2003; 300: 686-693). In the present study, we investigate the regulation of HABP1 expression in cisplatin induced apoptosis in HeLa cells. Apoptosis induced in HeLa cells at 24 h of cisplatin treatment was confirmed by nuclear fragmentation, increase in subdiploid population and the enhanced activation of ERK and upregulation of p53. In association with apoptosis induction, an upregulation of HABP1 expression was observed in HeLa cells at 18 and 24 h of cisplatin treatment. Quantification of HABP1 expression by flow cytometry confirmed a two-fold increase in total intracellular HABP1 expression at 24 h of cisplatin treatment. Under the same condition the HABP1 transcript level measured by semi quantitative RT PCR showed 2.5-fold increase ascertaining transcriptional regulation of HABP1 during cisplatin induced apoptosis. Further, in normal HeLa cells though a small amount of HABP1 can be detected in nucleus, but with apoptosis induction the protein is mainly concentrating around the nuclear periphery at 18 h of cisplatin treatment and is present both in the nucleus as well as in the cytosol at 24 h of treatment, suggesting its nuclear translocation during apoptosis. To substantiate our findings prior to the cisplatin treatment, the expression of HABP1 was reduced by small interfering RNA mediated knockdown. We observed a reduction in apoptotic cell population in cisplatin treated HeLa cells with disrupted HABP1 conferring resistance to cisplatin induced apoptosis. We report here that HABP1 upregulation in the cell is important for cisplatin induced apoptosis.
我们之前报道过，成纤维细胞中HABP1的过表达会导致细胞生长紊乱、广泛空泡化以及进入S期受限，最终导致细胞凋亡（《生物化学与生物物理研究通讯》，2003年；300: 686 - 693）。在本研究中，我们探究了顺铂诱导HeLa细胞凋亡过程中HABP1表达的调控机制。顺铂处理HeLa细胞24小时后诱导的细胞凋亡通过核碎裂、亚二倍体群体增加、ERK的激活增强以及p53的上调得以证实。与细胞凋亡诱导相关，在顺铂处理18小时和24小时的HeLa细胞中观察到HABP1表达上调。通过流式细胞术对HABP1表达进行定量分析，证实顺铂处理24小时后细胞内总HABP1表达增加了两倍。在相同条件下，通过半定量RT - PCR检测到的HABP1转录水平增加了2.5倍，确定了顺铂诱导细胞凋亡过程中HABP1的转录调控。此外，在正常HeLa细胞中，虽然在细胞核中可检测到少量HABP1，但随着细胞凋亡诱导，在顺铂处理18小时时该蛋白主要集中在核周边，处理24小时时则同时存在于细胞核和细胞质中，表明其在细胞凋亡过程中发生了核转位。为了在顺铂处理之前证实我们的发现，通过小干扰RNA介导的敲低降低了HABP1的表达。我们观察到，在顺铂处理的HeLa细胞中，HABP1被破坏后凋亡细胞群体减少，赋予了对顺铂诱导凋亡的抗性。我们在此报告，细胞中HABP1的上调对于顺铂诱导的细胞凋亡很重要。
