Supplementary Material for: p16 Gene Transfer Induces Centrosome Amplification and Abnormal Nucleation Associated with Survivin Downregulation in Glioma Cells

<b><i>Objective:</i></b> In human glioma cells, p16 gene transfer induced G1/S arrest, increased radiosensitivity and abnormal nucleation (especially bi- and multinucleation). Survivin suppression caused G2/M arrest, radiosensitization and an increase in aneuploidy accompanied by centrosome amplification. Abnormal nucleation and aneuploidy represent chromosome instability (CIN), and it is well known that centrosome amplification leads to CIN. However, little has been reported that suggests that transferring p16 causes centrosome overduplication during the G1/S phase. <b><i>Methods:</i></b> The p16 gene was transferred into p16-null human glioma cell lines (U251MG and D54MG) using adenovirus with or without irradiation. Centrosome amplification was evaluated by immunofluorescence. We also investigated the DNA replication licensing factor CDT1, its inhibitor geminin and survivin expression as regulators of chromosomal segregation. <b><i>Results:</i></b> p16 gene transfer with radiation initiated the greatest degree of centrosome overduplication. CDT1 showed low levels, geminin was unchanged and survivin decreased in Ax-hp16-infected cells with radiation. Those changes of factors affecting DNA licensing or chromosomal segregation might contribute to CIN. <b><i>Conclusion:</i></b> p16 transfer caused centrosome amplification even in G1/S phase-arrested cells. This suggests that p16 is involved in abnormal nucleation and radiosensitization in human glioma cells.

<b><i>研究目的：</i></b> 在人胶质瘤细胞中，p16基因（p16）转染可诱导G1/S期阻滞、提升放射敏感性并引发核异常（尤以双核及多核形成为著）；抑制生存素（Survivin）可导致G2/M期阻滞、产生放射增敏效应，同时伴随中心体扩增的非整倍体比例升高。核异常与非整倍体均属于染色体不稳定性（CIN），而中心体扩增可引发CIN已是学界共识，但目前鲜有研究报道p16基因转染会在G1/S期触发中心体过度复制。
<b><i>研究方法：</i></b> 本研究借助腺病毒载体，将p16基因（p16）转染至p16基因缺失的人胶质瘤细胞系U251MG与D54MG，并设置辐照与未辐照两组对照。通过免疫荧光法检测中心体扩增情况；同时探究DNA复制起始许可因子CDT1（CDT1）、其抑制剂geminin（geminin）及生存素（Survivin）的表达水平，以此作为染色体分离调控因子展开分析。
<b><i>研究结果：</i></b> 联合辐照的p16基因转染可诱导最显著的中心体过度复制。在经辐照且被Ax-hp16感染的细胞中，CDT1表达水平显著下调，geminin表达无明显变化，而Survivin表达量降低。上述影响DNA复制许可或染色体分离的因子变化，可能参与染色体不稳定性（CIN）的发生发展。
<b><i>研究结论：</i></b> 即便在G1/S期阻滞的细胞中，p16基因转染仍可引发中心体扩增。这提示p16基因参与了人胶质瘤细胞的核异常形成与放射增敏过程。