Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats.
Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats.
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DOI：
10.1186/1475-2867-2-2
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发表时间：
2002-04-04
影响因子：
5.8
通讯作者：
Costanzi-Strauss E
中科院分区：
文献类型：
--
作者：
Strauss BE;Fontes RB;Lotfi CF;Skorupa A;Bartol I;Cipolla-Neto J;Costanzi-Strauss E
p16INK4A 基因产物通过阻止 Rb 蛋白的磷酸化来阻止细胞增殖。 p16INK4a 基因在人多形性胶质母细胞瘤中经常被删除，从而导致不受控制的 Rb 磷酸化和快速细胞分裂。我们在此表明，使用 pCL 逆转录病毒系统转导人 p16INK4a cDNA 是阻止大鼠源性神经胶质瘤细胞系 C6 在组织培养和动物模型中增殖的有效方法。 C6 细胞用编码 p16INK4a、p53 或 Rb 基因的 pCL 逆转录病毒转导。通过集落形成测定来分析这些细胞。通过免疫组织化学和蛋白质印迹分析证实了p16INK4a的表达。通过衰老相关的 β-半乳糖苷酶测定进一步表征了表达 p16 的细胞形态的改变。通过立体定位注射植入离体感染的 C6 细胞以评估肿瘤形成。 p16INK4a 基因比 p53 或 Rb 更有效地抑制 C6 细胞。对 p16INK4a 基因的持续研究表明，大部分受感染的细胞表达 p16INK4a 蛋白，并且这些细胞的形态发生了改变。扩大、平坦和双极的形状表明衰老样状态，通过衰老相关的 β-半乳糖苷酶测定证实。动物模型显示，感染pCLp16病毒的细胞不会形成肿瘤。我们的结果表明，逆转录病毒介导的 p16INK4a 转移可阻止大鼠模型中神经胶质瘤的形成。这些结果证实了逆转录病毒介导的 p16INK4a 基因转移可能是阻止人类神经胶质瘤和胶质母细胞瘤的有效手段的观点。
The p16INK4A gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated β-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated β-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma.