Fueyo J, Gomez-Manzano C, Yung W K, Liu T J, Alemany R, Bruner J M, Chintala S K, Rao J S, Levin V A, Kyritsis A P
Department of Neuro-Oncology, The University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Neurology. 1998 May;50(5):1307-15. doi: 10.1212/wnl.50.5.1307.
This study was conducted to obtain evidence that restoration of the retinoblastoma protein function may have therapeutic application for gliomas.
The development of glioblastoma multiforme involves progressive inactivation of several tumor suppressor genes. Abnormalities of the retinoblastoma tumor suppressor gene are found in the majority of cancers, including at least 30% of malignant gliomas. No final evidence has been produced about the role of Rb in suppressing glioma growth.
To address this question, the Ad5CMV-Rb adenovirus carrying a 3.2-kb cDNA of the Rb gene was constructed. Expression of the exogenous protein was assessed by immunoblot and immunohistochemistry analyses. Growth curve assays were used to evaluate the effect of the Rb protein on glioma cell growth. Flow-cytometry analyses were used to analyze the phenotype of the cell cycle after the transfer of Rb. Human glioma xenografts implanted subcutaneously in nude mice were used for the tumorigenicity assay.
After the transfer of Rb, 80% of the treated cells expressed high levels of the retinoblastoma protein for at least 7 days. Within 5 days of treatment, the cells lost the neoplastic morphology and showed marked growth suppression. The majority of the Rb-expressing cells were arrested in the G1 phase of the cell cycle. In addition, the restoration of the retinoblastoma activity rendered the human glioma cells unable to form tumors in nude mice.
These findings provide direct evidence that inactivation of the retinoblastoma protein is a critical event in gliomas, and suggest that the restoration of wild-type retinoblastoma activity in these tumors may have therapeutic utility.
本研究旨在获取证据，证明视网膜母细胞瘤蛋白功能的恢复可能对胶质瘤具有治疗作用。
多形性胶质母细胞瘤的发生涉及多个肿瘤抑制基因的逐步失活。视网膜母细胞瘤肿瘤抑制基因的异常在大多数癌症中都有发现，包括至少30%的恶性胶质瘤。关于Rb在抑制胶质瘤生长中的作用尚未产生最终证据。
为解决这个问题，构建了携带Rb基因3.2 kb cDNA的Ad5CMV-Rb腺病毒。通过免疫印迹和免疫组织化学分析评估外源蛋白的表达。生长曲线分析用于评估Rb蛋白对胶质瘤细胞生长的影响。流式细胞术分析用于分析Rb转移后细胞周期的表型。将人胶质瘤异种移植皮下植入裸鼠用于致瘤性试验。
Rb转移后，80%的处理细胞至少7天表达高水平的视网膜母细胞瘤蛋白。在处理的5天内，细胞失去肿瘤形态并表现出明显的生长抑制。大多数表达Rb的细胞停滞在细胞周期的G1期。此外，视网膜母细胞瘤活性的恢复使人类胶质瘤细胞在裸鼠中无法形成肿瘤。
这些发现提供了直接证据，证明视网膜母细胞瘤蛋白的失活是胶质瘤中的关键事件，并表明在这些肿瘤中恢复野生型视网膜母细胞瘤活性可能具有治疗作用。
