Combined radiation and p53 gene therapy of malignant glioma cells
Combined radiation and p53 gene therapy of malignant glioma cells
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DOI：
10.1038/sj.cgt.7700009
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发表时间：
1999-03-01
影响因子：
6.4
通讯作者：
Boothman, DA
中科院分区：
文献类型：
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作者：
Badie, B;Goh, CS;Boothman, DA
据报道，超过一半的恶性胶质瘤在p53肿瘤抑制基因中存在改变。由于p53在细胞对DNA损伤剂的反应中起着关键作用，我们研究了p53基因治疗在电离辐射前培养的含有正常或突变p53的人脑胶质瘤细胞中的作用。用携带人p53（Adp 53）和大肠杆菌β-半乳糖苷酶基因（AdLacZ，对照病毒）的重组腺病毒载体，分别转染表达野生型（U87 MG，p53（wt））和突变型（A172和U373 MG，p53（mut））p53基因的3株人脑胶质瘤细胞系。通过Western免疫印迹法研究了p53，p21和Bar表达的变化，而通过流式细胞术和核染色法研究了细胞周期改变和凋亡。通过克隆形成试验评估存活率。在48小时的Adp 53暴露中，所有三种细胞系均在100的病毒感染复数下表现出p53表达。p21是p53诱导的下游效应基因，过表达，细胞停滞在G（1）期。Bar表达被认为在p53诱导的细胞凋亡中起作用，但辐射或Adp 53均未改变Bar表达。p53基因治疗后的细胞凋亡和存活率不同。U87 MC（p53（wt））细胞在Adp 53、照射或联合处理后显示出最小的凋亡。U373 MC（p53（mut））细胞在Adp 53处理48小时内发生大量凋亡并死亡，与照射无关。令人惊讶的是，A172（p53（mut））细胞在Adp 53暴露后表现出最小的凋亡;然而，与U373 MG细胞不同，凋亡随着辐射剂量增加;所有三种细胞系的存活在>10戈伊后显著降低。虽然Adp 53转导显著降低了U373 MC细胞的存活率并抑制了A172的生长，但它对U87 MC细胞系没有影响。转导与AdLacZ不影响细胞凋亡或细胞周期的进展，只有最低限度地影响所有细胞系的生存。我们的结论是，对p53基因治疗的反应是不同的胶质瘤，最有可能取决于细胞p53的状态和尚未明确的下游途径，涉及激活细胞周期调控和凋亡基因。
More than half of malignant gliomas reportedly have alterations in the p53 tumor suppressor gene. Because p53 plays a key role in the cellular response to DNA-damaging agents, we investigated the role of p53 gene therapy before ionizing radiation in cultured human glioma cells containing normal or mutated p53. Three established human glioma cell lines expressing the wild-type (U87 MG, p53(wt)) or mutant (A172 and U373 MG, p53(mut)) p53 gene were transduced by recombinant adenoviral vectors bearing human p53 (Adp53) and Escherichia coli beta-galactosidase genes (AdLacZ, control virus) before radiation (0-20 Gy). Changes in p53, p21, and Bar expression were studied by Western immunoblotting, whereas cell cycle alterations and apoptosis were investigated by flow cytometry and nuclear staining. Survival was assessed by clonogenic assays. With in 48 hours of Adp53 exposure, all three cell lines demonstrated p53 expression at a viral multiplicity of infection of 100. p21, which is a p53-inducible downstream effector ;gene, was overexpressed, and cells were arrested in the G(1) phase. Bar expression, which is thought to play a role in p53-induced apoptosis, did not change with either radiation or Adp53. Apoptosis and survival after p53 gene therapy varied. U87 MC (p53(wt)) cells showed minimal apoptosis after Adp53, irradiation, or combined treatments. U373 MC (p53(mut)) cells underwent massive apoptosis and died within 48 hours of Adp53 treatment, independent of irradiation. Surprisingly, A172 (p53(mut)) cells demonstrated minimal apoptosis after Adp53 exposure; however, unlike U373 MG cells, apoptosis increased with radiation dose; Survival of all three cell lines was reduced dramatically after >10 Gy. Although Adp53 transduction significantly reduced the survival of U373 MC cells and inhibited A172 growth, it had no effect on the U87 MC cell line. Transduction with AdLacZ did not affect apoptosis or cell cycle progression and only minimally affected survival in all cell lines. We conclude that responses to p53 gene therapy are variable among gliomas and most likely depend upon both cellular p53 status and as yet ill-defined downstream pathways involving activation of cell cycle regulatory and apoptotic genes.