Yang H L, Dong Y B, Elliott M J, Liu T J, McMasters K M
Department of Surgery, University of Louisville, James Graham Brown Cancer Center, Kentucky 40202, USA.
Clin Cancer Res. 2000 Apr;6(4):1579-89.
The prognosis for patients with esophageal cancer remains poor, prompting the search for new treatment strategies. Overexpression of E2F-1 has been shown to induce apoptosis in several cancer cell types. In the present study, the effect of adenovirus-mediated E2F-1 overexpression on human esophageal cancer cell lines Yes-4 and Yes-6 was evaluated. Cells were treated by mock infection, infection with an adenoviral vector expressing beta-galactosidase (Ad5CMV-LacZ), or E2F-1 (Ad5CMVE2F-1). Western blot analysis confirmed marked overexpression of E2F-1 in Ad5CMVE2F-1-infected cells. Overexpression of E2F-1 resulted in marked growth inhibition and rapid loss of cell viability due to apoptosis, although Yes-6 cells were somewhat more resistant to E2F-1-mediated growth inhibition than Yes-4 cells. Cell cycle analysis revealed that overexpression of E2F-1 led to G2 arrest, followed by apoptotic cell death. p53 expression remained undetectable in both cell lines after E2F-1 overexpression. The apoptosis inhibitor proteins of the Bcl-2 gene family, Bcl-2, Mcl-1, and BcI-XL, decreased at 48 h after infection in Yes-4 cells, but remained unchanged in Yes-6 cells. Levels of retinoblastoma gene product (pRb) declined at 48 h after E2F-1 infection in Yes-4 cells, at which apoptosis predominated, whereas pRb expression remained constant in Yes-6 cells. Expression of p14ARF did not change after E2F-1 infection in either cell line. Involvement of caspase 3 and caspase 6 in E2F-1-mediated apoptosis was demonstrated by cleavage of caspase 3/CPP32 and poly-ADP-ribose polymerase, as well as fragmentation of the caspase 6 substrate, lamin B. These results indicate that the sensitivity of esophageal cancer cells to E2F-1-mediated apoptosis may be related to differential expression of Bcl-2 family member proteins and suggest that the adenovirus-mediated E2F-1 gene therapy may be a promising treatment strategy for the treatment of this disease.
食管癌患者的预后仍然很差，这促使人们寻找新的治疗策略。研究表明，E2F-1的过表达可诱导多种癌细胞类型发生凋亡。在本研究中，评估了腺病毒介导的E2F-1过表达对人食管癌细胞系Yes-4和Yes-6的影响。细胞分别接受空感染、感染表达β-半乳糖苷酶的腺病毒载体（Ad5CMV-LacZ）或E2F-1（Ad5CMVE2F-1）处理。蛋白质免疫印迹分析证实，Ad5CMVE2F-1感染的细胞中E2F-1明显过表达。E2F-1的过表达导致显著的生长抑制，并因凋亡而使细胞活力迅速丧失，尽管Yes-6细胞对E2F-1介导的生长抑制的抗性比Yes-4细胞略强。细胞周期分析显示，E2F-1的过表达导致G2期阻滞，随后发生凋亡性细胞死亡。E2F-1过表达后，两种细胞系中均未检测到p53表达。Yes-4细胞在感染后48小时，Bcl-2基因家族的凋亡抑制蛋白Bcl-2、Mcl-1和BcI-XL水平下降，但在Yes-6细胞中保持不变。在Yes-4细胞中，E2F-1感染后48小时，视网膜母细胞瘤基因产物（pRb）水平下降，此时凋亡占主导，而在Yes-6细胞中pRb表达保持恒定。在两种细胞系中，E2F-1感染后p14ARF的表达均未改变。通过caspase 3/CPP32和聚ADP-核糖聚合酶的裂解，以及caspase 6底物核纤层蛋白B的片段化，证实了caspase 3和caspase 6参与了E2F-1介导的凋亡。这些结果表明，食管癌细胞对E2F-1介导的凋亡的敏感性可能与Bcl-2家族成员蛋白的差异表达有关，并提示腺病毒介导的E2F-1基因治疗可能是治疗该疾病的一种有前景的治疗策略。
