Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, UK.
Biochem Biophys Res Commun. 2014 Jul 18;450(1):240-6. doi: 10.1016/j.bbrc.2014.05.102. Epub 2014 Jun 2.
In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter is activated by E2F only in cancer cells and therefore may be more cancer cell-specific than E2F1 promoter to drive gene expression. We show here that the ARF promoter has lower activity in normal growing fibroblasts and shows higher cancer cell-specificity compared to the E2F1 promoter. We also demonstrate that adenovirus expressing HSV-TK under the control of the ARF promoter shows lower cytotoxicity than that of the E2F1 promoter, in normal growing fibroblasts but has equivalent cytotoxicity in cancer cell lines. These results suggest that the ARF promoter, which is specifically activated by deregulated E2F activity, is an excellent candidate to drive therapeutic cytotoxic gene expression, specifically in cancer cells.
在当前的癌症治疗方案中，如放射治疗和化学疗法，正常细胞的副作用是根治性治疗的主要障碍。为了避免这些副作用，需要一种针对癌细胞的方法。一种特异性靶向癌细胞的方法是利用癌细胞特异性启动子表达细胞毒性基因（自杀基因治疗）或病毒复制所需的病毒基因（溶瘤病毒治疗）。为此，所选启动子在正常细胞中应具有最小的活性以避免副作用，并且在广泛的癌症中具有高活性以获得最佳治疗效果。与 AFP、CEA 和 PSA 启动子相比，这些启动子仅在有限的肿瘤谱中具有高活性，E2F1 启动子在广泛的癌症中具有高活性。这是基于致癌机制。在几乎所有癌症中都观察到 RB 途径的缺陷和转录因子 E2F 的激活，E2F 是 RB 途径的主要靶标。因此，主要受 E2F 调节的 E2F1 启动子在广泛的癌症中具有高活性。然而，E2F 也被正常生长细胞中的生长刺激激活，这表明 E2F1 启动子也可能在正常生长细胞中具有高活性。相比之下，我们发现肿瘤抑制因子 ARF 启动子是由 RB 强制失活诱导的 E2F 活性失调激活的，但对生长刺激诱导的生理 E2F 活性没有反应。我们还发现，仅在癌细胞系中检测到激活 ARF 启动子的失调 E2F 活性。这些观察结果表明，ARF 启动子仅在癌细胞中被 E2F 激活，因此与 E2F1 启动子相比，它可能更具癌细胞特异性，以驱动基因表达。我们在这里表明，ARF 启动子在正常生长的成纤维细胞中的活性较低，并且与 E2F1 启动子相比表现出更高的癌细胞特异性。我们还证明，在正常生长的成纤维细胞中，受 ARF 启动子控制表达 HSV-TK 的腺病毒的细胞毒性低于 E2F1 启动子，但在癌细胞系中具有等效的细胞毒性。这些结果表明，ARF 启动子是一种由失调的 E2F 活性特异性激活的启动子，是在癌细胞中特异性表达治疗性细胞毒性基因的理想候选者。
