Murofushi Yoshiteru, Nagano Satoshi, Kamizono Junichi, Takahashi Tomoyuki, Fujiwara Hisayoshi, Komiya Setsuro, Matsuishi Toyojiro, Kosai Ken-ichiro
Division of Gene Therapy and Regenerative Medicine, Cognitive and Molecular Research Institute of Brain Diseases, Kurume University, Kurume 830-0011, Japan.
Int J Oncol. 2006 Sep;29(3):681-8.
Based on the finding that telomerase is reactivated solely in cancer cells, the human telomerase reverse transcriptase (hTERT) promoter has recently been used to target cancer cells by gene therapy. The recent, surprising observation that telomerase is physiologically activated even in normal somatic cells during S-phase has raised concerns as to the safety of this methodology. To clarify this issue, the present study carefully examined the changes in endogenous telomerase activities, hTERT mRNA expression, and hTERT promoter-based transgene expression in normal and cancer cells at synchronized phases of the cell cycle. Telomerase activity and hTERT expression were detected at variable, but relatively high, levels in all 12 cancer cell lines, while both were undetectable in the 11 normal cell lines. In HepG2 cancer cells, the highest levels of hTERT expression and telomerase activity, seen in the G(1)/S- and S-phases, were 2-3-fold higher than the lowest levels of both, observed in G(0)-phase and during asynchronization. No hTERT expression or telomerase activitiy could be detected in normal WI-38 fibroblasts at any phase of the cell cycle, including S-phase. Consequently, activity of the shorter hTERT promoter, which was transferred into HepG2 cancer cells via adenovirus transduction, was stronger than that of the longer hTERT promoter at all phases and that of two representatives of ubiquitously strong promoters, at both S-phase and asynchronization, but not at G(0)-phase. In contrast, neither of hTERT promoters induced detectable transgene expressions in normal WI-38 cells at any cell cycle phase, including S-phase. These results, particularly the lack of problematic levels of S-phase-specific activation of hTERT promoters in normal cells, have promising implications for hTERT promoter-based targeted gene therapy of cancer.
基于端粒酶仅在癌细胞中重新激活这一发现，人类端粒酶逆转录酶（hTERT）启动子最近已被用于通过基因治疗靶向癌细胞。最近令人惊讶的观察结果表明，即使在正常体细胞的S期，端粒酶也会被生理激活，这引发了对该方法安全性的担忧。为了阐明这个问题，本研究仔细检查了细胞周期同步阶段正常细胞和癌细胞中内源性端粒酶活性、hTERT mRNA表达以及基于hTERT启动子的转基因表达的变化。在所有12种癌细胞系中均检测到端粒酶活性和hTERT表达，其水平各不相同但相对较高，而在11种正常细胞系中均未检测到这两者。在肝癌细胞系HepG2中，G(1)/S期和S期观察到的hTERT表达和端粒酶活性最高水平比G(0)期和非同步化期间观察到的两者最低水平高2至3倍。在正常WI-38成纤维细胞的任何细胞周期阶段，包括S期，均未检测到hTERT表达或端粒酶活性。因此，通过腺病毒转导转入肝癌细胞系HepG2的较短hTERT启动子的活性在所有阶段均强于较长hTERT启动子，在S期和非同步化时也强于两个普遍强启动子的代表，但在G(0)期则不然。相比之下，在正常WI-38细胞的任何细胞周期阶段，包括S期，两种hTERT启动子均未诱导可检测到的转基因表达。这些结果，特别是正常细胞中hTERT启动子缺乏有问题的S期特异性激活水平，对于基于hTERT启动子的癌症靶向基因治疗具有积极意义。
