Department of Surgery, Walsgrave Hospital, Coventry, UK, Department of Biological Science, University of Warwick, Coventry, UK.
Colorectal Dis. 2000 Apr;2(2):106-12. doi: 10.1046/j.1463-1318.2000.00142.x.
Colorectal cancer represents the second leading cause of cancer-related deaths. Despite local tumour control, patients die from disseminated disease and improved therapy is clearly required. One possible new approach is inhibition of telomerase, a reverse-transcribing enzyme thought to be essential to prevent senescence of cells by synthesizing chromosomal telomeres, which is reactivated in 85-95% of colorectal cancers. The purpose of this study was to determine the degree of telomerase inhibition by known retroviral reverse-transcriptase inhibitors using concentrations that are not acutely toxic to cells. The concentrations of three drugs (azidothymidine (AZT); dideoxythymidine (ddT); and dideoxyguanidine (ddG)) needed to reduce the proliferation (ID50 ) of the colorectal cell line HT29 by 50% were determined. Extracts were made of cells exposed for 24-48 h to these concentrations of each prodrug, telomerase activity determined using the Telomerase Repeat Amplification Protocol (TRAP), quantifying polymerase chain reaction products generated by telomerase with a phosphorimager and ImageQuant(TM) software. The drug treatments reduced the activity of telomerase in these cells by 97% for AZT, 38% for ddT and 47% for ddG, compared with control extracts. In order to confirm that the drugs used were directly inhibitory to telomerase, extracts of control cells were exposed to the active (phosphorylated) drugs and telomerase activity determined: greater than 60% and 80% inhibition occurred at 0.02 m M and 0.04 m M concentration of ddT and ddG, respectively. (The phosphorylated form of AZT was not available.) Control experiments demonstrated that the action of the active drug was not at the PCR stage of the TRAP assay and so was directly exerted on telomerase. We conclude that reverse transcriptase inhibitors can directly inhibit telomerase in cells exposed to prodrug concentrations, which are not acutely toxic, and that the active drug does directly inhibit telomerase. We propose that such inhibitors may have a role in reducing the survival, by inducing senescence, of remaining malignant cells after potential curative surgery, thus reducing recurrence and improving the prognosis of the disease. In addition they may be used in high-risk susceptible patients and in early-stage cancers.
结直肠癌是癌症相关死亡的第二大主要原因。尽管局部肿瘤得到控制，但患者仍死于播散性疾病，显然需要更好的治疗方法。一种可能的新方法是抑制端粒酶，端粒酶是一种逆转录酶，被认为通过合成染色体端粒来防止细胞衰老至关重要，而 85-95%的结直肠癌中都会重新激活端粒酶。本研究的目的是确定已知逆转录病毒逆转录酶抑制剂在不使细胞急性中毒的浓度下对端粒酶的抑制程度。确定了三种药物（叠氮胸苷（AZT）；双脱氧胸苷（ddT）；双脱氧鸟苷（ddG））使结直肠细胞系 HT29 增殖减少 50%所需的浓度（ID50）。将细胞暴露于每种前药 24-48 小时后提取提取物，使用端粒酶重复扩增协议（TRAP）测定端粒酶活性，通过磷屏仪和 ImageQuant（TM）软件定量由端粒酶产生的聚合酶链反应产物。与对照提取物相比，药物处理将这些细胞中端粒酶的活性降低了 97%（AZT），38%（ddT）和 47%（ddG）。为了确认所使用的药物直接抑制端粒酶，将对照细胞的提取物暴露于活性（磷酸化）药物中，并测定端粒酶活性：在 0.02 mM 和 0.04 mM 浓度下，ddT 和 ddG 分别发生超过 60%和 80%的抑制。（无法获得 AZT 的磷酸化形式。）对照实验表明，活性药物的作用不是在 TRAP 测定的 PCR 阶段，因此直接作用于端粒酶。我们得出结论，逆转录酶抑制剂可以直接抑制暴露于前药浓度的细胞中端粒酶，而前药浓度没有急性毒性，并且活性药物确实直接抑制端粒酶。我们提出，此类抑制剂可能通过诱导衰老来减少潜在治愈性手术后残留恶性细胞的存活，从而降低疾病的复发率并改善预后。此外，它们可以在高危易感患者和早期癌症中使用。
