Horikawa I, Cable P L, Afshari C, Barrett J C
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Cancer Res. 1999 Feb 15;59(4):826-30.
Activation of telomerase is one of the rate-limiting steps in human cell immortalization and carcinogenesis Human telomerase is composed of at least two protein subunits and an RNA component. Regulation of expression of the catalytic subunit, human telomerase reverse transcriptase (hTERT), is suggested as the major determinant of the enzymatic activity. We report here the cloning and characterization of the 5'-regulatory region of the hTERT gene. The highly GC-rich content of the 5' end of the hTERT cDNA spans to the 5'-flanking region and intron 1, making a CpG island. A 1.7-kb DNA fragment encompassing the hTERT gene promoter was placed upstream of the luciferase reporter gene and transiently transfected into human cell lines of fibroblastic and epithelial origins that differed in their expression of the endogenous hTERT gene. Endogenous hTERT-expressing cells, but not nonexpressing cells, showed high levels of luciferase activity, suggesting that the regulation of hTERT gene expression occurs mainly at the transcriptional level. Additional luciferase assays using a series of constructs containing unidirectionally deleted fragments revealed that a 59-bp region (-208 to -150) is required for the maximal promoter activity. The region contains a potential Myc oncoprotein binding site (E-box), and cotransfection of a c-myc expression plasmid markedly enhanced the promoter activity, suggesting a role of the Myc protein in telomerase activation. Identification of the regulatory regions of the hTERT promoter sequence will be essential in understanding the molecular mechanisms of positive and negative regulation of telomerase.
端粒酶的激活是人类细胞永生化和癌变过程中的限速步骤之一。人类端粒酶至少由两个蛋白质亚基和一个RNA组分组成。催化亚基人端粒酶逆转录酶（hTERT）表达的调控被认为是酶活性的主要决定因素。我们在此报告hTERT基因5'调控区的克隆与特性分析。hTERT cDNA 5'端富含GC的区域延伸至5'侧翼区和内含子1，形成一个CpG岛。将包含hTERT基因启动子的1.7 kb DNA片段置于荧光素酶报告基因上游，并瞬时转染到内源性hTERT基因表达不同的成纤维细胞和上皮细胞系来源的人类细胞系中。内源性表达hTERT的细胞而非不表达的细胞显示出高水平的荧光素酶活性，这表明hTERT基因表达的调控主要发生在转录水平。使用一系列包含单向缺失片段的构建体进行的额外荧光素酶测定表明，一个59 bp的区域（-208至-150）对于最大启动子活性是必需的。该区域包含一个潜在的Myc癌蛋白结合位点（E盒），共转染c-myc表达质粒可显著增强启动子活性，这表明Myc蛋白在端粒酶激活中起作用。鉴定hTERT启动子序列的调控区域对于理解端粒酶正负调控的分子机制至关重要。
