Ducrest A L, Amacker M, Mathieu Y D, Cuthbert A P, Trott D A, Newbold R F, Nabholz M, Lingner J
Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland.
Cancer Res. 2001 Oct 15;61(20):7594-602.
Telomerase is required for the complete replication of chromosomal ends. In tumors, the human telomerase reverse transcriptase subunit (hTERT) is up-regulated, thereby removing a critical barrier for unlimited cell proliferation. To understand more about hTERT regulation, we measured hTERT RNA levels by quantitative reverse transcription (RT)-PCR. Telomerase-positive cell lines were found to contain between 0.2 and 6 molecules of spliced hTERT RNA per cell, whereas in telomerase-negative cells, the number of molecules was below the sensitivity of the assay (<0.004 molecules/cell). Intron-containing, immature hTERT RNA was observed only in nuclei of telomerase-positive cells, which suggests that hTERT RNA levels are transcriptionally regulated. Microcell transfer of a normal chromosome 3 into the human breast carcinoma cell line (21NT) abolishes telomerase activity and induces senescence. Endogenous hTERT transcripts were undetectable in the nuclei of 21NT-chromosome 3 hybrids, even in cells permanently expressing a transfected hTERT cDNA. However, chromosome 3 transfer did not affect the expression of green fluorescent protein reporter constructs driven by up to 7.4 kb of noncoding DNA flanking the 5' end of the hTERT gene. Because direct up-regulation of hTERT through c-Myc overexpression had previously been reported, we investigated whether chromosome 3 transfer affected c-Myc activity. An at least 30-fold reduction of immature intron-containing hTERT RNA was observed after the introduction of a normal chromosome 3, but expression levels of c-Myc, Mad1, and other c-Myc target genes were unchanged. Our results suggest that telomerase is regulated primarily at the level of hTERT transcription by complex mechanisms involving regulatory elements distant from the 5' flanking region, and that the putative hTERT repressor on chromosome 3 does not regulate the expression of hTERT through c-Myc or one of its coregulators.
端粒酶是染色体末端完全复制所必需的。在肿瘤中，人类端粒酶逆转录酶亚基（hTERT）上调，从而消除了无限细胞增殖的关键障碍。为了更深入了解hTERT的调控机制，我们通过定量逆转录（RT）-PCR测量了hTERT RNA水平。发现端粒酶阳性细胞系每个细胞含有0.2至6个剪接后的hTERT RNA分子，而在端粒酶阴性细胞中，分子数量低于检测灵敏度（<0.004分子/细胞）。仅在端粒酶阳性细胞的细胞核中观察到含内含子的未成熟hTERT RNA，这表明hTERT RNA水平受转录调控。将正常的3号染色体微细胞转移到人乳腺癌细胞系（21NT）中可消除端粒酶活性并诱导衰老。在21NT-3号染色体杂种细胞的细胞核中检测不到内源性hTERT转录本，即使在永久表达转染的hTERT cDNA的细胞中也是如此。然而，3号染色体转移并不影响由hTERT基因5'端侧翼长达7.4 kb的非编码DNA驱动的绿色荧光蛋白报告构建体的表达。由于先前已报道通过c-Myc过表达直接上调hTERT，我们研究了3号染色体转移是否影响c-Myc活性。引入正常的3号染色体后，观察到含未成熟内含子的hTERT RNA至少降低了30倍，但c-Myc、Mad1和其他c-Myc靶基因的表达水平未改变。我们的结果表明，端粒酶主要在hTERT转录水平受到复杂机制的调控，这些机制涉及远离5'侧翼区域的调控元件，并且3号染色体上假定的hTERT抑制因子不通过c-Myc或其共调节因子之一来调控hTERT的表达。
