Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation
Mass cultured human fibroblasts overexpressing hTERT encounter a growth crisis following an extended period of proliferation
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DOI：
10.1006/excr.2000.4982
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发表时间：
2000-09-15
影响因子：
3.7
通讯作者：
Moore, MAS
中科院分区：
文献类型：
--
作者：
MacKenzie, KL;Franco, S;Moore, MAS
在永生化的过程中，必须绕过至少两个死亡率检查点M1和M2。绕过M1（衰老）的细胞寿命延长，但不一定是不朽的。最近的研究表明，端粒酶催化亚单位（hTERT）的异位表达使正常人细胞绕过衰老（M1）和癌基因转化细胞避免危机（M2），成为不朽的。然而，目前还不清楚hTERT表达是否足以使正常人成纤维细胞克服M1和M2并变得永生。我们研究了端粒酶在永生化中的作用，通过维持大量培养hTERT转导的原代人胎肺成纤维细胞（MRC-5细胞）很长一段时间（超过2年）。在目前的研究中，高达70%的MRC-5细胞用表达hTERT的逆转录病毒载体转导。hTERT-转导的细胞表现出高水平的端粒酶活性，端粒的延长，和增殖超过衰老。然而，在增殖超过36个群体倍增（PDL）超过衰老之后，表达hTERT的细胞的总体生长速率下降。在这些生长减少的时期，hTERT转导的MRC-5细胞表现出危机细胞的典型特征，包括细胞死亡率和多倍性增加。在某些情况下，非常晚的传代细胞获得衰老样表型，其特征在于细胞周期的G1期停滞并大大减少DNA合成。在危机开始时，hTERT转导的细胞表达高水平的端粒酶，并具有非常长的端粒，范围高达30 kb。并不是所有的细胞都屈服于危机，因此，一些培养物已经增殖超过240个PDL，而另一种培养物似乎在160个PDL处永久停滞。晚期传代MRC-5细胞，包括危机后细胞，没有显示恶性转化的迹象。我们的研究结果与端粒酶和端粒延长大大延长细胞寿命而不诱导恶性变化的模型一致。然而，这些研究也表明，hTERT表达细胞可能会经历危机后，延长寿命和永生不是hTERT表达在正常二倍体成纤维细胞的普遍结果。(C)北京大学出版社.
During the process of immortalization, at least two mortality checkpoints, M1 and M2, must be bypassed. Cells that have bypassed M1 (senescence) have an extended life span, but are not necessarily immortal. Recent studies have shown that ectopic expression of the catalytic subunit of telomerase (hTERT) enables normal human cells to bypass senescence (M1) and oncogene transformed cells to avert crisis (M2) and become immortal. However, it is unclear whether hTERT expression is sufficient for normal human fibroblasts to overcome both M1 and M2 and become immortal. We have investigated the role of telomerase in immortalization by maintaining mass cultures of hTERT-transduced primary human fetal lung fibroblasts (MRC-5 cells) for very long periods of time (more than 2 years). In the present studies, up to 70% of MRC-5 cells were transduced with retroviral vectors that express hTERT. hTERT-transduced cells exhibited high levels of telomerase activity, elongation of telomeres, and proliferation beyond senescence. However, after proliferating for more than 36 population doublings (PDLs) beyond senescence, the overall growth rate of hTERT-expressing cells declined. During theses periods of reduced growth, hTERT-transduced MRC-5 cells exhibited features typical of cells in crisis, including an increased rate of cell death and polyploidy. In some instances, very late passage cells acquired a senescence-like phenotype characterized by arrest in the G1 phase of the cell cycle and greatly reduced DNA synthesis. At the onset of crisis, hTERT-transduced cells expressed high levels of telomerase and had very long telomeres, ranging up to 30 kb. Not all cells succumbed to crisis and, consequently, some cultures have proliferated beyond 240 PDLs, while another culture appears to be permanently arrested at 160 PDLs. Late passage MRC-5 cells, including postcrisis cells, displayed no signs of malignant transformation. Our results are consistent with the model in which telomerase and telomere elongation greatly extends cellular life span without inducing malignant changes. However, these investigations also indicate that hTERT-expressing cells may undergo crisis following an extended life span and that immortality is not the universal outcome of hTERT expression in normal diploid fibroblasts. (C) 2000 Academic Press.