Filatov L, Golubovskaya V, Hurt J C, Byrd L L, Phillips J M, Kaufmann W K
Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 27599-7295, USA.
Oncogene. 1998 Apr 9;16(14):1825-38. doi: 10.1038/sj.onc.1201711.
Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin fibroblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53. Chromosome aberrations were enumerated during in vitro aging of isogenic control (F5neo) and HPV-16E6-expressing (F5E6) fibroblasts. We found that structural and numerical aberrations in chromosomes were significantly increased in F5E6 cells during aging in vitro and fluorescence in situ hybridization (FISH) analysis using chromosome-specific probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5E6 cells. Flow cytometry and karyotypic analyses revealed increased polyploidy and aneuploidy in F5E6 cells only at passages > 16, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was confirmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo fibroblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal beta-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce beta-galactosidase at high passage. Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridization and by quantitative FISH analysis on an individual cell level. Telomeric signals diminished continuously as F5neo cells aged in vitro being reduced by 80% near the time of replicative senescence. Telomeric signals detected by FISH also decreased continuously during aging of telomerase-negative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P < 0.05) with both loss of telomeric signals and inactivation of G2 checkpoint function. The results suggest that chromosomal stability depends upon a complex interaction among the systems of telomere length maintenance and cell cycle checkpoints.
细胞周期检查点和肿瘤抑制基因功能似乎是维持增殖细胞基因组稳定性所必需的。在本研究中，监测了与端粒结构、寿命控制和G2检查点功能相关的染色体不稳定情况。通过表达人乳头瘤病毒16型（HPV-16）E6癌蛋白使p53失活，从而在人皮肤成纤维细胞的传代培养中使复制性衰老失活。在同基因对照（F5neo）和表达HPV-16E6的（F5E6）成纤维细胞的体外老化过程中，对染色体畸变进行了计数。我们发现，在体外老化过程中，F5E6细胞中染色体的结构和数量畸变显著增加，并且使用染色体特异性探针的荧光原位杂交（FISH）分析表明，在遗传不稳定的F5E6细胞中发生了涉及4号和6号染色体的重排。流式细胞术和核型分析显示，仅在传代次数>16时，F5E6细胞中的多倍体和非整倍体增加，尽管这些细胞在传代5次和16次时表现出与p53失活相关的有丝分裂纺锤体检查点功能缺陷。在表达E6的细胞的体外老化过程中，证实G2检查点功能逐渐但持续失活。通过诱导衰老相关标志物pH 6.0溶酶体β-半乳糖苷酶，记录了F5neo成纤维细胞在体外传代过程中的老化情况。F5E6细胞表现出体外寿命延长，并且在高传代时不诱导β-半乳糖苷酶。通过Southern杂交和在单个细胞水平上的定量FISH分析，证明了端粒酶阴性的F5neo细胞在体外老化过程中端粒的侵蚀。随着F5neo细胞在体外老化，端粒信号持续减少，在复制性衰老时减少了80%。在端粒酶阴性的F5E6细胞老化过程中，通过FISH检测到的端粒信号也持续减少，但当表达端粒酶时，端粒似乎在第34代时稳定下来。表达E6的细胞中的染色体不稳定性与端粒信号的丧失和G2检查点功能的失活均相关（P<0.05）。结果表明，染色体稳定性取决于端粒长度维持系统和细胞周期检查点系统之间的复杂相互作用。
