Control of Replicative Life Span in Human Cells: Barriers to Clonal Expansion Intermediate Between M1 Senescence and M2 Crisis

The accumulation of genetic abnormalities in a developing tumor is driven, at least in part, by the need to overcome inherent restraints on the replicative life span of human cells, two of which—senescence (M1) and crisis (M2)—have been well characterized. Here we describe additional barriers to clonal expansion (M(int)) intermediate between M1 and M2, revealed by abrogation of tumor-suppressor gene (TSG) pathways by individual human papillomavirus type 16 (HPV16) proteins. In human fibroblasts, abrogation of p53 function by HPVE6 allowed escape from M1, followed up to 20 population doublings (PD) later by a second viable proliferation arrest state, M(int)E6, closely resembling M1. This occurred despite abrogation of p21(WAF1) induction but was associated with and potentially mediated by a further ∼3-fold increase in p16(INK4a) expression compared to its level at M1. Expression of HPVE7, which targets pRb (and p21(WAF1)), also permitted clonal expansion, but this was limited predominantly by increasing cell death, resulting in a M(int)E7 phenotype similar to M2 but occurring after fewer PD. This was associated with, and at least partly due to, an increase in nuclear p53 content and activity, not seen in younger cells expressing E7. In a different cell type, thyroid epithelium, E7 also allowed clonal expansion terminating in a similar state to M(int)E7 in fibroblasts. In contrast, however, there was no evidence for a p53-regulated pathway; E6 was without effect, and the increases in p21(WAF1) expression at M1 and M(int)E7 were p53 independent. These data provide a model for clonal evolution by successive TSG inactivation and suggest that cell type diversity in life span regulation may determine the pattern of gene mutation in the corresponding tumors.

正在进展的肿瘤中遗传异常的积累，至少部分源于人类细胞需要突破其复制寿命固有的限制机制：其中两种——衰老（senescence，M1期）与危机（crisis，M2期）——已得到充分表征。本文报道了介于M1与M2之间的克隆扩增额外障碍（命名为M(int)），该发现源自通过单个人类乳头瘤病毒16型（human papillomavirus type 16，HPV16）蛋白分别失活肿瘤抑制基因（tumor-suppressor gene，TSG）通路的实验。在人类成纤维细胞中，经HPV E6蛋白抑制p53功能后，细胞可逃逸M1期阻滞，随后在约20次群体倍增（population doublings，PD）后进入第二种可存活的增殖停滞状态——M(int)E6，其表型与M1期高度相似。尽管此时p21(WAF1)的诱导已被阻断，但与M1期相比，细胞内p16(INK4a)的表达水平进一步升高约3倍，这一变化可能介导了该状态的形成。靶向pRb（同时也作用于p21(WAF1)）的HPV E7蛋白同样可促进克隆扩增，但该过程主要因细胞死亡增加而受限，最终形成M(int)E7表型，其特征与M2期类似，但所需群体倍增数更少。该表型与核内p53含量及活性升高相关，且至少部分由其介导——这种变化在表达E7的年轻细胞中并未出现。在另一种细胞类型——甲状腺上皮细胞（thyroid epithelium）中，E7蛋白同样可促进克隆扩增，最终形成与成纤维细胞中M(int)E7相似的状态。但与之不同的是，该细胞中并未发现p53调控通路的参与：HPV E6蛋白未产生任何效应，且M1期与M(int)E7状态下p21(WAF1)的表达上调均不依赖p53。本研究为通过依次失活肿瘤抑制基因实现克隆进化提供了模型，并提示寿命调控机制的细胞类型多样性，可能决定了对应肿瘤中基因突变的发生模式。