Tetraploid cells produced by absence of substrate adhesion during cytokinesis are limited in their proliferation and enter senescence after DNA replication

Tetraploidy has been proposed as an intermediate state in neoplastic transformation due to the intrinsic chromosome instability of tetraploid cells. Despite the identification of p53 as a major factor in growth arrest of tetraploid cells, it is still unclear whether the p53-dependent mechanism for proliferation restriction is intrinsic to the tetraploid status or dependent on the origin of tetraploidy. Substrate adherence is fundamental for cytokinesis completion in adherent untransformed cells. Here we show that untransformed fibroblast cells undergoing mitosis in suspension produce binucleated tetraploid cells due to defective cleavage furrow constriction that leads to incomplete cell abscission. Binucleated cells obtained after loss of substrate adhesion maintain an inactive p53 status and are able to progress into G1 and S phase. However, binucleated cells arrest in G2, accumulate p53 and are not able to enter mitosis as no tetraploid metaphases were recorded after one cell cycle time. In contrast, tetraploid metaphases were found following pharmacological inhibition of Chk1 kinase, suggesting the involvement of the ATR/Chk1 pathway in the G2 arrest of binucleated cells. Interestingly, after persistence in the G2 phase of the cell cycle, a large fraction of binucleated cells become senescent. These findings identify a new pathway of proliferation restriction for tetraploid untransformed cells that seems to be specific for loss of adhesion-dependent cytokinesis failure. This involves Chk1 and p53 activation during G2. Inhibition of growth and entrance into senescence after cytokinesis in suspension may represent an important mechanism to control tumor growth. In fact, anchorage independent growth is a hallmark of cancer and it has been demonstrated that binucleated transformed cells can enter a cycle of anchorage independent growth.

四倍体（tetraploidy）因四倍体细胞固有的染色体不稳定性，被提出为肿瘤转化过程中的中间状态。尽管已确认p53是介导四倍体细胞生长停滞的关键因子，但p53依赖的增殖限制机制究竟是四倍体状态本身固有，还是取决于四倍体的产生来源，目前仍不明确。底物黏附对于贴壁未转化细胞完成胞质分裂（cytokinesis）至关重要。本研究发现，在悬浮状态下进行有丝分裂的未转化成纤维细胞，会因分裂沟收缩缺陷导致细胞脱落不全，进而产生双核四倍体细胞。因失去底物黏附而形成的双核细胞，其p53状态维持失活，并可顺利进入G1期与S期。然而，双核细胞会在G2期发生停滞，积累p53，且无法进入有丝分裂——在一个细胞周期后未观察到四倍体中期细胞。与之相反，经Chk1激酶（Chk1 kinase）药理学抑制后可检测到四倍体中期细胞，这提示ATR/Chk1通路参与了双核细胞的G2期阻滞。有趣的是，在细胞周期G2期持续停留一段时间后，大量双核细胞会发生衰老。上述研究结果明确了一条针对未转化四倍体细胞的新型增殖限制通路，该通路似乎特异性对应依赖底物黏附的胞质分裂失败事件，其过程涉及G2期的Chk1与p53激活。悬浮状态下胞质分裂失败后出现的生长抑制与衰老诱导，或许是控制肿瘤发生的重要机制。事实上，锚定非依赖生长（anchorage independent growth）是癌症的标志性特征之一，且已有研究证实，双核转化细胞可进入锚定非依赖生长周期。