Hauser P J, Agrawal D, Pledger W J
Department of Cell Biology, Vanderbilt University, Nashville, Tennesse 37240, USA.
Oncogene. 1998 Dec 17;17(24):3083-92. doi: 10.1038/sj.onc.1202235.
In order to understand the mechanism through which loss of anchorage inhibits growth, we have investigated the events that occur in murine keratinocytes upon substratum detachment utilizing both primary cells and established immortalized cell lines. Our data has revealed that while both primary and immortalized cells undergo growth arrest in suspension, the nature of this arrest is markedly different. Primary cells exhibit a growth arrest that is characterized by rapid cessation of DNA synthesis resulting in a static S phase population. In contrast, an immortalized non-tumorgenic cell line, Balb MK, exhibits growth arrest as measured by thymidine incorporation, but does not prevent cells that have entered S phase from continuing into G2/M, and accumulating as a 4N population. In contrast to both primary and MK cells, the tumorigenic SLC-1 cell line did not accumulate in a specific cell cycle interval and were able to undergo continuous growth in suspension. Examination of cyclin A protein and its associated activity revealed that cyclin A protein levels decreased in primary but not MK cells; suggesting the continued presence of cyclin A may allow continued DNA synthesis observed in MK cells. Furthermore, we demonstrate the accumulation of suspension cultured MK cells as a 4N population correlated with the loss of cyclin A/cdk2 kinase activity, which in turn occurred through the accumulation of p27kip1, whereas neither p27kip1 accumulation nor loss of cyclin A activity was observed in SLC-1 cells. Our results clearly reveal that the process of growth inhibition in suspension cultured cells may occur in several forms with distinct characteristics that are dependent on the status of cyclin/cdk complexes and CKI proteins. Tumor derived cells in suspension did not lose cyclin A dependent kinase activity and thus continued to grow and divide.
为了了解失去锚定作用抑制生长的机制，我们利用原代细胞和已建立的永生化细胞系，研究了鼠角质形成细胞在脱离基质后发生的事件。我们的数据显示，虽然原代细胞和永生化细胞在悬浮培养时都会发生生长停滞，但这种停滞的性质明显不同。原代细胞表现出的生长停滞特征是DNA合成迅速停止，导致S期细胞群体静止。相比之下，一种永生化的非致瘤细胞系Balb MK，通过胸苷掺入法测量显示出生长停滞，但并不阻止已进入S期的细胞继续进入G2/M期，并积累为4N群体。与原代细胞和MK细胞不同，致瘤性SLC-1细胞系不会在特定的细胞周期阶段积累，并且能够在悬浮培养中持续生长。对细胞周期蛋白A及其相关活性的检测表明，细胞周期蛋白A的蛋白水平在原代细胞中下降，但在MK细胞中没有下降；这表明细胞周期蛋白A的持续存在可能允许MK细胞中观察到的持续DNA合成。此外，我们证明悬浮培养的MK细胞积累为4N群体与细胞周期蛋白A/cdk2激酶活性的丧失相关，而这又通过p27kip1的积累发生，而在SLC-1细胞中未观察到p27kip1的积累或细胞周期蛋白A活性的丧失。我们的结果清楚地表明，悬浮培养细胞中的生长抑制过程可能以几种具有不同特征的形式发生，这些特征取决于细胞周期蛋白/cdk复合物和CKI蛋白的状态。悬浮培养的肿瘤衍生细胞没有丧失细胞周期蛋白A依赖性激酶活性，因此继续生长和分裂。
