Molecular biological study on the significance of keratinocyte-substratum interaction for their existence and proliferation-comparison with cancer cells-

除血细胞外，培养中的正常细胞不能在不附着于底物的情况下生长。这是一种被称为锚定依赖的现象。已知有几种恶性细胞是锚定非依赖性的。粘着斑激酶(FAK)是最近发现的一种非受体型蛋白酪氨酸激酶，定位于细胞-基质附着部位，称为粘着斑。当细胞附着在细胞外基质上并传递生长和分化信号时，FAK被认为是被激活的，可能是通过酪氨酸自动磷酸化。在这项研究中，我们检测了FAK在培养的正常人角质形成细胞和其恶性对应的人表皮癌细胞系(DJM-1)中的表达及其酪氨酸磷酸化，以阐明细胞-基质相互作用和FAK在其生存和生长中的作用。经弹性酶分离和解离的DJM-1细胞存活至少3周。存活细胞(约占总数的30%)…从脱离状态释放后，增殖速度更快。脱离后一周的FAK活性较附着井时略有下降。大部分被弹性酶分离的角质形成细胞在一周内消失。分离3天后，细胞即失去增殖能力。加入弹性蛋白酶24小时，角质形成细胞中的FAK几乎不被酪氨酸磷酸化。这些事实表明，持续的FAK酪氨酸磷酸化与它们在DJM-1中的存活时间延长密切相关。分离的DJM-1细胞表面表达层粘连蛋白以及整合素α_2β_1、α_3β_1和α_6β_4，它们均介导角质形成细胞与层粘连蛋白的黏附。因此，它们在细胞膜上的相互作用可能在DJM-1的FAK酪氨酸磷酸化中起作用。奇怪的是，分离的DJM-1具有真菌样的外观。这项研究的结果可以极大地促进人类根除癌症的努力。较少

Normal cells in culture other than blood cells can not grow without attachment to the substratum. This is a phenomenon called anchorage-dependency. Several malignant cells are known to be anchorage-independent. Focal adhesion kinase (FAK) is a recently described non-receptor type protein-tyrosine kinase that localizes at cell-substratum attachment sites named focal adhesion. FAK is supposed to be activated, possibly by tyrosine autophosphorylation, when cells attach to extracellular matrix and transmit signals for growth and differentiation. In this study we examined FAK expression and its tyrosine phosphorylation in both attached and detached forms of cultured normal human keratinocytes and their malignant counterpart, human epidermal carcinoma cell line (DJM-1), in order to clarify how cell-substratum interaction and FAK are involved in their survival and growth. DJM-1 kept detached and dissociated by elastase survived at least 3 weeks. Survived cells (approximately 30% of the total) … More rapidly proliferated when released from detachment. Their FAK assayd at one week in detachment was tyrosine-phosphorylated in a degree slightly decreased than that when they were attached to wells. Majority of keratinocytes kept detached by elastase disappeared within a week. They lost the ability to proliferate when kept detached only for 3 days. FAK in keratinocytes detached by addition of elastase for 24h was hardly tyrosine-phosphorylated. These facts suggested a close association of sustained FAK tyrosine phosphorylation with their prolonged survival in detached DJM-1. Detached DJM-1 expressed laminin on their surfaces as well as integrins alpha_2beta_1, alpha_3beta_1 and alpha_6beta_4, all of which mediate keratinocyte adhesion to laminin. Thus their interactions on cell membrane might have playd a role in FAK tyrosine phosphorylation in detached DJM-1. Curiously, detached DJM-1 had fungus-like appearance. Results obtained in this study could greatly contribute to human efforts to eradicate cancers. Less