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

培养中的正常细胞除了血细胞以外，如果不附着在基质上就不能生长。这种现象被称为锚定依赖。已知几种恶性细胞是锚定非依赖性的。粘着斑激酶（Focal adhesion kinase，FAK）是近年来发现的一种非受体型蛋白酪氨酸激酶，定位于细胞与基质的粘着斑。FAK被认为是激活的，可能是通过酪氨酸自磷酸化，当细胞附着到细胞外基质和传输信号的生长和分化。在这项研究中，我们研究了FAK的表达和酪氨酸磷酸化的附着和分离形式的培养的正常人角质形成细胞和他们的恶性对应物，人表皮癌细胞系（DJM-1），以澄清如何细胞-基质相互作用和FAK参与他们的生存和生长。保持DJM-1的分离状态和弹性蛋白酶的解离状态，DJM-1至少存活3周。存活细胞（约占总数的30%） 关于我们 当脱离时迅速增殖。脱落1周后FAK酪氨酸磷酸化程度较附着威尔斯孔时略有降低。经弹性蛋白酶处理的角质形成细胞大多数在一周内消失。它们在保持分离仅3天时就失去了增殖能力。加入弹性蛋白酶处理24小时后，角质形成细胞中FAK几乎不发生酪氨酸磷酸化。这些事实表明持续的FAK酪氨酸磷酸化与它们在脱离的DJM-1中的延长存活密切相关。分离的DJM-1在其表面表达层粘连蛋白以及整合素α_2 β_1、α_3 β_1和α_6 β_4，所有这些都介导角质形成细胞与层粘连蛋白的粘附。因此，它们在细胞膜上的相互作用可能在脱落的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。这项研究所获得的结果可能会大大有助于人类根除癌症的努力。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