蛋白酪氨酸磷酸化水平与乳腺癌细胞株抗失巢凋亡特性的关系

背景与目的：正常上皮或内皮细胞脱离与细胞外基质的联系时会发生失巢凋亡(anoikis),而大多数来源于上皮或内皮的肿瘤细胞则丧失了这种特性,多数相关研究表明肿瘤细胞的抗失巢凋失特性与细胞信号转导的异常密切相关。本研究初步探讨信号分子的蛋白酪氨酸磷酸化水平与肿瘤细胞抗失巢凋亡特性之间的关系。方法：用琼脂糖凝胶电泳检测DNA梯状带,用流式细胞术以及软琼脂集落形成实验等方法观察正常狗肾脏上皮细胞株MDCK(Madin-Darby canine kidney)和3种乳腺癌细胞株MCF-7、Bcap-37以及MDA-MB-231的抗失巢凋亡特性。同时分析广谱蛋白酪氨酸激酶抑制剂染料木黄酮(genistein)对肿瘤细胞这种特性的抑制作用,并通过western blot检测3种乳腺癌细胞在悬浮培养与贴壁培养时总体蛋白酪氨酸磷酸化水平的差异。结果：3种乳腺癌细胞均表现出明显的抗失巢凋亡特性,进一步的研究证明这种特性可被染料木黄酮所抑制。Western blot的结果显示与贴壁培养时相比,在悬浮培养的MDcK细胞中所有的蛋白酪氨酸的磷酸化水平均呈现下降趋势;而在肿瘤细胞中,尽管大多数蛋白质酪氨酸的磷酸化水平下降,但是可见有酪氨酸磷酸化水平异常增高的蛋白条带(幅度3．5～6．5倍)。结论：3种乳腺癌细胞均具有不同程度的抗失巢凋亡特性,而信号蛋白酪氨酸的异常磷酸化与这些肿瘤细胞的抗失巢凋亡特性有关。

Relationship between protein tyrosine phosphorylation level and anoikis resistance of breast tumor cell lines

BACKGROUND &OBJECTIVES: Normal epithelial or endothelial cells can undergo anoikis, a type of apoptosis, when they are detached from their extracel lular matrices (ECM), while most tumor cells derived from epithelial or endothel ial tissues lose this feature. Most of the studies indicate that anoikis resista nce of tumor cells is closely related to abnormal signal transductioin. The aim of this research was to screen out the tumor cell lines that are anoikis resista nt, then to investigate the relationship between the protein tyrosine phosphoryl ation of signaling molecules and anoikis resistance feature of tumor cells. METH ODS:Anoikis resistance of breast tumor cell lines MCF-7, Bcap-37, and MDA-MB -231 was determined by DNA ladder assay, flow cytometry analysis, and soft agar assay upon suspending culture. A normal epithelial cell line MDCK (Madin-Darby canine kidney) was taken as a control. The inhibitory effect of genistein, a ge neral protein tyrosine kinase inhibitor, on anoikis resistance of tumor cells wa s analyzed at the same time. The differences of total protein tyrosine phosphory lation levels between suspended and attached cultural conditions of three breast cancer cell lines were examined by Western blot analysis. RESULTS:All the thre e tumor cell lines were distinctly anoikis resistant. Further study showed that these features could be suppressed by genistein. The results of Western blot ana lysis showed that the general level of tyrosine phosphorylation in suspended MDC K cells was decreased compared with that in attached cells, while tyrosine phosp horylation level of some proteins increased (more than 3.56.5 folds) aberrantl y in suspended tumor cells. CONCLUSION: All the three breast tumor cells in this study are anoikis resistant respectively to some extent, and aberrant tyrosine phosphorylation of signaling proteins may play a role in the process of anoikis resistance of these tumor cells.