Behrens P, Meissner C, Hopfer H, Schümann J, Tan M I, Ellerbrake N, Strunck E, Vollmer G
Institut für Biochemische Endokrinologie, Medizinische Universität, Lübeck, Germany.
Biochem Cell Biol. 1996;74(6):875-86. doi: 10.1139/o96-093.
In vitro studies on endometrial carcinogenesis have been hampered by limited differentiation of the cells in culture. Using the endometrial carcinoma cell lines HEC 1B and its subclone HEC 1B(L), we established and characterized cell culture conditions that preserve a more differentiated state of the tumor cells. Randomly seeded HEC 1B(L) cells, if grown in a serum-free defined medium on top of a reconstituted basement membrane (Matrigel), within a few hours assembled themselves to web-like structures. In a thick layer of Matrigel, they showed an even more pronounced morphological differentiation. Functionally, two additional secretory proteins, about 31 and 77 kDa in size, became apparent as a response to matrigel. To further investigate the regulatory role of the extracellular matrix in the process of in vitro differentiation of endometrial adenocarcinoma cells, we addressed two specific problems. First, we investigated if the capacity of in vitro differentiation is a specific feature of HEC 1B(L) cells or if it is common to all endometrial adenocarcinoma cells. Second, we tried to identify the Matrigel component(s) responsible for in vitro differentiation. The assembly of HEC 1B and HEC 1B(L) cells into spatially organized web-like structures and the expression of the 77 kDa protein were thereby used as an assay. All endometrial adenocarcinoma cell lines tested to a variable degree formed web-like structures on Matrigel. Although the pattern of de novo synthesized secretory proteins changed as a response to Matrigel, only HEC 1A, HEC 1B, HEC 1B(L), and Ishikawa cells responded to culture on Matrigel by an increased expression of the 77 kDa protein. Functionally, polyclonal anti-laminin antibodies, but not anti-collagen type IV antibodies, disrupted formation of web-like structures by HEC 1B cells. The laminin-specific peptides YIGSR and SIKVAV but none of the RGD-peptides RGDS, GRGDSP, or GRADSP affected the three-dimensional assembly of these cells in vitro. Both anti-laminin antibodies and laminin-specific peptides suppressed Matrigel-induced formation of the 77-kDa secretory protein by HEC 1B cells. These findings suggest the involvement of laminin in the in vitro differentiation of the HEC 1B endometrial adenocarcinoma cell line. In a mechanistic view, laminin appears to play a crucial role in the regulation of this in vitro differentiation process.
体外培养中细胞分化有限，这阻碍了对子宫内膜癌发生的体外研究。利用子宫内膜癌细胞系HEC 1B及其亚克隆HEC 1B(L)，我们建立并描述了能使肿瘤细胞保持更分化状态的细胞培养条件。随机接种的HEC 1B(L)细胞，如果在无血清限定培养基中于重组基底膜（基质胶）上生长，几小时内就会自行组装成网状结构。在厚层基质胶中，它们表现出更明显的形态分化。在功能上，作为对基质胶的反应，另外两种分泌蛋白变得明显，大小约为31 kDa和77 kDa。为了进一步研究细胞外基质在子宫内膜腺癌细胞体外分化过程中的调节作用，我们解决了两个具体问题。第一，我们研究体外分化能力是HEC 1B(L)细胞的特有特征还是所有子宫内膜腺癌细胞共有的特征。第二，我们试图确定负责体外分化的基质胶成分。因此，将HEC 1B和HEC 1B(L)细胞组装成空间有序的网状结构以及77 kDa蛋白的表达用作检测方法。所有测试的子宫内膜腺癌细胞系在不同程度上都在基质胶上形成了网状结构。尽管新生合成的分泌蛋白模式因基质胶而改变，但只有HEC 1A、HEC 1B、HEC 1B(L)和石川细胞对在基质胶上培养的反应是77 kDa蛋白表达增加。在功能上，多克隆抗层粘连蛋白抗体而非抗IV型胶原抗体破坏了HEC 1B细胞形成网状结构。层粘连蛋白特异性肽YIGSR和SIKVAV，但RGD肽RGDS、GRGDSP或GRADSP均不影响这些细胞在体外的三维组装。抗层粘连蛋白抗体和层粘连蛋白特异性肽均抑制了HEC 1B细胞基质胶诱导的77 kDa分泌蛋白的形成。这些发现表明层粘连蛋白参与了HEC 1B子宫内膜腺癌细胞系的体外分化。从机制角度看，层粘连蛋白似乎在这一体外分化过程的调节中起关键作用。
