Hoevel Thorsten, Macek Robert, Mundigl Olaf, Swisshelm Karen, Kubbies Manfred
Roche Diagnostics GmbH, Pharma Research Oncology, Department of Cell Analytics, Penzberg, Germany.
J Cell Physiol. 2002 Apr;191(1):60-8. doi: 10.1002/jcp.10076.
Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.
紧密连接（TJs）的主要跨膜蛋白是闭合蛋白（Claudins）和闭锁蛋白（occludin）。我们之前已鉴定出小鼠Cldn1的人类同源物CLDN1（SEMP1），它在正常的乳腺来源上皮细胞中表达，但在大多数人类乳腺癌细胞系中不存在。为了研究CLDN1蛋白在肿瘤细胞和正常上皮细胞中的潜在功能，我们构建了一种I-NGFR逆转录病毒载体和单克隆抗CLDN1抗体。在亚汇合和汇合的乳腺癌细胞MDA-MB-435和MDA-MB-361中，通过蛋白质免疫印迹分析或定量逆转录-聚合酶链反应（RT-PCR），未检测到抗CLDN1单克隆抗体所识别的内源性CLDN1表达。当用CLDN1逆转录病毒转导CLDN1阴性的乳腺癌细胞系时，定量RT-PCR结果显示细胞持续表达CLDN1 mRNA。使用抗CLDN1单克隆抗体对CLDN1逆转录病毒转导的乳腺肿瘤细胞进行免疫荧光分析，结果显示CLDN1在细胞间接触位点的亚细胞分布类似于内源性表达CLDN1的T47-D细胞中的CLDN1归巢模式。CLDN1在细胞间接触处的共定位在未表达occludin蛋白（MDA-MB-361和MDA-MB-435）且ZO-1蛋白表达相对较少（MDA-MB-435）的CLDN1转导的乳腺肿瘤细胞中很明显，这表明可能有其他蛋白负责将CLDN1靶向细胞间接触位点。尽管MDA-MB-361肿瘤细胞中不存在occludin，但CLDN1的重新表达降低了3 kDa和40 kDa葡聚糖的细胞旁通量。我们的研究结果表明，在CLDN1阴性的乳腺肿瘤细胞中，CLDN1蛋白生理性归巢的基础蛋白伴侣需求是完整的，并且即使在没有其他紧密连接相关蛋白（如occludin）的情况下，CLDN1基因转移和蛋白表达本身可能足以在转移性肿瘤细胞中发挥紧密连接介导的闸门功能。
