Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells.
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.
Hoevel
Thorsten
T
Roche Diagnostics GmbH, Pharma Research Oncology, Department of Cell Analytics, Penzberg, Germany.
Macek
Robert
R
Mundigl
Olaf
O
Swisshelm
Karen
K
Kubbies
Manfred
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000503:physiopathology; D002450:Cell Communication / Q000502:physiology; D002460:Cell Line; D062445:Claudin-1; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D018390:Gene Targeting; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D008565:Membrane Proteins / Q000172:deficiency* / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D062826:Zonula Occludens-1 Protein
D000911:Antibodies, Monoclonal; C568108:CLDN1 protein, human; D062445:Claudin-1; C568113:Cldn1 protein, mouse; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein