Reexpression of the TJ protein CLDN1 induces apoptosis in breast tumor spheroids

Members of the claudin family together with occludin are the major constituents of the tight junction (TJ) complex. The human homologue of the murine CLDN1, previously called SEMP1, was identified by differential expression analysis, and the CLDN1 mRNA was found to be downregulated or completely lost in human breast cancer cells in vitro. Retroviral-induced CLDN1 reexpression in breast cancer cells results in plasma membrane homing of the protein and reconstitution of paracellular flux inhibition, which is not dependent on the presence of occludin protein. In this report, we investigated the physiologic role of CLDN1 in CLDN1-transduced MDA-MB 361 breast tumor cells in adherent 2D and suspension 3D spheroid cell cultures. Retroviral-transduced bulk cultures were FACS-sorted to enrich for 100% CLDN1-positive clonal derivatives with similar expression levels of CLDN1 mRNA and protein. There was no difference in proliferation and cell death characteristics in 2D adherent cell cultures of CLDN1-positive compared to control CLDN1-negative and mock-transduced cell cultures. In contrast, the majority of the CLDN1-transduced derivatives displayed a significant elevation of apoptosis that became evident as early as 2 days after 3D spheroid culture onset. This elevated apoptosis was independent of the volume of established spheroids. The cellular immunofluorescence analysis of CLDN1 protein expression in transduced bulk cultures revealed a CLDN1-positive subfraction with a heterogeneous pattern of membrane and cytosolic immunostaining. In the clonal MDA-MB 361 CLDN1-positive cultures, we found that a more prominent cell membrane localization correlated with a pronounced increase of apoptosis in tumor spheroids. In parallel, inhibition of the paracellular flux rate was observed. These findings support a potential role of the TJ protein CLDN1 in restricting nutrient and growth factor supplies in breast cancer cells, and they indicate that the loss of the cell membrane localization of the TJ protein CLDN1 in carcinomas may be a crucial step during tumor progression. © 2003 Wiley-Liss, Inc.