br The expression of molecules
The expression of molecules related to epithelial-mesenchymal transition (EMT) was examined in the MIA PaCa-2-A and MIA PaCa-2-R CAY10683 by DNA microarray (Supplement Table 2). CDH1, an epithelial marker, was expressed in the MIA PaCa-2-A cells, but not in the MIA PaCa-2-R cells. However, the expression of mesenchymal markers and cancer stem-like cell markers did not differ between the MIA PaCa-2-A and MIA PaCa-2-R cells.
The MIA PaCa-2-A cells were more proliferative than the MIA PaCa-2-R cells (Fig. 2A). The inoculation of MIA PaCa-2-A cells into SCID mice produced rapidly growing tumors; however, the inocu-lation of the MIA PaCa-2-R cells produced tumors that grew at a significantly slower rate (Fig. 2B). Furthermore, the MIA PaCa-2-A cells were more resistant to gemcitabine, a standard chemothera-peutic drug used in the treatment of pancreatic cancer, than the MIA PaCa-2-R cells (Fig. 2C). These results indicate that the MIA PaCa-2-A and MIA PaCa-2-R cells represent populations with high and low malignant characteristics, respectively.
Fig. 1. Morphologically and functionally distinct clonal populations were isolated from the MIA PaCa-2 cell line. (A) Left panels, MIA PaCa-2 cells; middle panels, MIA PaCa-2-A cells isolated by the limited dilution of MIA PaCa-2 cells; right panels, MIA PaCa-2-R cells isolated by the limited dilution of MIA PaCa-2 cells; upper panels: MIA PaCa-2-A and MIA PaCa-2-R cells directly after establishment; lower panels, MIA PaCa-2-A and MIA PaCa-2-R cells after maintenance for >4 years. (B) DNA short tandem repeat (STR) patterns of the MIA PaCa-2-A and MIA PaCa-2-R cells. (C) Western blot analysis of cell signaling pathways. M, MIA PaCa-2 cells (mixture of epithelial-like cells and non-epithelial-like cells); A, MIA PaCa-2-A cells (epithelial-like cells); R, MIA PaCa-2-R cells (non-epithelial-like cells). p-, phosphorylated.
CLDN7 expression is high in the MIA PaCa-2-A cells, but low in the MIA PaCa-2-R cells
DNA microarray analysis demonstrated that the expression of several genes differed markedly between the MIA PaCa-2-A and MIA PaCa-2-R cells (Fig. 3A). The genes that were expressed in the MIA PaCa-2-A cells, but not in the MIA PaCa-2-R cells and corre-sponded to the Gene Ontology process of ‘adhesion’ were selected as the MIA PaCa-2-A-specific adhesion-related genes and are listed in Table 1. According to the difference in the morphology between the MIA PaCa-2-A cells and MIA PaCa-2-R cells, the expression of molecules composing tight junctions is also considered to differ . As CLDN was the gene mostly associated with tight junctions among the molecules listed in Table 1 , we focused CLDN 7 as a target molecule. Furthermore, CLDN 7 exhibited the greatest dif-ference in expression between the MIA PaCa-2-A and MIA PaCa-2-R
cells among the CLDN family members. (Supplement Table 3). RT-PCR analysis of CLDN7 expression also revealed a higher CLDN7 expression in the MIA PaCa-2-A cells compared with the MIA PaCa-2-R cells (Fig. 3B). Western blot analysis demonstrated that CLDN7 protein expression was also high in the MIA PaCa-2-A cells, but low in the MIA PaCa-2-R cells (Fig. 3C). CLDN7 protein expression was examined in several human pancreatic cancer cell lines other than the MIA PaCa-2, MIA PaCa-2-A and MIA PaCa-2-R cells. Human pancreatic cancer cell lines, Panc-1, BxPC, ASPC, Capan-1 and Capan-2, also expressed CDLN7 by western blot analysis (Fig. 3C).
Knockdown of CLDN7 gene in the MIA PaCa-2-A cells suppresses proliferation
Treatment of the MIA PaCa-2-A cells with siRNA specific to CLDN7 decreased the protein and mRNA expression of CLDN7
Fig. 2. Different proliferative capacities and drug sensitivities of the MIA PaCa-2, MIA PaCa-2-A and MIA PaCa-2-R cells. (A) The MIA PaCa-2, MIA PaCa-2-A or MIA PaCa-2-R cells were seeded in 96-well culture plates (5 102 cells/well). Following incubation for 48, 72, 120 and 168 h, cell numbers were determined using Cell Count Reagent SF. The results are presented as the mean values from 3 independent experiments, and the bars indicate standard deviation; *p < 0.01; n.s., not significant. (B) The MIA PaCa-2 cells, MIA PaCa-2-A or MIA PaCa-2-R cells were subcutaneously inoculated into SCID mice (106 cells/mouse, n ¼ 6). Tumor size (long diameter x short diameter, mm2) was determined 4 weeks after the inoculation by measuring each tumor with a caliper. The same experiment was repeated twice, and similar results were obtained; *p < 0.01. (C) The MIA PaCa-2 cells, MIA PaCa-2-A or MIA PaCa-2-R cells were seeded in 96-well culture plates (5 103/well). Following 24 h of incubation, the cells were untreated or treated with gemcitabine at indicated concentrations for 72 h. The cell number was then determined using Cell Count Reagent SF. The number of viable cells after each treatment was expressed as a ratio to that of untreated cells. The results are shown as the average of those in 3 independent experiments and the bars indicate standard deviation; *p < 0.01.