T-Cadherin Negatively Regulates the Proliferation of Cutaneous Squamous Carcinoma Cells
2005; Elsevier BV; Volume: 124; Issue: 4 Linguagem: Inglês
10.1111/j.0022-202x.2005.23660.x
ISSN1523-1747
AutoresYohei Mukoyama, Shuxia Zhou, Yoshiki Miyachi, Norihisa Matsuyoshi,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoT-cadherin is a unique member of the cadherin superfamily that lacks the transmembrane and cytoplasmic domains, and is instead linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. We previously reported that T-cadherin was specifically expressed on the basal keratinocytes of the epidermis, and the expression of T-cadherin was significantly reduced in invasive cutaneous squamous cell carcinoma (SCC) and in the lesional skin of psoriasis vulgaris. In this study, to obtain an insight into the role of T-cadherin in keratinocytes, we used transfection methods and examined the effect of overexpression or knockdown of T-cadherin in immortalized keratinocyte cell lines derived from SCC. T-cadherin overexpressed cells showed clearly reduced cell proliferation, but the influence of cell–cell adhesiveness and cell mobility was not detected. Using a tetracycline-regulated expression system, we also confirmed that the suppression of cell proliferation was dependent on the expression level of T-cadherin. Cell cycle analysis demonstrated that over expression of T-cadherin induced a delay in the G2/M phase. Our findings suggest that T-cadherin acts as an endogenous negative regulator of keratinocyte proliferation and its inactivation is the cause for keratinocyte hyperproliferation in SCC or in psoriasis vulgaris. T-cadherin is a unique member of the cadherin superfamily that lacks the transmembrane and cytoplasmic domains, and is instead linked to the cell membrane via a glycosyl-phosphatidylinositol anchor. We previously reported that T-cadherin was specifically expressed on the basal keratinocytes of the epidermis, and the expression of T-cadherin was significantly reduced in invasive cutaneous squamous cell carcinoma (SCC) and in the lesional skin of psoriasis vulgaris. In this study, to obtain an insight into the role of T-cadherin in keratinocytes, we used transfection methods and examined the effect of overexpression or knockdown of T-cadherin in immortalized keratinocyte cell lines derived from SCC. T-cadherin overexpressed cells showed clearly reduced cell proliferation, but the influence of cell–cell adhesiveness and cell mobility was not detected. Using a tetracycline-regulated expression system, we also confirmed that the suppression of cell proliferation was dependent on the expression level of T-cadherin. Cell cycle analysis demonstrated that over expression of T-cadherin induced a delay in the G2/M phase. Our findings suggest that T-cadherin acts as an endogenous negative regulator of keratinocyte proliferation and its inactivation is the cause for keratinocyte hyperproliferation in SCC or in psoriasis vulgaris. squamous cell carcinoma tetracycline Cadherins are a family of cell adhesion molecules that exhibit calcium-dependent, homophilic binding. Each member of the cadherins shows specific distribution in various kinds of organs and is known to be involved not only in cell adhesion but also in many biological processes, such as cell recognition, cell signaling, cell communication, morphogenesis, as well as pathological conditions such as carcinoma (Angst et al., 2001Angst B.D. Marcozzi C. Magee A.I. The cadherin superfamily: Diversity in form and function.J Cell Sci. 2001; 114: 629-641Crossref PubMed Google Scholar). It is easy to imagine that a reduced expression of cadherin triggers the release of carcinoma cells from the tumor mass. In fact, strong E-cadherin expression is recognized in well-differentiated carcinomas, which maintain their cell–cell adhesiveness and are less invasive, but E-cadherin expression is reduced in undifferentiated carcinomas, which have lost their cell–cell adhesion and show a strong invasive tendency (Hirohashi and Kanai, 2003Hirohashi S. Kanai Y. Cell adhesion system and human cancer morphogenesis.Cancer Sci. 2003; 94: 575-581Crossref PubMed Scopus (353) Google Scholar). T-cadherin (CDH 13, H-cadherin) is a unique member of the cadherin superfamily that lacks the transmembrane and cytoplasmic domains, and is instead linked to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor (Ranscht and Dours-Zimmermann, 1991Ranscht B. Dours-Zimmermann M.T. T-cadherin, a novel cadherin cell adhesion molecule in the nervous system lacks the conserved cytoplasmic region.Neuron. 1991; 7: 391-402Abstract Full Text PDF PubMed Scopus (255) Google Scholar). The fact that the cytoplasmic domain of classical cadherin is crucial for its cell–cell adhesion suggests the existence of another function of T-cadherin (Nagafuchi and Takeichi, 1988Nagafuchi A. Takeichi M. Cell binding function of E-cadherin is regulated by the cytoplasmic domain.EMBO J. 1988; 7: 3679-3684Crossref PubMed Scopus (652) Google Scholar; Stappert and Kemler, 1994Stappert J. Kemler R. A short core region of E-cadherin is essential for catenin binding and is highly phosphorylated.Cell Adhes Commun. 1994; 2: 319-327Crossref PubMed Scopus (195) Google Scholar; Yap et al., 1998Yap A.S. Niessen C.M. Gumbiner B.M. The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn.J Cell Biol. 1998; 141: 779-789Crossref PubMed Scopus (452) Google Scholar). Furthermore, T-cadherin does not contain the highly conserved His–Ala–Val motif in the extracellular domain (Ranscht and Dours-Zimmermann, 1991Ranscht B. Dours-Zimmermann M.T. T-cadherin, a novel cadherin cell adhesion molecule in the nervous system lacks the conserved cytoplasmic region.Neuron. 1991; 7: 391-402Abstract Full Text PDF PubMed Scopus (255) Google Scholar). Since this motif is crucial for the homophilic interactions of classical cadherin (Blaschuk et al., 1990Blaschuk O.W. Sullivan R. David S. Pouliot Y. Identification of a cadherin cell adhesion recognition sequence.Dev Biol. 1990; 139: 227-229Crossref PubMed Scopus (325) Google Scholar; Nose et al., 1990Nose A. Tsuji K. Takeichi M. Localization of specificity determining sites in cadherin cell adhesion molecules.Cell. 1990; 61: 147-155Abstract Full Text PDF PubMed Scopus (401) Google Scholar), the lack of this motif in T-cadherin also supports this hypothesis. Like other GPI-anchored proteins, T-cadherin is enriched in minor detergent-insoluble plasma membrane domains, called lipid rafts, and co-localizes with signaling molecules such as Src family kinases (Doyle et al., 1998Doyle D.D. Goings G.E. Upshaw-Earley J. Page E. Ranscht B. Palfrey H.C. T-cadherin is a major glycophosphoinositol-anchored protein associated with noncaveolar detergent-insoluble domains of the cardiac sarcolemma.J Biol Chem. 1998; 273: 6937-6943Crossref PubMed Scopus (62) Google Scholar; Philippova et al., 1998Philippova M.P. Bochkov V.N. Stambolsky D.V. Tkachuk V.A. Resink T.J. T-cadherin and signal-transducing molecules co-localize in caveolin-rich membrane domains of vascular smooth muscle cells.FEBS Lett. 1998; 429: 207-210Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These features of T-cadherin indicate that T-cadherin may be involved in signal transduction rather than cell–cell adhesion. We previously reported that T-cadherin is expressed on keratinocytes and is specifically localized in the basal layer of the epidermis, where keratinocyte proliferation takes place (Zhou et al., 2002Zhou S. Matsuyoshi N. Liang S.B. Takeuchi T. Ohtsuki Y. Miyachi Y. Expression of T-cadherin in Basal keratinocytes of skin.J Invest Dermatol. 2002; 118: 1080-1084Crossref PubMed Scopus (28) Google Scholar). Furthermore, we also proved that T-cadherin expression is downregulated in invasive cutaneous squamous cell carcinoma (SCC) and in the lesional skin of psoriasis vulgaris (Takeuchi et al., 2002Takeuchi T. Liang S.B. Matsuyoshi N. Zhou S. Miyachi Y. Sonobe H. Ohtsuki Y. Loss of T-cadherin (CDH13, H-cadherin) expression in cutaneous squamous cell carcinoma.Lab Invest. 2002; 82: 1023-1029Crossref PubMed Scopus (57) Google Scholar; Zhou et al., 2003Zhou S. Matsuyoshi N. Takeuchi T. Ohtsuki Y. Miyachi Y. Reciprocal altered expression of T-cadherin and P-cadherin in psoriasis vulgaris.Br J Dermatol. 2003; 149: 268-273Crossref PubMed Scopus (21) Google Scholar). Another study showed that T-cadherin suppressed the proliferation of neuroblastoma cells and glioma cells (Takeuchi et al., 2000Takeuchi T. Misaki A. Liang S.B. Tachibana A. Hayashi N. Sonobe H. Ohtsuki Y. Expression of T-cadherin (CDH13, H-Cadherin) in human brain and its characteristics as a negative growth regulator of epidermal growth factor in neuroblastoma cells.J Neurochem. 2000; 74: 1489-1497Crossref PubMed Scopus (148) Google Scholar; Huang et al., 2003Huang Z.Y. Wu Y. Hedrick N. Gutmann D.H. T-cadherin-mediated cell growth regulation involves G2 phase arrest and requires p21CIP1/WAF1 expression.Mol Cell Biol. 2003; 23: 566-578Crossref PubMed Scopus (77) Google Scholar). The function of T-cadherin in the skin, however, still remains unclear; these results permit us to speculate that the role of T-cadherin in the skin is the negative regulation of keratinocyte proliferation, and inactivation of T-cadherin is, conversely, the cause for keratinocyte hyperproliferation. In this study, to obtain an insight into the role of T-cadherin in keratinocytes, we used transfection methods and examined the effect of overexpression or knockdown of T-cadherin in immortalized keratinocyte cell lines derived from SCC. In T-cadherin overexpressing cells, we clearly observed reduced cell proliferation, whereas the influence of cell–cell adhesiveness and cell mobility was not detected. Using a tetracycline (Tet)-regulated expression system, we also confirmed that the suppression of cell proliferation was dependent on the expression level of T-cadherin. Cell cycle analysis demonstrated that overexpression of T-cadherin induced a delay in the G2/M phase. We determined the expression level of T-cadherin on native HSC-1 cells and the transfectants, the HSC-1 RNAI cell line (T-cadherin expression is downregulated by the RNAi method), the HSC-1 TCAD cell line (overexpression of T-cadherin, which is driven by the cytomegalovirus promoter), and the HSC-1 TCAD RNAI cell line (overexpressed T-cadherin is downregulated by the RNAi method), with western blotting (Figure 1a). The results demonstrated that a weak band was detected from native HSC-1 cells and no signal was observed from HSC-1 RNAI (Figure 1a). HSC-1 TCAD showed two thick bands, which were considered to be mature protein (lower band) and precursor (upper band) of T-cadherin. These two bands were thinner in HSC-1 TCAD RNAI, which possessed the characteristic that the overexpressed T-cadherin was downregulated by the RNAi method. The expression levels of E-and P-cadherin were not changed by overexpression or knockdown of T-cadherin (Figure 1a). Similar results were obtained from immunofluorescence staining. HSC-1 cells showed a weak signal, no signal was detected from HSC-1 RNAI, a stronger signal than the native HSC-1 cells was detected in HSC-1 TCAD, and a weaker signal than HSC-1 TCAD was observed in HSC-1 TCAD RNAI (Figure 1b). In HSC-1 cells, it was also demonstrated that T-cadherin was diffusely distributed on both cell–cell boundaries and the cell surface (Figure 1b1), whereas E-and P-cadherin were concentrated mainly at the cell–cell boundaries (Figure 1b5,6). These observations were similar to their transfectants (data not shown). To analyze the effect of T-cadherin on cutaneous squamous carcinoma cell proliferation, we counted the native HSC-1 cells, HSC-1 RNAI, HSC-1 TCAD, and HSC-1 TCAD RNAI, which expressed different levels of T-cadherin, and compared the difference in proliferation activity. HSC-1 cells proliferated well and after 96 h the cell number increased 13.8 times (Figure 2a). On the other hand, the T-cadherin overexpressing cell line, HSC-1 TCAD, only proliferated up to 5.6 times (Figure 2a). HSC-1 TCAD RNAI restored the proliferation ability of HSC-1 TCAD to 11.1 times (Figure 2a). To know the effect of T-cadherin in native HSC-1 cells, we compared the proliferation curve of native HSC-1 cells and HSC-1 RNAI. But the proliferation curve of HSC-1 RNAI was almost the same as that of native HSC-1 cells (Figure 2a). To confirm the difference in the proliferation between native HSC-1 and T-cadherin overexpressing HSC-1 TCAD, other proliferation assays were performed. Both the WST-1 cell proliferation assay and BrdU incorporation assay revealed that overexpression of T-cadherin led to the suppression of cell proliferation (Figure 2b and Figure 3c). To analyze the relationship between cell proliferation and the T-cadherin expression level, we used Tet-inducible T-cadherin overexpressing cells. HSC-1 Trex TCAD showed three bands, which consisted of the precursor (upper), mature protein (middle), and a degradation product (lower) of T-cadherin, and these bands were upregulated by Tet in a concentration-dependent manner (Figure 3a). Although the addition of Tet to native HSC-1 cells did not affect cell proliferation, its addition to HSC-1 Trex TCAD suppressed cell proliferation concentration-dependently, which means the expression level of T-cadherin negatively regulates cell proliferation in HSC-1 cells. Similar results were obtained using a different cutaneous squamous carcinoma cell line, DJM-1, except that only mature T-cadherin was detected in DJM-1 Trex TCAD (Figure 3c,d). The difference between HSC-1 Trex TCAD and DJM-1 Trex TCAD was probably due to the expression efficacy of T-cadherin. To explore the mechanism of T-cadherin-mediated downregulation of proliferation, cell cycle analysis was performed. Both cell lines, native HSC-1 cells and HSC-1 TCAD, showed G1/G0 (2N) synchronization by serum starvation and a similar pattern of the cell cycle within 12 h of serum restimulation (Figure 4). Although HSC-1 cells showed a continuous reduction in the number of cells in the G2/M (4N) phase until 12–24 h after serum restimulation, a larger number of HSC-1 TCAD were still retained in the G2/M phase at 24 h after serum restimulation (Figure 4). These results clearly showed that overexpression of T-cadherin affects the progress of the cell cycle at the point of G2 or M phase, and consequently delayed the cell cycle. To investigate the effect of T-cadherin on cell–cell adhesiveness or cell mobility in cutaneous squamous carcinoma cells, we used HSC-1, HSC-1 RNAI, and HSC-1 TCAD, which expressed a different amount of T-cadherin, and performed a cell aggregation assay and cell migration assay. HSC-1 TCAD tended to show a slightly higher aggregation index than native HSC-1, but the difference was not significant. HSC-1 RNAI exhibited almost the same rates as native HSC-1 (Figure 5a). In the cell mobility assay, there was no significant difference in the migration rates between native HSC-1 and HSC-1 TCAD. The aim of this study was to obtain an insight into the role of T-cadherin in keratinocytes. We examined the effect of overexpression or knockdown of T-cadherin in immortalized keratinocyte cell lines derived from SCC, and the results clearly demonstrate that T-cadherin negatively regulates the proliferation of cutaneous squamous carcinoma cells. We previously reported the loss of T-cadherin expression in SCC (Takeuchi et al., 2002Takeuchi T. Liang S.B. Matsuyoshi N. Zhou S. Miyachi Y. Sonobe H. Ohtsuki Y. Loss of T-cadherin (CDH13, H-cadherin) expression in cutaneous squamous cell carcinoma.Lab Invest. 2002; 82: 1023-1029Crossref PubMed Scopus (57) Google Scholar) and hypothesized that T-cadherin was one of the tumor suppressor factors. Similar results were obtained from breast, lung, ovarian, and bladder carcinomas (Lee, 1996Lee S.W. H-cadherin, a novel cadherin with growth inhibitory functions and diminished expression in human breast cancer.Nat Med. 1996; 2: 776-782Crossref PubMed Scopus (177) Google Scholar; Kawakami et al., 1999Kawakami M. Staub J. Cliby W. Hartmann L. Smith D.I. Shridhar V. Involvement of H-cadherin (CDH13) on 16q in the region of frequent deletion in ovarian cancer.Int J Oncol. 1999; 15: 715-720PubMed Google Scholar; Maruyama et al., 2001Maruyama R. Toyooka S. Toyooka K.O. et al.Aberrant promoter methylation profile of bladder cancer and its relationship to clinicopathological features.Cancer Res. 2001; 61: 8659-8663PubMed Google Scholar; Toyooka et al., 2001Toyooka K.O. Toyooka S. Virmani A.K. et al.Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas.Cancer Res. 2001; 61: 4556-4560PubMed Google Scholar). Other studies showed that T-cadherin suppressed proliferation of glioma cells and neuroblastoma cells (Takeuchi et al., 2000Takeuchi T. Misaki A. Liang S.B. Tachibana A. Hayashi N. Sonobe H. Ohtsuki Y. Expression of T-cadherin (CDH13, H-Cadherin) in human brain and its characteristics as a negative growth regulator of epidermal growth factor in neuroblastoma cells.J Neurochem. 2000; 74: 1489-1497Crossref PubMed Scopus (148) Google Scholar; Huang et al., 2003Huang Z.Y. Wu Y. Hedrick N. Gutmann D.H. T-cadherin-mediated cell growth regulation involves G2 phase arrest and requires p21CIP1/WAF1 expression.Mol Cell Biol. 2003; 23: 566-578Crossref PubMed Scopus (77) Google Scholar), but the function of T-cadherin in cutaneous tissue was still unclear. In the previous study, we also demonstrated that the expression of T-cadherin was significantly downregulated in the lesional skin of psoriasis vulgaris (Zhou et al., 2003Zhou S. Matsuyoshi N. Takeuchi T. Ohtsuki Y. Miyachi Y. Reciprocal altered expression of T-cadherin and P-cadherin in psoriasis vulgaris.Br J Dermatol. 2003; 149: 268-273Crossref PubMed Scopus (21) Google Scholar), which does not exhibit a malignant phenotype, and is considered to be a T lymphocyte-mediated keratinocyte hyperproliferative disease. Further, in normal skin tissue, T-cadherin was specifically localized in the basal layer, where keratinocyte proliferation takes place (Zhou et al., 2002Zhou S. Matsuyoshi N. Liang S.B. Takeuchi T. Ohtsuki Y. Miyachi Y. Expression of T-cadherin in Basal keratinocytes of skin.J Invest Dermatol. 2002; 118: 1080-1084Crossref PubMed Scopus (28) Google Scholar). Here, we showed that T-cadherin negatively regulated the cell proliferation of at least two kinds of immortalized keratinocyte cell lines, HSC-1 and DJM-1, in accordance with the T-cadherin expression level. This finding suggests that T-cadherin acts as an endogenous negative regulator of keratinocyte proliferation at the basal layer of the epidermis. The epidermis is a constantly self-renewing tissue and balancing the cell loss from the cornified layer and the cell division in the basal layer is essential for the maintenance of skin structure. This regulation is thought to be achieved by growth control at the basal layer cell including two different keratinocyte subpopulations: the stem cells and transient amplifying cells (TAC) (Lavker and Sun, 2000Lavker R.M. Sun T.T. Epidermal stem cells: Properties, markers, and location.Proc Natl Acad Sci USA. 2000; 97: 13473-13475Crossref PubMed Scopus (342) Google Scholar; Watt, 2001Watt F.M. Stem cell fate and patterning in mammalian epidermis.Curr Opin Genet Dev. 2001; 11: 410-417Crossref PubMed Scopus (201) Google Scholar). The relation between T-cadherin and the stem cell or TAC is a very interesting issue that remains to be elucidated. The next question is how T-cadherin suppresses the cell proliferation of cutaneous squamous carcinoma cells.Huang et al., 2003Huang Z.Y. Wu Y. Hedrick N. Gutmann D.H. T-cadherin-mediated cell growth regulation involves G2 phase arrest and requires p21CIP1/WAF1 expression.Mol Cell Biol. 2003; 23: 566-578Crossref PubMed Scopus (77) Google Scholar reported that T-cadherin overexpressing glioma C6 cells resulted in p21CIP1/WAF1-mediated G2 phase arrest and aneuploidy (>4N) in cells. They also showed that T-cadherin overexpressing cells exhibited significantly increased numbers of cells in the G2/M phase after synchronization by serum starvation, compared with the vector-transfected cells that were synchronized in the G1/G0 phase by serum starvation. In our studies, T-cadherin overexpressing HSC-1 cells were able to be induced into the G1/G0 phase by serum starvation, similar to native HSC-1 cells. After serum restimulation, T-cadherin overexpressing cells resulted in a delay but not in an arrest at the G2/M phase and there were no aneuploid cells (>4N). Although the precise mechanism of T-cadherin-mediated suppression of proliferation is still unknown, it may depend on the difference of cell type. Additionally, we confirmed that overexpression of T-cadherin induced no apoptosis in HSC-1 cells (data not shown). In the epidermis, E- and P-cadherin are the major components of intercellular adherence junctions (Nose and Takeichi, 1986Nose A. Takeichi M. A novel cadherin cell adhesion molecule: Its expression patterns associated with implantation and organogenesis of mouse embryos.J Cell Biol. 1986; 103: 2649-2658Crossref PubMed Scopus (345) Google Scholar), and are involved in morphogenesis and maintaining the structure of skin (Hirai et al., 1989Hirai Y. Nose A. Kobayashi S. Takeichi M. Expression and role of E- and P-cadherin adhesion molecules in embryonic histogenesis. II. Skin morphogenesis.Development. 1989; 105: 271-277PubMed Google Scholar). It is reported that the expressions of E- and P-cadherin are reduced in SCC, melanoma, and Paget's disease and reduction of cadherin expression is considered to be related to the detachment of carcinoma cells from the tumor mass (Shirahama et al., 1996Shirahama S. Furukawa F. Wakita H. Takigawa M. E- and P-cadherin expression in tumor tissues and soluble E-cadherin levels in sera of patients with skin cancer.J Dermatol Sci. 1996; 13: 30-36Abstract Full Text PDF PubMed Scopus (34) Google Scholar; Furukawa et al., 1997Furukawa F. Fujii K. Horiguchi Y. et al.Roles of E- and P-cadherin in the human skin.Microsc Res Technol. 1997; 38: 343-352Crossref PubMed Scopus (67) Google Scholar). The contribution of T-cadherin to keratinocyte cell–cell adhesion, however, is not yet known.Vestal and Ranscht, 1992Vestal D.J. Ranscht B. Glycosyl phosphatidylinositol-anchored T-cadherin mediates calcium-dependent, homophilic cell adhesion.J Cell Biol. 1992; 119: 451-461Crossref PubMed Scopus (132) Google Scholar showed, using T-cadherin cDNA-transfected CHO cells, that T-cadherin induced calcium-dependent, homophilic adhesion, like E- or P-cadherin. In our present data from the cell aggregation assay, overexpression of T-cadherin tended to show slightly increased cell–cell adhesiveness, but the difference was not significant. In addition, T-cadherin was detected at both the cell–cell borders and the cell surface, whereas E- and P-cadherin were concentrated mainly at the cell–cell borders. These results indicate that the homophilic binding activity of T-cadherin is relatively weaker than that of E- or P-cadherin. This phenomenon may be explained by the specific structure of T-cadherin, which lacks the cytoplasmic domain and His–Ala–Val motif. So, we concluded that, in the epidermis, the major function of E- and P-cadherin is cell–cell adhesion, and that of T-cadherin must be signal transduction or cell recognition, rather than cell–cell adhesion. In conclusion, this study has provided new information on the functional significance of T-cadherin in cutaneous squamous carcinoma cells. T-cadherin negatively regulates the proliferation of cutaneous squamous carcinoma cells, and downregulation of T-cadherin may be involved in the pathogenesis of SCC and psoriasis vulgaris. Further studies on the expression and regulation of T-cadherin in hyperproliferative skin diseases will provide insights into not only the pathogenesis but also the development of novel therapeutic targets. Human cutaneous squamous carcinoma cell lines, HSC-1 (Kondo and Aso, 1981Kondo S. Aso K. Establishment of a cell line of human skin squamous cell carcinoma in vitro.Br J Dermatol. 1981; 105: 125-132Crossref PubMed Scopus (50) Google Scholar) and DJM-1 (Kitajima et al., 1987Kitajima Y. Inoue S. Yaoita H. Effects of pemphigus antibody on the regeneration of cell–cell contact in keratinocyte cultures grown in low to normal Ca++ concentration.J Invest Dermatol. 1987; 89: 167-171Abstract Full Text PDF PubMed Google Scholar), were used in this study. The cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 10 ng per mL streptomycin, and 100 units per mL penicillin G at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were trypsinized and subcultured when they were approaching subconfluency. The number of cells was determined using a Coulter Counter (Beckman Coulter, Fullerton, California). To assess the cell proliferation, 2 × 103 cells per cm2 were seeded in DMEM containing 1% FBS onto culture dishes for direct cell counting or 96-well plates for a WST-1 cell proliferation assay and BrdU incorporation assay. WST-1, which is one of the tetrazolium salts, was used to determine cell proliferation according to the procedure provided by the manufacturer (Takara bio, Shiga, Japan). The BrdU incorporation assay was performed using a Cell Proliferation Biotrack ELISA System, ver2 (Amersham Bioscience, Piscataway, New Jersey) according to the protocol provided with the kit. The primary antibodies used in this study included the rat anti-T-cadherin monoclonal antibody, TCD-1, generated in our laboratory as outlined below. Rats were immunized by injecting purified glutathione S-transferase (GST) fusion protein containing a human T-cadherin extracellular domain (residues 210–350) to the footpad. After the immunization, lymph node cells from the rats were fused with myeloma cells P3U1 (Matsuyoshi et al., 1997Matsuyoshi N. Toda K. Horiguchi Y. Tanaka T. Nakagawa S. Takeichi M. Imamura S. In vivo evidence of the critical role of cadherin-5 in murine vascular integrity.Proc Assoc Am Physicians. 1997; 109: 362-371PubMed Google Scholar). Screening was carried out by ELISA, and a hybridoma clone, designated TCD-1, was established. Its culture supernatant was used in the following experiments. The mouse anti-human E-cadherin monoclonal antibody HECD-1 and mouse anti-human P-cadherin NCC-CAD-299 were also used (Shimoyama et al., 1989Shimoyama Y. Hirohashi S. Hirano S. Noguchi M. Shimosato Y. Takeichi M. Abe O. Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas.Cancer Res. 1989; 49: 2128-2133PubMed Google Scholar). HRP-conjugated goat anti-rat IgG (DakoCytomation, Glostrup, Denmark), HRP-conjugated goat anti-mouse IgG (DakoCytomation), Cy3-conjugated goat anti-rat IgG (CHEMICON International, Temecula, California), and Cy3-conjugated goat anti-mouse IgG (CHEMICON International) were used as secondary antibodies. Total RNA from HSC-1 cells was purified using ISOGEN (Nippon Gene, Tokyo, Japan), and cDNA was synthesized by reverse transcriptase Super Scripts II (Invitrogen, Carlsbad, California) using oligo dT primers. PCR was carried out to obtain full-length cDNA of human T-cadherin using the forward primer, 5′-cgg gcg ctt cta gtc gga caa aat gca gcc-3′, and reverse primer, 5′-agt caa gct tca gac gtc agg agt tct cac-3′, and the product was subsequently cloned to the mammalian expression vector pIRES neo2 (BD Biosciences Clontech, Palo Alto, California). To construct the siRNA-human T-cadherin, the sense template, 5′-gat ccc ggt agt cga tag tga cag gtt caa gag acc tgt cac tat cga cta cct ttt ttg gaa a-3′ (italic: BamHI cohesive end, bold: sense strand, underline: antisense strand); and antisense template, 5′-agc ttt tcc aaa aaa ggt agt cga tag tga cag gtc tct tga acc tgt cac tat cga cta ccg g-3′ (italic: HindIII cohesive end) were annealed and ligated into a pSilencer 3.1-H1 hygro siRNA expression vector (Ambion, Austin, Texas). The pIRESneo2-human T-cadherin was digested with EcoRV and NotI restriction endonucleases and the insert was ligated to the appropriate sites of pcDNA 5/TO (Invitrogen), Tet-regulated expression vector. All plasmid constructs were confirmed by sequencing using the ABI PRISM BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, California). The cells were transfected with various plasmid constructs using lipofectamine plus reagent (Invitrogen). HSC-1 cells were transfected with human T-cadherin-pIRESneo2 and selected with 250 μg per mL G418. Single cell clones were analyzed for T-cadherin expression using western blotting, and HSC-1 TCAD expressing the highest level of T-cadherin was established. HSC-1 cells and HSC-1 TCAD cells were transfected with human T-cadherin-siRNA-pSilencer. Cells were selected for 250 μg per mL hygromycin and screened with western blotting. HSC-1 RNAI and HSC-1 TCAD RNAI with the lowest level of T-cadherin expression were established. HSC-1 cells and DJM-1 cells were co-transfected with human T-cadherin-pcDNA5/TO and pcDNA6/TR (Invitrogen) that express the Tet repressor. Cells were selected with 250 μg per mL hygromycin and 5 μg per mL blasticidin double antibiotic resistance, and cultured in the presence or absence of 1 μg per mL Tet for 48 h. Cell lysates were analyzed with western blotting for T-cadherin expression; HSC-1 Trex TCAD and DJM-1 Trex TCAD with Tet-inducible T-cadherin expression were established. Confluent cells were maintained for 48 h with serum free medium for G0 synchronization. Then, the cells were trypsinized, cultured in DMEM containing 1% FBS for 6, 12, 18, 24 h, and collected. The DNA content was determined by a CycleTEST PLUS DNA Reagent Kit (Beckton Dickinson, San Jose, California), using the propidium iodide staining method, according to the instructions provided by the manufacturer. Assays were performed with a flow cytometer (Epics XL-MCL, Beckman Coulter) and analyzed with EXPO32 software. Cadherin-mediated cell aggregation was assayed as described (Matsuyoshi et al., 1992Matsuyoshi N. Hamaguchi M. Taniguchi S. Nagafuchi A. Tsukita S. Takeichi M. Cadherin-mediated cell–cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts.J Cell Biol. 1992; 118: 703-714Crossref PubMed Scopus (448) Google Scholar). Briefly, cells were treated with 0.01% trypsin in the presence of 1 mM CaCl2 at 37°C for 20 min, and then washed with Ca2+- and Mg2+-free Hepes-buffered (pH 7.4) HBSS (HMF) to obtain single-cell suspensions. 5 × 104 cells in 0.5 mL HMF with 1 mM CaCl2 were incubated in albumin-coated 24-well plates on a rotary shaker (80 r.p.m.) at 37°C for 20 min. The degree of cell aggregation was represented by the aggregation index (N0-N20)/N0, where N0 is the total cell number per well and N20 is the total particle number per well after 20 min of incubation. Cover slips were coated with albumin and treated with ethanol. After air drying, these cover slips were further coated with colloidal gold particles as described (Albrecht-Buehler and Goldman, 1976Albrecht-Buehler G. Goldman R.D. Microspike-mediated particle transport towards the cell body during early spreading of 3T3 cells.Exp Cell Res. 1976; 97: 329-339Crossref PubMed Scopus (44) Google Scholar). 1 × 103 cells were seeded on these cover slips placed into six-well plates and cultured for 24 h. Pictures of the migrated cells were taken and the areas where the cells had removed the colloidal gold particles were measured with Adobe Photoshop software (Adobe Systems, San Jose, California). The cells cultured on chamber slides were fixed with PLP (10 mM sodium periodate, 75 mM lysine, and 2.14 mg per mL paraformaldehyde) for 10 min at room temperature, followed by blocking with 5% skim milk in 0.01 M pH 7.4 phosphate-buffered saline (PBS) for 30 min at room temperature. The chamber slides were incubated with primary antibody for 60 min at room temperature. After being washed with PBS three times, cells were incubated with Cy3-conjugated anti-rat or anti-mouse secondary antibody diluted with 5% skim milk in PBS for 60 min. The slides were washed again with PBS three times, and mounted with glycerol. The slides were viewed on a Nikon microphot-SA fluorescence microscope (Nikon, Tokyo, Japan). Cells were lysed with 2% SDS lysis buffer containing 5%-mercaptoethanol, and boiled for 5 min. Protein concentrations were measured by means of the Bio-Rad DC protein assay (BIO-RAD, Hercules, California). Samples were separated by SDS-PAGE, followed by transfer to a nitrocellulose membrane sheet. The membranes were blocked with 5% skim milk in PBS. After incubation with the primary antibody, the membrane was washed with PBS for 5 min, three times, and then incubated with the HRP-conjugated secondary antibody. Signals were detected by using the ECL system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Statistical analyses were performed using Student's t test and the Kruskal–Wallis rank test with Scheffe's multiple comparison test or Dunnett's multiple comparison test. Significance was defined as a value of p<0.05. We are grateful to Professor S. Kondo for kindly providing us with the human cutaneous squamous carcinoma cell line, HSC-1. We also would like to thank Dr S. Hirohashi and Professor M. Takeichi for the gifts of the mouse anti-human E-cadherin antibody, HECD-1, and the anti-human P-cadherin antibody, NCC-CAD-299.
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