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We previously isolated the 5' upstream sequences of the mouse P-cadherin gene, in which putative binding sites for several transcription factors were identified between nt-101 and +30. In the study reported here, the promoter activity of the postulated 5' cis-acting sequences of the P-cadherin promoter, and the activity of the proximal E-cadherin promoter were investigated in several murine keratinocyte cell lines showing different levels of P- and E-cadherin expression as well as different morphology and tumorigenic behavior. Cell-type specificity and optimal activity of P-cadherin expression in murine keratinocytes was conferred by 5' sequences located between nt -200 and +30, and the GC-rich region (nt -101 to +80) and a CCAAT box element (nt -65) had a major regulatory role. The cell-type specificity of the E-cadherin promoter, on the other hand, was mediated by a combination of positive regulatory elements, a GC-rich region (nt -58 to -24), and a CCAAT box (nt -65) and repressor elements inside the E-pal sequence. Interestingly, the maximum repressor effect of the E-pal element was observed in non-expressing undifferentiated spindle cells. In vitro binding studies indicated that the GC-rich region of the P-cadherin promoter was mainly recognized by Sp1-related nuclear factors, whereas both AP2- and Sp1-related factors were involved in the interaction of the GC-rich region of the E-cadherin promoter. Common factors (probably related to the CP1 family) seemed also to be involved in the recognition of the CCAAT-box element of both the E- and P-cadherin promoters, but additional specific factors participated in the interaction with the CCAAT box of the E-cadherin promoter. Our studies also support the hypothesis that loss or modification of some of the regulatory factors occurs during mouse skin tumor progression.

The HaCa4 cell line, derived from a mouse skin carcinoma induced by Harvey murine sarcoma virus, is highly tumorigenic when injected into nude mice and produces multiple metastases in the lungs. HaCa4 cells express high levels of viral Ha-ras oncogene products, anomalously synthesize the embryonic/simple epithelial keratin K8, and have lost the expression of the cell-cell adhesion receptor E-cadherin (E-CD). E-CD(+) cell clones (E62 and E24), obtained by transfection of an exogenous E-CD cDNA into HaCa4 cells, had a decreased ability to migrate through type IV collagen matrices. However, the E-CD (+) E62 clone remained as metastatic as the parental cell line, whereas the E24 clone, which does not take up the exogenous cDNA but spontaneously switches on the endogenous E-CD gene, suppressed the metastatic phenotype although it maintained its tumorigenicity. E24 cells had fivefold to sixfold lower levels of viral Ha-ras mRNA and p21 protein than the other cell lines. In addition, they did not synthesize K8 but rather switched on keratin K19. The comparison of E-CD proteins synthesized by E62 and E24 cell lines revealed no structural or functional differences because both localized at cell-cell contacts and associated with alpha-catenin, beta-catenin, and plakoglobin. Furthermore, E-CD was still expressed in metastatic lung nodules produced by E62 cells. These results suggest that suppression of the metastatic phenotype in E24 cells occurs independently of E-CD expression and correlates with decreased levels of the oncogenic ras p21 protein.

The influence of plakoglobin on the phenotype and tumorigenicity of murine spindle carcinoma cells was analyzed by stable transfection of plakoglobin cDNA in the presence or absence of E-cadherin expression. In either situation, overexpression of plakoglobin was unable to modify the fibroblastic phenotype or to completely suppress the tumorigenic behavior of the spindle cells, but a moderate reduction in the growth rate of the tumors was induced by plakoglobin and was further enhanced by E-cadherin. Coexpression of E-cadherin and plakoglobin induced a mutual stabilization, increasing the half-life of both molecules in the double transfectants more than 5- and 30-fold, respectively, with a turnover rate similar to that observed in control keratinocytes. The stabilization of E-cadherin, as well as that of plakoglobin, was maintained in the tumors induced by the double transfectants, in contrast to the unstable expression of E-cadherin observed in tumors induced in plakoglobin-deficient cells. The E-cadherin/catenin complexes present in the double transfectants were functional in calcium- dependent aggregation assays and similar in composition to those of control keratinocytes. However, most of the components of the complexes of the transfectants were solubilized by non-ionic detergents, indicating a weak interaction with the actin cytoskeleton. These results indicated that restoration of E-cadherin/catenin complexes was not sufficient to induce the transition of the fibroblastic cells to an epithelial phenotype or to completely suppress the tumorigenicity of mouse skin spindle carcinoma cells., This work was supported by grants from the Spanish Comisión Interministerial de Ciencia y Tecnología (SAF92-0146 and SAF95-0818) to AC. During the conduct of this study, EL was a recipient of a predoctoral fellowship from the Spanish Ministry of Education and Science.

The adhesive properties and the expression of extracellular matrix receptors of the β1-integrin subfamily were analyzed in transformed epidermal keratinocyte cell lines of different stages of mouse skin carcinogenesis. One- and two-dimensional analyses of the immunoprecipitates obtained with anti-β1- and specific anti-α-integrin subunits showed qualitative and quantitative changes in the expression of β1 integrins by the different cell lines. The polyvalent α3β1 integrin was expressed by all analyzed cell lines, although the levels detected in undifferentiated spindle CarC cells were lower than those present in the rest of keratinocyte cell lines. In contrast, spindle cells expressed high levels of the specific fibronectin receptor α5β1, whereas this integrin was absent or expressed at very reduced levels in the other epithelial cell lines. Expression of α5β1 integrin in spindle cells appeared organized in cell-substratum contact areas on spread cells. In addition, high and homogenous expression of α5β1 was detected in fully undifferentiated tumors induced in nude mice by three independent spindle cell lines. These results suggest that the expression of α5β1 integrin is upregulated during the development of spindle cell carcinomas that occur in the last stages of mouse skin carcinogenesis and can be associated with the acquisition of the fibroblastoid phenotype of spindle cells. On the other hand, expression of the collagen receptor α2β1 was demonstrated in a transformed cell line (PDV), and it was apparently also expressed in two other malignant keratinocyte cell lines (PDVC57 and HaCa4). The expression of α2β1 was correlated with the increased adhesion to collagen type I and collagen type IV exhibited by the tumorigenic cell lines., Funded by: Comunidad Autónoma de Madrid (CAM). Grant Number: (C075/91) and Comisión Interministerial de Ciencia y Tecnología. Grant Number: (SAF 92-0146).