Properties of carcinogen altered mouse epidermal cells resistant to calcium-induced terminal differentiation

Abstract

Eight cell lines exhibiting resistance to Ca2+ induced terminal differentiation were derived from primary mouse epidermal cultures and their properties analyzed. The lines developed either spontaneously (2 lines) or after exposure of primary cultures to carcinogens or carcinogens and tumor promoter. All but one of the lines were of epithelial or epitheloid morphology but 3 of the 8 lines lacked desmosomes, keratin filaments and immunoprecipitable keratin proteins, and thus could not be defined as keratinocytes. Two of the 5 keratinocyte lines were tumorigenic in syngeneic Balb/c newborns after 4 months in medium containing 1.2 mM Ca2+, and 3 lines remained non-tumorigenic even after 11 months in 1.2 mM Ca2+. All three of the non-keratinizing lines were tumorigenic. Tumorigenic potential of the 5 keratinocyte lines did not correlate with ploidy (as determined by DNA content), transglutaminase activity or growth in soft agar. However, the 2 tumorigenic keratinocyte lines contained cells which stained intensely red for gamma glutamyl transpeptidase activity, while the non-tumorigenic keratinocyte lines did not. Only those lines lacking desmosomes and keratin filaments grew in soft agar, but these lines were negative for gamma glutamyl transpeptidase activity. Ploidy and transglutaminase activity did not correlate with tumorigenicity in these non-keratinizing lines. These results show that cell lines derived from cultured mouse epidermal cells and selected on the basis of their resistance to Ca2+ induced terminal differentiation may be preneoplastic. Furthermore the association of additional markers with malignant change in these cell lines depended on whether or not the cells were keratinizing.

Ectopic Expression of PA2.26 Antigen in Epidermal Keratinocytes Leads to Destabilization of Adherens Junctions and Malignant Progression

“…To analyze the biological function of PA2.26 in epidermal cells, we transfected the mouse PA2.26 cDNA in MCA3D keratinocytes, which do not express the antigen. MCA3D cells were derived from primary epidermal cultures treated with the carcinogen 7,12-dimethylbenz(a)anthracene and selected on the basis of their resistance to Ca 2ϩ -induced terminal differentiation (Kulesz-Martin et al, 1983). MCA3D keratinocytes have normal Ras, p53, p15 INK4B , and p16 INK4A genes and are nontumorigenic upon injection in nude mice (Burns et al, 1991;Linardopoulos et al, 1995;Quintanilla et al, 1991).…”

“…MCA3D keratinocytes have normal Ras, p53, p15 INK4B , and p16 INK4A genes and are nontumorigenic upon injection in nude mice (Burns et al, 1991;Linardopoulos et al, 1995;Quintanilla et al, 1991). However, they have a tetraploid content of DNA and have been found to spontaneously develop tumors in skin graft experiments (Kulesz-Martin et al, 1983;Strickland et al, 1988). Therefore, MCA3D can be considered a premalignant cell line.…”

The activation and composition of FiRE (an FGF-inducible response element) differ in a cell type- and growth factor-specific manner

“…However, additional phosphorylation of the AP-1 complex also by FGF-2 in ®broblasts can not be ruled out. Interestingly, the MCA3D keratinocytes which do not di erentiate upon calcium or growth factor treatment (Kulesz-Martin et al, 1983), did not show binding of other Jun-family members than JunD. JunD has been shown to be constitutively expressed in di erentiating keratinocytes while e.g.…”

“…Since syndecan-1 is strongly induced in keratinocytes in vivo (Elenius et al, 1991), we studied whether the syndecan-1 expression could be regulated by growth factors in these cells. We used a keratinocyte cell line MCA3D (Kulesz-Martin et al, 1983) and stimulated the cells with EGF, KGF or FGF-2, growth factors known to stimulate biological responses in keratinocytes. The MCA3D cells were grown in absence of serum for two days and treated with growth factors for 5 h. A Northern blot was performed followed by quanti®cation of the syndecan-1 RNA levels by scanning and correlation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) loading control.…”

Extracellular Matrix-dependent Activation of Syndecan-1 Expression in Keratinocyte Growth Factor-treated Keratinocytes

“…Cell Culture and Growth Factor Treatments-MCA-3D is a keratinocyte cell line derived from carcinogen-treated mouse skin (24). It is not, however, tumorigenic in nude mice (17).…”