Reexpression of fetal characters in simian virus 40-transformed human keratinocytes.

Normal human keratinocytes are able to stratify, form cornified squames, and terminally differentiate in tissue culture. These properties are frequently impaired by malignant transformation. In the present paper, we show that, in addition, transformation by SV40 results in the coordinate reexpression of a whole set of fetal characters. Moreover, a comparison of two SV40-transformed human keratinocyte cell lines, one still showing a certain degree of stratification and terminal differentiation (HE-SV) and the other almost completely unable to differentiate (SVK14), suggests that the impairment of differentiation and the intensity of reexpression of fetal markers are correlated. Particularly, a set of three keratin polypeptides, absent in adult stratified epithelia but normally found in the nonstratified fetal epidermis, is present in much larger amounts in SVK14 cells than in HE-SV cells. On the other hand, the inability of SVK14 cells, in contrast to HE-SV cells, to form cornified envelopes seems to be due to the inability of those cells to accumulate involucrin.

Mesenchymal-epithelial conversions induced by 5-azacytidine: appearance of cytokeratin Endo-A messenger RNA.

When mouse-teratocarcinoma-derived fibroblasts (1246 cell line) are subjected to treatment with the inhibitor of DNA methylation, 5-Azacytidine (5 AzaC), they transiently express at 55-kilodalton intermediate-filament protein recognized by the epithelial-specific monoclonal antibody, TROMA-1, although they retain a fibroblastic morphology. However, rare clones (e.g., the 1339 cell line) that permanently express the antigen recognized by TROMA-1 can be derived from the 5 AzaC-treated 1246 population, and these clones have an epithelial phenotype. In the present study, we used cloned DNA probes to demonstrate that, in 1246 fibroblasts, 5 AzaC induces the appearance of Endo-A mRNA. High levels of Endo-A mRNA were also detected in the epithelial derivative, cell line 1339. In both cases, the capping site of the Endo-A mRNA was found to be the same as that in epithelial cells which normally express this RNA.

Sequence of a cDNA encoding human keratin No 10 selected according to structural homologies of keratins and their tissue-specific expression.

We present here the nucleotide sequence of a 1700 bp-long cDNA encoding human epidermal keratin No. 10 (56.5 kDa). cDNA clones of the acidic keratin family were first isolated from a pBR322 human epidermal cDNA library by hybridization with a probe coding for keratin No. 14. Differential hybridization using total cDNA probes prepared from poly(A)+ RNA extracted either from epidermis (which contains keratin No. 10) and from squamous carcinoma or hepatoma cell lines (which do not express keratin No. 10) made possible the selection of clones potentially coding for keratin No. 10. The 1.7 kb sequence exhibits the characteristic features of an acidic keratin with a constant central rod domain and C-terminal variable structures. Moreover, the sequence shows extensive homologies with the cDNA of murine keratin No. 10.

Retinoic acid receptor isoform RAR gamma 1: an antagonist of the transactivation of the RAR beta RARE in epithelial cell lines and normal human keratinocytes.

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR beta RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR beta RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR alpha 1 and RAR beta 2 isoforms, but not by RAR gamam 1. On the contrary, this latter isoform behaved towards the RAR beta RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR alpha 1 and RAR beta 2. RAR gamma 1 also behaved as an antagonist of the transactivation produced by cotransfected RXR alpha. The natural RAR beta gene promoter or RAR beta RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR gamma 1. It was, however, possible to activate to a certain extent RAR beta RARE-reporter constructs in NHK by co-transfecting RAR alpha 1, RAR beta 2 or RXR alpha. The antagonist behavior of RAR gamma 1 towards the RAR beta RARE may explain why in certain cell types such as keratinocytes, RAR beta is neither expressed nor induced by RA.

Sequence analysis of murine cytokeratin endo A (no. 8) cDNA. Evidence for mRNA species initiated upstream of the normal 5' end in PCC4 cells.

Keratin 8 (Endo A) is expressed in simple epithelia, together with keratin 18 (Endo B). Filaments formed by this keratin pair are the first cytoskeletal elements induced during mouse embryogenesis. We have isolated Endo A cDNA clones from lambda gt11 libraries prepared with mRNA isolated from PCC4 embryonal carcinoma (EC) cells. Sequencing of three overlapping cDNAs and of a genomic clone allowed us to determine the complete sequence of the Endo A message. Analysis of the protein sequence deduced showed that the Endo A protein presented all the characteristics of intermediate filaments, including an alpha-helical central rod domain and nonhelical N- and C-termini. In the rod domain, the degree of similarity to the other members of the basic keratin family was high. A high degree of homology to keratin 8 of other species was observed, even in the non-helical domains. During these analyses, we found clones extending upstream of the normal 5' end of the mRNA. Sequence comparison between these cDNAs and the 5' upstream region of the Endo A gene suggested that they corresponded to transcripts initiated at an upstream alternative promoter. These observations supported previous results showing the presence of Endo A transcripts initiated upstream of the normal 5' end in mouse morulae and blastocysts.

A keratin of fetal skin is reexpressed in human keratinocytes transformed by SV40 virus or treated with the tumor promoter TPA.

SV40-transformation as well as treatment with tumor promoters produce alterations in morphology, differentiation and keratinization of human keratinocytes. Two cell lines of SV40-transformed keratinocytes and primary cultures of keratinocytes treated with the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) were found to contain an additional protein of 52.5 kD molecular weight (MW). This protein was identified by its reactivity with the monoclonal antibody TROMA-I as being keratin no. 8, a keratin normally present only in simple epithelia. Since this keratin is present in fetal epidermis but disappears gradually when fetal skin becomes multilayered after week 13 of development (Moll et al., Differentiation 23 (1982) 170. [23]), it suggests that SV40 virus and TPA are able to induce in human keratinocytes the reexpression of fetal characters.