Research Article: H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

Date Published: November 19, 2009

Publisher: Public Library of Science

Author(s): Melville B. Vaughan, Ruben D. Ramirez, Capri M. Andrews, Woodring E. Wright, Jerry W. Shay, Mikhail V. Blagosklonny.

Abstract: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.

Partial Text: Two major types of skin cancer, squamous cell carcinoma and basal cell carcinoma, affect the primary skin epidermal cell type termed the keratinocyte. While basal cell carcinomas locally invade, the ability of squamous cell carcinomas to migrate and spread to other areas of the body is of great concern. One of the hallmarks of metastatic carcinoma cells is the epithelial to mesenchymal transition (EMT), characterized by changes in the cytoskeleton and cell-to-cell connections [1]–[2] including decreased E-cadherin and increased vimentin expression. These protein changes primarily affect the integrity of the stratified epithelial cells’ attachment to each other. However, extracellular changes will disrupt the entire architecture. Inappropriate proliferation and extracellular matrix degradation produce a tissue mosaic instead of a compartmentalized, stratified epithelium. While it appears that there are many cellular changes necessary to produce a skin carcinoma, at least two major pathways are likely to be involved [3]. One such pathway involves Ras activation signaling [4]; specifically, H-Ras has been shown to affect proliferation [5] and matrix degradation [6]–[7]. While it remains unknown if a partial or complete EMT program underlies the invasive/metastatic phenotype of all high-grade human tumors, cell-based assays such as those presented here may permit addressing this central question.

The goal of this study was to determine whether H-Ras expression in immortalized keratinocytes affected the normal stratification and differentiation of keratinocytes placed in organotypic 3D culture. First, we demonstrated that cdk4 and hTERT immortalized keratinocytes terminally differentiated normally and by all morphological and biochemical criteria were similar to unimmortalized keratinocytes used in skin equivalents. This provided a stable keratinocyte cell line reagent for additional cancer progression experiments. Ras expression in combination with cdk4 and hTERT was sufficient to disorganize the lower strata of the developing epidermis. This was apparent by the presence of involucrin-staining cells surrounded by keratin-14-staining cells.



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