Basement membrane proteins promote progression of intraepithelial neoplasia in 3-dimensional models of human stratified epithelium

We have developed novel 3-dimensional in vitro and in vivo tissue models that mimic premalignant disease of human stratified epithelium in order to analyze the stromal contribution of extracellular matrix and basement membrane proteins to the progression of intraepithelial neoplasia. Three-dimensional, organotypic cultures were grown either on a de-epidermalized human dermis with pre-existing basement membrane components on its surface (AlloDerm), on a Type I collagen gel that lacked basement membrane proteins or on polycarbonate membranes coated with purified extracellular matrix proteins. When tumor cells (HaCaT-II4) were mixed with normal keratinocytes (4:1/normals:HaCaT-II4), tumor cells selectively attached, persisted and proliferated at the dermal-epidermal interface in vitro and generated dysplastic tissues when transplanted to nude mice only when grown in the presence of the AlloDerm substrate. This stromal interface was permissive for tumor cell attachment due to the rapid assembly of structured basement membrane. When tumor cells were mixed with normal keratinocytes and grown on polycarbonate membranes coated with individual extracellular matrix or basement membrane components, selective attachment and significant intraepithelial expansion occurred only on laminin 1 and Type IV collagen-coated membranes. This preferential adhesion of tumor cells restricted the synthesis of laminin 5 to basal cells where it was deposited in a polarized distribution. Western blot analysis revealed that tumor cell attachment was not due to differences in the synthesis or processing of laminin 5. Thus, intraepithelial progression towards premalignant disease is dependent on the selective adhesion of cells with malignant potential to basement membrane proteins that provide a permissive template for their persistence and expansion.     

Association of deficient DNA repair during G2 phase with progression from benign to malignant state in a line of human skin keratinocytes transfected with ras oncogene

Human skin keratinocytes after malignant neoplastic transformationby infection with Kirsten murine sarcoma virus (KiMSV) or transfectionwith pSV² ras (containing an activated c-Ha-ras oncogene) showeda DNA repair deficiency(ies). The repair deficiency was manifestas an abnormally high frequency of chromatid breaks and gapspersisting after X-ray-induced DNA damage inflicted during theG₂ phase of the cell cycle. Non-tumorigenic control cells atthat time were clearly repair-efficient. By analyzing benignand malignant tumorigenic HaCaT-ras clones, we could excluderas p21 oncoprotein expression as the causal mechanism for repairdeficiency, since both clone types expressed similar levelsof the mutated protein and only the malignant tumorigenlc cellsshowed repair deficiency. The results suggest that mutated p21ras provided the human keratinocytes with a growth advantagein vivo (benign tumor growth), but acquisition of repair deficiencyis required for progression from benign to malignant state.     

Expression and function of connexin in normal and transformed human keratinocytes in culture

We have studied the gap junctional intercellular communication(GJIC) of immortalized and tumourigenic human keratinocyte celllines and of a spontaneously immortalized non-tumourigenic anda highly differentiating keratinocyte cell line (HaCaT) as thecontrol. In homologous cultures, the GJIC capacity of five squamouscell carcinoma-derived cell lines was 1–27% that of theHaCaT cells. Ha-ras-transfected HaCaT cells with tumourigenicpotential and an SV40 DNA-immortalized cell line had markedlyreduced GJIC capacities. Northern analysis and immunohisto-chemistryshowed that connexin (Cx) 43 is the major gap junction proteinexpressed in the communicating cells. They do not express Cx26 or 32. The low or absent communication observed in certaincell lines was due in some to a lack of Cx 43 gene expression,but in others to aberrant localization of the gap junction protein.GJIC of these cell lines, as well as that of primary normalhuman epidermal keratinocytes, was susceptible to 12-O-tetra-decanoylphorbol-13-acetate-mediatedinhibition. Moreover, GJIC of HaCaT cells and their tumourigenicderivatives is Ca²⁺-dependent. These results, when comparedwith those previously obtained for mouse keratinocyte cell lines,reveal that GJIC of human keratinocytes was correlated to thedegree of differentiation and is controlled in a similar wayto that of murine keratinocytes. Aberrant GJIC seems to be acommon feature of human and murine skin carcinogenesis.