Cultures Of Normal Keratinocytes Research Articles

Monoclonal antibodies specific for subsets of epidermal keratins: biochemical and immunocytochemical characterization--applications in pathology and cell culture.

Keratin composition has been widely used as a biochemical marker of differentiation in normal epithelia, cell culture systems and tumours of epithelial tissues. We have been developing a model system for the study of human squamous epithelial cell differentiation, and among a panel of monoclonal antibodies we have generated for analysing this system are two antibodies recognizing subsets of epidermal keratins. The two antibodies, designated LICR-LON-16a and LICR-LON-29b, were raised to the human squamous carcinoma cell line LICR-LON-HN-5, and we describe here their biochemical and immunocytochemical characterization. Antibody 16a reacts with only epidermal basal cells in normal human skin and shows specificity for the 45 and 46 kdalton keratins. Antibody 29b stains all living layers of the epidermis, and reacts with a broad range of ketain polypeptides, (45-56 kdaltons) in immunoblotting analyses. We have investigated the alterations of cellular staining that occur in chronic hyperproliferative skin diseases and carcinomas and compared this with the staining of multilayered cultures of normal keratinocytes and the HN-5 cell line. We show that in squamous cell carcinomas and in HN-5 cell xenografts 16a and 29b stain only the well-differentiated cell types. Furthermore we found that the basal cell specificity of 16a was lost in all of the hyperproliferative skin lesions examined including psoriasis and eczema. This transition to suprabasal staining pattern was also seen in the cultures of normal keratinocytes and HN-5 cells. We conclude that aberrant keratin synthesis or abnormal post-translational processing of keratins associated with an increased rate of cell turnover could account for the altered expression of the epitope recognized by antibody 16a.

Effect of polyamine antimetabolites on cultured human keratinocytes from normal and uninvolved psoriatic skin.

We describe the effect of two polyamine antimetabolites on polyamine and macromolecule synthesis of cultured human keratinocytes obtained by suction blisters from normal skin and the uninvolved skin of psoriatic patients. The concentrations of spermidine and spermine steadily increased during the culture of normal keratinocytes in vitro, whereas the putrescine concentration showed only a transient rise at the beginning of the active growth phase. Treatment with difluoromethylornithine decreased the concentrations of putrescine and spermidine in both normal and uninvolved psoriatic keratinocytes, but had no effect on either DNA or protein synthesis. Methylglyoxal bis(guanylhydrazone) marginally decreased the levels of spermidine and spermine and significantly inhibited the DNA and protein synthetic activities. Pretreatment of uninvolved psoriatic keratinocytes with difluoromethylornithine enhanced the accumulation of methylglyoxal bis(guanylhydrazone), resulting in a profound inhibition of cellular macromolecule synthesis. This synergistic effect was not seen in normal keratinocytes. Thus, although no statistically significant difference was observed between the cells derived from normal and uninvolved psoriatic epidermis, the psoriatic keratinocytes appeared to be more sensitive to the action of polyamine antimetabolites. The inhibition of DNA and protein synthesis by methylglyoxal bis(guanylhydrazone) was prevented by concomitant treatment with spermidine.

A subpopulation of cultured human keratinocytes which is resistant to the induction of terminal differentiation-related changes by phorbol, 12-myristate, 13-acetate: evidence for an increase in the resistant population following transformation.

We have studied the action of phorbol, 12-myristate, 13-acetate (PMA) on human keratinocytes grown with lethally-irradiated 3T3 cells using medium supplemented with hydrocortisone, cholera toxin and epidermal growth factor. Normal keratinocyte cultures show a heterogeneous response to PMA; 90-93% of the colony-forming cells lose their colony-forming ability and form cornified envelopes when treated for 24 h with doses of 100 nM or less, but the remainder are resistant to doses of 1000 nM. The resistant cells are the precursors of the sensitive ones and heterogeneity is restored to those cells and their progeny after 8 days culture in the absence of PMA. Cultures of 3 squamous cell carcinoma lines, a SV40-transformed human keratinocyte line, and three clones of these lines were found to contain 3-17 times more PMA-resistant keratinocytes than the normal strains, and the size of the PMA-resistant fraction in each line was inversely related to the competence of that line to lose colony-forming efficiency when placed in suspension culture (which is the first detectable change in an ordered programme of events resembling terminal differentiation of the keratinocyte). The number of cells with cornified envelopes in surface cultures of normal human keratinocytes increased from approximately 3% in control cultures to approximately 70% in those treated for 6 days with 100 nM PMA. The transformed human keratinocyte cultures showed a 3-25-fold smaller increase in cornified envelope formation when treated with 100 nM PMA, and the increase in envelope formation by each line when exposed to this dose of PMA was related to the competence of that line to lose cloning efficiency in suspension culture. No relationship was found between the ability of any human keratinocyte strain or line we studied to metabolically inactivate PMA and their resulting response to the compound. The results are discussed in relation to the mechanism of action of PMA as a promoter of epidermal carcinogenesis.