H Green

Colony-forming human epidermal cells are heterogeneous in their capacity for sustained growth. Once a clone has been derived from a single cell, its growth potential can be estimated from the colony types resulting from a single plating, and the clone can be assigned to one of three classes. The holoclone has the greatest reproductive capacity: under standard conditions, fewer than 5% of the colonies formed by the cells of a holoclone abort and terminally differentiate. The paraclone contains exclusively cells with a short replicative lifespan (not more than 15 cell generations), after which they uniformly abort and terminally differentiate. The third type of clone, the meroclone, contains a mixture of cells of different growth potential and is a transitional stage between the holoclone and the paraclone. The incidence of the different clonal types is affected by aging, since cells originating from the epidermis of older donors give rise to a lower proportion of holoclones and a higher proportion of paraclones.

Different stratified squamous epithelia, whether they bear a stratum corneum or not, are shown by immunofluorescence to possess the precursor protein of the cross-linked envelope that is characteristic of epidermal s. corneum. This protein, involucrin, is not present in the deepest epithelial cells but appears in the course of their outward migration. The boundary at which involucrin first appears can sometimes by correlated with a visible boundary between zones of large and small cells. Cultured keratinocytes, derived from all stratified squamous epithelia (epidermal, corneal, conjuctival, esophageal, lingual, and vaginal), form colonies that grow together to form a stratified epithelium. The cells of the basal layer are nearly always free of detectable involucrin, but, in contrast to the natural epithelium, this protein usually makes its appearance in the cells immediately above the basal layer. When a cultured epithelium derived from epidermal keratinocytes is detached and applied as a graft to animals, the cells flatten and the distinctness of the basal layer is at first reduced; but with time the organization of the epithelium becomes more characteristic of epidermis. Cell size and shape become more orderly along the cell migration pathway, and involucrin first appears at some distance from the basal layer, instead of in immediately suprabasal cells, as in the cultured epithelium. The progeny of dissociated and cultured keratinocytes are therefore able, when grafted, to reassemble an epidermis in which the timing of specific gene expression is restored to that of the original tissue.

Late in terminal differentiation, human epidermal keratinocytes form an insoluble protein envelope on the cytoplasmic side of the plasma membrane. Involucrin, a soluble protein precursor of the envelope, is synthesized at an earlier stage of differentiation, both in the natural epithelium and in cultured keratinocytes. Because keratinocytes are known to enlarge during differentiation, we looked for a correlation between involucrin synthesis and cell size, using antiserum raised against the purified protein. We found that virtually no cultured epidermal keratinocytes with a diameter less than or equal to 14 micrometer contained involucrin, but most cells greater than 17 micrometer did. Using density gradient centrifugation, we were able to isolate a population of small cells containing almost no involucrin, as judged by immunodiffusion, PAGE, and immunoprecipitation. Large cells possessed translatable mRNA for involucrin, whereas small cells did not. We conclude that when cultured keratinocytes reach a certain size (approximately 14 micrometer in diameter) the specific mRNA for involucrin begins to accumulate and synthesis of the protein begins.

When serially cultivated human epidermal keratinocytes are placed in suspension culture they stop growing and form, beneath the plasma membrane, an insoluble envelope consisting of protein cross-linked by ε- (γ-glutamyl)lysine. The formation of envelopes in suspended cells is preceded by a sharp decline in the rate of protein synthesis, and most envelopes appear only after the average rate of protein synthesis has fallen to a very low level. If protein synthesis is reduced over 98 percent with cycloheximide or emetine at the time that surface-grown cells are placed in suspension culture, cross-linked envelopes form in most of the cells. This shows that the precursor of the envelope and the cross-linking enzyme are already in the cytoplasm in most cells of growing surface cultures. The process of envelope formation by suspension cultures is actually accelerated by the inhibitors of protein synthesis; an increased number of cells with cross-linked envelopes is observable within 4-6 h after the addition of cycloheximide. The inhibitor also induces a large fraction of the cells of surface cultures to form enveloped within a few days. These findings suggest that arrest of protein synthesis leads to activation of the cross-linking process. Agents known to inhibit transglutaminase-mediated protein cross-linking-putrescine, iodoacetamide, and ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA)- also prevent envelope formation. Though the activity of the cross-linking transglutaminase depends on the presence of cellular Ca++, we have not been able to activate the cross-linking process by high external Ca++ concentration or ionophores.