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New born mouse epidermal cells proliferate when cultured in 0.5 mM Ca++ medium and terminally differentiate when the Ca++ is increased to about 1.2 mM, the level found in most cell culture media. We found that hyaluronic acid and proteoglycans were synthesized by isolated cultured newborn mouse epidermal cells and that quantitative and qualitative changes in these macromolecules appeared when proliferating epidermal cultures were induced to differentiate by calcium. A major change that occurred with differentiation was a reduction in synthesis of hyaluronic acid while synthesis of proteoglycans and glycoproteins increased. The proteoglycans synthesized in these cultures were heparan sulfate-proteoglycan (90%) and chondroitin sulfate-proteoglycan (10%), regardless of the calcium level. [ABSTRACT FROM AUTHOR]

Studies performed on mouse skin have indicated that chemical carcinogenesis can be subdivided into two distinct stages, initiation and promotion. Initiation results from exposure to a classical mutagenic carcinogen and is irreversible even after a single exposure. The permanently altered initiated cell and its progeny may never form a tumor or in any way be recognizable in the target tissue, Exposure to tumor promoters permits the expression of the neoplastic change in initiated cells, and tumors develop. In contrast to initiators, promoters must be given repeatedly to be effective; individual exposures are reversible, A similar biology is suggested by epidemiologic studies of certain human cancers, particularly lung, breast, colon, and uterine malignancies. Studies in mouse skin cell culture have provided new insights into the changes associated with initiation and promotion. Initiated cells appear to be resistant to signals for terminal differentiation and can proliferate under conditions where normal epidermal cells are obligated to cease proliferation and begin their maturation program. This change is essential for an epithelial tumor cell since it provides the ability to grow away from a basement- membrane attachment site, In cultured epidermal cells, tumor promoters are capable of selectively stimulating the growth of certain cells, including initiated cells, while simultaneously inducing terminal differentiation in other epidermal cells, The net effect of these responses to promoters is the clonal expansion of cells stimulated to proliferate. In this way, promoters are capable of increasing the clone size of initiated cells. These cell culture data provided a biological framework for under- standing initiation and promotion in terminally differentiating epithelial tissues. [ABSTRACT FROM AUTHOR]

Skin tumors chemically induced in mice have provided an important experimental model for studying carcinogenesis and for bioassaying carcinogenic agents. The information obtained from this model suggests that the events leading to tumor formation can be divided into at least two stages, initiation and promotion. A single small. dose of carcinogen produces initiation which appears to be irreversible. These initiating agents may have to be metabolically activated and can interact with cellular macromolecules. The extent to which they bind to DNA correlates well with their carcinogenicity. Increased DNA replication at the time of or during the first day after these agents have been applied appears to enhance carcinogenesis. Unlike initiation, promotion appears to be reversible and the promoting agents must be applied repeatedly before tumors are formed. Promoters interact with membranes, stimulate and alter genetic expression, and increase the rate of cell proliferation. The knowledge gained from these studies in mouse skin has immeasurably helped the entire field of chemical carcinogenesis. But efforts to determine the cellular and molecular mechanisms involved in the carcinogenic process, particularly in the skin, have been hampered by the difficulties of working on whole animals and by the special problems associated with the biologic and biochemical methods required for this target organ. Such problems, however, can be solved by the use of cell cultures of mouse epidermis which can metabolize and bind carcinogens just as is done in vivo. The fact that epidermal cells in vitro proliferate synchronously should facilitate the study of the relation between the cell cycle and carcinogenesis. These cells repair chemically induced DNA damage by at least two mechanisms, excision repair and base-specific repair. [ABSTRACT FROM AUTHOR]

Mixed cultures of epidermal and dermal cells from term fetuses of Balb/cAn mice were exposed to high concentrations (50 μg/ml) of 7. 12-dimethylbenz(a)anthracene (DMBA) in medium containing Tween-80 or to medium with Tween-80 alone for 45 min. Within 5 weeks the cultures exposed to DMBA began to exhibit accelerated growth in vitro and an epithelioid morphology. These same changes occurred in the Tween-80-treated group starting around 15 weeks in culture. Both sets of cultures remained epithelioid in appearance and rapidly growing for over 9 months. Injections of cells into syngeneic hosts, beginning approximately 21 weeks after treatment. gave rise to undifferentiated tumors. Animals receiving carcinogen-treated cells had more tumors than those receiving vehicle-treated cells. Ultrastructural studies of the transformed cells in vitro suggested that they were of keratinocyte origin, but DMBA + Tween-80-treated cells were more differentiated than cells treated with Tween-80 alone. 80-100 Å cytofilaments, presumably identical to tonofilaments in vivo, were organized into bundles which traversed both the cellular endoplasm and ectoplasm, either terminating on adherens-like junctional structures, or looping back into the cytoplasm after attaching peripherally to such structures. After transplantation into syngeneic hosts, transformed cells from both groups produced tumors which ultrastructurally resembled anaplastic squamous-cell carcinomas. When highly undifferentiated tumors derived from DMBA + Tween-80-treated cells were recultured, more differentiated features reappeared. This observation indicates an important role for the cellular millieu in the determination of phenotypic expression. [ABSTRACT FROM AUTHOR]

During the final stage of epidermal differentiation, activation of keratinocyte transglutaminase results in covalent cross-linking of a variety of proteins to form highly protective cornified cell envelopes. We have studied the regulation of keratinocyte transglutaminase (TGK) gene expression in murine epidermal keratinocytes induced to terminally differentiate in vitro by increasing the level of extracellular Ca++ or treatment with the protein kinase C (PKC) activator 12- O-tetradecanoylphorbol-13-acetate (TPA). Raising extracellular Ca++ induces squamous differentiation of cultured keratinocytes and elicits a concentration-dependent increase in expression of TGK mRNA; keratinocytes grown for 24 h in 0.12 mM Ca++ medium express ∼12 times as much TGK mRNA as basal cells (grown in 0.05 mM Ca++ medium), whereas cultures exposed to 1.4 mM Ca++ express ∼17 times as much. TPA induces squamous differentiation and TGK mRNA even in basal keratinocyte cultures grown in 0.05 mM Ca++ medium, suggesting that expression of this differentiation marker is regulated by the PKC signaling pathway. Induction of TGK mRNA in response to TPA treatment is transient, reaching a peak at 6-8 h and returning to baseline by 24 h. In contrast, elevation of TGK mRNA levels in response to Ca++ persists for at least 24 h. The increased abundance of TGK mRNA reflects increased transcription of the TGK gene, based on nuclear run-on analysis of Ca++- and TPA-treated keratinocytes. Induction of TGK mRNA by either TPA or Ca++ is blocked in the presence of cycloheximide, suggesting that a PKC-dependent protein factor is required for TGK gene expression in response to both stimuli. Furthermore, the accumulation of TGK mRNA in keratinocytes treated with TPA or CA++ is blocked in cells treated with the PKC inhibitor GF 109203X or bryostatin. These results suggest that the induction of TGK gene expression by CA++ is dependent on PKC, providing further support for the hypothesis that PKC plays a central role in regulating the late stages of epidermal differentiation. [ABSTRACT FROM AUTHOR]

During embryonic development presumptive hair follicle cells of epithelial and mesenchymal origin are determined in defined body locations. This is followed by rapid proliferation of epithelial cells and associated penetration into the dermis in response to as yet undetermined signals. A collagen matrix culture system, which maintains the three-dimensional relationships of hair follicle cells to each other, was developed to study the regulation of the enlargement of immature hair follicles and the accompanying remodeling of the dermis. In studies with a heterogeneous dermis-derived preparation of murine hair follicles, ranging in size from the earliest down-growing budding cell mass to hair-forming follicles, we had previously shown that cell proliferation was stimulated by cholera toxin and epidermal growth factor, but only the epidermal growth factor-stimulated proliferation was accompanied by digestion of the collagen matrix due to release of collagenolytic enzymes. Further studies revealed that transforming growth factor-α also stimulated hair follicle cell proliferation and collagenase release. However, although transforming growth factor-β inhibited the transforming growth factor-α-stimulated proliferation, it enhanced the release and activation of collagenases and other gelatin-degrading enzymes detectable by gelatin zymography. Stimulation of collagenolytic activity depended on the three-dimensional hair follicle structure and did not occur in monolayer cultures of hair follicle cells. Comparison of hair follicle buds with more developed dermis-derived hair follicles, plated at the same cell density (based on DNA content), suggested that a greater fraction of cells in the bud-stage follicle responded to the growth factors by release of collagenases. Possibly only the cells in the advancing portion of growing hair follicles that are closest to the dermal papilla cell cluster produce the collagenases in response to growth factors. To examine the participation of dermal papilla cells in collagenase release and activation, several immortalized rat whisker dermal papilla cell liners were co-cultured with mouse hair follicle buds. Co-culture resulted in a marked enlargement of follicles as well as activation of the 92-kDa type IV collagenase, produced by hair follicle buds, that correlated with ability of the dermal papilla cells to stimulate hair formation in grafts of hair follicle buds on nude mice. Dermal papilla cells cultured alone produced the 72-kDa type IV collagenase, which was also activated during co-culture with hair follicle buds. Thus, two activities, both relevant for hair follicle development, namely, cell proliferation and release and activation of collagenases, have been stimulated in immature hair follicle buds by either growth-factor supplementation or interaction with dermal papilla cells. The collagen matrix culture system allows investigation of the mechanism by which these activities are controlled under defined in vitro conditions. [ABSTRACT FROM AUTHOR]

A method for the monolayer culture of fetal mouse skin cells and the behavior of primary cultures have been described. The early cultures consist of discrete colonies of epidermoid cells with individual fibroblasts scattered between these foci. The cell colonies begin to produce a material which may be keratin by the third in vitro day. After one week in vitro the majority of epidermoid foci have degenerated and the cultures consist largely of elongated rapidly multiplying cells. When cells from cultures in vitro five days or less are transplanted back to the panniculus carnosus of F1 hosts, donor skin grows. This skin is hyperplastic but contains normal appendages and produces hair. These grafts have remained recognizable and stable for over four months. [ABSTRACT FROM AUTHOR]

Mouse keratinocytes can he grown at clonal densities in dermal fibroblast conditioned medium with a calcium concentration of 0.02 MM. Colony forming efficiencies of approximately 1-3% can be achieved with primary and secondary cultures and up to 6% with selected subclones. A substrate of frozen-thawed dermal fibroblasts enhances colony formation over plastic. Colony size increases more rapidly in the presence of epidermal growth factor in conditioned medium, Conditioning of medium by fibroblasts is optimum after day 6 of culture and conditioned medium can be stored at -20°C for at least 4 mo. [ABSTRACT FROM AUTHOR]