Henry Hennings

Calcium regulation of growth and differentiation of mouse epidermal cells in culture

Modification of the ionic calcium concentration in the culture medium markedly alters the pattern of proliferation and differentiation in cultured mouse epidermal cells. When medium calcium is lowered to 0.05--0.1 mM, keratinocytes proliferate rapidly with a high growth fraction and do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured and cloned in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces, abundant microvilli and perinuclear organization of tonofilaments and cellular organelles. Desmosomes are absent. Epidermal cells growing as a monolayer in low Ca++ can be induced to terminally differentiate by adding calcium to the level normally found in the culture medium (1.2 mM). Cell-to-cell contact occurs rapidly and desmosomes form within 2 hr. The cells stratify by 1--2 days and terminally differentiate with cell sloughing by 3--4 days. After Ca++ addition, DNA synthesis decreases with a lag of 5--10 hr and is totally inhibited within 34 hr. In contrast, RNA and protein synthesis continue at 40--50% of the low Ca++ level at day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch.

Calcium regulation of cell-cell contact and differentiation of epidermal cells in culture. An ultrastructural study.

Calcium modulation of keratinocyte growth in culture was studied by both transmission (TEM) and scanning electron microscopy (SEM). Under standard culture conditions (1.2-1.8 mM calcium), cells were connected by desmosomes and stratified to 4-6 cell layers. Many aspects of in vitro epidermal maturation were analogous to the in vivo process, with formation of keratohyalin granules, loss of nuclei, formation of cornified envelopes and shedding of cornified cells containing keratin filaments. When the medium calcium concentration was lowered to 0.02-0.1 mM, the pattern of keratinocyte growth was strikingly changed. Cells grew as a monolayer with no desmosomal connections and proliferated rapidly, shedding largely non-cornified cells into the medium. Large bundles of keratin filaments were concentrated in the perinuclear cytoplasm. The elevation of extracellular calcium to 1.2 mM induced low calcium keratinocytes to stratify, keratinize and cornify in a manner analogous to that seen when plated in standard calcium medium. The earliest calcium-induced ultrastructural change was the asymmetric formation of desmosomes between adjacent cells. Desmosomal plaques with associated tonofilaments were observed 5 min after calcium addition; symmetric desmosomes were formed within 1-2 h. This system is presented as a useful model for the study of the regulation of desmosome assembly and disassembly.

Malignant conversion of mouse skin tumours is increased by tumour initiators and unaffected by tumour promoters.

Multi-stage carcinogenesis (initiation–promotion) was first demonstrated in mouse skin1,2. The first stage, initiation, is accomplished by a low dose of carcinogen that causes no tumours. Promotion by repeated treatment of initiated mice with certain non-carcinogenic hyperplastic agents results in the rapid production of numerous benign papillomas, a few of which progress to squamous cell carcinomas. Although this model system produces mostly benign tumours, many of the concepts concerning carcinogenesis in epithelial tissues have been derived from mouse skin studies. The permanent change in growth potential accomplished by tumour initiators is generally considered to be a mutagenic event3; cell selection and clonal expansion of initiated cells may be involved in promotion4. In initiation–promotion experiments, more than 90% of the squamous cell carcinomas develop from papillomas5,6, but the conversion rate is low. The factors necessary for this conversion of benign to malignant tumours have not been defined but tumour promoters have been assumed to be involved. However, we report here that the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is ineffective in the conversion of papillomas to carcinomas whereas three initiators, urethane, N-methyl-N′-nitro-N-nitrosoguanidine ((MNNG) and 4-nitroquinoline-N-oxide (4-NQO) are effective. This suggests that malignant conversion may result from a further genetic change in papilloma cells and that the ineffectiveness of TPA may be due to its inactivity as a mutagen.

Critical Aspects of Initiation, Promotion, and Progression in Multistage Epidermal Carcinogenesis

Abstract Carcinogenesis in mouse skin can be divided into three distinct stages: initiation, promotion, and progression (malignant conversion). Initiation, induced by a single exposure to a genotoxic carcinogen, can result from a mutation in a single critical gene (e.g., rasHa), apparently in only a few epidermal cells. The change is irreversible. Promotion, resulting in the development of numerous benign tumors (papillomas), is accomplished by the repeated application of a nonmutagenic tumor promoter. The effects of single applications of tumor promoters are reversible since papillomas do not develop after insufficient exposure of initiated skin to promoters or when the interval between individual promoter applications is increased sufficiently. The reversibility of promotion suggests an epigenetic mechanism. Promoter treatment provides an environment that allows the selective clonal expansion of foci of initiated cells. The conversion of squamous papillomas to carcinomas (termed progression or malignant conversion) occurs spontaneously at a low frequency. The rate of progression to malignancy can be significantly increased by treatment of papilloma-bearing mice with certain genotoxic agents. These progressor agents or converting agents are likely to act via a second genetic change in papillonias already bearing the initiating mutation. Progression in the skin is characterized by genetic changes that result in several distinct changes in the levels or activity of structural proteins, growth factors, and proteases. The mechanisms involved in progression are being studied in epidermal cell culture. In order to determine the in vivo phenotype of cultured cells, a grafting system was developed in which the cells were transferred from culture to a prepared skin bed in athymic mice. Introduction of an activated v-fos oncogene into initiated cells bearing an activated ras Ha gene produced cells with a carcinoma phenotype, of Initiation i.e., carcinomas formed when the cells were grafted as part of reconstituted skin. Grafted keratinocytes containing the ras Ha gene alone produced papillomas; with v-fos alone, normal skin formed when grafted. The ras Ha/fos carcinomas showed changes in differentiation markers characteristic of chemically induced carcinomas. A cell culture assay utilizing cells initiated by the introduction of an activated ras Ha oncogene was developed to study progression. After exposure of initiated cells to progressor agents under conditions in which the proliferation of the ras Ha-initiated cells was suppressed, proliferating foci developed, with a good correlation of activity in the assay with activity in the progression stage in vivo. The cell culture assay provides a quantitative model to study chemically induced neoplastic progression and may be useful to identify potential progressor agents.

Phorbol ester tumor promoters induce epidermal transglutaminase activity

Abstract Epidermal basal cells in culture have low levels of epidermal transglutaminase, the enzyme responsible for the formation of the cross-linked envelope in differentiated cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and other active (but not inactive) phorbol ester skin tumor promoters induce transglutaminase activity. Sloughing of differentiated cells accompanies the rise in transglutaminase activity. Phorbol esters do not affect transglutaminase activity when added directly to cell lysates. Corticosteroids have little influence on transglutaminase induction by phorbol esters. Retinoic acid induces transglutaminase activity, but activity does not further increase when basal cells are treated with both retinoic acid and 12-O-tetradecanoylphorbol-13-acetate.

Signal transduction for proliferation and differentiation in keratinocytes.

In mouse and human epidermis, the Ca2+ environment of the basal cell layer is substantially below serum Ca2+, while that of the granular cell layer is unusually high. Reduction of extracellular Ca2+ concentration (Cao) in the medium of keratinocyte cultures maintains a basal cell phenotype while serum Ca2+ concentrations induce terminal differentiation. Measurements of intracellular Ca2+ (Cai) by the use of Fura 2 and digital imaging technology reveal that Cai increases 10-20-fold in response to an increase in Cao and remains elevated. Concomitant with the rise in Cai is an increase in the metabolism of phosphatidylinositol (PI) to yield inositol phosphates and diacylglycerol. PI metabolism is also stimulated by calcium ionophores suggesting that a rise in Cai is directly responsible. The consequent increase in diacylglycerol and Cai would activate protein kinase C, an event known to trigger epidermal differentiation. Specific Cao and Cai determine the expression of individual markers of keratinocyte differentiation in vitro. These findings may account for the importance of the Ca2+ gradient for maintaining regulated growth and differentiation of the epidermis in vivo.

Calcium induction of transglutaminase and the formation of ɛ(γ-glutamyl)lysine cross-links in cultured mouse epidermal cells

Summary Terminal differentiation can be induced in cultured mouse epidermal cells by increasing the extracellular calcium concentration from 0.07 mM to 1.2 mM. An ultrastructural characteristic of this process is the formation of a cornified envelope in which membrane proteins are cross-linked by ɛ(γ-glutamyl) lysine bonds. This process is catalyzed by the calcium-requiring enzyme transglutaminase. Elevation of medium calcium levels increases both the activity of transglutaminase and the fraction of lysine residues involved in ɛ(γ-glutamyl)lysine cross-links.

Fluocinolone acetonide: a potent inhibitor of mouse skin tumor promotion and epidermal DNA synthesis.

Abstract The relationship between the inhibition of mouse skin tumor promotion and the inhibition of epidermal DNA synthesis by the steroidal anti-inflammatory agent, fluocinolone acetonide (FA), was investigated. Simultaneous doses of either 10, 1, or 0.1 μg of FA and phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), resulted in an almost complete inhibition of promotion, whereas 0.01 and 0.001 μg of FA resulted in inhibition rates of 82% and 15%, respectively. Likewise, simultaneous doses of 10 or 1 μg of fluclorolone acetonide (FCA) and TPA caused a nearly complete inhibition of promotion, whereas 0.1 μg of FCA decreased promotion by 62%. In general, as the dose of both steroids was increased, an increase in the tumor latency period was observed. With the exception of the borderline effect of 0.001 μg of FA, the above doses of FA inhibited epidermal DNA synthesis by at least 60% for a 24-h period. Topical treatment with 10 μg of FA resulted in an almost complete inhibition of DNA synthesis for 6 days. The administration of 10 μg of FA 24 h after TPA treatment brought about a maximal inhibition of DNA synthesis of 65%, as compared with a 98% inhibition in control mice whose DNA synthesis had not been prestimulated. That is, FA was not quite as effective on S-phase cells as on G-1 cells. There appears to be a relationship between the inhibition of tumor promotion and epidermal DNA synthesis.

Inducible Cutaneous Inflammation Reveals a Protumorigenic Role for Keratinocyte CXCR2 in Skin Carcinogenesis

Transgenic mice that overexpress PKCalpha in the epidermis (K5-PKCalpha mice) exhibit acute CXCR2-mediated intraepidermal neutrophilic inflammation and a strong epidermal hyperplasia in response to application of 12-O-tetradecanoylphorbol-13-acetate (TPA). We now show that hyperplasia is independent of infiltrating neutrophils. Furthermore, when K5-PKCalpha mice were initiated with 7,12-dimethylbenz(a)anthracene (DMBA) and promoted with a low dose of TPA, 58% of K5-PKCalpha mice developed skin papillomas that progressed to carcinoma, whereas wild-type mice did not develop tumors. We confirmed that CXCR2 is expressed by keratinocytes and showed that transformation by oncogenic ras (a hallmark of DMBA initiation) or TPA exposure induced all CXCR2 ligands. Ras induction of CXCR2 ligands was mediated by autocrine activation of epidermal growth factor receptor and nuclear factor-kappaB, and potentiated by PKCalpha. Oncogenic ras also induced CXCR2 ligands in keratinocytes genetically ablated for CXCR2. However, ras transformed CXCR2 null keratinocytes formed only small skin tumors in orthotopic skin grafts to CXCR2 intact hosts, whereas transformed wild-type keratinocytes produced large tumors. In vitro, CXCR2 was essential for CXCR2 ligand-stimulated migration of ras-transformed keratinocytes and for ligand activation of the extracellular signal-regulated kinase (ERK) and Akt pathways. Both migration and activation of ERK and Akt were restored by CXCR2 reconstitution of CXCR2 null keratinocytes. Thus, activation of CXCR2 on ras-transformed keratinocytes has both promigratory and protumorigenic functions. The up-regulation of CXCR2 ligands after initiation by oncogenic ras and promotion with TPA in the mouse skin model provides a mechanism to stimulate migration by both autocrine and paracrine pathways and contribute to tumor development.

Growth and Differentiation of Mouse Epidermal Cells in Culture: Effects of Extracellular Calcium

The pattern of proliferation and differentiation in cultured mouse epidermal cells in markedly altered by modifying the ionic calcium concentration in the culture medium. When medium calcium is lowered from 1.44 mM to 0.05-0.1 mM, keratinocytes proliferate rapidly with a high growth fraction, do not stratify, but continue to synthesize keratin. The cells grow as a monolayer for several months and can be subcultured in low Ca++ medium. Ultrastructural examination of cells cultured under low Ca++ conditions reveals widened intercellular spaces with an absence of desmosomes. Microvilli are numerous, and tonofilaments and cellular organelles are organized perinuclearly. Epidermal cells growing as a monolayer in low Ca++ can be identified to terminally differentiate by adding calcium to the level normally found in the culture medium. Contact between cells occurs rapidly and desmosomes form within 2 hours. The cells stratify in 1-2 days and terminally differentiate in 3-4 days. After Ca++ addition, DNA synthesis decreases after a lag of 5-10 hours and is totally inhibited within 36 hours. In contrast, RNA and protein synthesis continue at 40-50% of the control level at Day 3, a time when many cells are detaching from the culture dish. Keratin synthesis is unaffected by the Ca++ switch. Manipulation of epidermal proliferation and differentiation by altering extracellular calcium levels should enhance the usefulness of epidermal cell cultures in the study of differentiation and carcinogenesis.