Growth Of Epidermal Keratinocytes Research Articles

Low dose TGF-β1 can improve vohwinkel syndrome by promoting the proliferation of keratinocytes

Vohwinkel syndrome (VS) is a very rare autosomal dominant disorder that can cause disability and deformity in severe cases. Mutations of the LOR (loricrin) and GJB2 (Cx26) genes have been found in VS so far. Many studies have indicated that the differentiation and growth of epidermal keratinocytes are regulated by mutant Cx26, and it may explain the pathogenesis of VS. It has been found that transforming growth factor β1 (TGF-β1) expression was lower in G130V (OE1) and D66H (OE2) mutant keratinocytes in the VS model with GJB2 mutation as compared to normal keratinocytes (NC). TGF-β is a cytokine involved in the regulation of processes like cell proliferation and differentiation in different types of cells. At present, many in vitro studies focus on TGF- β 1 inhibition of keratinocyte growth.However, the relationship between TGF-β1 and VS remains unknown. This study aimed at elucidating the role and potential pathogenic mechanism of TGF-β in VS. The results indicated that the down-regulation expression of TGF-β1 in VS was linked to cell proliferation inhibition through p-Smad3/c-myc. In contrast, low-dose TGF-β1 treatment of VS keratinocytes can improve their proliferation inhibition and up-regulate the expression Cyclin D1. This suggests that low doses of TGF-β1 can improve the proliferation of VS and provide new insights into its treatment.

Abstract LB-091: Tumor suppressor WW domain-containing oxidoreductase controls epidermal keratinocyte growth

Abstract Human fragile WWOX gene encodes a candidate tumor suppressor WW domain-containing oxidoreductase. Ectopically overexpressed WWOX protein induces apoptosis in cancer cells and suppresses tumor growth both in vitro and in vivo. Functional suppression of WWOX by antisense mRNA, dominant negatives, and small interfering RNA protects cells from apoptosis induced by tumor necrosis factor, anticancer drugs, UV light, and ectopic p53 in vitro. Interestingly, substantial studies have revealed that progression of various cancers to a pre-metastatic stage positively correlates with upregulation of WWOX protein expression, suggesting that WWOX may act more than a tumor suppressor. In normal human epidermis, WWOX is abundant in proliferating epithelial cells of the skin, and its expression is increased in intensity toward the superficial layers. In this study, we determined a reduced number of Ki67-positive proliferating keratinocytes in the epidermis of our newly developed Wwox gene knockout (Wwox-/-) mice. A marked reduction in the basal (K5-positive) and suprabasal (K10-positive) keratinocyte numbers was examined in the keratinized squamous epithelium of the Wwox-/- mouse skin. Apparent decreases in Wwox-/- epidermal and dermal thickness were found. To further verify whether WWOX regulates keratinocyte growth, we detected significantly decreased cell proliferation in both Wwox-/- primary keratinocyte cultures and WWOX-knockdown HaCaT cells as compared to the control cells. No significant increases in the number of TUNEL-positive cells were observed in the Wwox-/- epidermal tissues. Taken together, our results clearly demonstrated a crucial role of tumor suppressor WWOX in regulating normal keratinocyte proliferation for maintaining skin homeostasis. Note: This abstract was not presented at the meeting. Citation Format: Li-Jin Hsu. Tumor suppressor WW domain-containing oxidoreductase controls epidermal keratinocyte growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-091.

An optimization protocol for Swiss 3T3 feeder cell growth-arrest by Mitomycin C dose-to-volume derivation strategy.

Feeder cell functionality following growth-arrest with the cost-effective Mitomycin C vis-à-vis irradiation is controversial due to several methodological variables reported. Earlier, we demonstrated variability in growth arrested Swiss 3T3 feeder cell life-span following titration of feeder cell densities with Mitomycin C concentrations which led to the derivation of doses per cell. Alternatively, to counter the unexpected feeder regrowth at high exposure cell density, we proposed titration of a fixed density with arithmetically derived volumes of Mitomycin C solution that corresponded to permutations of specific concentrations and doses per cell. We now describe an experimental procedure of inducing differential feeder cell growth-arrest by titrating with such volumes and validating the best feeder batch through target cell growth assessment. A safe cell density of Swiss 3T3 tested for the exclusion of Mitomycin C resistant variants was titrated with a range of volumes of a Mitomycin C solution. The differentially growth-arrested feeder batches generated were tested for short-term and long-term viability and human epidermal keratinocyte growth supporting ability. The feeder cell extinction rate was directly proportional to the volume of Mitomycin C solution within a given concentration per se. The keratinocyte colony forming efficiency and the overall growth in mass cultures were maximal with a median extinction rate produced by an intermediate volume, while the faster and slower extinction rates by high and low volumes, respectively, were suboptimal. The described method could counter the inadequacies of growth-arrest with Mitomycin C.

Occurrence and control of sporadic proliferation in growth arrested Swiss 3T3 feeder cells.

Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum.

Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis

AbstractGene therapy approaches involving vascular endothelial growth factor (VEGF) to promote therapeutic angiogenesis are under consideration for conditions ranging from ischemic heart disease to nonhealing skin ulcers. Here we make the surprising observation that the transgenic delivery of VEGF to the skin results in a profound inflammatory skin condition with many of the cellular and molecular features of psoriasis, including the characteristic vascular changes, epidermal alterations, and inflammatory infiltrates. Even longstanding psoriatic disease remains dependent on the transgenic VEGF in this model because it can be effectively reversed by the addition of VEGF Trap, a potent VEGF antagonist. Previous attempts to faithfully replicate the psoriatic phenotype through the transgenic delivery of epidermal keratinocyte growth factors or inflammatory mediators generated phenotypes with only partial resemblance to human psoriasis, leaving unanswered questions about the etiology of this disease. The ability of transgenic VEGF to induce a psoriasiform phenotype suggests a new etiology and treatment approach for this disease and further substantiates emerging concerns about possible proinflammatory adverse effects that might be associated with therapeutic attempts to deliver VEGF. (Blood. 2003;102:161-168)

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma.

Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.

1,25-Dihydroxyvitamin D 3, phospholipase D and protein kinase C in keratinocyte differentiation

1,25-Dihydroxyvitamin D 3, thought to be a physiological regulator of epidermal keratinocyte growth and differentiation, also elicits the complete differentiative program in vitro, with expression of various genes/proteins characteristic of both early and late differentiation. 1,25-Dihydroxyvitamin D 3 functions by interacting with an intracellular receptor that binds to DNA at vitamin D response elements (VDRE) thereby affecting transcription. 1,25-Dihydroxyvitamin D 3 has been demonstrated to alter the expression of several enzymes involved in signal transduction, and presumably this is the mechanism through which the hormone regulates differentiation. It has recently been shown that 1,25-dihydroxyvitamin D 3 specifically increases the expression/activity of phospholipase D-1, an enzyme that hydrolyzes phospholipids to generate lipid messengers, such as diacylglycerol (DAG). DAG, in turn, is known to activate several members of the protein kinase C (PKC) family. It has been proposed that this signaling pathway mediates late differentiation events in epidermal keratinocytes. In this article the data supporting a role for PKC and phospholipase D in keratinocyte differentiation, as well as in the pathogenesis of skin diseases, are reviewed and a model is proposed for the signaling pathways that regulate this process upon exposure to 1,25-dihydroxyvitamin D 3.

Accelerated proliferation of epidermal keratinocytes by the transgenic expression of the platelet-activating factor receptor.

Transgenic mice overexpressing platelet-activating factor receptor (PAFR) have abnormal pigmentation of the ear and the tail, which can progress to melanocytic tumors as the mice age. Histologically, epidermal hyperproliferation and increases in dermal melanocytes are evident. Examination of these transgenic mice at various ages revealed hyperproliferation of the epidermis even 2 weeks after birth which developed as the mice aged. Dermal melanocytes also increased in number with growth. Expression of the PAFR transgene was found in keratinocytes and not in melanocytes, thereby suggesting that PAF does not play a direct role in proliferation of melanocytes. Topical application of a cream containing WEB2086, a specific PAFR antagonist, to the ear and the dorsal skin significantly suppressed the number of BrdU-positive cells in PAFR transgenic mice. These results suggest that PAF plays a modulatory role in the growth of epidermal keratinocytes. PAFR transgenic mice would be a useful model for investigations of skin diseases related to altered proliferation of epidermal keratinocytes including psoriasis.

Sustained phospholipase D activation is associated with keratinocyte differentiation.

Our previous results and data in the literature have suggested a potential role for phospholipase D (PLD) in the regulation of epidermal keratinocyte growth and differentiation. Therefore, we investigated the effect of agents reported to modulate keratinocyte growth and differentiation on PLD activation. The purported protein kinase C (PKC) 'inhibitor', staurosporine (Stsp), has been reported to activate PKC in keratinocytes, eliciting many of the same effects as active tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). Stsp also induces a programmed pattern of differentiation similar to that seen in keratinocytes in vivo; TPA, on the other hand, appears to preferentially elicit markers consistent with late (granular) differentiation. In contrast, bradykinin is reported to stimulate keratinocyte proliferation. We found that these three agents had different effects on PLD activation in primary mouse epidermal keratinocytes. TPA increased PLD activity acutely and in a sustained fashion. In contrast, Stsp did not acutely activate PLD and inhibited acute TPA-induced activation of PLD. However, treatment of keratinocytes with Stsp for longer time periods (3-5 h) induced sustained PLD activation and this long-term effect was additive with that of TPA. Bradykinin activated PLD acutely but transiently. Both TPA and Stsp increased transglutaminase activity, a marker of late differentiation, whereas bradykinin had little or no effect on either cell proliferation or transglutaminase activity. These results suggest that a sustained activation of PLD is associated with the induction of keratinocyte differentiation. We hypothesize that PLD activity mediates late keratinocyte differentiation through generation of diacylglycerol and activation of specific PKC isoforms. Furthermore, we propose that the profound and immediate TPA-induced stimulation of PLD activity 'drives' the keratinocytes to late differentiation steps. However, the less efficacious (and more gradual) sustained activation of PLD by Stsp may allow a patterned differentiation more like that observed in skin.

EGF-Receptor Tyrosine Kinase Inhibition Induces Keratinocyte Growth Arrest and Terminal Differentiation

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.

Fas Signal Transduction Triggers Either Proliferation or Apoptosis in Human Fibroblasts

Although shown to be highly expressed by the epidermis in inflammatory skin disease, the ability of the Fas protein to trigger apoptosis in the distinct cell subpopulations of cutaneous tissue, particularly with regard to receptor density and the degree of crosslinking, has not been fully characterized. We therefore determined the effect of Fas cross-linking in primary human dermal fibroblasts at both high and low levels of Fas receptor expression. First, we examined the effects of the anti-Fas monoclonal antibody, CH-11, on fibroblasts expressing low basal levels of Fas. In these cells Fas aggregation stimulated proliferation by 160 +/- 10% over untreated controls. In contrast, the same concentration of CH-11 had an inhibitory effect on epidermal keratinocyte growth. Because Fas is upregulated in inflamed skin, we next examined the effects of Fas cross-linking on fibroblasts expressing augmented levels ofFas. Fibroblasts were either transfected with plasmids for overexpression of full length or bioengineered Fas receptors or were transduced with a retroviral Fas expression vector. In these cells Fas oligomerization triggered the morphologic changes indicative of apoptosis regardless of whether or not the Fas-signaling domain was tethered to the plasma membrane. These studies indicate that Fas oligomerization in dermal fibroblasts may initiate dual signaling programs, either proliferation or apoptosis, and that the chosen outcome may depend upon the magnitude of Fas aggregation.

Interferon-gamma-mediated inactivation of transcription of the 230-kDa bullous pemphigoid antigen gene (BPAG1) provides novel insight into keratinocyte differentiation.

Interferon-gamma (IFN-gamma) has been shown to regulate epidermal keratinocyte growth and differentiation. In this study, we examined the effects of recombinant human IFN-gamma on the expression of the gene encoding the 230-kDa bullous pemphigoid antigen (BPAG1), a marker of the mitotic basal cell phenotype in the epidermis. Northern analysis revealed a dose- and time-dependent suppression of BPAG1 expression by IFN-gamma in cultured human keratinocytes from several different donors, and incubation of the cells with IFN-gamma in the presence of cycloheximide demonstrated that this effect required ongoing protein synthesis. The inhibition of BPAG1 gene expression was also demonstrated at the protein level by indirect immunofluorescence using a monoclonal antibody recognizing the human 230-kDa bullous pemphigoid antigen. Transient transfections of cultured keratinocytes with BPAG1 promoter-chloramphenicol acetyltransferase reporter gene plasmids indicated marked suppression of the promoter activity by IFN-gamma, and deletion constructs were able to identify a defined region containing the responsive element (IFN-gamma inhibitory element). Reduced transcription of the BPAG1 gene by IFN-gamma was also demonstrated by in vitro nuclear run-on assays. These data, which indicate inactivation of transcription of a basal keratinocyte-specific gene of transcription of a basal keratinocyte-specific gene (BPAG1) by IFN-gamma, provide novel insight into the mechanisms of IFN-gamma-mediated keratinocyte gene regulation and epidermal differentiation in inflammatory diseases.

Regulation of fos-lacZ fusion gene expression in primary mouse epidermal keratinocytes isolated from transgenic mice.

The expression of a fos-lacZ fusion gene was studied in primary mouse epidermal keratinocytes obtained from transgenic mice. This gene construct contains the entire upstream regulatory sequence of c-fos, and expression of the endogenous and fusion gene was shown by Northern analysis to correlate upon induction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Using a chromogenic substrate of beta-galactosidase, we also demonstrated that expression of the fusion gene product, like that of Fos, was localized to the cell nucleus. In addition, we showed that epidermal keratinocytes responded to dialysed fetal bovine serum (FBS), TPA and high-calcium medium with enhanced Fos-lacZ expression and an inhibition of proliferation. The time course of induction of Fos-lacZ expression was similar for dialysed FBS and TPA, with a peak approximately 2 h after exposure. Exposure for approximately 24 h to an elevated extracellular calcium concentration was required to elicit an increase in Fos-lacZ expression. The lack of an immediate effect of raising medium calcium levels on Fos-lacZ expression contrasted with the rapidity of its effect on DNA synthesis, which was significantly inhibited within 6-8 h. In addition, we found that the protein kinase C inhibitor Ro 31-7549 blocked Fos-lacZ expression induced by TPA but had little or no effect on that elicited by high calcium levels. Thus, although our results indicate that the fos gene product may be involved in mediating epidermal keratinocyte growth arrest in response to differentiative agents such as FBS, TPA and high medium calcium levels, the exact role of this gene product remains unclear.

Cell cycle arrest induced by epidermal growth factor on human squamous cell carcinoma cell lines

Although human squamous cell carcinoma (SCC) cell lines frequently contain an elevated number of epidermal growth factor (EGF) receptor accompanied with amplification of EGF receptor/c-erbB gene, it is well known that EGF inhibits the growth of these cells in culture at doses that stimulate the growth of epidermal keratinocytes and dermal fibroblasts. To study this growth inhibitory effect of EGF on the SCC cell lines, 3H-thymidine incorporation into DNA and cell cycle distribution were analyzed. In HSC-1 and NA cells, which contain the highest number of EGF receptor among these SCC cell lines, the inhibition of 3H-thymidine incorporation was apparent 2 to 4 hours after treatment with 100 ng/ml of EGF and reached more than 95% inhibition after 24 hours. Two-color cell cycle analysis using fluorescein isothiocyanate (FITC)-conjugated anti-Bromodeoxyuridine (BrdU) antibody and propidium iodide revealed that this inhibitory effect was due to cell cycle arrest not only in G1 but also in G2 phase. This effect was well correlated to the sensitivity to the growth inhibitory effect of EGF among the 4 SCC cell lines. These observations suggest that the SCC cells contain altered machineries which regulate the normal cell growth in both G1 and G2 phases, and the EGF affects these machineries via overexpressed its receptor.

Expression of p53 protein in benign and malignant epidermal pathologic conditions

p53 is an oncogene and a tumor-suppressor gene whose gene product regulates the cell growth cycle. Mutations in p53 are the most common genetic alterations in human cancer. Our purpose was to define p53 expression and subcellular localization in normal and pathologic epidermis. We examined sections of normal skin, psoriasis, squamous cell carcinoma (SCC), basal cell carcinoma (BCC), keratoacanthoma, and cultured epithelial cell lines with five antibodies to p53. Monoclonal antibody MAb421 and MAb1801 stained the cytoplasm of normal basal keratinocytes, suggesting that subcellular localization or sequestration of wild-type p53 regulates its activity. Polyclonal antibody reacted throughout normal epidermis, suggesting heterogeneity of species or conformational forms of p53 protein. Cytoplasmic reactivity to MAb421 was similar in normal epidermis, psoriasis, and cultured keratinocytes but was, diminished in SCC, BCC, and keratoacanthoma. CM-1 reactivity persisted in these tumors. Putative p53 mutations detected by MAb240 reactivity were present in 44% of SCC specimens. Epidermis expresses p53 and the gene may regulate epidermal keratinocyte growth and carcinogenesis.

Computerized microassay of keratinocyte cell-plastic attachment and proliferation for assessing net stimulatory, inhibitory and toxic effects of compounds on nonimmortalized cell lines.

Testing of pharmacological agents that affect growth of epidermal keratinocytes (EK) requires a standardized assay. We have developed an assay measuring net effects of stimulatory (e.g. growth factors), inhibitory (e.g. methotrexate) or toxic (e.g. Triton X-100) compounds. The amount of crystal violet staining viable EK attached to the wells of standard 96-well microplates is measured in situ using an ELISA plate reader. Optical density readings are directly converted into cell counts by computer software. Counts obtained by this method strongly correlate with the results obtained using the [3H]thymidine uptake assay and direct cell counts. The assay standardizes measurements of nonimmortalized EK lines with different innate proliferative properties and allows accurate quantitation of EK numbers in the range of 2,500-500,000 EK/well.

A New in Vitro Model for Study of Wound Healing

Summary This new in vitro wound model consists of layers of human keratinocytes grown on a very thin transparent silicone disc in which a central hole is cut before placing it on a layer of collagen gel containing human fibroblasts (simulating a dermis). The growth of epidermal keratinocytes across the ‘wound' can easily be visualised and photographed by inverted light microscopy, at frequent intervals, to monitor the stimulating or adverse effect of many substances presently designed for wound treatment.

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

The effect of cyclosporin on human epidermal keratinocytes in vitro

Cyclosporin A has been shown to be effective in the treatment of severe, recalcitrant psoriasis, but it is uncertain whether the mode of action is primarily by immune suppression or by other mechanisms. Cyclosporin-dependent growth-inhibition has recently been demonstrated in vitro using several non-human and transformed epithelial cell lines. In this study the effect of cyclosporin on human epidermal keratinocytes and skin fibroblasts was investigated. Secondary cultures of human epidermal keratinocytes were grown on collagen-coated dishes in the presence of increasing concentrations of cyclosporin. Inhibition of growth was observed at 6-8 microM. An almost identical dose-response curve was obtained for the cytotoxic drug, cis-platin. Short-term exposure (I h) to cyclosporin did not have any effect on epidermal cell growth, suggesting that direct membrane-related effects were not involved. Analysis of cellular proteins by SDS-PAGE indicated no effect of continuous cyclosporin exposure on in vitro differentiation. The observation that human epidermal keratinocyte growth is inhibited by cyclosporin suggests that a topical form of therapy for psoriasis may be an effective alternative to oral treatment.

Effects of cyclosporin A on cultured human epidermal keratinocytes. Growth and 5-bromo-2'-deoxyuridine incorporation.

We have studied the in vitro effect of CsA at various doses (0.5, 1, 5, 10 micrograms/ml) on the growth and 5-bromo-2'-deoxyuridine (BrdU) incorporation by cultured normal human epidermal keratinocytes (EK). CsA was found to reduce the growth of EK at all doses used after 24, 48 and 72 h in culture, but the difference with appropriate controls became statistically significant for the dose of 10 micrograms/ml after a 48- and 72-h culture. On the other hand, CsA-treated EK comprised a reduced fraction of BrdU + (S-phase) cells, the difference being statistically significant for the dose of 10 micrograms/ml at 24, 48 and 72 h. Ultrastructural examination of CsA-treated EK, despite the presence of cytoplasmic vacuoles also observed in control EK did not show signs of severe cytoplasmic or nuclear damage. These results confirm the antiproliferative effect of CsA on cultured EK and suggest that at the concentration used, CsA acts through a cytostatic rather than a cytotoxic mechanism, most likely by blocking EK in an early phase (G0/G1) of the cell cycle.