Induced G2/M arrest and apoptosis in human epidermoid carcinoma cell lines by semisynthetic drug Ukrain.

Exposure of ME180 and A431 carcinoma cells to Ukrain (NSC-631570), a novel semisynthetic drug from Chelidonium majus L, results in cell growth inhibition which is concomitant with reversible G2/M cell cycle arrest and apoptosis at doses as low as 7 microM. In contrast, the same drug concentrations were not affective towards normal human keratinocytes. In order to investigate whether cell cycle control mechanisms are effected in response to Ukrain, we analyzed cell cycle distribution and levels of cyclins and cyclin-dependent kinases in drug treated carcinoma cells. We found alterations in levels of mitotic cyclins A and B1, and cyclin-dependent kinases CDK1 and CDK2, after treatment. We also observed an upregulation of CDK inhibitor p27 in both cancer cell lines which may lead to the G2/M cells accumulation.

Induced apoptosis in human prostate cancer cell line LNCaP by Ukrain.

Exposure of LNCaP prostate cancer cells to Ukrain (NSC-631570), a novel semisynthetic drug from Chelidonium majus L., results in cell growth inhibition which is concomitant with apoptosis. After 24 h treatment with 3.5 microM of Ukrain as many as 73% cells were found in the G2/M phase. However, at higher drug concentrations (7 microM and 17.5 microM) the changes in cell phase distribution were less dramatic but cell accumulation in the G2/M phase was still evident. The rate of apoptotic cells rose steadily with increased drug concentration in a dose-dependent manner and reached 20% at a dosage of 17.5 microM. To investigate whether the cell cycle control mechanisms are affected in response to Ukrain, we analyzed the expression levels of some cyclins, cyclin-dependent kinases (CDK) and apoptosis-related proteins in drug treated cancer cells. Western blot experiments revealed alterations in levels of CDK1 and CDK2, after treatment. Up-regulation of the CDK inhibitor p27 was observed, which may lead to G2/M cell accumulation, but no substantial changes in expression of Bcl-2 and Bax proteins were found.

Bcl-2 overexpression protects human keratinocyte cells from Ukrain-induced apoptosis but not from G2/M arrest.

Exposure of ME180 and A431 carcinoma cells to Ukrain (NSC-631570), a semisynthetic compound consisting of alkaloids isolated from Chelidonium majus L. (Papaveracea), results in cell cycle arrest at the G2/M phase. Ukrain selectively inhibits growth of ME180 and A431 cells at a concentration range from 3.5 microM to 7.0 microM and induces apoptosis. In contrast, normal human keratinocytes showed no difference in the kinetics of progression through the cell cycle in response to this compound. We found that at a concentration of 7.0 microM of this drug Bcl-2 protein overexpression protected HaCaT cell line keratinocytes against apoptosis induced by Ukrain but did not prevent G2/M arrest. Following exposure of normal keratinocytes to Ukrain, we detected an increase in Bcl-2 protein levels and a significant change in protein modification as suggested by observation of its different isoform with shifted electrophoretic mobility. Bcl-2 protein expression and its isoform distribution did not change substantially in ME180 and A431 carcinoma cells. We also suggest that drug-induced mitotic arrest and apoptosis represent dual Ukrain action on cell cycle progression machinery and Bcl-2-involved program cell death in the cell.

B7-1 overexpression by thymic epithelial cells results in transient and long-lasting effects on thymocytes and peripheral T helper cells but does not result in immunodeficiency.

The B7-1 (CD80) molecule provides costimulatory function for the activation of T helper lymphocytes upon encounter with antigen. To investigate the role of this molecule in thymocyte maturation, we have generated transgenic (Tg) mice in which CD80 expression is driven by the keratin 14 promoter (K14). This overexpression of CD80 resulted in the loss of detectable cell surface CD28 expression on thymocytes and a significant reduction in both the surface T cell receptor expression and the ratio of CD4(+) to CD8(+) single-positive thymocytes in Tg animals compared to nontransgenic (non-Tg) controls. While many of these defects were transient, the significant decrease in CD4(+) versus CD8(+) T cell ratio persisted peripherally. Peripheral T cells from these Tg mice were found to be significantly hyporesponsive to T cell mitogens and in mixed leukocyte reaction, effects that our data indicate are due to reduced IL-2 production by Tg T cells upon activation. Despite these functional defects, immunization with both complex and simple protein antigens produced no differences in the proliferative or humoral responses to these antigens between Tg and non-Tg groups. These data indicate that thymic CD80 signaling results in the deletion of significant numbers of CD4(+) T cells but does not culminate in antigen-specific immunodeficiency.

Induction of TR4 orphan receptor by retinoic acid in human HaCaT keratinocytes.

Human TR4 orphan receptor (TR4) can modulate the transcriptional activity of the reporter gene containing an AGGTCA direct repeat-hormone response element. Here we studied the potential role of TR4 in human HaCaT keratinocytes. Using a chloramphenicol acetyl-transferase reporter gene assay, it was shown that TR4 can suppress retinoic acid-induced transactivation by 47.3% in human HaCaT keratinocytes. Electrophoretic mobility shift assay indicated that this suppression may be due to TR4 binding with higher affinity to the retinoic acid response element than retinoid receptors. Western blot analysis further suggested that retinoic acid can increase the expression of TR4 protein in human HaCaT keratinocytes, indicating that TR4 acts as a negative feedback modulator for retinoic acid action. Interestingly, TR4 expression is increased in normal human keratinocytes when substituting a low calcium medium with a high calcium medium. Together, our data suggested, for the first time, that an orphan receptor, such as TR4, may play an important part in retinoid-mediated signaling pathways in human keratinocytes, providing a new insight into keratinocyte biology.

Apoptosis: a role in skin aging?

Prevailing theories view aging as an outcome of both programmed and stochastic events that occur over the lifetime of the individual. In this context, aging can be defined as a progressive decline in homeostasis and a period characterized by the inability of the organism to respond adaptively to stress. Apoptosis thus stands out as a potential key cellular process that may be affected during aging because apoptosis is both an important homeostatic mechanism and a protective cellular response to stress. In this paper we introduce the topic of apoptosis, its regulation, and its roles in epidermal homeostasis as a feature of normal keratinocyte differentiation and as a cellular endpoint of stress. To probe the question of whether apoptosis contributes to the process of skin aging, we present evidence for apoptotic dysregulation during aging in nonskin systems and discuss some findings suggesting that decreased efficiency of apoptosis may contribute to the alterations characteristic of intrinsic (chronologic) aging and extrinsic (photoaging) skin aging.

Autocrine nerve growth factor protects human keratinocytes from apoptosis through its high affinity receptor (TRK): a role for BCL-2.

Normal human keratinocytes synthesize and release nerve growth factor (NGF) and express both the low- and the high-affinity NGF receptor. Because NGF has been shown to rescue certain cell types from programmed cell death, we investigated the role of endogenous NGF in preventing keratinocyte apoptosis. We report here that apoptosis is induced in normal human keratinocytes in culture by blocking endogenous NGF signaling with either anti-NGF neutralizing antibody or K252, a specific inhibitor of the tyrosine kinase high-affinity NGF receptor. Apoptosis was assessed by DNA laddering, electron microscopy, and in situ nick end labeling technique. In anti-NGF-treated keratinocytes, the apoptotic process starts at 96 h, and is maximal at 120 h. After K252 treatment, apoptosis starts at 48 h and peaks at 120 h. Because the product of the bcl-2 proto-oncogene protects many cell types from apoptosis, we measured the levels of this protein in apoptotic keratinocytes. We found that both K252 and anti-NGF antibody strikingly downregulate bcl-2 expression, starting at 72 h. Furthermore, HaCat keratinocytes stably transfected with a plasmid containing bcl-2 cDNA fail to undergo apoptosis when treated with K252. These findings show that autocrine NGF acts as a survival factor for human keratinocytes in vitro through its high-affinity NGF receptor, possibly by maintaining constant levels of Bcl-2.

Induction of apoptosis through the PKC pathway in cultured dermal papilla fibroblasts.

The dermal papilla (DP) consists of a discrete population of specialized fibroblasts that are important in the morphogenesis of the hair follicle in the embryo and in the control of the hair growth cycle in the adult. This mitotically quiescent and long-lived cell population expresses gene products that promote cell survival such as Bcl-2, and thus normally might be protected from apoptosis. We investigated whether cultured DP fibroblasts are able to undergo apoptosis by treatment with the protein kinase inhibitor staurosporine. Involvement of the PKC signaling pathway in DP fibroblast survival/death was investigated by inhibition (staurosporine and Bisindolylmaleimide (Bis) treatment) or activation (TPA; 12-O-tetradecanoylphorbol-13-acetate treatment) of PKC and characterization of DP-expressed PKC isoforms by RT-PCR. We determined that cultured DP fibroblasts undergo apoptosis, in a dose-related manner, when treated with staurosporine but not when treated with Bis, an inhibitor with narrow PKC isoform specificity. TPA confers partial and transient resistance to staurosporine-induced DP apoptosis. Staurosporine and Bis each induced G1 arrest, whereas TPA treatment of cultured DP resulted in increased entry into S-phase. The differential responses to individual inhibitors and activators of PKC may be related to the multiple PKC isoforms that DP fibroblasts express. Flow cytometric analysis indicates that the mechanism of staurosporine-induced apoptosis may be through decrease of Bcl-2 in treated DP cells or through modulation of cell cycle regulators. Correlation between sensitivity to induction of apoptosis and proliferation suggests that dermal papilla cells may normally be protected from apoptosis in vivo by their mitotically quiescent state.

Incomplete differentiation of fetal keratinocytes in the skin equivalent leads to the default pathway of apoptosis.

The program of epidermal morphogenesis and differentiation changes dramatically during development of human fetal skin. Keratinocytes derived from fetal basal cells at early stages undergo only an incomplete keratinization and are eventually replaced by newly formed fetal keratinocytes that complete the terminal differentiation program and form the first stratum granulosum and stratum corneum. Once established, this program is reiterated throughout life, as the epidermis continually renews. To test the developmental potential of early fetal keratinocytes, we have cultured them in the physiologic skin equivalent (SE) system in the presence of varied retinoic acid (RA) concentrations and have compared them to neonatal keratinocytes cultured under the same conditions. The responses of fetal and neonatal SEs have been characterized by analysis of epidermal morphology, the presence and distribution of RA-responsive and differentiation-specific keratins and filaggrin, proliferation, and apoptosis. Our study shows that fetal basal keratinocytes already are programmed to form the granular layer and incomplete stratum corneum, even when isolated from a stage prior to formation of these layers. Fetal keratinocytes respond differently than neonatal keratinocytes to RA in terms of modulation of both epidermal morphology and expression of differentiation markers. Modulation of RA-responsive K1 and K19 occurs in both fetal SE and neonatal SE but the fetal keratinocyte responds at lower RA concentrations in the medium. In contrast, fetal keratinocytes appear to be less responsive than neonatal keratinocytes in terms of filaggrin expression and stratum corneum formation. These differences in the differentiation and RA response in vitro may be related to inherent stage-specific differences between fetal and neonatal keratinocytes in RA-signaling pathways including expression of the retinoic acid receptor, RARbeta. Furthermore, high rates of apoptosis in the fetal SE suggest that apoptosis is the default pathway that is taken in the absence of complete keratinocyte differentiation.

UV-induced apoptosis in skin equivalents: inhibition by phorbol ester and Bcl-2 overexpression.

To determine the role of apoptosis in epidermal homeostasis and to identify its regulators in skin, we have developed and characterised a physiologically relevant in vitro model of epidermal apoptosis. First, we show that keratinocyte cell death can be induced by ultraviolet irradiation within the stratified epidermis of the skin equivalent in an in vivo-like manner. DNA fragmentation and changes in the patterns of expression of p53 and Bcl-2 suggest that the mechanisms operating in UV-induced apoptosis in the skin equivalent are controlled by these factors. Secondly, we demonstrate that apoptosis in this model is amenable to modulation by exogenous factors present in the culture medium, such as phorbol ester, and by tranfected genes, as shown by overexpression of bcl-2. These studies show that the skin equivalent is a valuable model in which to determine the controllable steps of the apoptotic pathway independently of the immune system and to correlate apoptosis to the physiologic state of the keratinocyte.

Apoptosis: the skin from a new perspective.

In this review we present skin biology from the perspective of apoptosis. We stress that apoptosis acts as an important homeostatic and defence mechanism in the developing and mature epidermis. Programmed cell death functions in establishing the architecture of the human epidermis and its appendages during development by deletion of stage-specific cells and in the adult epidermis by elimination of excess and abnormal cells. Arguments are presented to support the hypothesis that known regulators of keratinocyte growth may act as survival factors which suppress the cell death pathway. Surviving cells continue to divide until they encounter anti-proliferative factors. Then, unless cells are severely injured and die of necrosis, they will terminally differentiate to death or will die by apoptosis. The mechanisms controlling keratinocyte maturation are co-ordinated with cell position within the epidermal strata. Inappropriate regulatory signals or response of a cell inappropriate to its state will activate apoptosis. Parallels between terminally differentiating keratinocytes and apoptotic cells imply that terminal differentiation and apoptosis proceed along the same death pathway. For terminally differentiating cells, however, this pathway is more elaborate because it allows expression of tissue- and differentiation-specific genes. A model is presented that integrates apoptosis and keratinocyte growth and differentiation.

Apoptosis in human skin development: morphogenesis, periderm, and stem cells.

During human skin development, embryonic- and fetal-specific periderm cells and incompletely keratinized cells are replaced by keratinocytes that differentiate while stratifying to form the fully functional epidermis. Proliferating basal cells of fetal skin also develop into epidermal appendages such as hair follicles and glands. We demonstrate that programmed cell death, not emphasized in conventional epidermal biology, has an important function in establishing the final architecture of the human epidermis and its appendages. Immunohistochemical localization of transglutaminases in fetal periderm, intermediate epidermal cells, and within appendages coincides with DNA fragmentation indicating that apoptosis is involved in deletion of these stage-specific cells and remodeling of appendages. The data also suggest that terminal differentiation of epidermal cells might be a specialized form of apoptosis. The pattern of expression of bcl-2, a gene associated with survival of some cells, is exclusive of the distribution patterns of markers of the cell death pathway. Bcl-2 protein is correlated with specific morphogenetic events in hair follicles and eccrine sweat glands, and its presence in single cells of the hair follicle bulge suggests that Bcl-2 may be a stem cell marker.

Cell death by apoptosis in epidermal biology.

Homeostasis in continually renewing tissues is maintained by a tightly regulated balance between cell proliferation, cell differentiation, and cell death. Until recently, proliferation was thought to be the primary point of control in the regulation of normal tissue kinetic homeostasis and as such has been the major focus of both understanding the etiology of disease and developing therapeutic strategies. Now, physiologic cell death, known as apoptosis (a-pop-to' sis, a-po-to' sis [Thomas CL (ed.): Taber's Cyclopedic Medical Dictionary. F.A. Davis, Co., Philadelphia, 1989)] has gained scientific recognition as an active regulatory mechanism, complementary, but functionally opposite, to proliferation with important roles in shaping and maintaining tissue size and prevention of disease. In this review we will describe the concept of apoptosis and discuss possible molecular mechanisms of its regulation that may have implications for skin biology.

Keratinocytes regulate melanocyte number in human fetal and neonatal skin equivalents.

To determine if keratinocytes influence melanocyte number and position in the developing epidermis we have experimentally recombined keratinocytes and melanocytes from epidermis of different stages of differentiation in the skin equivalent (SE) system. Previously we showed that developmental differences in the position and number of melanocytes characteristic of the epidermis in vivo were preserved in fetal and neonatal skin equivalents. In the present study we have combined cultured fetal or neonatal keratinocytes with age-matched or non-age-matched cultured melanocytes on the dermal equivalent. The ratio of basal keratinocytes to melanocytes (BK/M) present in multiple high-power fields was determined after localization of melanocytes by staining with the melanocyte-specific monoclonal antibody, HMB-45. The BK/M ratio in SE composed of neonatal keratinocytes and either fetal (n = 4) or neonatal (n = 5) melanocytes was 26.2 and 21.5, respectively. The BK/M ratio in SE composed of fetal keratinocytes and either fetal (n = 8) or neonatal (n = 5) melanocytes was 9.2 and 7.7, respectively. In each case, the BK/M ratio was dependent on the keratinocytes rather than the melanocytes. With either type of melanocyte, ratios in SE composed of neonatal keratinocytes were significantly greater than those with fetal keratinocytes. These results establish that keratinocytes regulate the BK/M ratio in this model and suggest that developmental differences between fetal and neonatal keratinocytes may be responsible for determining melanocyte numbers in the epidermal-melanin unit in vivo. The precise mechanisms that control the organization and number of melanocytes in the epidermis are unknown although keratinocytes may interact with melanocytes via growth factors, cell surface molecules, or other factors related to proliferation and differentiation of the epidermis.

Physiologic distribution and differentiation of melanocytes in human fetal and neonatal skin equivalents.

There is evidence that epidermal keratinocytes play a critical role in melanocyte position and differentiation in the epidermis, although little is known about the molecular mechanisms involved. We have used an in vitro skin equivalent as a model system in which to study keratinocyte/melanocyte interactions in both fetal and neonatal skin. Because the skin equivalent model has been shown to closely simulate the morphologic and biochemical features of differentiated epidermis we hypothesized that the factors that influence melanocyte position and differnetiation would also function in this system. Localization of melanocytes in skin equivalents, using the monoclonal antibody HMB-45, established that melanocytes in fetal skin equivalents are grouped and distributed both basally and suprabasally, whereas melanocytes in neonatal skin equivalents are singly distributed among basal epidermal keratinocytes, similar to the distributions of fetal and neonatal melanocytes, respectively, in vivo. Similarly, in fetal and neonatal skin equivalents the patterns of expression of a number of melanoma/melanocyte-associated antigens closely parallels that seen in vivo. These results suggest that the skin equivalent model is an excellent system in which to study the dynamic factors that regulate melanocyte migration, proliferation, and differentiation during ontogeny and post-natal differentiation of the skin.

Retention of differentiated characteristics in human fetal keratinocytes in vitro.

Many of the morphologic and biochemical changes that occur during human fetal skin development have been described, yet there has been little experimental analysis of the processes that regulate the development of human fetal skin. This is due in part to difficulties in culturing human fetal epidermal keratinocytes. We have successfully cultured fetal keratinocytes in two different in vitro systems; in a serum-free keratinocyte growth medium (KGM) on tissue culture plastic and cocultured with dermal fibroblasts as spheroidal aggregates. To characterize these fetal keratinocytes in vitro we have assessed their ability to express several markers of epidermal differentiation. Human fetal keratinocytes grown on plastic in KGM stratify and express some of the components of the differentiated epidermis, such as involucrin and the high molecular weight keratins. However, these keratinocytes co-express keratins and vimentin and do not form a structured basement membrane. More characteristics of fetal skin are preserved in mixed aggregates of epidermal keratinocytes and dermal fibroblasts, including epidermal stratification, synthesis of basement membrane components, tissue-specific expression of intermediate filaments, involucrin, and expression of high molecular weight keratins. The maintenance of human fetal epidermal keratinocytes in these two in vitro systems and their ability to express many differentiated characteristics suggests that these cultures will be valuable for studies of the molecular mechanisms that regulate the regionally specific differentiation of the human fetal epidermis.

Differences in the histogenesis and keratin expression of avian extraembryonic ectoderm and endoderm recombined with dermis.

The responses of the chorionic ectoderm and allantoic endoderm (from 8-day chick embryos) to dermal induction were compared through tissue recombinants grafted onto the chorioallantoic membrane. The chorionic epithelium formed the appropriate epidermis with a fully developed stratum corneum in response to both spur and scutate scale dermises. Analysis of these recombinant epidermal tissues by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that tissue-specific expression of the alpha (alpha) and beta (beta) keratin polypeptides occurred. In addition, indirect immunofluorescence studies with antisera to alpha or beta keratins showed that the beta stratum, which characterizes the epidermis of spurs and scutate scales, was formed, and the alpha keratins were distributed as in the normal epidermal tissues. In contrast, although the allantoic endoderm became stratified in association with either spur or scutate scale dermis, a stratum corneum with a beta stratum did not develop. SDS-PAGE analysis demonstrated that while the characteristic beta keratins of scutate scales and spur were not detected, most of the alpha keratins normally elaborated by these structures were present, suggesting that even without histogenesis of a stratum corneum the expression of alpha keratins of endoderm could be regulated in a tissue-specific manner by dermis. This study also demonstrated that there are differences in the abilities of the chorionic and allantoic epithelia to respond to the same dermal cues, which may reflect earlier restrictions in their developmental potentials.

Avian feather development: relationships between morphogenesis and keratinization.

Morphogenesis and expression of the alpha and beta keratin polypeptides are controlled by epidermal-dermal interactions during development of avian skin derivatives. We have examined the relationship between morphogenesis of the embryonic feather and expression of the feather alpha and beta keratins by routine histology, indirect-immunofluorescence, and SDS-PAGE. Initially beta keratins are expressed only in the feather sheath. Following barb ridge morphogenesis beta keratins can be detected in the barb ridge, coincident with the differentiation of barb ridge cells into eight distinct morphological types. Beta keratinization occurs in gradients; from feather apex to base, and from periphery of the barb ridge to the interior. The onset of beta keratinization in the barb ridges is paralleled by an increase in the major feather beta keratin polypeptides, as detected by SDS-PAGE. The alpha keratins are present in both the periderm and feather sheath at early stages of feather development, but become greatly reduced after hatching, when the down feather emerges from the sheath.