Search

Monohydroxy acids (HETEs) and leukotriene B4 (LTB4) metabolites of arachidonic acid were measured in skin of healthy volunteers after ultraviolet B irradiation, and in the uninvolved skin of psoriatics after topical dithranol application. Exudate was collected from suction bullae on control and inflamed abdominal skin, and analysed for 12-HETE and PGE2 by GC-MS and LTB4 by bioassay. 12-HETE and PGE2 were raised at 24 h but not at 72 h after u.v.B irradiation: control and 24 h values were 13.7 and 41.5 ng ml-1 (P less than 0.05, n = 6) for 12-HETE respectively, and 4.5 and 30.2 ng ml-1 (P less than 0.01, n = 6) for PGE2. Dithranol application raised PGE2 levels from 23.1 ng ml-1 in control exudate to 62 ng ml-1 (P less than 0.01, n = 6) at 24 h before declining to base levels at 72 h. However, 12-HETE was raised at 72 h (200 ng ml-1, P less than 0.01, n = 5) but not at 24 h (104 ng ml-1) compared to control levels (50 ng ml-1, n = 5). The levels of the LTB4 were low (less than 100 pg ml-1), and no significant increases were observed. Arachidonic acid in inflamed skin can be metabolised by the cyclo-oxygenase and lipoxygenase pathway. It is probable that the lipoxygenase product 12-HETE is involved in these inflammatory reactions. [ABSTRACT FROM AUTHOR]

Retinoids modulate the growth and differentiation of cancer cells presumably by activating gene transcription via the nuclear retinoic acid receptor (RAR) alpha, beta, and gamma and retinoid X receptor (RXR) alpha, beta, and gamma. We analyzed the effects of 38 RAR-selective and RXR-selective retinoids on the proliferation of 10 human head and neck squamous cell carcinoma (HNSCC) cell lines. All of these cell lines expressed constitutively all of the receptor subtypes except RARbeta, which was detected in only two of them. Most of the RAR-selective retinoids inhibited the growth of HNSCC cells to varying degrees, whereas the RXR-selective retinoids showed very weak or no inhibitory effects. Three RAR antagonists suppressed growth inhibition by RAR-selective agonists, as well as by RAR/RXR panagonists such as 9-cis-retinoic acid. Combinations of RXR-selective and RAR-selective retinoids exhibited additive growth-inhibitory effects. Furthermore, we found that CD437, the most potent growth-inhibitory retinoid induced apoptosis and up-regulated the expression of several apoptosis-related genes in HNSCC cells. These results indicate that: (a) retinoid receptors are involved in the growth-inhibitory effects of retinoids; (b) RXR-RAR heterodimers rather than RXR-RXR homodimer are the major mediators of growth inhibition by retinoids in HNSCC cells; and (c) induction of apoptosis can account for one mechanism by which retinoids such as CD437 inhibit the growth of HNSCC cells. Finally, these studies identified several synthetic retinoids, which are much more effective than the natural RAs and can be good candidates for chemoprevention and therapy of head and neck cancers.

The addition of all-trans-retinoic acid has been found to mediate a G1 cell cycle phase arrest but not apoptosis in normal mammary epithelial cells. We have now found that addition of the novel retinoid 6-[3-(1-adamantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which appears to function through a pathway independent of retinoic acid nuclear receptors, results in an S-phase arrest that is preceded by a 4-fold elevation in the levels of the cyclin-cyclin dependent kinase (cdk) inhibitor p21WAF1/CIP1. Failure to inhibit E2F-1 activation of genes through its phosphorylation by the cyclin cdk2 kinase has been shown to result in S-phase arrest and apoptosis in a number of cell types. Although exposure of the normal mammary cells to CD437 does not result in modulation of cyclin A or cdk2 levels, an increase in E2F-1 levels and a marked inhibition of cyclin A/cdk2 kinase activity are observed. Exposure to CD437 results in enhanced E2F-1 binding to its DNA consensus sequences and transcriptional activity during S phase. We hypothesize that this enhanced E2F-1 transcriptional activity results in S-phase arrest and subsequent apoptosis that has been observed in other systems.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been recently identified to be a potent inducer of apoptosis in human non-small cell lung carcinoma (NSCLC) cells through a nuclear retinoic acid receptor independent mechanism. To approach the mechanism by which CD437 induces apoptosis in NSCLC cells, we investigated the involvement of c-Myc in CD437-induced apoptosis. CD437 (1 microM) up-regulated the expression of c-Myc and of its downstream target genes ornithine decarboxylase (ODC) and cdc25A in all three NSCLC cell lines (i.e., H460, SK-MES-1 and H1792) used. These effects were correlated with cellular susceptibilities to induction of apoptosis by CD437. Furthermore, CD437-induced apoptosis could be blocked by the ODC inhibitor difluoromethylornithine, the caspase inhibitors Z-VAD FMK and Z-DEVD FMK, and c-Myc antisense oligodeoxynucleotide, respectively. These data indicate that c-Myc gene plays an important role in mediating CD437-induced apoptosis in human NSCLC cells.

CD437 is a novel retinoid that can induce apoptosis in a variety of tumor cell types by an unknown mechanism. We found that CD437 up-regulated the expression of p21(WAF1/CIP1), Bax, and Killer/DR5 and induced G1 arrest and rapid apoptosis in three human non-small cell lung carcinoma cell lines with wild-type p53 but not in five cell lines with mutant p53, suggesting a role for p53 in the effects of CD437. Using H460 cells in which wild-type p53 protein was degraded by transfection of the human papillomavirus 16 E6 (HPV-16 E6) gene and H460 cells transfected with a control plasmid only, we found that CD437 increased p53, p21(WAF1/CIP1), Bax, and Killer/DR5 in the control transfectants. In contrast, the constitutive p53 protein level was suppressed, and the ability of CD437 to increase p53 and its downstream genes was compromised in E6 transfectants. In addition, CD437 induced G1 arrest and apoptosis in the control transfectants but not in the E6-transfected cells. These results indicate that p53 plays a role in CD437-induced growth inhibition and apoptosis in human non-small cell lung carcinoma cells.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been shown to induce apoptosis in various tumor cell lines including human non-small cell lung carcinoma (NSCLC) cells, which are resistant to the natural all-trans retinoic acid and to many synthetic receptor-selective retinoids. Although the mechanism of this effect was not elucidated, it was found to be independent of nuclear retinoid receptors. In the present study, we analysed the mechanisms by which CD437 induces apoptosis in two human NSCLC cell lines: H460 with wild-type p53 and H1792 with mutant p53. Both cell lines underwent apoptosis after exposure to CD437, although the cell line with wild-type p53 (H460) was more sensitive to the induction of apoptosis. CD437 increased the activity of caspase in both cell lines, however, the effect was much more pronounced in the H460 cells. The caspase inhibitors (Z-DEVD-FMK and Z-VAD-FMK) suppressed CD437-induced CPP32-like caspase activation and apoptosis in both cell lines. CD437 induced the expression of the p53 gene and its target genes, p21, Bax, and Killer/DR5, only in the H460 cells. These results suggest that CD437-induced apoptosis is more extensive in NSCLC cells that express wild-type p53, possibly due to the involvement of the p53 regulated genes Killer/DR5, and Bax although CD437 can also induce apoptosis by means of a p53-independent mechanism. Both pathways of CD437-induced apoptosis appear to involve activation of CPP32-like caspase.

Using an in vitro lung carcinogenesis model consisting of normal, premalignant, and malignant human bronchial epithelial (HBE) cells, we analyzed the growth inhibitory effects of 26 novel synthetic retinoic acid receptor (RAR)- and retinoid X receptor (RXR)-selective retinoids. RAR-selective retinoids such as CD271, CD437, CD2325, and SR11364 showed potent activity in inhibiting the growth of either normal or premalignant and malignant HBE cells (IC50s mostly <1 microM) and were much more potent than RXR-selective retinoids. Nonetheless, the combination of RAR- and RXR-selective retinoids exhibited additive effects in HBE cells. As the HBE cells became progressively more malignant, they exhibited decreased or lost sensitivity to many retinoids. The activity of the RAR-selective retinoids, with the exception of the most potent retinoid, CD437, could be suppressed by an RAR panantagonist. These results suggest that: (a) RAR/RXR heterodimers play an important role in mediating the growth inhibitory effects of most retinoids in HBE cells; (b) CD437 may act through an RAR-independent pathway; (c) some of the RAR-selective retinoids may have the potential to be used in the clinic as chemopreventive and chemotherapeutic agents for lung cancer; and (d) early stages of lung carcinogenesis may be responsive targets for chemoprevention by retinoids, as opposed to later stages.

6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor gamma-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21(WAF1/CIP1). In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.

Retinoids are promising agents for cancer chemoprevention and therapy. Nuclear retinoic acid receptors (RARs; RARalpha, -beta, and -gamma) and retinoid X receptors (RXRs; RXRalpha, -beta, and -gamma) are thought to mediate most of retinoids' effects on cell growth and differentiation. Because the majority of human non-small cell lung carcinoma (NSCLC) cell lines are resistant to all-trans-retinoic acid, we searched for more potent retinoids. Therefore, we examined the effects of 37 natural and synthetic retinoids that exhibit specific binding to and transactivation of individual RARs or RXRs on the proliferation of eight human NSCLC cell lines. All of these cells expressed mRNAs of the three RXRs; however, they expressed varying levels of RARalpha and RARgamma, and only three of the eight cell lines expressed RARbeta mRNA. Cellular retinoic acid-binding proteins (CRABPs) I and II were detected in one and three of the eight cell lines, respectively. Only 8 of the 37 retinoids exhibited growth-inhibitory activity (IC50, < 10 microM) against at least two of the eight NSCLC cell lines. The active retinoids included one (TD550) of five RARalpha-selective, one (Ch55) of three RARbeta-selective, three (CD437, CD2325, and SR11364) of six RARgamma-selective, and one (CD271) of four RARbeta/gamma-selective retinoids. The potency of these retinoids was low (IC50, > 1 microM), except for CD437, which was very potent (IC50, 0.1-0.5 microM). The six RXR-selective retinoids were mostly inactive even at 10 microM. However, combinations of RAR-selective and RXR-selective retinoids exhibited additive effects. There appeared to be no simple correlation among the histological type of the NSCLC (adeno- or squamous), the levels of nuclear receptors or CRABPs, and the response of the cells to the growth-inhibitory effects of retinoids. Nevertheless, in contrast with former studies with natural retinoids, these results suggest that several synthetic retinoids do exhibit inhibitory activity against NSCLC cells, and some of them may be useful clinically.

Retinoic acid (RA) plays a major role in embryogenesis of the nervous system and has been reported to induce differentiation in neuroblastoma cell lines. To identify RA signaling pathways involved in such differentiation processes, two RA-sensitive neuroblastoma cell lines (LA-N-5 and SH-SY5Y) were extensively studied. Northern blot experiments determined that of the three RAR mRNAs, only RARalpha was significantly expressed, with respectively weak or undetectable levels of RARgamma and RARbeta. RXRs (alpha and beta) receptors were weakly expressed. Western blotting analysis confirmed the constitutive expression of RARalpha and absence of RARbeta and weak levels of RXRalpha. Treatment with all-trans-RA up-regulated RARalpha and induced a drastic increase of RARbeta (both at the RNA and protein level). To further characterize the function of RARalpha, RARbeta and RXRalpha in NB cells, nuclear extracts from LA-N-5 cells were analysed by EMSA studies. Three specific retarded complexes were observed which were significantly decreased or shifted in the presence of monoclonal antibodies to RARalpha, RARbeta and RXRalpha. RA treatment dramatically induced a DR5-binding RXRalpha-RARbeta heterodimer. Treatment with combinations of RARalpha or RARbeta agonists with a RXRalpha agonist or with a RARalpha agonist alone, induced neurite-outgrowth supporting the probability that both RXRalpha-RARalpha or RXRalpha-RARbeta heterodimers are involved in RA-mediated differentiation of NB cells. The availability of novel synthetic RA-specific receptor ligands should provide the possibility of tissue specific therapeutic regimes.

Trans retinoic acid (RA) has proven to be a potent therapeutic agent in the treatment of acute promyelocytic leukemia. Unfortunately, other subtypes of acute myelogenous leukemia are resistant to the antiproliferative and differentiating effects of RA. In this report, we describe a novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN; CD437) that not only totally inhibits the proliferation of RA-resistant leukemic cell lines HL-60R and K562 but also induces apoptosis in these cells. Exposure of HL-60R to CD437 results in the rapid (within 30 minutes) increase of the cyclin-dependent kinase inhibitor p21(waf1/cip1) as well as GADD45 mRNA. Manifestations of CD437-mediated programmed cell death are noted within 2 hours, as indicated by both the cleavage and activation of the CPP32 protease and cleavage of poly (ADP-ribose) polymerase. This is followed by cleavage of bcl-2 and internucleosomal DNA degradation. HL-60R cells do not express the retinoid nuclear receptor RAR beta and RAR gamma and express a truncated RAR alpha. Thus, CD437 induction of p21(waf1/cip1) and GADD45 mRNAs and apoptosis occurs through a unique mechanism not involving the retinoid nuclear receptors. CD437 represents a unique retinoid with therapeutic potential in the treatment of myeloid leukemia.

p21WAF1/CIP1 plays a major role in the induction of G1 arrest following DNA damage. Although p21WAF1/CIP1 expression is regulated by the tumor suppressor p53, induction of p21WAF1/CIP1 expression through p53-independent pathways has been described in numerous cell types. In this report, we describe the mechanism by which the retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induces p21WAF1/CIP1 in breast carcinoma cells possessing either a wild-type (MCF-7 cells) or mutated (MDA-MB-468 cells) p53. Exposure of MDA-MB-468 cells to this retinoid results in an approximately 10-fold increase in p21WAF1/CIP1 mRNA levels, whereas less than a 2-fold increase in p21WAF1/CIP1 gene transcription was observed as indicated by transient transfection experiments utilizing a p21WAF1/CIP1 promoter firefly luciferase reporter gene construct and nuclear run-off studies. We found similar results in the MCF-7 cells (Z-M. Shao et al., Oncogene, 11: 493-504, 1995). We have now found that while enhancing p21WAF1/CIP1 gene transcription minimally, this retinoid increases p21WAF1/CIP1 mRNA stability by 3-fold in both cell types. We also demonstrate that approximately 1.5 kb of the 3' untranslated region causes enhanced instability of p21WAF1/CIP1 mRNA. The retinoid-dependent increase in p21WAF1/CIP1 mRNA stability is accompanied by an increase in p21WAF1/CIP1 protein expression, as indicated by Western blot experiments utilizing anti-p21WAF1/CIP1 monoclonal antibody. This increase in p21WAF1/CIP1 is subsequently followed by the onset of programmed cell death in both cell types. Thus, CD437 is a novel retinoid which enhances p21WAF1/CIP1 mRNA levels through stabilization of the message regardless of the p53 status of the cell.

The biologic activities of retinoic acid and 3,4-didehydroretinoic acid, two endogenous vitamin A derivatives in various tissues, were compared to their affinities for the nuclear retinoic acid receptors and their ability to induce transcriptional activation. Both retinoids were equipotent inducers of differentiation of F9 teratocarcinoma cells. In a morphologic assay, using reconstructed skin, retinoic acid and 3,4-didehydroretinoic acid inhibited keratinization at a concentration of 100 nM. In cultured keratinocytes, a 50% inhibition of the production of the keratinocyte transglutaminase enzyme was achieved with about 20 nM for both retinoids. The in vitro binding to the nuclear retinoic acid receptors alpha, beta, and gamma showed that retinoic acid and 3,4-didehydroretinoic acid had almost equal affinities for the receptors with Kds ranging from 3 to 47 nM. The transcriptional activation resulting from the addition of the two retinoids to cells co-transfected with alpha, beta, or gamma retinoic acid receptor expression vectors and a retinoic acid responsive element linked to the chloramphenicol acetyltransferase reporter gene was similar. Finally, it was demonstrated that retinoic acid did not metabolize to 3,4-didehydroretinoic acid, and a slow conversion of 3,4-didehydroretinoic acid into retinoic acid was not sufficient to explain the biologic effects produced by the former compound. In conclusion, the present study demonstrates that retinoic acid and 3,4-didehydroretinoic acid have the same activity in several different test systems, but their metabolism differs depending on the cell type used.

A new synthetic retinoid analogue, adapalene (6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid, CD271), which is relatively selective for retinoic acid receptor beta, was noted to be an effective comedolytic agent in the rhino mouse model and to have clinical efficacy against acne. In pursuit of this observation, we studied the effects of CD271 on the development of erythema, spongiosis, and epidermal hyperplasia as well as other well-characterized markers of in vivo retinoid action after 4 d of occluded topical treatment. The objective of the study was to elucidate further those parameters associated with potential clinical efficacy. Twenty-five subjects were treated with 0.1% all-trans retinoic acid cream, all-trans retinoic acid vehicle, 0.1% CD271 gel, or CD271 vehicle under occlusion for 4 d. Only all-trans retinoic acid induced erythema (p < 0.01 versus all other treatments). Similarly, histologic analysis revealed that epidermal hyperplasia and spongiosis were induced only by all-trans retinoic acid (p < 0.01 versus all other treatments). By immunohistochemical analysis: all-trans retinoic acid increased expression of epidermal transglutaminase, involucrin, and calgranulin (p < 0.05 versus all other treatments). In contrast to these data, both CD271 and all-trans retinoic acid caused marked and significant (p < 0.05) elevation of cellular retinoic acid-binding protein-II (CRABP-II) messenger ribonucleic acid steady-state levels as judged by quantitative RNA blot analysis. Although CD271 treatment did not lead to erythema or affect epidermal morphology, its ability to induce a marker of retinoid action (i.e., CRABP-II) was 70% the potency of all-trans retinoic acid. This study suggests that CRABP-II gene expression may be a more sensitive indicator of retinoid biologic activity in skin than are erythema or changes in epidermal morphology and differentiation.

The area of UV erythema produced by a small beam head was found to increase with increasing doses. The aim was to investigate whether measurement of the area could be a more useful indicator of UV-induced damage than classic visual grading. Topical pretreatment with all-trans retinoic acid (tretinoin) and betamethasone valerate was used to test the applicability of the method in pharmacological studies. We used a round outlet head (5 mm2) connected by optical fibre to a monochromatic irradiator, and doses ranging from 0.05 to 0.2 Joule of 300 mm UV light were applied to the skin of 6 healthy subjects. Erythemal area was calculated by the measurement of two diameters, and intensity was graded visually (0-6 scale). The area of the erythema correlated with the increase in intensity up to score 6. Area measurement was less subject to intra-investigators' variability than the intensity score. By multiplying intensity by area, a good indicator of UV-induced reactivity was obtained. Pretreatment with betamethasone valerate decreased the area of erythema, as did tretinoin 12 h after irradiation. Thus, area measurement of erythema is a useful adjunct to visual grading of UV-induced skin reactions.

The monoclonal antibody F12, raised against epidermal cells from a psoriatic lesion, decorated antigens highly expressed in psoriatic epidermis and in cultured normal human keratinocytes. In normal human skin, F12 reacted only with follicular keratinocytes. Characterization of the immunoprecipitated antigens by two-dimensional gel electrophoresis revealed their identity with calgranulin A and B. A semiquantitative study with various established epithelial cell lines demonstrated that the expression of calgranulin A and B in hyperproliferative keratinocytes correlates with their potential to undergo terminal differentiation. In epidermis reconstructed in vitro, the antigen expression was stimulated by retinoids and suppressed under vitamin A starvation.

Plasma membrane-bound transglutaminase (TGm) catalyzes the formation of cornified envelopes (CE) in terminally differentiating keratinocytes. The recent cloning of cDNA encoding rabbit TGm allows detailed studies of its gene expression and regulation. In the present paper, we describe the localization of TGm mRNA in rabbit tissues, as well as in normal and psoriatic human skin, as assessed by in situ hybridization. Furthermore, we correlate TGm mRNA localization with the distribution of the TGm protein detected by immunohistochemistry with a specific monoclonal antibody. In rabbit epidermis, TGm mRNA was expressed in suprabasal cells. The TGm protein was detected in the upper stratum spinosum and stratum granulosum. In rabbit esophagus, TGm mRNA and protein were already expressed to a high level in the first suprabasal cell layer, and their expression decreased in the more differentiated cells. In normal human skin, a small amount of TGm mRNA, restricted to the stratum granulosum, was found, whereas psoriatic skin samples contained high amounts of TGm mRNA in the suprabasal layers with a decreasing gradient into the rete ridges, i.e., the involutions of the epidermis into the dermal compartment. The TGm protein was absent from the rete ridges and confined to several cell layers expressing high levels of mRNA. There was virtually no difference between uninvolved psoriatic and normal epidermis.

The role played by cytosolic retinoic acid-binding proteins (CRABP) in the control of differentiation and morphogenesis by retinoids remains unclear, which contrasts with the presence of these binding proteins in tissues known to be targets for retinoic acid effects. Human epidermis represents a good system to address this question because 1) the effect of retinoids on keratinocyte differentiation is well documented; 2) epidermis contains CRABP, and the amount of these proteins is modulated both by keratinization and retinoids; 3) the architecture of epidermis obtained in vitro by growing adult human keratinocytes on a dermal substrate can be modulated by retinoids added to the culture medium in a dose-dependent manner; and 4) most markers of epidermal differentiation are also modulated by retinoids in a dose-dependent manner. In this study, we compared, in dose-response experiments, the biologic activities of retinoic acid and CD271, a substance unable to bind to CRABP, but able to bind to nuclear retinoic acid receptors (RAR). Our results show that retinoic acid and CD271 exert similar controls on epidermal morphogenesis and keratinocyte differentiation, as shown by the inhibition of the synthesis of suprabasal keratins, filaggrin, and transglutaminase. Therefore, we exclude a qualitative role for CRABP in the control exerted by retinoids on the differentiation and morphogenesis of cultured human keratinocytes. Instead of being involved in the pathway via which retinoids control epidermal gene expression, CRABP might regulate the amount of intracellular-active retinoic acid and thus control quantitatively the intensity of biologic effects.

The inhibition of erythema by drugs applied topically after irradiation with 0.2-0.8 J of 313 nm has been studied in healthy volunteers. Indomethacin and piroxicam markedly inhibited the erythema at 6 to 24 h after irradiation but erythema reappeared at 45 h. The results reported suggest that the UVB response is biphasic with an early phase responsive to nonsteroid anti-inflammatory drugs and a late phase which is not responsive. No effect on the intensity of the erythema was seen with betamethasone chloroquine and cetirizine. Oral intake of aspirin before and/or after irradiation did not influence the UV response.

When cultured human epidermal keratinocytes (NHK) reach confluence they start to differentiate and an increase in the total cellular cholesterol content is observed. This increase parallels the appearance of a characteristic feature of terminal keratinocyte differentiation, the spontaneous formation of cornified envelopes (CE). Synthesis of CE is catalyzed by the plasma membrane-associated transglutaminase (TGm). Supplementation of the medium with inhibitors of cholesterologenesis suppressed increase in cholesterol levels and CE formation but did not interfere with TGm expression or TGm activity. Modulation of the plasma membrane cholesterol-phospholipid ratio of confluent NHK cultures using either pure phospholipid liposomes or liposomes enriched in cholesterol strongly affected spontaneous CE formation. Pure phospholipid liposomes completely inhibited CE formation, whereas cholesterol-enriched liposomes ensured envelope formation, even in the presence of inhibitors of cholesterol synthesis. From these results we conclude that in differentiating NHK an increase in the cellular cholesterol level is part of the differentiation program and is essential for the spontaneous CE formation.

The metabolic capacity of reconstituted epidermis from the outer root sheath cells of human hair follicles was determined. It was found that this epidermis possesses enzymes involved in both phase I (oxidation) and phase II (conjugation) reactions for drug biotransformation. The use of model substrates allowed the characterization of several isoenzymes. The homogenate fraction contained membrane-bound mixed-function oxydases (cytochrome P-450 dependent) involved in the O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzoxyresorufin, NADPH cytochrome c (P-450) reductase, testosterone 5 alpha-reductase, and UDP-glucuronosyltransferases, which conjugate 1-naphthol and bilirubin. One isoform of each glutathione S-transferase, steroid-, and arylsulfatases, acting on estrone- and 4-methylumbelliferone sulfates, was detected. Additionally, the activity of two distinct forms of epoxide hydrolases, which hydrate cis- and trans-stilbene oxides, could be measured. The presence of these drug metabolizing enzymes in the reconstituted epidermis indicates that it has a potential to serve as a model to study epidermal drug metabolism in vitro.

Psoriatic and control human hair follicle keratinocytes were cultured on bovine eye lens capsules in Epicult dishes for a period of 5-6 weeks and examined using light microscopy. The following morphological differences between cultures were observed: 1. The lower cell layers contained predominantly flattened cells in psoriatic cultures instead of roundish in control cultures. 2. The differentiation pattern was irregular in psoriatic cultures instead of regular in control cultures. 3. The differentiated zone of psoriatic cultures was more compact and thicker in comparison to normal cultures. These differences might allow discrimination between normal and psoriatic cultures.

Cross-linked envelope formation in the transformed human keratinocyte line SV-K14 requires treatment of the cells with a Ca2+ ionophore. Depending on the culture conditions, different extracellular Ca2+ concentrations are necessary to trigger the process which is catalyzed by the enzyme transglutaminase. Confluent cells grown in the presence of serum express only the cytosoluble form of the enzyme and need 5 mM Ca2+ for optimum protein cross-linking, whereas serum-starved cells which additionally contain the plasma membrane associated form of the enzyme require only 1 mM Ca2+. The envelope-like structures thus synthesized are morphologically and biochemically distinct.

When confluent cultures of the transformed human keratinocyte line SV-K14 are shifted to serum-free medium the cells achieve, within 4 days, the ability to synthesize a cornified envelope after challenge with the Ca2+ ionophore A23187. During these 4 days the enzyme transglutaminase (EC 2.3.2.13), which catalyses the cross-linking of different envelope precursor proteins, is partially transferred from the cytosolic pool into the plasma membrane. The association of the enzyme with the plasma membrane proves to be an essential step in the envelope formation since a direct correlation between plasma membrane-bound transglutaminase and envelope competence is observed. Retinoids block the insertion of the enzyme and therefore prevent envelope formation.

The transglutaminases of cultured normal and transformed human keratinocytes (line SV-K14) are characterized. Both cell types display two forms of the enzyme, one of which is cytosoluble (TGc) and the other which is associated with the plasma membrane (TGm). Normal keratinocytes contain predominantly TGm, and SV-K14 cells mainly TGc. The ratio of TGm to TGc can be modulated by the culture conditions and correlates with the competence of the cells to form a cornified envelope. TGm and TGc differ in their biochemical and immunological properties. SDS electrophoresis reveals apparent molecular weights of 92 and 85 kD, respectively. Only the activity of TGc is inhibited in the presence of guanosine 5'-triphosphate. Their response to Ca2+ is different: TGc exhibits a sigmoidal activation kinetics with an A50 value of about 200 microM, whereas the kinetics for TGm is hyperbolic with an A50 value of 75 microM. TGm reacts with a monoclonal antibody raised against epidermal "particulate" transglutaminase, and TGc with a polyclonal antibody raised against guinea pig liver transglutaminase. These reactions are very specific and no cross-reaction occurs. The coappearance of TGm with a proteolytic fragment (Mr 82,000) in the cytosol and intracellular particulate fraction of normal human keratinocytes is probably a preparation artifact.

The formation of a cornified envelope (CE) is a major event in the terminal differentiation of epidermal cells. Nomarski contrast microscopy of the envelopes purified from different sources reveals the existence of two major, but morphologically distinct classes: the very irregularily shaped fragile type CEf, and the polygonal rigid type CEr. Human keratinocytes in submerged culture are only able to produce type CEf. Specimens from healthy human epidermis contain largely type CEr. Psoriatic scales from different patients show both types in varying proportions. Tape stripping of normal epidermis reveals that type CEf is present in the lowermost layers of the stratum corneum and type CEr is present in the upper layers, indicating that the two types represent a different stage of maturation. Cyanogen bromide peptide mapping of electrophoretically purified envelopes reveals striking differences between cultured keratinocytes, normal epidermis, and psoriatic scales but also slight interindividual variations. This variability supports the view that the molecular CE composition is not strictly determined. On the other hand, no difference could be detected in the peptide maps of CEf and CEr obtained after tape stripping from the same healthy volunteer indicating that CE maturation within the stratum corneum does not involve the provision of qualitatively new proteins.

Human epidermal keratinocytes were cultured until sub-confluence in low Ca2+ (0.15 mM) serum-free synthetic MCDB 153 medium. Raising the Ca2+ concentration to 1.15 mM caused an increase in envelope competence as well as plasma membrane associated transglutaminase (TGm) activity. This increase was not observed when the high Ca2+ medium contained retinoic acid. Immunofluorescence studies as well as immunoblotting with the TGm-specific monoclonal antibody B.C1 revealed that retinoic acid inhibits expression of TGm. Isolation and in vitro translation of mRNA with subsequent immunoprecipitation showed that retinoic acid inhibits TGm expression at the pretranslational level.

SV-40 transformed human foreskin keratinocytes (line SV-K14) develop under conditions of serum starvation the competence to form cornified envelopes that are characteristic of terminally differentiating epidermal cells. In this cell line, the final assembly of the envelope does not occur spontaneously but must be induced using a calcium ionophore. Five potential precursor proteins with molecular weights of 140K, 90K, 61K, 53K, and 36K, respectively, could be detected in the extracts of envelope competent and noncompetent cells. The 61 kD and the 36 kD precursors were specifically decorated in immunoblots when using an antiserum directed against the purified cornified envelope of SV-K14 cells. The 140 kD protein was identified as involucrin by means of a commercial anti-involucrin antibody. Part of the 61 kD protein was found to be inserted into the plasma membrane after the cells gained envelope competence. The set of precursor proteins used by SV-K14 cells differed markedly from those described in the literature for epidermal cells in vivo and for normal human keratinocytes in vitro. Furthermore, cyanogen bromide cleavage of purified envelopes from transformed and normal keratinocytes revealed a completely different peptide pattern. This indicates that the exact molecular composition of the cornified envelope may not be strictly determined and may vary according to the availability of potential substrate proteins at the very moment when the cross-linking enzyme, the plasma membrane associated transglutaminase, becomes functional.