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CELLULAR AND MOLECULAR

A Novel Benzodiazepine Selectively Inhibits Keratinocyte Proliferation and Reduces Retinoid-Induced Epidermal Hyperplasia in Organ-Cultured Human Skin

Departments of Pathology (J.V., N.B., K.C.N., H.S., K.F., K.J.), Chemistry (A.E.B., G.D.G.), Dermatology (S.K.), and Obstetrics and Gynecology (A.W.O.), University of Michigan, Ann Arbor, Michigan

Received August 10, 2004; accepted December 1, 2004.

	Abstract

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Bz-423 is a new benzodiazepine that has cytotoxic and cytostatic effects against a number of cell types in culture, and recent studies have shown efficacy in experimental lupus models in rodents. The present study demonstrates that treating human skin in organ culture with Bz-423 suppresses retinoid-induced epidermal hyperplasia. Bz-423 suppresses hyperplasia in organ culture at concentrations that also inhibit keratinocyte proliferation in monolayer culture but that are not cytotoxic for keratinocytes and do not inhibit fibroblast growth. Under conditions in which keratinocyte proliferation is inhibited, there is no measurable effect on epidermal growth factor receptor activation, but there is reduced signaling at the level of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase. Suppression of keratinocyte growth by Bz-423 is associated with generation of intracellular oxidants. However, antioxidant treatment reduces keratinocyte cytotoxicity that occurs at high concentrations of Bz-423, but it does not inhibit growth suppression. Together, these data suggest that Bz-423 has the potential to limit the untoward effects associated with topical retinoid treatment, and in addition, may have therapeutic effects against other forms of epidermal hyperplasia.

Previously, we identified a novel antiproliferative and proapoptotic 1,4-benzodiazepine, termed Bz-423 (Blatt et al., 2002; Boitano et al., 2003a). Bz-423 and congeners with similar activity differ from benzodiazepines currently in clinical use by the presence of a hydrophobic substituent at the C3 position, which blocks binding to the central benzodiazepine receptor and renders binding to the peripheral benzodiazepine receptor weak (Boitano et al., 2003b). The antiproliferative and cytotoxic properties of Bz-423 are evident against malignant B and T cell lines in vitro. In vivo, this compound reduces pathologically expanded populations of B and T lymphocytes in autoimmune-prone (NZB x NZW) F1 and MRL-lpr strains of mice, respectively (Blatt et al., 2002; Bednarski et al., 2003). Reduction of activated lymphocytes by Bz-423 results in less autoimmune-mediated kidney damage (glomerulonephritis), preserves renal function, and in MRL-lpr mice, reduces autoimmune arthritis specific to this strain. Experiments with normal immune mice show no evidence of toxicity against normal lymphocyte populations, bone marrow cells, or solid tissues, including liver, heart, gastrointestinal mucosa, and skin.

The potent antiproliferative actions of Bz-423, its effectiveness at limiting disease manifestations in lupus, and its low index of toxicity in animals led us to consider whether this agent might be used to treat hyperplastic skin diseases such as psoriasis. As a first step, we used a model of retinoid-induced epidermal hyperplasia in human skin organ culture as the test system. Past studies have demonstrated that although the etiologies are different, retinoid hyperplasia (Varani et al., 2001), like the hyperplasia in psoriasis (Gottlieb et al., 1988; Elder et al., 1989; Cook et al., 1992; Piepkorn et al., 1998, 2003; Varani et al., 1998), involves intraepidermal production of ligands for the epidermal growth factor (EGF) receptor, and autocrine or paracrine EGF receptor activation. The results presented here demonstrate significant reduction in retinoid-stimulated hyperplasia without detrimental effects on normal keratinocyte proliferation or fibroblast function.

	Materials and Methods

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Benzodiazepines and Other Reagents. Bz-423 and Bz-OMe (Fig. 1), a congener of Bz-423 that differs from Bz-423 only by substitution of the phenolic hydroxyl group with a methoxy group, were synthesized as described previously (Bednarski et al., 2003). Three molecules used in this study for comparative purposes, including clonazepam and 4-chlorodiazepam [benzodiazepines that tightly bind the central and peripheral (mitochondrial) benzodiazepine receptors, respectively] and PK11195 an isoquinoline that binds tightly to the peripheral benzodiazepine receptor, were obtained from Sigma-Aldrich (St. Louis, MO). Each of the compounds was dissolved in dimethyl sulfoxide (DMSO) at 20 mg/ml and diluted in culture medium at the time of use. The concentration of DMSO in the final mixture was always less than 0.05 µl/ml, well below concentrations at which biological effects are observed in monolayer cultures of human skin cells or organ cultures of human skin under the conditions used here (Varani et al., 1994).

View larger version (11K): [in this window] [in a new window]   Fig. 1. Structure of Bz-423. Arrow indicates the phenolic hydroxyl group that is substituted with a methyl ester in Bz-OMe.

RA was obtained from Sigma-Aldrich. The retinoid was diluted in DMSO at 20 mg/ml and stored frozen. At the time of use, the RA stock solution was diluted in culture medium and used in organ culture studies at a final concentration of 1.0 µg/ml. DMSO was present at a final concentration of 0.5% (v/v) or less in all experiments. Reagents used in intracellular signaling studies included antibodies to total and phosphorylated forms of the EGF receptor and antibodies to total and phospho-Erk 1/2 (obtained from Cell Signaling Technology Inc., Beverly, MA). Mouse monoclonal anti-human Ki-67 antibody was from DakoCytomation Inc. (Carpinteria, CA). The superoxide dismutase mimetic manganese (III) meso-tetrakis (4-benzoic acid) porphyrin (MnTBAP), was obtained from Sigma Aldrich.

Human Skin Organ Cultures. Replicate 2-mm full-thickness punch biopsies of sun-protected hip skin were obtained from young adult volunteers. This project was approved by the University of Michigan Institutional Review Board. All subjects provided written informed consent before participation in the study. Immediately upon biopsy, the tissue was immersed in culture medium consisting of keratinocyte basal medium (KBM) (Cambrex Bio Science Walkersville, Inc., Walkersville, MD). KBM is a low-Ca²⁺, serum-free modification of MCDB-153 medium. It was supplemented with CaCl₂ to bring the final Ca²⁺ concentration to 1.4 mM. After transport to the laboratory on ice, the biopsies were incubated in wells of a 24-well dish containing 400 µl of Ca²⁺-supplemented KBM with or without additional treatments [i.e., RA and Bz-423 (or DMSO control)] as described under Results. Cultures were incubated at 37°C in an atmosphere of 95% air and 5% CO₂. Other than to maintain the tissue in a minimal volume of medium, nothing further was done to ensure a strict air-liquid interface. Incubation was for 8 days, with change of medium and fresh treatments every second day. At the end of the incubation period, tissue was fixed in 10% buffered formalin and examined histologically after staining with hematoxylin and eosin. Routinely, three to six tissue sections were prepared from each block. Epidermal thickness measurements were made at four sites in each tissue section (distance from the dermal-epidermal juncture to the top of the viable portion of the epithelium) and averaged. Average thickness values for each group of tissues were determined. The organ culture procedure has been described previously (Varani et al., 1993, 1994).

Human Epidermal Keratinocytes and Dermal Fibroblasts in Monolayer Culture. Epidermal keratinocytes were isolated as described previously (Varani et al., 1994). Primary and early passage cells were maintained in keratinocyte growth medium (KGM) (Cambrex Bio Science Walkerville, Inc.). KGM contains the same basal medium as KBM but is further supplemented with a mixture of growth factors, including 0.1 ng/ml EGF, 0.5 µg/ml insulin, and 0.4% bovine pituitary extract. Fibroblasts obtained from the same tissue were grown in monolayer culture using Dulbecco's modified minimal essential medium supplemented with nonessential amino acids and 10% fetal bovine serum. Both keratinocytes and fibroblasts were maintained at 37°C in an atmosphere of 95% air and 5% CO₂. Cells were subcultured by exposure to EDTA and used at passage 2 to 4.

Proliferation Assays. Keratinocyte proliferation was assessed by seeding 4 x 10⁴ cells/well in a 24-well plate using KGM as culture medium. After the cells had attached (overnight), they were washed, and triplicate samples were harvested for zero time counts. The remaining cells were then incubated in KGM with different concentrations of Bz-423, Bz-OMe, or control compounds as indicated in Fig. 2 and Table 2. DMSO served as a negative control. Proliferation was measured on day 3 by releasing the cells with trypsin/EDTA and enumerating them using a particle counter (Beckman Coulter, Inc., Fullerton, CA). The use of KGM [growth factor-supplemented, low-Ca²⁺ (0.15 mM) medium] with keratinocytes at a starting concentration of approximately 2 x 10⁴ cells/cm² of surface area (subconfluent but higher than clonal density) is designed for optimal growth and to prevent differentiation from occurring over a 3-day period (Varani et al., 1989). Fibroblast proliferation studies were conducted in the same manner, except KBM supplemented with 1.4 mM Ca²⁺ was used as culture medium.

View larger version (19K): [in this window] [in a new window]   Fig. 2. Bz-423 inhibits proliferation of rapidly growing keratinocytes and fibroblasts. Keratinocytes were plated at 4 x 104 cells/well in KGM. After the cells had attached, wells were harvested and counted for time zero (T0) values. The remaining wells were washed and incubated for 72 h in KGM with the indicated amounts of Bz-423. At the end of the incubation period, samples were harvested and counted. The values shown represent mean number of cells per well ± standard errors based on five separate experiments with duplicate or triplicate samples per data point. Fibroblast proliferation was determined the same way except the initial plating medium was Dulbecco's modified minimal essential medium supplemented with nonessential amino acids and 10% fetal bovine serum, and the incubation medium was Ca2+-supplemented KBM. Fibroblast data are based on triplicate experiments. Statistical significance was determined by ANOVA, followed by paired group comparisons. *, statistically significant difference from the vehicle alone (0.05% DMSO) value at p < 0.05 levels.

Cytotoxicity Assay. Keratinocytes were seeded at 4 x 10⁴ cells/well in a 24-well plate using KGM as culture medium. After the cells had attached, they were washed and triplicate samples were harvested for zero time counts. The remaining cells were then incubated in KGM with different concentrations of Bz-423. Four hours later, cells were harvested and counted. Cells were replated in wells of a 24-well dish using KGM alone as culture medium. After incubation for an additional 18 h, the cells that had attached were harvested and counted. The percentage of cells that attached after exposure to varying concentrations of Bz-423 was compared with the percentage of untreated cells that attached. Cytotoxicity was determined from these data.

Preparation of Cell Lysates and Immunoblot Analysis of Signaling Intermediates. Keratinocytes were plated at 3 x 10⁵ cells/well in wells of a six-well dish using KGM as culture medium. The cells were allowed to attach overnight. The next day, they were washed and then incubated for 1 h in KBM with or without Bz-423 (0.5 or 1.0 µg/ml). After this, 10 ng of EGF was added. After an additional incubation for 5 or 15 min, cells were lysed in 1x cell lysis buffer consisting of 20 mM Tris-HCl, pH 7.4, 2 mM sodium vanadate, 1.0 mM sodium fluoride, 100 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 25 µg/ml each of aprotinin, leupeptin, and pepstatin, and 2 mM EDTA and EGTA. Lysis was performed at 4°C by scraping the cells into lysis buffer and sonicating the samples. Cell lysates were incubated on ice for 15 min and then cleared by microcentrifugation at 16,000g for 15 min. The supernatant fluids were collected and protein concentrations estimated using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA).

Western blotting for total and phosphorylated forms of the EGF receptor and for total and phosphorylated forms of Erk 1/2 was carried out as described previously (Zeigler et al., 1999; Bhagavathula et al., 2004). Briefly, samples were separated in SDS-polyacrylamide gel electrophoresis under denaturing and reducing conditions and transferred to nitrocellulose membranes. After blocking with a 5% nonfat milk solution in Tris-buffered saline with 0.1% Tween (TTBS) at 4°C overnight, membranes were incubated for 1 h at room temperature with the desired antibody, diluted 1:1000 in 5% nonfat milk/0.1% TTBS. Thereafter, the membranes were washed with TTBS, and bound antibody was detected using the Phototopehorseradish peroxidase Western blot detection kit (Cell Signaling Technology Inc.). A Kodak 1000 X-OMAT processor was used to capture the positive images of the Western blots, and these positive images were scanned and digitized. The digitized images were quantitated using NIH Image analysis software.

Detection of Intracellular Reactive Oxygen Species (ROS). 2',7'-Dichlorodihydrofluorescin diacetate (DCFH-DA; Molecular Probes, Eugene, OR) was prepared as a 10 mM stock solution in DMSO before each use. Cells growing in 48-well plates were loaded (30 min, 37°C) with DCFH-DA (100 µM) added directly to culture media. The cells were then washed and placed in fresh media before treatment. After the indicated treatments, the fluorescence of the oxidized product 2',7'-dichlorofluorescin (DCF) was monitored by flow cytometry using a FACSCalibur (BD Bioscience, San Diego, CA). For each sample, 10,000 events were recorded and the data analyzed to determine median fluorescence intensity.

Statistical Analysis. Differences between groups in experiments with multiple groups were analyzed for statistical significance by ANOVA followed by paired-group comparisons. Where there were only two groups, the Student's t test was used. Data were analyzed as paired or unpaired as appropriate.

	Results

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Keratinocytes Are More Sensitive Than Fibroblasts to the Antiproliferative Effects of Bz-423. In vitro keratinocyte and fibroblast proliferation assays were used to test the hypothesis that Bz-423 inhibits keratinocyte proliferation selectively with respect to fibroblasts. Human epidermal keratinocytes and human dermal fibroblasts were maintained in monolayer culture and examined for growth under control conditions and in response to Bz-423. The dose-dependent effects of Bz-423 on proliferation of both cell types over a 72-h period are shown in Fig. 2. These results demonstrate that keratinocytes are more sensitive than fibroblasts to the antiproliferative effects of Bz-423. The results also indicate that although Bz-423 at 0.5 to 2 µM concentrations reduced the net number of cells compared with control conditions, there was still a net cell increase compared with the number of cells present at time zero. Additionally, when the cultures were examined microscopically for dead cells (trypan blue-positive cells, cell ghosts, and cell debris) after incubation for 72 h, virtually none were detected. This suggests that those concentrations of Bz-423 were growth inhibitory but not cytotoxic. In contrast, at 4 and 10 µM concentrations, the number of cells present at the end of the incubation period was reduced compared with the number at time zero, and the culture medium contained many dead cells and much cell debris.

To confirm the concentration-dependent differences in growth inhibition versus cell death as detected in the proliferation assay, the capacity of Bz-423 to lethally injure keratinocytes was formally examined in a 4-h cytotoxicity assay. Consistent with results from the proliferation assays, Bz-423 at 1 and 2 µM did not inhibit the ability of treated cells to replate successfully after exposure (i.e., was not cytotoxic), whereas at 4 µM, ability of the treated cells to replate was greatly reduced (Table 1).

To determine whether the effects of Bz-423 on keratinocyte and fibroblast proliferation were specific for Bz-423, we compared the response to Bz-423 with responses to other selected benzodiazepines, and PK11195, which binds tightly to the peripheral benzodiazepine receptor. One of the compounds examined was Bz-OMe, a congener of Bz-423 that differs from Bz-423 only by methylation of the phenolic hydroxyl group (Fig. 1). Bz-OMe has potent antiproliferative and minimal cytotoxic activities against lymphoid cells (Boitano et al., 2003a). As expected, Bz-423 proved to be a more potent inhibitor of keratinocyte proliferation than the other compounds tested (Table 2). Bz-OMe was intermediary and the other benzodiazapines were inactive against keratinocytes and fibroblasts at concentrations up to 10 µM.

Bz-423 Increases ROS in Keratinocytes and Fibroblasts. In lymphoid cells, Bz-423 increases intracellular superoxide within 1 h of treatment, and this oxidant response is necessary for the cytotoxic and antiproliferative effects that follow (Blatt et al., 2002; Boitano et al., 2003a). To determine whether the antiproliferative and cytotoxic effects of Bz-423 in keratinocytes and fibroblasts similarly involve ROS generation, intracellular ROS levels in control and Bz-423-treated cells were measured. As shown in Fig. 3, a significant ROS response, assessed as median cell fluorescence from the ROS-sensitive indicator DCF, was observed in both cell types treated with Bz-423. ROS was detected at a dose of Bz-423 as low as 0.5 µM, and the response increased in a dose-dependent manner in both cell types. At all concentrations of Bz-423 tested, keratinocytes generated a greater ROS response than fibroblasts. As a second way to assess ROS generation, the percentage of cells with fluorescence values above baseline levels was determined. Similar differences between keratinocytes and fibroblasts were observed when ROS generation was evaluated in this manner (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Bz-423 increases ROS in cells within 1 h of treatment. Keratinocytes and fibroblasts were plated in their respective growth media at 2 x 104 cells/well in 48-well dishes. After the cells had attached, they were loaded with the ROS-specific indicator DCFH for 30 min and then washed. After this, the cells were incubated with vehicle alone (0.05% DMSO) or with Bz-423 at the indicated concentrations for 1 h and then analyzed by flow cytometry. Average DCF fluorescence intensity values ± standard deviations in a single experiment with triplicate data points are displayed. Each cell type was assessed four times with consistent results.

In subsequent experiments, proliferation assays were carried out with keratinocytes after treatment with Bz-423 in the presence of MnTBAP, a superoxide dismutase mimetic that blocks Bz-423-induced intracellular superoxide accumulation (Bednarski et al., 2003; Boitano et al., 2003a). Keratinocytes were treated for 30 min with MnTBAP (1 µM) before Bz-423 and then exposed to Bz-423 for 72 h. When Bz-423 was used at a concentration of 1 or 2 µM (concentrations that inhibit keratinocyte proliferation but are not cytotoxic), MnTBAP did not block the antiproliferative effects of Bz-423 (Fig. 4). In contrast, when keratinocytes were treated with cytotoxic concentrations of Bz-423 (4 µM), significant protection, albeit partial, was observed (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4. MnTBAP inhibits cytotoxicity but not growth suppression in Bz-423-treated, proliferating keratinocytes. Keratinocytes were plated at 4 x 104 cells/well in KGM. After the cells had attached, wells were harvested and counted for time zero (T0) values. The remaining wells were washed and pretreated for 30 min with 1 µM MnTBAP. After this, they were incubated for 72 h in KGM with the indicated amounts of Bz-423 and MnTBAP. At the end of the incubation period, cells were harvested and counted. The values shown represent mean number of cells per well ± standard errors based on four separate experiments with duplicate or triplicate samples per data point. Statistical significance was determined by ANOVA, followed by paired-group comparisons. *, statistically significant difference from the vehicle alone (0.05% DMSO); value at p < 0.05 levels.

Effects of Bz-423 on EGF Receptor Expression and Erk Phosphorylation in Keratinocytes. EGF receptor activation and downstream signaling through MAP kinase pathways: 1) are engaged in response to stimuli that induce keratinocyte proliferation and 2) play an important role in the pathogenesis of epidermal hyperplasia. Therefore, we hypothesized that Bz-423 treatment might affect EGF receptor activation and/or MAP kinase (Erk 1/2) signaling. To test these possibilities, total and phosphorylated forms of the EGF receptor were measured in untreated and Bz-423 (0.5 and 1 µM)-treated keratinocytes after mitogen stimulation. No differences between Bz-423 treated and control cells in the levels of total or phosphorylated EGF receptor were found (Fig. 5). In a like manner, the phosphorylation status of Erk 1/2 before and shortly after (5 min) mitogen stimulation of keratinocytes was assessed in the presence or absence of Bz-423. Although no change was observed in total Erk 1/2 protein, EGF-induced Erk phosphorylation was significantly reduced by Bz-423 (Fig. 6). Together, these results indicate that the antiproliferative action of Bz-423 in keratinocytes is associated with reduced Erk 1/2 activation but that the effect seems to be mediated downstream of (or independent of) EGF receptor expression/activation.

View larger version (32K): [in this window] [in a new window]   Fig. 5. EGF receptor activation in Bz-423-treated, proliferating keratinocytes. Keratinocytes were plated at 3 x 105 cells/well in wells of a six-well dish using KGM as culture medium. After the cells had attached, they were washed and then incubated for 1 h in KBM with or without Bz-423 (0.5 or 1.0 µM). After this, 10 ng of EGF was added. After an additional incubation for 5 or 15 min, cells were lysed. Lysates prepared from replicate samples just prior to EGF addition, 5 and 15 min after EGF stimulation were analyzed for total and phosphorylated EGF receptor expression. Top, Western blots showing equivalent amounts of phosphorylated EGF receptor in the absence or presence of Bz-423. Bottom, quantification of Western blots. Blots were digitized, scanned, and quantified using NIH Image software. The experiment was repeated three times, and the values shown represent means and standard deviations based on the three experiments. Values shown for the 5-min control in each experiment were arbitrarily set at 1.0, and the other values were normalized to the control. T0 control refers to cells incubated in 0.05% DMSO and harvested before EGF addition. In these cells, densitometry-scanned images were never above background.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Erk 1/2 activation in Bz-423-treated, proliferating keratinocytes. Experimental design was as indicated in the legend to Fig. 5, except that lysates were analyzed for total and phosphorylated Erk 1/2 expression. Top, Western blots showing reduced amounts of phosphorylated Erk 1/2 but equivalent amounts of total Erk 1/2 in the absence or presence of Bz-423. Bottom, quantification of Western blots. Blots were digitized, scanned, and quantified using NIH Image software. The experiment was repeated three times, and the values shown represent means and standard deviations based on the three experiments. Values shown for the T0 control in each experiment were arbitrarily set at 1.0, and the other values were normalized to the control. T0 control refers to cells incubated in 0.05% DMSO and harvested before EGF addition. *, statistically significant difference from the control value at each time point at the p < 0.05 level.

Bz-423 Reduces Epidermal Thickness of RA-Treated Human Skin in Organ Culture. In a final series of experiments, 2-mm punch biopsies of human skin from healthy volunteers were incubated in organ culture for 8 days. Tissue incubated under control conditions (i.e., in the absence of both RA and Bz-423) maintained histological features of normal skin (Fig. 7, A and D). Consistent with what occurs in vivo after retinoid application (Kang et al., 1995) and consistent with past observations in organ culture (Varani et al., 2001), epidermal hyperplasia developed in biopsies from the same subjects upon incubation in the presence of RA [1 µg/ml in 0.01% (v/v) DMSO vehicle] (Fig. 7, B and E). When biopsy specimens were cultured in media containing both RA (1 µg/ml) and Bz-423 (1 µM), the hyperproliferative response of the epithelium was substantially reduced (Fig. 7, C and F). Average epidermal thickness measurements of skin from five separate human donors revealed a highly significant reduction in RA-induced epidermal thickening by Bz-423 (Fig. 7, bottom). In untreated skin, the average distance from the dermal-epidermal junction to the top of the viable portion of the epidermis was 92 ± 12 µm. In the presence of RA (1 µg/ml), epidermal thickness increased to 200 ± 16 µm, whereas in the presence of RA (1 µg/ml) plus Bz-423 (1 µM), epidermal thickness was 132 ± 13 µm (p < 0.05). Careful microscopic evaluation of biopsy specimens treated with Bz-423 failed to show any additional histological changes that could be ascribed to Bz-423. In particular, no differences in the cellularity or structure of the dermis, no changes in the dermal-epidermal interface, and no effects on keratinocyte differentiation and keratinization were evident. In addition, Bz-423-treated specimens were notable for the lack of increased apoptotic cells. In additional studies (not shown), RA-exposed skin was treated with Bz-423 at different concentrations and examined for epidermal thickness. At 0.5 µM, epidermal thickening was also reduced but this concentration was less effective than 1 µM. A reduction in epidermal thickness was also observed at higher concentrations of Bz-423 (i.e., at 5 µM), but necrosis was also observed.

View larger version (77K): [in this window] [in a new window]   Fig. 7. Bz-423 reduces retinoid-induced epidermal hyperplasia. Top, histological appearance. Organ cultures of skin were incubated for 8 days as described under Materials and Methods and examined by light microscopy after staining with hematoxylin and eosin. A and D, untreated skin maintains normal histological appearance. B and E, skin cultured in the continuous presence of RA (1 µg/ml) demonstrates marked epidermal hyperplasia. C and F, RA-induced epidermal thickening is substantially reduced in specimens cultured in media containing RA (1 µg/ml) and Bz-423 (1 µM). (A–C, 42x, D–F, 84x). Bottom, quantitative data: epidermal thickness measurements were made at four sites in three to six sections per tissue and averaged. Values shown are means and standard errors based on organ cultures from six different subjects. Statistical significance was assessed by ANOVA followed by paired group comparisons. Difference between untreated control and RA values were statistically different at the p < 0.05 level. Difference between RA alone and RA plus Bz-423 values were statistically different at the p < 0.05 level.

In addition to using epidermal thickness as a measure, we also assessed the effects of Bz-423 on epidermal expression of Ki-67. Tissue was obtained from four donors and incubated for 8 days in the presence of 1 µg/ml RA with or without 1 µM Bz-423. At the end of the incubation period, reactivity with an antibody to the proliferation-associated antigen, Ki-67 was assessed. Consistent with past findings (Varani et al., 2001), there were a large number of Ki-67-positive cells in sections treated with RA alone (9.4 ± 1.5 per 400x high power field; mean ± S.E.M.; n = 3). In contrast, fewer reactive cells were observed in tissue sections that had been treated with RA and Bz-423 (5.7 ± 2.1 per high-power field).

Finally, 2-mm skin biopsies from four donors were incubated in the absence of RA but treated with 1 µM Bz-423. In the normal skin, minimal change in histological structure was observed. Likewise, there was minimal change in epidermal thickness (Fig. 8).

View larger version (64K): [in this window] [in a new window]   Fig. 8. Histological appearance of control organ-cultured skin in the presence of Bz-423. Top, histological appearance. Organ cultures of skin were incubated for 8 days as described under Materials and Methods and examined by light microscopy after staining with hematoxylin and eosin. A, untreated control skin maintains normal histologic appearance. B, Bz-423 (1 µM)-treated control skin maintains normal histological appearance. A and B (115x), bottom, quantitative data: epidermal thickness measurements were made at four sites in three to six sections per tissue and averaged. Values shown are means and standard errors based on organ cultures from four different subjects.

	Discussion

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Bz-423 was initially discovered as a proapoptotic agent that has the capacity to effectively treat autoimmune disease in several animal models (Blatt et al., 2002; Bednarski et al., 2003). Other work has demonstrated the compound's ability to block proliferation independently of cytotoxicity in cells derived from malignant B cell neoplasms (Boitano et al., 2003a). The present study demonstrates that Bz-423 has the capacity to inhibit (retinoid-induced) hyperplastic growth in the epidermis of human skin without affecting normal skin physiology.

Capacity to interfere with retinoid-induced epidermal hyperplasia without affecting normal epidermal growth or dermal function provides a significant therapeutic opportunity. It is generally accepted that hyperplasia in skin after topical application of RA accounts for the attendant skin irritation that accompanies retinoid treatment in many individuals (Kang et al., 1995). The cellular and molecular events that underlie the irritation response are not fully understood. Elaboration of interleukin-1 and other cytokines within the rapidly proliferating keratinocyte microenvironment is thought to be involved (Wood et al., 1996; Maas-Szabowski et al., 2000). These cytokines produce localized changes in vascular function (Nguyen et al., 2001), which, in turn, promote edema and inflammatory cell influx into the skin. Concomitantly, shedding of excess epithelium produces the "skin flaking" commonly observed in retinoid-treated skin. At one time, it was believed that retinoid action in the epidermis and dermis occurred through the same pathways. As such, the beneficial effects of retinoid treatment in the dermis (i.e., fibroblast activation, increased procollagen production and decreased elaboration of matrix metalloproteinases) (Griffiths et al., 1993; Fisher et al., 1996; Varani et al., 2000) and skin irritation were thought to be inseparable. The present studies are consistent with recent findings suggesting that may not be entirely the cases (Varani et al., 2001).

An agent with the capacity to interfere with epidermal hyperplasia may also find use as an antipsoriatic agent. A number of approaches have led to the conclusion that the triggering event in psoriasis is an immune system defect (Valdimarsson et al., 1995; Austin et al., 1999). Nevertheless, the downstream events that precipitate psoriatic hyperplasia (like those in retinoid hyperplasia), involve keratinocyte growth regulation (Gottlieb et al., 1988; Elder et al., 1989; Cook et al., 1992; Piepkorn et al., 1998, 2003; Varani et al., 1998). Utility of agents that target abnormal keratinocyte proliferation for treatment of hyperplastic skin diseases will, ultimately, require formulation for topical delivery. Although it is not yet known whether Bz-423 and congeners can be successfully formulated for topical delivery, the physical properties of Bz-423 and related compounds (e.g., formula weight <500 molecular weight units) provide a highly favorable context from which to pursue this goal.

The mechanism by which Bz-423 suppresses hyperplastic epidermal growth is not fully understood. Past studies conducted with malignant B-lymphocytes demonstrated that low-level generation of intracellular ROS was correlated with growth inhibition and generation of higher amounts of ROS with cytotoxicity (Boitano et al., 2003a). Similarly, it was shown in the present study that concentrations of Bz-423 that induced ROS production in epidermal keratinocytes also reduced keratinocyte proliferation in monolayer culture and suppressed hyperplasia in organ culture. Furthermore, epidermal keratinocytes, which were more sensitive to the growth-inhibiting effects of Bz-423 than dermal fibroblasts, generated greater amounts of ROS at lower concentrations of Bz-423 than did fibroblasts. Arguing against this, however, is the fact that growth inhibition was not suppressed by MnTBAP, which blocks accumulation of superoxide in cells treated with Bz-423. Additional studies will be necessary to completely elucidate the role of intracellular ROS as an intracellular signal leading to keratinocyte growth inhibition.

Past studies have shown that epidermal hyperplasia (including both retinoid hyperplasia and psoriasis) involves intracutaneous production of ligands for the EGF receptor, leading to autocrine or paracrine EGF receptor activation (Gottlieb et al., 1988; Elder et al., 1989; Cook et al., 1992; Pittelkow et al., 1993; Piepkorn et al., 1998, 2003; Varani et al., 1998, 2001), and downstream signaling through MAP kinase pathways (Alpin et al., 1998). In the present study, we did not observe a change in phosphorylation status of the EGF receptor as a consequence of treatment with Bz-423. On the other hand, Erk 1/2 phosphorylation was reduced by the same treatment. Erk 1/2 activation (evidenced by phosphorylation) is a down-stream target of EGF receptor activation, but also occurs in response to multiple other receptor-ligand interactions (Alpin et al., 1998). It is reasonable to suggest that Bz-423 uncouples signaling events linking EGF receptor activation to Erk 1/2 but might also act to interfere with signals originating from other sources that also induce Erk 1/2 activation. Of interest in this regard, past studies have shown that thiazoladinediones also inhibit Erk 1/2 phosphorylation in keratinocytes after EGF stimulation (Bhagavathula et al., 2004). Similar to BZ-423, the thiazoladinediones also suppress epidermal hyperplasia in organ culture. More importantly, these agents also reduce epidermal thickness of psoriatic lesional skin transplanted to SCID mice (Ellis et al., 2000) and have proven clinically useful in treatment of psoriasis (Ellis et al., 2000; Malhotra et al., 2002). Perhaps Bz-423 will also prove to be beneficial in psoriasis. It should be noted that it is not necessary to completely suppress Erk 1/2 activation to prevent epidermal hyperplasia. In fact, complete suppression of Erk phosphorylation is associated with cytotoxicity rather than cytostasis (Zeigler et al., 1999).

In summary, Bz-423 is a benzodiazepine that has cytotoxic and cytostatic effects on a number of cell types in culture. In the present study, it is demonstrated that treatment of human skin in organ culture with Bz-423 suppresses epidermal hyperplasia, resulting from concomitant retinoid treatment. Ability to suppress retinoid-induced hyperplasia in human skin organ culture argues for the potential of this class of molecules to mitigate the retinoid irritation response in skin. Beneficial effects against other forms of epidermal hyperplasia are also suggested.

	Footnotes

 
This study was supported in part by Grants DK59169, AR49621, and AI47450 from the U.S. Public Health Service.

doi:10.1124/jpet.104.075929.

ABBREVIATIONS: RA, all-trans retinoic acid; EGF, epidermal growth factor; PK11195, 1-(2-chlorophenyl)-N-(1-methylpropyl)-3-isoquinolinecarboxamide; DMSO, dimethyl sulfoxide; Erk, extracellular signal-regulated kinase; MnTBAP, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin; KBM, keratinocyte basal medium; KGM, keratinocyte growth medium; TTBS, Tris-buffered saline with 0.1% Tween; ROS, reactive oxygen species; DCFH-DA, 2',7'-dichlorodihydrofluorescin diacetate; DCF, 2',7'-dichlorofluorescin; ANOVA, analysis of variance; MAP, mitogen-activated protein.

Address correspondence to: Dr. James Varani, Department of Pathology, The University of Michigan, 1301 Catherine Rd./Box 0602, Ann Arbor, MI 48109. E-mail: varani{at}umich.edu

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