Circulating cytokines and monocyte subpopulations as biomarkers of outcome and biological activity in sunitinib-treated patients with advanced neuroendocrine tumours.

BACKGROUND: Sunitinib is approved worldwide for treatment of advanced pancreatic neuroendocrine tumours (pNET), but no validated markers exist to predict response. This analysis explored biomarkers associated with sunitinib activity and clinical benefit in patients with pNET and carcinoid tumours in a phase II study. METHODS: Plasma was assessed for vascular endothelial growth factor (VEGF)-A, soluble VEGF receptor (sVEGFR)-2, sVEGFR-3, interleukin (IL)-8 (n=105), and stromal cell-derived factor (SDF)-1α (n=28). Pre-treatment levels were compared between tumour types and correlated with response, progression-free (PFS), and overall survival (OS). Changes in circulating myelomonocytic and endothelial cells were also analysed. RESULTS: Stromal cell-derived factor-1α and sVEGFR-2 levels were higher in pNET than in carcinoid (P=0.003 and 0.041, respectively). High (above-median) baseline SDF-1α was associated with worse PFS, OS, and response in pNET, and high sVEGFR-2 with longer OS (P⩽0.05). For carcinoid, high IL-8, sVEGFR-3, and SDF-1α were associated with shorter PFS and OS, and high IL-8 and SDF-1α with worse response (P⩽0.05). Among circulating cell types, monocytes showed the largest on-treatment decrease, particularly CD14+ monocytes co-expressing VEGFR-1 or CXCR4. CONCLUSIONS: Interleukin-8, sVEGFR-3, and SDF-1α were identified as predictors of sunitinib clinical outcome. Putative pro-tumorigenic CXCR4+ and VEGFR-1+ monocytes represent novel candidate markers and biologically relevant targets explaining the activity of sunitinib.

Sunitinib in combination with docetaxel and prednisone in chemotherapy-naive patients with metastatic, castration-resistant prostate cancer: a phase 1/2 clinical trial.

BACKGROUND: This phase 1/2 study assessed sunitinib combined with docetaxel (Taxotere) and prednisone in chemotherapy-naive metastatic, castration-resistant prostate cancer (mCRPC) patients. PATIENTS AND METHODS: To determine the recommended phase 2 dose (RP2D), 25 patients in four dose escalation cohorts received 3-week cycles of sunitinib (2 weeks on, 1 week off), docetaxel and prednisone, preceded by a 4-week sunitinib 50 mg/day lead in. RP2D was evaluated in 55 additional patients. The primary end point was prostate-specific antigen (PSA) response rate. RESULTS: One phase 1 dose-limiting toxicity occurred (grade 3 hyponatremia). The RP2D was sunitinib 37.5 mg/day, docetaxel 75 mg/m(2) and prednisone 5 mg b.i.d. During phase 2, confirmed PSA responses occurred in 31 patients [56.4% (95% confidence interval 42.3-69.7)]. Median time to PSA progression was 9.8 months. Forty-one patients (75%) were treated >3 months, 12 (22%) completed the study (16 cycles) and 43 (78%) discontinued (36% for disease progression and 27% adverse events). The most frequent treatment-related grade 3/4 adverse events were neutropenia (53%; 15% febrile) and fatigue/asthenia (16%). Among 33 assessable patients, 14 (42.4%) had confirmed partial response. Median progression-free and overall survivals were 12.6 and 21.7 months, respectively. CONCLUSION: This combination was moderately well tolerated, with promising response rate and survival benefit, justifying further investigation in mCRPC.

Clinical evaluation of continuous daily dosing of sunitinib malate in patients with advanced gastrointestinal stromal tumour after imatinib failure.

AIMS: To assess the antitumour activity, safety, pharmacokinetics and pharmacodynamics of continuous daily sunitinib dosing in patients with imatinib-resistant/intolerant gastrointestinal stromal tumour (GIST) and to assess morning dosing versus evening dosing. PATIENTS AND METHODS: In this open-label phase II study, patients were randomised to receive morning or evening dosing of sunitinib 37.5mg/day. The primary end-point was clinical benefit rate (CBR; percent complete responses+partial responses [PRs]+stable disease [SD] 24 weeks). Secondary end-points included progression-free survival (PFS), overall survival (OS), safety, pharmacokinetic parameters and plasma biomarker levels. RESULTS: Sixty of 61 planned patients received treatment (30 per dosing group); 26 completed the study. Overall, the CBR was 53% (95% exact CI, 40-66): eight patients (13%) achieved objective PRs; 24 (40%) achieved SD 24 weeks. Median PFS was 34 weeks (95% CI, 24-49); median OS was 107 weeks (95% CI, 72 - not yet calculable). Most adverse events (AEs) were of grade 1 or 2 in severity, and were manageable through dose modification or standard interventions. No new AEs were apparent compared with the approved intermittent dosing schedule. Antitumour activity and safety were generally similar with morning and evening dosing. Continuous daily sunitinib dosing achieved and sustained effective drug concentrations without additional accumulation across cycles. Decreases from baseline in plasma levels of soluble KIT after 20 and 24 weeks of dosing correlated with longer OS. CONCLUSION: For patients with imatinib-resistant/intolerant GIST, continuous daily sunitinib dosing appears to be an active alternative dosing strategy with acceptable safety.

Evidence that activation of protein kinase A inhibits human hair follicle growth and hair fibre production in organ culture and DNA synthesis in human and mouse hair follicle organ culture.

We have investigated the possibility that protein kinase A (PKA) may play a part in regulating the activity of human and mouse hair follicles in whole organ culture. Human hair follicles were isolated from facial skin by microdissection, and hair follicle and hair fibre length measurements were made daily during suspension culture. Incubation of human hair follicles with dibutyryl-cAMP (db-cAMP) resulted in a dose-dependent inhibition of total cumulative follicle growth (IC50 = 100 mumol/L, 85% inhibition at 1 mmol/L). db-cAMP (0.5 mmol/L) also caused a rapid, partial inhibition of follicular DNA synthesis (20.3% inhibition at 6 h, 48.0% inhibition at 24 h). Human hair follicle growth was inhibited by the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and Ro 20-1724, and by the adenylate cyclase activator, forskolin. In addition, db-cAMP inhibited DNA synthesis in organ cultures of whisker follicles isolated from neonatal mice by microdissection. Taken together, these findings indicate that agents which increase cAMP levels are potent inhibitors of human and mouse hair follicle growth, and suggest that PKA may play a part in the regulation of hair follicle activity in vivo.

Interleukin-1beta is differentially expressed by human dermal papilla cells in response to PKC activation and is a potent inhibitor of human hair follicle growth in organ culture.

The dermal papilla plays an important role in the regulation of hair follicle matrix cell proliferation and hair fiber production, at least in part through mesenchymal-epithelial interactions. In the present study, we have investigated the regulation of interleukin-1 (IL-1) production by protein kinase C in cultured human dermal papilla cells. Treatment of dermal papilla cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) elicited the rapid and transient production of mature (17 kDa) cytosolic IL-1beta protein, but not IL-1alpha, with maximal levels achieved after 12 h. Rapid secretion of IL-1beta into the medium occurred subsequent to increased intracellular cytokine levels, after which medium IL-1beta protein levels were stable for 4 days. Northern blot analysis showed that TPA treatment elicited a transient induction of IL-1beta mRNA expression, maximal after 12 h, indicating that TPA regulates dermal papilla cell IL-1beta production at the transcriptional level. Pretreatment of dermal papilla cells with Ro 31-7549, a selective protein kinase C inhibitor, dose dependently and completely reversed phorbol-induced IL-1beta protein production. In addition, we demonstrated that IL-1beta is a highly potent inhibitor of the growth of human hair follicles in whole-organ culture, with an IC50 value of approximately 5 pg/ml. These findings, taken together with a previous report that follicular matrix cells express type I IL-1 receptors but dermal papilla cells do not, raise the possibility that dermal papilla cell-derived IL-1beta may act as a negative paracrine factor in the regulation of matrix cell proliferation.

Bisindolylmaleimide protein-kinase-C inhibitors delay the decline in DNA synthesis in mouse hair follicle organ cultures.

We have used a series of bisindolylmaleimide selective protein-kinase C (PKC) inhibitors to investigate the role of this enzyme in the regulation of cell proliferation in mouse hair follicle organ cultures. Mouse whisker follicles were isolated by microdissection, and rates of DNA synthesis during culture were determined from 3H-thymidine incorporation. The bisindolylmaleimides Ro 31-7549, Ro 31-8161, Ro 31-8425 and Ro 31-8830 inhibit isolated brain PKC with IC50 values of 8-80 nM, are > 60-fold less potent against protein kinase A, and inhibit PKC-mediated protein phosphorylation in platelets with IC50 values in the range 0.25-4.4 microM. These PKC inhibitors were found to increase levels of mouse hair follicle DNA synthesis, with EC50 values in the range 1-4 microM and maximal levels in the range 151-197% of control. Ro 31-7549 had an IC50 value 50-fold lower than that of minoxidil, while the maximal level of DNA synthesis for the PKC inhibitor was 86% higher. Incubation of mouse hair follicles with Ro 31-7549 resulted in a delay of approximately 24 h in the onset of decline in follicular DNA synthesis rates. Ro 31-6045 and Ro 31-7208, bisindolylmaleimides without activity in the platelet PKC assay, did not affect mouse hair follicle DNA synthesis rates. Taken together, these findings show that PKC mediates, at least in part, the rapid loss of proliferative activity that occurs in mouse whisker follicles in culture, and provide further evidence that PKC plays a role as a negative proliferative signal in hair follicles.

Thapsigargin induces rapid, transient growth inhibition and c-fos expression followed by sustained growth stimulation in mouse keratinocyte cultures.

Although the sesquiterpene lactone thapsigargin has been shown to possess hyperplastic and tumor-promoting activities when applied topically to mouse skin in vivo, the cellular mechanism(s) which underlie these effects are unclear. We show here that thapsigargin treatment of Primary mouse epidermal keratinocytes increased intracellular free Ca2+ concentration (Cai) in a concentration-dependent manner. Thapsigargin induced a rapid, transient elevation in keratinocyte Cai, in part due to the release of Ca2+ from intracellular stores. This response was followed by a sustained elevation in Ca2+, resulting entirely from calcium influx. Thapsigargin elicited a biphasic effect on keratinocyte DNA synthesis: a rapid inhibitory effect (50-60% inhibition at 4-8 h), followed by a very marked and sustained elevation. Prolonged treatment of keratinocytes with thapsigargin at relatively high concentrations resulted in cytotoxicity (inhibition of neutral red uptake). The rapid antiproliferative effect of thapsigargin was not associated with cytotoxicity, as determined by either neutral red uptake or by trypan blue exclusion, and was not blocked by pretreatment with Ro 31-7349, a selective inhibitor of protein kinase C. The rapid antiproliferative effect of thapsigargin was associated with rapid, transient activation of keratinocyte c-fos expression and rapid inhibition of total protein synthesis. Taken together, these findings raise the possibility that the hyperplastic and tumor-promoting activities of thapsigargin on epidermis in vivo result from direct keratinocyte growth stimulation as a consequence of a prolonged elevation in levels of Cai.

Regulation of human dermal papilla cell production of insulin-like growth factor binding protein-3 by retinoic acid, glucocorticoids, and insulin-like growth factor-1.

Insulin-like growth factor-1 (IGF1) has been reported to stimulate hair elongation and to facilitate maintenance of the hair follicle in anagen phase. However, little is known about IGF1 signaling in the hair follicle. In this study we investigate the effects of IGF1, glucocorticoids, and retinoids on dermal papilla (DP) cell production of insulin-like growth factor binding proteins (IGFBPs). IGFBPs comprise a family of IGF binding proteins that are produced and released by most cell types. They bind to IGFs to either enhance or inhibit IGF activity. In the present report we identify IGFBP-3 as being produced and released by cultured human dermal papilla (DP) cells. IGFBP-3 levels are increased fivefold by retinoic acid, eightfold by dexamethasone, and tenfold by IGF1. DP cells are known to produce IGF1, and so the observed stimulation of DP cell IGFBP-3 production by IGF1 is consistent with the idea that DP cells possess the IGF transmembrane receptor kinase and are autoregulated by IGFs. The level of another IGFBP, tentatively identified as IGFBP-2, is, in contrast, not regulated by these agents. IGFBP-3 has been shown to inhibit the activity of IGFs in a variety of systems. Our results are consistent with a model in which retinoids and glucocorticoids inhibit IGF action on DP cells and surrounding matrix cells by stimulating increased DP cell production of IGFBP-3. The IGFBP-3, in turn, forms a complex with free IGF1 to reduce the concentration of IGF1 available to stimulate hair elongation and maintenance of anagen phase.

Protein kinase C inhibits human hair follicle growth and hair fibre production in organ culture.

In this study we have used a human hair follicle whole-organ culture system to examine the effects of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a potent activator of protein kinase C (PKC), on hair follicle growth and hair fibre production. Anagen hair follicles were isolated from human facial skin by microdissection and placed in suspension culture in supplemented Williams E medium. Hair follicle and hair fibre lengths were measured daily using an inverted microscope and cumulative growth values were calculated. Treatment with TPA resulted in a potent, dose-dependent inhibition of total cumulative hair follicle growth (IC50 = 1 nM). Hair follicles grew at a comparable rate for 4 days in the presence or absence of 10 nM TPA, after which growth of TPA-treated follicles ceased while control follicles grew by a further 0.8 mm over the subsequent 6 days. In contrast, 10 nM TPA treatment did not affect hair fibre elongation for a period of 8 days, after which TPA-treated fibre production ceased while control fibres grew by a further 0.79 mm over the subsequent 7 days. Incubation of hair follicles with TPA resulted in a 41% inhibition of hair fibre protein synthesis, as measured biochemically from the incorporation of 3H-leucine using a differential akali extraction method. The inhibitory effect of TPA on follicle growth was partially prevented by preincubation with the selective PKC inhibitor H-7, and almost completely prevented by preincubation with the more potent PKC inhibitor Ro 31-7549. Neither agent alone significantly affected follicle growth at concentrations that reversed the TPA response. These findings indicate that PKC is a negative regulator of hair follicle growth, and suggest that PKC may play a part in the transduction of follicular growth-inhibitory signals.

Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation.

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has been proposed as a physiologic regulator of keratinocyte growth and differentiation. Utilizing a proliferative serum-free culture system, we have found that a physiologic (picomolar) concentrations this hormone stimulated proliferation of primary mouse epidermal keratinocytes; at higher (nanomolar to micromolar) doses, growth was inhibited by 1,25(OH)2D3. We investigated the nature of the signal transduction mechanism underlying the response to 1,25(OH)2D3 and observed little or no effect of either low or high concentrations of the hormone on cytosolic calcium levels or Fos expression. Furthermore, the protein kinase C inhibitor, Ro 31-7549, had very little effect on the growth inhibition induced by a high dose (1 microM) of 1,25(OH)2D3. This lack of rapid signal transduction events was consistent with the inability of a short (4-hour) exposure to 1,25(OH)2D3 to initiate a complete growth-inhibitory response as measured using [3H]thymidine incorporation. Our results indicate that physiologic concentrations of 1,25(OH)2D3 are required for optimal keratinocyte growth. Furthermore, we found no evidence of rapid effects of 1,25(OH)2D3 and suggest that in mouse epidermal keratinocytes, the response to this hormone is mediated by a slow transduction pathway, such as that activated by the intracellular 1,25(OH)2D3 receptor (VDR).

Evidence that diazoxide promotes calcium influx in mouse keratinocyte cultures by membrane hyperpolarization.

We have used fura-2/AM to investigate the effect of diazoxide on the cytosolic calcium concentration (Cai) in primary mouse keratinocyte cultures. Treatment of keratinocytes with 100 microM diazoxide induced a transient peak in Cai, followed by a sustained elevation. Depletion of medium calcium by addition of EGTA abolished the diazoxide-induced Cai response, indicating that the agent promoted calcium influx without release of calcium from intracellular stores. The diazoxide-induced rise in Cai was inhibited both by addition of 60 mM KCl to the assay buffer and by preincubation with glibenclamide, a specific K+ channel blocker. In addition, studies with the membrane potential-sensitive fluorescent probe bis-oxonol demonstrated that diazoxide hyperpolarized keratinocyte membranes. These findings suggest that keratinocytes possess K+ channels, and that the previously reported proliferation effects of K+ channel openers such as diazoxide on keratinocytes may result from hyperpolarization-induced elevation of Cai.

Biphasic effect of 1,25-dihydroxyvitamin D3 on human hair follicle growth and hair fiber production in whole-organ cultures.

We have used a whole-organ culture system to investigate the effects of 1,25(OH)2D3 on human hair follicle growth and hair fiber production. Relatively low concentrations (1-10 nM) of 1,25(OH)2D3 stimulated the cumulative growth of hair follicles and hair fibers, by 52% and 36%, respectively (concentration producing 50% of the maximal response [EC50] values of 0.3 nM). The initial rates of follicle and fiber growth were increased, whereas the respective growth periods were unaffected. At higher concentrations of 1,25(OH)2D3, there was a dose-dependent inhibition of both follicle and fiber growth (IC50 values of 100 nM), in part due to reduction in the growth periods. There was a marked delay between the onset of 1,25(OH)2D-induced hair follicle and hair fiber growth inhibition. Incubation of hair follicles with 100 nM 1,25(OH)2D3 resulted in a rapid, transient inhibition of DNA synthesis (55% inhibition at 24 h), followed by a gradual return to control levels at day 4. Prolonged (> 5 h), incubation in the presence of 100 nM of 1,25 (OH)2D3 was required for follicle growth inhibition to be manifest. Ro 31-7549, a selective inhibitor of protein kinase C, did not prevent 1,25(OH)2D3-induced inhibition of hair follicle growth. These data suggest that 1,25(OH)2D3 may play a physiologic role in maintaining optimal hair follicle activity, and that elevation of 1,25(OH)2D3 may inhibit hair growth in vivo.

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

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

Hair fibre production by human hair follicles in whole-organ culture.

We have used both pulse-chase radiolabelling and morphometric techniques to investigate the ability of whole-organ cultures of human hair follicles to synthesize hair fibre. Anagen hair follicles were obtained from human facial skin by microdissection. Follicles were pulse-labelled with 3H-leucine, and radioactivity in alkali-soluble protein (ASP) and alkali-insoluble hair fibre protein (HFP) fractions was determined by differential extraction. There was a slow decline in ASP radioactivity over 7 days of culture, with a half-life of 4 days. In contrast, leucine incorporation into HFP increased linearly from day 1 to day 5, following an initial delay of 17 h. Morphometric analysis revealed that hair follicles and hair fibres grew at the same rate (0.25 mm/day) for 4 days in culture, after which the rate of hair follicle growth declined progressively, whereas fibre growth continued at the initial rate for a further 3 days. Concurrent with the onset of the decline in follicle growth, the base of the hair fibre began to descend towards the follicle base, until hair fibre occupied almost the full length of the hair follicle at day 15. The decline in follicle growth was preceded by a decline in the rate of follicular 3H-thymidine incorporation, with a delay of approximately 1 day. Overall, these data demonstrate that human hair follicles in whole-organ culture are capable of the production of hair fibre, and suggest that cessation of hair growth in vitro results from depletion of the pool of differentiated follicular keratinocytes, secondary to a loss of matrix cell proliferative activity.

IL-1 alpha inhibits human hair follicle growth and hair fiber production in whole-organ cultures.

In this study we have used a recently developed human hair follicle whole-organ culture system to investigate the effect of IL-1 alpha on hair follicle growth and hair fiber production. In the presence of 10 ng/ml IL-1 alpha, the growth of cultured human hair follicles ceased within 2-4 days, whereas control hair follicles grew for a period of 7-10 days. IL-1 alpha also inhibited hair fiber growth, but with an onset which occurred 3 days later than that of follicle growth inhibition. An IC50 value of approximately 30 pg/ml was obtained for IL-1 alpha inhibition of follicle growth. Incubation of hair follicles with IL-1 alpha resulted in a rapid, transient reduction in the rate of whole-follicle DNA synthesis. 1000-fold molar excess of IL-1 receptor antagonist prevented IL-1-induced follicle growth inhibition, while antagonist alone was without effect. The selective PKC inhibitor, Ro 31-7549, augmented IL-1-induced inhibition of hair follicle growth, but did not itself affect hair follicle growth. These findings indicate that IL-1 alpha exerts a rapid antiproliferative effect on hair follicles, and that inhibition of hair fiber growth is a secondary response. Thus, IL-1 may play a role in the pathophysiology of inflammatory hair loss conditions, such as alopecia areata, through a direct growth-inhibitory effect on hair follicles.

Effects of the selective protein kinase C inhibitor, Ro 31-7549, on the proliferation of cultured mouse epidermal keratinocytes.

We have investigated the effects of Ro 31-7549, a selective protein kinase C (PKC) inhibitor, on DNA synthesis and proliferation in two primary mouse epidermal keratinocyte culture systems. In differentiating keratinocytes incubated in medium containing 10% serum and high calcium (approximately 0.5 mM), Ro 31-7549 blocked the inhibitory effect of the phorbol ester 12-0-tetradecanoyl-13-acetate (TPA) (a PKC activator) on keratinocyte DNA synthesis at 24 h [50% maximal response concentration (EC50) = 1 microM], consistent with inhibition of PKC-mediated differentiation. Continuous treatment of the differentiative culture system with the PKC inhibitor resulted in a marked (fourfold) stimulation of [3H]thymidine incorporation at day 7 of exposure, with an EC50 of 0.25 microM. The potencies of these effects of Ro 31-7549 are comparable to that reported for inhibition of TPA-induced platelet 47-kD protein phosphorylation [50% inhibitory concentration (IC50) = 4.4 microM]. The time course of [3H]thymidine incorporation indicated that Ro 31-7549 did not directly stimulate DNA synthesis but instead prevented the loss of proliferative capacity associated with continued culture in this medium. Maximal stimulation (2.6 times) of DNA synthesis was observed on day 4, whereas DNA synthesis at day 1 was unaffected. In a highly proliferative culture system using serum-free medium containing 25 microM calcium, TPA dose-dependently inhibited proliferation with an IC50 of approximately 0.3 mM. This antiproliferative effect of TPA was largely reversed by 0.1 microM Ro 31-7549. In the proliferative culture system, 0.75 microM Ro 31-7549 also essentially reversed the inhibition of proliferation caused by switching to high (1.0 mM) calcium. These results suggest that the loss of proliferative capacity in differentiating epidermal keratinocyte cultures may be mediated, at least in part, by PKC.

Potassium channel openers stimulate DNA synthesis in mouse epidermal keratinocyte and whole hair follicle cultures.

We have conducted studies using primary mouse epidermal keratinocyte and whole hair follicle cultures to investigate the mechanism of the hypertrichotic activity of potassium channel openers. In a time course study, the extent of stimulation of epidermal keratinocyte DNA synthesis by minoxidil increased as the rate of DNA synthesis in control cultures declined. Minoxidil stimulation of DNA synthesis in 7-day cultures required prolonged (> 1 day) exposure to the agent. Pinacidil and diazoxide also stimulated DNA synthesis in mouse epidermal keratinocyte cultures. In addition, minoxidil, pinacidil, diazoxide, and cromakalim stimulated DNA synthesis in whole-organ cultures of mouse hair follicles. These results suggest that potassium channel openers retard the loss of proliferative activity of differentiating keratinocytes and support the hypothesis that these agents stimulate hair growth through a direct effect on hair follicles.

Barrier function of human keratinocyte cultures grown at the air-liquid interface.

Stratum corneum (SC), the outermost and least permeable layer of skin, is the major barrier to passive transepidermal water loss. In the research described in this paper, we have used human keratinocyte cultures, grown at the air-liquid (A/L) interface, to examine the relationship between epidermal differentiation (including SC formation) and barrier function. Histologically, the A/L culture showed several markers of complete differentiation, including the presence of well-organized and defined epidermal cell layers, keratohyalin granules, and a multilayered SC. The permeability of tritiated water through epidermal cultures, which had grown for 3 weeks at the A/L interface, was measured with a microdiffusion apparatus. The results of these experiments demonstrated that: a) the human keratinocyte cultures developed a substantial barrier (i.e., a multilayered SC) to water diffusion across the entire surface. If the relative humidity of the culturing environment was lowered from 100% to around 75%, the barrier was significantly improved; b) the differentiation promoter, 1.25-dihydroxy-vitamin-D3, increased the number of SC layers and reduced water permeation through the culture; c) the nature of the keratinocyte support matrix could be altered to improve the morphology as well as the barrier function of the epidermal cultures. Overall, the observations are consistent with the relationship that is believed to exist between SC intercellular lipid content and percutaneous penetration. Confirmation of this hypothesis will further the considerable potential of human keratinocyte A/L cultures as a valuable and relevant model in which to study drug absorption and metabolism.

The water permeability of primary mouse keratinocyte cultures grown at the air-liquid interface.

In order to study the development of the epidermal permeability barrier in vitro, tritiated water (HTO) flux was measured across murine keratinocytes cultured at the air-liquid interface. Using a micro-diffusion technique, it was shown that air-liquid cultures form areas where the water diffusion is comparable to that of intact neonatal mouse skin. When water permeability is measured over a large area of the culture surface, however, significantly higher flux is obtained. These results show that under the culture conditions used, areas of water barrier comparable to intact neonatal mouse skin coexist with regions of less complete barrier formation.