Skin stem cell hypotheses and long term clone survival--explored using agent-based modelling.

Epithelial renewal in skin is achieved by the constant turnover and differentiation of keratinocytes. Three popular hypotheses have been proposed to explain basal keratinocyte regeneration and epidermal homeostasis: 1) asymmetric division (stem-transit amplifying cell); 2) populational asymmetry (progenitor cell with stochastic fate); and 3) populational asymmetry with stem cells. In this study, we investigated lineage dynamics using these hypotheses with a 3D agent-based model of the epidermis. The model simulated the growth and maintenance of the epidermis over three years. The offspring of each proliferative cell was traced. While all lineages were preserved in asymmetric division, the vast majority were lost when assuming populational asymmetry. The third hypothesis provided the most reliable mechanism for self-renewal by preserving genetic heterogeneity in quiescent stem cells, and also inherent mechanisms for skin ageing and the accumulation of genetic mutation.

Dandruff/seborrhoeic dermatitis is characterized by an inflammatory genomic signature and possible immune dysfunction: transcriptional analysis of the condition and treatment effects of zinc pyrithione.

BACKGROUND: Dandruff/seborrhoeic dermatitis is a common scalp condition that is characterized by flakes, pruritus and sometimes mild erythema. These symptoms reflect tissue level events that are poorly understood at the molecular level. OBJECTIVES: The purpose of this work was: (i) to compare gene expression profiles in subjects with dandruff vs. those of subjects without dandruff to determine the key physiological disruptions manifest in the condition; and (ii) to determine the effect on this profile of treatment with a shampoo containing potentiated zinc pyrithione (ZPT). METHODS: In study 1, scalp biopsies were taken from 16 normal subjects and from involved and uninvolved sites in 15 subjects with dandruff. In study 2, 30 subjects with dandruff were treated for 3 weeks with a commercial ZPT shampoo (n = 15) or a vehicle (n = 15), and scalp lesional biopsies were collected at baseline and end of study for transcriptomic analysis. RNA was extracted from all biopsies and Affymetrix gene chips were used to analyse transcriptomic profiles, followed by bioinformatic analysis. RESULTS: Analysis of study 1 biopsies revealed more than 7000 individual probes differentially regulated in dandruff lesional skin relative to normal. Enriched Gene Ontology categories included: lipid metabolism, immune response, response to stimulus, apoptosis, cell proliferation, and epidermal development. The most striking feature of lesional skin relative to normal was the reciprocal expression of induced inflammatory genes and repressed lipid metabolism genes. Induced inflammatory genes were also enriched in dandruff uninvolved skin, suggesting the existence of predisposing factors associated with inflammation. Many genes increased in lesional skin were increased at the level of protein in stratum corneum samples (e.g. IL-1RA, S100A8, S100A9, S100A11, IL-8). Under conditions known to improve overall scalp condition, the ZPT shampoo treatment in study 2 produced a transcriptomic profile resembling that of normal scalp skin. CONCLUSIONS: These data provide novel insights into the nature of dandruff and the therapeutic action of potentiated ZPT-containing shampoo, and provide a basis to explore many new mechanistic questions related to these topics.

Sex differences in the relationship between activity and weight loss in the rat.

Access to a running wheel combined with restricted feeding produced body weight loss at an equivalent rate in male and female litter-mate rats (Experiment 1). Thus, despite weighing less and running more, females were not more vulnerable to this procedure. When factors influencing weight loss were varied, no sex difference was found in adaptation to a new feeding schedule or in the effect of single versus group housing (Experiment 2). The apparent critical difference was that body weight loss increased running in males but not in females (Experiment 3). In all rats, rapid recovery of body weight occurred when food access was no longer restricted (Experiment 1), suggesting that "activity-based anorexia" is a misnomer for weight loss by rats in a running wheel.

Regulation of retinoid-induced differentiation in embryonal carcinoma PCC4.aza1R cells: effects of retinoid-receptor selective ligands.

Retinoic acid (RA) is a potent inducer of differentiation of embryonal carcinoma PCC4.aza1R cells into mesenchymal stem cells. Induction of Hoxa-1, Hoxa-5, cellular retinoic acid-binding protein (CRABP) I and II, and retinoic acid receptor (RAR)-beta expression occurs early in this multistage program of differentiation. RA is also a potent inducer of these genes in the differentiation-defective mutant PCC4(RA)-1; however, RA is much less effective in the mutant cell line PCC4(RA)-2. The up-regulation of several of these genes by RA is, at least in part, due to increased transcription. It is likely that some of these changes are mediated either directly or indirectly by nuclear retinoid receptors. Previously, we characterized the expression of RARs in PCC4.aza1R and (RA)-1 and (RA)-2 cells. In this study, we show that these cells also express retinoid X receptor (RXR)-alpha, RXR-beta, and RXR-gamma and that RA treatment down-regulates the expression of RXR-gamma. No large differences were found in RXR mRNA expression between parental and mutant cell lines except that PCC4(RA)-1 cells expressed an 8-fold higher level of RXR gamma mRNA than the parental cells. To obtain more insight into the retinoid signaling pathways involved in the regulation of this pathway of differentiation, we examined the action of two retinoid receptor-selective agonists and one antagonist. The RAR-selective retinoid SRI-6751-84 is a very effective inducer of transactivation of beta RARE-tk-LUC, but not of RXRE-tk-CAT, in PCC4.aza1R cells and is a very potent inducer of morphological differentiation and Hoxa-1, Hoxa-5, CRABP II, and RAR-beta expression. In contrast, the RXR-selective retinoid SR11,217, which transactivates the RXRE-tk-CAT effectively, but beta RARE-tk-LUC poorly, is unable to induce differentiation and has little effect on the expression of these early genes. The RAR-alpha-selective antagonist Ro 41-5253, which inhibits RARE-dependent transcriptional activation, has by itself no effect on the differentiation of PCC4.aza1R cells. However, this antagonist is able to block the induction of morphological differentiation by the RAR-selective retinoid as well as the expression of Hoxa-1, Hoxa-5, CRABP II, and RAR-beta. Our data suggest that the activation of RAR signaling pathways is important in initiating the cascade of changes in gene expression that result in the differentiation of PCC4.aza1R into mesenchymal stem cells. In addition, we demonstrate that the two mutant cell lines, PCC4(RA)-1 and PCC4(RA)-2, are defective at different stages of the differentiation program.

Evidence for the involvement of retinoic acid receptor RAR alpha-dependent signaling pathway in the induction of tissue transglutaminase and apoptosis by retinoids.

In this study, we show that all-trans-retinoic acid (RA) is a potent inducer of tissue transglutaminase (TGase II) and apoptosis in the rat tracheobronchial epithelial cell line SPOC-1. We demonstrate that these cells express the retinoid receptors RAR alpha, RAR gamma, and RXR beta. To identify which of these receptors are involved in regulating these processes, we analyzed the effects of several receptor-selective agonists, an antagonist, and a dominant-negative RAR alpha. We show that the RAR-selective retinoid SRI-6751-84 strongly increased TGase II expression at both the protein and mRNA levels, whereas the RXR-selective retinoid SR11217 had little effect. The RAR alpha-selective retinoid Ro40-6055 was also able to induce TGase II, whereas the RAR gamma-selective retinoid CD437 was inactive. The induction of TGase II by the RAR-selective retinoid was completely inhibited by the RAR alpha-antagonist Ro41-5253. Overexpression of a truncated RAR alpha gene with dominant-negative activity also inhibited the induction of TGase II expression. The increase in TGase II is associated with an induction of apoptosis as revealed by DNA fragmentation and the generation of apoptotic cells. We demonstrate that apoptosis is affected by retinoids in a manner similar to TGase II. Our results suggest that the induction of TGase II expression and apoptosis in SPOC-1 cells are mediated through an RAR alpha-dependent signaling pathway.

Diacylglycerol is an effector of the clonal expansion of cells containing activated Ha-ras genes.

Diacylglycerols (DAG) are lipid second messengers which are generated during phospholipase-catalyzed hydrolysis of phospholipids. The model DAG, sn-1,2-didecanoylglycerol (DIC10), is an effective topical tumor promoter in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. We now report that 11/12 of DMBA-initiated/DIC10-promoted papillomas examined contain an A-->T mutation in the 61st codon of the Ha-ras gene, suggesting that DAGs affect the clonal expansion of activated Ha-ras-containing cells. To explore further the DIC10-induced clonal expansion of activated Ha-ras-containing cells, we have examined the tumor-promoting effect of DIC10 in the skin of transgenic TG.AC mice, which harbor a v-Ha-ras transgene. By 9 weeks of promotion, 100% of the TG.AC mice developed squamous papillomas and by 15 weeks these mice developed > 20 papillomas/mouse. Because fatty acids are known to participate in signal transduction pathways, and since cellular lipases could cleave the fatty acid side chains present in DIC10, we have examined the tumor promoting activity of n-decanoic acid to verify the specificity of promotional activity of DIC10. n-Decanoic acid did not function as a tumor promoter. These data implicate DAG as an effector of the clonal expansion of mutated Ha-ras-containing cells, and support a mechanism whereby an increase in endogenous DAG could contribute to the clonal expansion of cells containing a Ha-ras oncogene.

Alterations in protein kinase C isozymes alpha and beta 2 in activated Ha-ras containing papillomas in the absence of an increase in diacylglycerol.

The levels of protein kinase C (PKC) activity, PKC isozymes, as well as the level of endogenous diacylglycerols (DAG) were examined in early emergence mouse skin papillomas and compared to the levels in the epidermis. The papillomas were derived from a two-stage carcinogenesis protocol in which mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted twice weekly for only 12 weeks with 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected, greater than 90% of these early emergence papillomas contained an activated Ha-ras gene with an A----T transversion in the 61st codon. There was a TPA-independent, irreversible decrease in total PKC activity (70%) in the early emergence papillomas compared to that in the epidermis. Immunoblot analysis of epidermis and papillomas taken 4 weeks following the cessation of TPA treatment, a time when PKC catalytic activity has completely recovered to control level in epidermis but not in papillomas, revealed that the levels of PKC-alpha and PKC-beta 2 were dramatically decreased in the cytosol of the papillomas, while the levels of these two isozymes in the particulate fraction were approximately equal to the epidermis. PKC-delta, -epsilon and -zeta immunoreactive proteins were present in both epidermis and papillomas and only minor changes were observed in the papillomas. PKC-delta and PKC-epsilon displayed a particulate fraction localization in both the epidermis and papillomas, while PKC-zeta was found in both subcellular fractions. We were unable to detect PKC-gamma in mouse epidermis or papillomas. Since the level of DAG has been shown to be elevated in some ras-transformed cells, we examined DAG levels in the papillomas, as an increased DAG level could explain the constitutive decreases in the levels of PKC. Measurements of cellular DAG indicated that there was no elevation in the total pool of DAG in the early emergence papillomas. These data demonstrate an irreversible decrease in and alteration of the subcellular distribution of PKC-alpha and beta 2 in DMBA-initiated/TPA-promoted papillomas. These changes are TPA-independent, and occur in the absence of an elevation in the total pool of endogenous DAG. These alterations of PKC isozymes may be important early events in multistage tumorigenesis.

Synthetic lipid second messenger sn-1,2-didecanoylglycerol: a complete tumor promoter in mouse skin.

sn-1,2-Didecanoylglycerol, a synthetic lipid second messenger and model diacylglycerol, was evaluated as a complete skin tumor promoter in CD-1 mice. In addition, sn-1,2-dioctanoylglycerol, sn-1,2-didecanoylglycerol, the second stage tumor promoter mezerein, and the complete tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined for their ability to stimulate epidermal protein kinase C activity in vitro. All four compounds stimulated epidermal protein kinase C activity utilizing lysine-rich histone as the phosphate acceptor substrate. sn-1,2-Dioctanoylglycerol and sn-1,2-didecanoylglycerol stimulated epidermal protein kinase C activity to a maximum velocity similar to that obtained when the enzyme was stimulated with TPA; however, about 1000 times greater concentration of the sn-1,2-diacylglycerols was required. sn-1,2-Didecanoylglycerol was evaluated as a complete skin tumor promoter in CD-1 mice utilizing a dosing regimen demonstrated to produce epidermal hyperplasia. Mice were initiated with 200 nmol 7,12-dimethylbenz[a]anthracene. One week later the mice received twice daily topical applications of 1 nmol TPA, 2 mumol sn-1,2-didecanoylglycerol or 5 mumol sn-1,2-didecanoylglycerol, 5 days/week. Additional initiated mice received twice weekly topical applications of 2 or 5 nmol TPA. Initiated mice treated with 5 nmol TPA twice weekly or with 1 nmol TPA twice daily for 5 days/week (cumulative weekly doses of 10 nmol TPA) responded similarly, based on the tumor incidence and the average number of tumors per mouse. Initiated mice treated with 2 or 5 mumol sn-1,2-didecanoylglycerol twice daily developed tumors in a dose-dependent manner. Initiated mice treated with 5 mumol sn-1,2-didecanoylglycerol twice daily developed many tumors, and at 20 weeks there was a 74% tumor incidence and an average of 6.0 tumors/mouse. At 20 weeks, 24% of the initiated mice treated with 2 mumol sn-1,2-didecanoylglycerol twice daily developed tumors, with an average of 1.1 tumors/mouse. Mice which were not initiated but treated twice daily with 5 mumol sn-1,2-didecanoylglycerol for 20 weeks did not develop any tumors. These data demonstrate that the representative synthetic lipid second messenger sn-1,2-didecanoylglycerol, like TPA, is a complete tumor promoter in DMBA-initiated mouse skin.

Comparison of epidermal protein kinase C activity, ornithine decarboxylase induction and DNA synthesis stimulated by TPA or dioctanoylglycerol in mouse strains with differing susceptibility to TPA-induced tumor promotion.

The purpose of this study was to examine the activity and associated kinetic parameters of epidermal protein kinase C (PKC) following stimulation by sn-1,2-dioctanoylglycerol (DIC8) or 12-O-tetradecanoylphorbol-13-acetate (TPA) and to examine the relationship between levels of epidermal PKC activity and the induction of ornithine decarboxylase by these agents, utilizing various stocks and strains of mice. Importantly, the mouse strains and stock used in this study have known differing susceptibilities to undergo TPA-induced tumor promotion: the CD-1 stock and the DBA/2 strain (both sensitive to TPA-induced tumor promotion) and the C57BL/6 strain (resistant to TPA-induced tumor promotion). TPA-stimulated protein kinase C activity was measured in the 10(5)g supernatant fraction of epidermal homogenates using lysine-rich histone as a phosphate acceptor substrate. The maximal velocities for TPA-stimulated epidermal PKC activity in CD-1, DBA/2 and C57BL/6 were 0.28, 0.29 and 0.27 nmol PO4-histone/mg 10(5)g protein/min, respectively. TPA-stimulated epidermal PKC from CD-1, DBA/2 and C57BL/6 had similar theoretical Vmax values and the apparent concentrations of TPA yielding half-maximal stimulation of PKC were also similar. DiC8-stimulated PKC activity to a greater Vmax; however, the concentration required to yield half-maximal stimulation of PKC was one thousand times greater than that of TPA. There were no strain differences in these parameters when the enzyme was stimulated with DiC8. Thus, the levels of epidermal PKC activity in CD-1, DBA/2 and C57BL/6 mice exhibit no strain differences when stimulated by TPA or DiC8 using lysine-rich histone as a phosphate acceptor substrate. Since sn-1,2-diacylglycerols are known effective inducers of epidermal ornithine decarboxylase (ODC) activity, the induction of epidermal ODC was examined in each mouse strain 5 h after topical application of 2 nmol TPA, 5 nmol TPA or 2.5 mumol DiC8. After topical treatment with TPA, C57BL/6 demonstrated an unexpected 2- and 4-fold increase in ODC activity over CD-1 and DBA/2 mice. After treatment with DiC8, C57BL/6 demonstrated a 6- and 10-fold increase in ODC activity over CD-1 and DBA/2, respectively. Thus, the resistant strain (C57BL/6) demonstrated a 'hyperinducibility' of epidermal ODC activity by TPA or DiC8. The time course for the induction of epidermal ODC was examined in each strain, and at every time point measured (3-15 h), the C57BL/6 strain exhibited this 'hyperinducibility' of ODC relative to the other strains. Epidermal DNA synthesis was stimulated to a similar extent in C57BL/6 and CD-1 mice.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of the effect of sn-1,2-didecanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate on cutaneous morphology, inflammation and tumor promotion in CD-1 mice.

Since sn-1,2-didecanoylglycerol mimics 12-O-tetradecanoylphorbol-13-acetate (TPA) by inducing ornithine decarboxylase activity and stimulating DNA synthesis in mouse epidermis [Smart, R.C., Huang, M.-T. and Conney, A.H. Carcinogenesis, 7, 1865 (1986)], we have investigated morphological changes induced by TPA and sn-1,2-didecanoylglycerol in the epidermis and we have also examined sn-1,2-didecanoylglycerol as a possible complete tumor promoter. It was determined that topical application of 2.5 or 10 mumol of sn-1,2-didecanoylglycerol induced epidermal ornithine decarboxylase activity to about the same extent as the application of 1 or 2 nmol of TPA respectively. Therefore, these doses of TPA and sn-1,2-didecanoylglycerol were used in most of our studies. Single or multiple application (2 X/week for 4 weeks) of 1, 2 or 5 nmol of TPA to the skin of CD-1 mice produced a dose-dependent increase in the number of epidermal non-cornified cell layers, epidermal thickness, leukocyte infiltration and intracellular edema. In contrast, neither single nor multiple application (2 X/week for 4 weeks) of 2.5 or 10 mumol sn-1,2-didecanoylglycerol produced any of these responses. However, when 5 mumol sn-1,2-didecanoylglycerol was applied topically twice a day (10 mumol/day) for 5 days there was a significant increase in the number of epidermal non-cornified cell layers and epidermal thickness. The effects of TPA and sn-1,2-didecanoylglycerol were compared using the mouse ear inflammation model. Application of TPA caused edema, but sn-1,2-didecanoylglycerol had little or no effect. sn-1,2-Didecanoylglycerol was then evaluated as a complete tumor promoter utilizing the mouse skin two-stage model. CD-1 mice were initiated with 200 nmol 7,12-dimethylbenz[a]anthracene and then treated with 1 nmol TPA or 2.5 mumol sn-1,2-didecanoylglycerol twice a week for 28 weeks. A 28 weeks, 28% of the mice treated with TPA had developed tumors, while none of the mice treated with 2.5 mumol sn-1,2-didecanoylglycerol developed tumors. The data indicate that topical application of 2.5 mumol sn-1,2-didecanoylglycerol induced ornithine decarboxylase activity to the same extent as a tumor-promoting dose of 1 nmol TPA, but it did not cause morphological changes in the epidermis when applied once or when applied twice a week for 4 weeks and did not function as a complete tumor promoter when applied twice a week for 28 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)

Listeners' discrimination of real and human-imitated animal sounds.

To determine if listeners can accurately distinguish between real and human-imitated animal sounds, a total of 165 recorded sounds (55 real and 110 human-imitated) of cats, cows, dogs, pigs, and sheep were randomly arranged on a master tape and presented to 30 listeners for discriminative judgments. Results indicate that, in general, listeners can accurately discriminate real from human-imitated animal sounds. Suggestions for future research are discussed.

Listeners' identification of human-imitated animal sounds.

To determine whether listeners can accurately identify human-imitated animal sounds, 20 speakers (10 females and 10 males) recorded their imitations of cows, cats, dogs, pigs, and sheep. These recordings were randomly arranged on a master tape and presented to 30 judges for identification. Analysis indicates that listeners can accurately identify various human-imitated animal sounds. Implications and suggestions for future research are discussed.