A hybrid gravity and route choice model to assess vector traffic in large-scale road networks.

Human traffic along roads can be a major vector for infectious diseases and invasive species. Though most road traffic is local, a small number of long-distance trips can suffice to move an invasion or disease front forward. Therefore, understanding how many agents travel over long distances and which routes they choose is key to successful management of diseases and invasions. Stochastic gravity models have been used to estimate the distribution of trips between origins and destinations of agents. However, in large-scale systems, it is hard to collect the data required to fit these models, as the number of long-distance travellers is small, and origins and destinations can have multiple access points. Therefore, gravity models often provide only relative measures of the agent flow. Furthermore, gravity models yield no insights into which roads agents use. We resolve these issues by combining a stochastic gravity model with a stochastic route choice model. Our hybrid model can be fitted to survey data collected at roads that are used by many long-distance travellers. This decreases the sampling effort, allows us to obtain absolute predictions of both vector pressure and pathways, and permits rigorous model validation. After introducing our approach in general terms, we demonstrate its benefits by applying it to the potential invasion of zebra and quagga mussels (Dreissena spp.) to the Canadian province British Columbia. The model yields an R 2-value of 0.73 for variance-corrected agent counts at survey locations.

Mitochondrial respiratory uncoupling promotes keratinocyte differentiation and blocks skin carcinogenesis.

Decreased mitochondrial oxidative metabolism is a hallmark bioenergetic characteristic of malignancy that may have an adaptive role in carcinogenesis. By stimulating proton leak, mitochondrial uncoupling proteins (UCP1-3) increase mitochondrial respiration and may thereby oppose cancer development. To test this idea, we generated a mouse model that expresses an epidermal-targeted keratin-5-UCP3 (K5-UCP3) transgene and exhibits significantly increased cutaneous mitochondrial respiration compared with wild type (FVB/N). Remarkably, we observed that mitochondrial uncoupling drove keratinocyte/epidermal differentiation both in vitro and in vivo. This increase in epidermal differentiation corresponded to the loss of markers of the quiescent bulge stem cell population, and an increase in epidermal turnover measured using a bromodeoxyuridine (BrdU)-based transit assay. Interestingly, these changes in K5-UCP3 skin were associated with a nearly complete resistance to chemically-mediated multistage skin carcinogenesis. These data suggest that targeting mitochondrial respiration is a promising novel avenue for cancer prevention and treatment.

The role of the EP receptors for prostaglandin E2 in skin and skin cancer.

One of the most common features of exposure of skin to ultraviolet (UV) light is the induction of inflammation, a contributor to tumorigenesis, which is characterized by the synthesis of cytokines, growth factors and arachidonic acid metabolites, including the prostaglandins (PGs). Studies on the role of the PGs in non-melanoma skin cancer (NMSC) have shown that the cyclooxygenase-2 (COX-2) isoform of the cyclooxygenases is responsible for the majority of the pathological effects of PGE(2). In mouse skin models, COX-2 deficiency significantly protects against chemical carcinogen- or UV-induced NMSC while overexpression confers endogenous tumor promoting activity. Current studies are focused on identifying which of the G protein-coupled EP receptors mediate the tumor promotion/progression activities of PGE(2) and the signaling pathways involved. As reviewed here, the EP1, EP2, and EP4 receptors, but not the EP3 receptor, contribute to NMSC development, albeit through different signaling pathways and with somewhat different outcomes. The signaling pathways activated by the specific EP receptors are context specific and likely depend on the level of PGE(2) synthesis, the differential levels of expression of the different EP receptors, as well as the levels of expression of other interacting receptors. Understanding the role and mechanisms of action of the EP receptors potentially offers new targets for the prevention or therapy of NMSCs.

IKKα represses a network of inflammation and proliferation pathways and elevates c-Myc antagonists and differentiation in a dose-dependent manner in the skin.

Inhibitor of nuclear factor κB kinase-α (IKKα) is required for maintaining skin homeostasis and preventing skin tumorigenesis. However, its signaling has not been extensively investigated. In the present study, we generated two mouse lines that expressed different levels of transgenic IKKα in the basal epidermis under the control of keratin-5 promoter and further evaluated their effects on the major pathways of inflammation, proliferation, and differentiation in the skin. Regardless of the transgenic IKKα levels, the mice develop normally. Because IKKα deletion in keratinocytes blocks terminal differentiation and induces epidermal hyperplasia and skin inflammation, we depleted the endogenous IKKα in these transgenic mice and found that the transgenic IKKα represses epidermal thickness and induces terminal differentiation in a dose-dependent manner. Also, transgenic IKKα was found to elevate expression of Max dimer protein 1 (Mad1) and ovo-like 1, c-Myc antagonists, but repress activities of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK), Jun-amino-terminal kinases, c-Jun, signal transducer and activator of transcription 3 (Stat3), and growth factor levels in a dose-dependent fashion in the skin. Moreover, EGFR reduction represses IKKα deletion-induced excessive ERK, Stat3 and c-Jun activities, and skin inflammation. These new findings indicate that elevated IKKα expression not only represses epidermal thickness and induces terminal differentiation, but also suppresses skin inflammation by an integrated loop. Thus, IKKα maintains skin homeostasis through a broad range of signaling pathways.

Highly sensitive C-reactive protein and male gender are independently related to the severity of coronary disease in patients with metabolic syndrome and an acute coronary event.

Patients with metabolic syndrome are at high-risk for development of atherosclerosis and cardiovascular events. The objective of this study was to examine the major determinants of coronary disease severity, including those coronary risk factors associated with metabolic syndrome, during the early period after an acute coronary episode. We tested the hypothesis that inflammatory markers, especially highly sensitive C-reactive protein (hsCRP), are related to coronary atherosclerosis, in addition to traditional coronary risk factors. Subjects of both genders aged 30 to 75 years (N = 116) were prospectively included if they had suffered a recent acute coronary syndrome (acute myocardial infarction or unstable angina pectoris requiring hospitalization) and if they had metabolic syndrome diagnosed according to the National Cholesterol Education Program/Adult Treatment Panel III. Patients were submitted to a coronary angiography and the burden of atherosclerosis was estimated by the Gensini score. The severity of coronary disease was correlated (Spearman's or Pearson's coefficient) with gender (r = 0.291, P = 0.008), age (r = 0.218, P = 0.048), hsCRP (r = 0.256, P = 0.020), ApoB/ApoA ratio (r = 0.233, P = 0.041), and carotid intima-media thickness (r = 0.236, P = 0.041). After multiple linear regression, only male gender (P = 0.046) and hsCRP (P = 0.012) remained independently associated with the Gensini score. In this high-risk population, male gender and high levels of hsCRP, two variables that can be easily obtained, were associated with more extensive coronary disease, identifying patients with the highest potential of developing new coronary events.

Highly sensitive C-reactive protein and male gender are independently related to the severity of coronary disease in patients with metabolic syndrome and an acute coronary event

Patients with metabolic syndrome are at high-risk for development of atherosclerosis and cardiovascular events. The objective of this study was to examine the major determinants of coronary disease severity, including those coronary risk factors associated with metabolic syndrome, during the early period after an acute coronary episode. We tested the hypothesis that inflammatory markers, especially highly sensitive C-reactive protein (hsCRP), are related to coronary atherosclerosis, in addition to traditional coronary risk factors. Subjects of both genders aged 30 to 75 years (N = 116) were prospectively included if they had suffered a recent acute coronary syndrome (acute myocardial infarction or unstable angina pectoris requiring hospitalization) and if they had metabolic syndrome diagnosed according to the National Cholesterol Education Program/Adult Treatment Panel III. Patients were submitted to a coronary angiography and the burden of atherosclerosis was estimated by the Gensini score. The severity of coronary disease was correlated (Spearman’s or Pearson’s coefficient) with gender (r = 0.291, P = 0.008), age (r = 0.218, P = 0.048), hsCRP (r = 0.256, P = 0.020), ApoB/ApoA ratio (r = 0.233, P = 0.041), and carotid intima-media thickness (r = 0.236, P = 0.041). After multiple linear regression, only male gender (P = 0.046) and hsCRP (P = 0.012) remained independently associated with the Gensini score. In this high-risk population, male gender and high levels of hsCRP, two variables that can be easily obtained, were associated with more extensive coronary disease, identifying patients with the highest potential of developing new coronary events.

Overexpression of the prostaglandin E2 receptor EP2 results in enhanced skin tumor development.

We previously showed that the EP2 knockout mice were resistant to chemically induced skin carcinogenesis. The purpose of this study was to investigate the role of the overexpression of the EP2 receptor in mouse skin carcinogenesis. To determine the effect of overexpression of EP2, we used EP2 transgenic (TG) mice and wild-type (WT) mice in a DMBA (7,12-dimethylbenz[alpha]anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate) two-stage carcinogenesis protocol. EP2 TG mice developed significantly more tumors compared with WT mice. Overexpression of the EP2 receptor increased TPA-induced keratinocyte proliferation both in vivo and in vitro. In addition, the epidermis of EP2 TG mice 48 h after topical TPA treatment was significantly thicker compared to that of WT mice. EP2 TG mice showed significantly increased cyclic adenosine monophosphate levels in the epidermis after prostaglandin E2 (PGE2) treatment. The inflammatory response to TPA was increased in EP2 TG mice, as demonstrated by an increased number of macrophages in the dermis. Tumors and 7 x TPA-treated and DMBA-TPA-treated (6 weeks) skins from EP2 TG mice produced more blood vessels than those of WT mice as determined by CD-31 immunostaining. Vascular endothelial growth factor (VEGF) protein expression was significantly increased in squamous cell carcinoma (SCC) samples from EP2 TG mice compared that of WT mice. There was, however, no difference in the number of apoptotic cells in tumors from WT and EP2 TG mice. Together, our results suggest that the overexpression of the EP2 receptor plays a significant role in the protumorigenic action of PGE2 in mouse skin.

Oxidative metabolism of linoleic acid modulates PPAR-beta/delta suppression of PPAR-gamma activity.

Peroxisome proliferator-activated receptors (PPARs) are transcription factors that strongly influence molecular events in normal and cancer cells. PPAR-beta/delta (PPAR-b/d) overexpression suppresses the activity of PPAR-gamma (PPAR-g) and PPAR-alpha. This interaction has been questioned, however, by studies with synthetic ligands of PPARs in PPAR-b/d-null cells, and it is not known whether an interaction between PPAR-b/d and PPAR-g exists, especially in relation to the signaling by natural PPAR ligands. Oxidative metabolites of linoleic and arachidonic acids are natural ligands of PPARs. 13-S-hydroxyoctadecadienoic acid (13-S-HODE), the main product of 15-lipoxygenase-1 (15-LOX-1) metabolism of linoleic acid, downregulates PPAR-b/d. We tested (a) whether PPAR-b/d expression modulates PPAR-g activity in experimental models of the loss and gain of PPAR-b/d function in colon cancer cells and (b) whether 15-LOX-1 formation of 13-S-HODE influences the interaction between PPAR-b/d and PPAR-g. We found that (a) 15-LOX-1 formation of 13-S-HODE promoted PPAR-g activity, (b) PPAR-b/d expression suppressed PPAR-g activity in models of both loss and gain of PPAR-b/d function, (c) 15-LOX-1 activated PPAR-g by downregulating PPAR-b/d, and (d) 15-LOX-1 expression induced apoptosis in colon cancer cells via modulating PPAR-b/d suppression of PPAR-g. These findings elucidate a novel mechanism of the signaling by natural ligands of PPARs, which involves modulating the interaction between PPAR-b/d and PPAR-g.

Ultraviolet-B irradiation alters the cell cycle machinery in murine epidermis in vivo.

Ultraviolet radiation of mouse skin leads to epidermal hyperplasia, inflammation, and subsequent tumor development. In this study we determined to what extent the cell cycle machinery is altered during epidermal proliferation after ultraviolet B radiation. A minimal erythema dose, 90 mJ per cm2, increased the protein expression of the G1 phase cyclins, cyclin D1 and E, by 12 h. The majority of epidermal cells entered S phase between 18 and 24 h as determined by 5'-bromo-2'-deoxyuridine incorporation, proliferating cell nuclear antigen, and cyclin A immunohistochemistry. An increase in cyclin-dependent kinase 2 (cdk-2) protein expression occurred after 12 h, but no changes in cdk-4 or cdk-6 protein levels were observed. The increase in cyclin D1, E, and A protein expression was associated with an increase in cyclin D1-cdk-4, cyclin E-cdk-2, and cyclin A-cdk-2 complex formation. p53 protein expression was elevated through 48 h, and the cdk inhibitor protein p21(Cip1/WAF1) was elevated 6-fold to 7.5-fold between 12 and 24 h. The elevated p21(Cip1/WAF1) protein contributed to an enhanced association with cdk-2 and cdk-4 at 3-24 h and 6-24 h post-ultraviolet B irradiation, respectively. These data indicate that 90 mJ per cm2 of ultraviolet B irradiation induces a DNA damage response, by increasing p53 and p21(Cip1/WAF1) protein expression, but also induces a rapid and sustained increase in S phase by 18 h.

Alignment delays in the N = Z nuclei (72)Kr, (76)Sr, and (80)Zr.

The ground state rotational bands of the N = Z nuclei (72)Kr, (76)Sr, and (80)Zr have been extended into the angular momentum region where rotation alignment of particles is normally expected. By measuring the moments of inertia of these bands we have observed a consistent increase in the rotational frequency required to start pair breaking, when compared to neighboring nuclei. (72)Kr shows the most marked effect. It has been widely suggested that these "delayed alignments" arise from np-pairing correlations. However, alignment frequencies are very sensitive to shape degrees of freedom and normal pairing, so the new experimental observations are still open to interpretation.

Distribution of conjugated linoleic acid and metabolites in different lipid fractions in the rat liver.

Conjugated linoleic acid (CLA) is known to provide certain health benefits in experimental animal models. The major CLA isomer in food is c 9,t11-CLA. A primary objective of this study was to investigate the uptake of c 9,t11-CLA and its downstream metabolites into various lipid fractions in the liver of rats fed either a high or low CLA diet (containing 0.1 or 0.8 g CLA/100 g diet, respectively). As expected, the levels of all conjugated diene (CD) fatty acids (CD 18:2 + CD 18:3 + CD 20:3 + CD 20:4) were elevated about 8-fold in the high CLA diet group. However, there was no change in the distribution of CLA and CLA metabolites into various lipid fractions due to CLA intake. Unlike linoleic acid or gamma-linolenic acid, which were distributed mainly in phospholipids, CD 18:2, CD 18:3, and CD 20:3 were incorporated primarily in neutral lipid. Furthermore, the incorporation of all nonconjugated unsaturated fatty acids was not perturbed by CLA. Regardless of the level of CLA in the diet, CD 20:4 was predominantly enriched in phosphatidylserine and phosphatidylinositol. In contrast, arachidonic acid was primarily enriched in phosphatidylcholine and less so in phosphatidylethanolamine. The above findings may have potential implication regarding the role of CLA in modulating eicosanoid metabolism.

Inhibition of peroxisome-proliferator-activated receptor (PPAR)alpha by MK886.

Although MK886 was originally identified as an inhibitor of 5-lipoxygenase activating protein (FLAP), recent data demonstrate that this activity does not underlie its ability to induce apoptosis [Datta, Biswal and Kehrer (1999) Biochem. J. 340, 371--375]. Since FLAP is a fatty-acid binding protein, it is conceivable that MK886 may affect other such proteins. A family of nuclear receptors that are activated by fatty acids and their metabolites, the peroxisome-proliferator-activated receptors (PPARs), have been implicated in apoptosis and may represent a target for MK886. The ability of MK886 to inhibit PPAR-alpha, -beta and -gamma activity was assessed using reporter assay systems (peroxisome-proliferator response element--luciferase). Using a transient transfection system in monkey kidney fibroblast CV-1 cells, mouse keratinocyte 308 cells and human lung adenocarcinoma A549 cells, 10--20 microM MK886 inhibited Wy14,643 activation of PPAR alpha by approximately 80%. Similar inhibition of PPAR alpha by MK886 was observed with a stable transfection reporter system in CV-1 cells. Only minimal inhibitory effects were seen on PPAR beta and PPAR gamma. MK886 inhibited PPAR alpha by a non-competitive mechanism as shown by its effects on the binding of arachidonic acid to PPAR alpha protein, and a dose-response study using a transient transfection reporter assay in COS-1 cells. An assay assessing PPAR ligand-receptor interactions showed that MK886 prevents the conformational change necessary for active-complex formation. The expression of keratin-1, a protein encoded by a PPAR alpha-responsive gene, was reduced by MK886 in a culture of mouse primary keratinocytes, suggesting that PPAR inhibition has functional consequences in normal cells. Although Jurkat cells express all PPAR isoforms, various PPAR alpha and PPAR gamma agonists were unable to prevent MK886-induced apoptosis. This is consistent with MK886 functioning as a non-competitive inhibitor of PPAR alpha, but may also indicate that PPAR alpha is not directly involved in MK886-induced apoptosis. Although numerous PPAR activators have been identified, the results show that MK886 can inhibit PPAR alpha, making it the first compound identified to have such an effect.

Nonsteroidal anti-inflammatory drugs induce apoptosis in esophageal cancer cells by restoring 15-lipoxygenase-1 expression.

In previous studies, we have found that expression of 15-lipoxygenase-1 (15-LOX-1) and its main product, 13-S-hydroxyoctadecadienoic acid, are decreased in human colorectal cancers and that nonsteroidal anti-inflammatory drugs (NSAIDs) can therapeutically induce 15-LOX-1 expression to trigger apoptosis in human colorectal cancer cells. NSAIDs similarly induce apoptosis in esophageal cancer cells, although the mechanisms of these effects remain to be defined. In the present study, we tested whether 15-LOX-1 is down-regulated in human esophageal cancers using paired normal and tumor human surgical samples and whether NSAIDs can up-regulate 15-LOX-1 to restore apoptosis in esophageal cancer cells. We found that: (a) 15-LOX-1 was down-regulated in human esophageal carcinomas; (b) NSAIDs induced 15-LOX-1 expression during apoptosis in esophageal cancer cells; and (c) 15-LOX-1 inhibition suppressed NSAID-induced apoptosis, which was restored by 13-S-hydroxyoctadecadienoic acid but not by its parent compound, linoleic acid. These findings demonstrate that 15-LOX-1 is down-regulated in human esophageal carcinomas and that NSAIDs induce apoptosis in esophageal cancer cells via up-regulation of 15-LOX-1. They also support the concept that the loss of the proapoptotic role of 15-LOX-1 in epithelial cancers is not limited to human colorectal cancers.

Transcriptional regulation of intracellular IL-1 receptor antagonist gene by IL-1 alpha in primary mouse keratinocytes.

The inflammatory cytokine IL-1alpha mediates inflammatory reactions in skin and up-regulates the expression of other proinflammatory genes. We previously found that IL-1alpha also increases steady state mRNA levels for intracellular IL-1 receptor antagonist (icIL-1Ra) in primary mouse keratinocytes; however, the mechanism for this was unknown. Here we show that increased expression in primary keratinocytes is due to increased rates of transcription. To study the transcriptional regulation of icIL-1Ra expression induced by IL-1alpha, we functionally characterized 4.5 kb of the 5'-flanking region of the human icIL-1Ra gene. Deletion analysis showed that regulatory elements were contained in the -598- and -288-bp region upstream of the transcription start site. Then we investigated cis- and trans-acting factors required for icIL-1Ra expression and found that a NF-IL-6 site and a NF-kappaB site in the icIL-1Ra promoter were responsible for IL-1alpha-induced icIL-1Ra expression. Moreover, gel shift assays and cotransfection experiments showed that CCAAT/enhancer-binding proteins alpha, beta, and p65 bind to the NF-IL-6 site and NF-kappaB site, respectively, and functionally trans-activate the icIL-1Ra promoter. Finally, mutational analysis confirmed that these elements were both essential for maximal transcription induced by IL-1alpha.

Role of intracellular interleukin-1 receptor antagonist in skin carcinogenesis.

Interleukin 1 (IL-1) is a major mediator of inflammation and exerts pleiotropic effects on many systems. To elucidate the role of its endogenous inhibitor, intracellular IL-1 receptor antagonist (icIL-1Ra), in mouse skin, we produced an icIL-1Ra-overexpressing skin carcinoma cell line (icIL-1Ra-JWF2). Altered expression of icIL-1Ra did not change IL-1alpha mRNA levels in these transfected cells. In icIL-1Ra-JWF2 cells, however, cyclooxygenase-2 mRNA levels were dramatically reduced and shown to be transcriptionally regulated by icIL-1Ra. To determine the effect of icIL-1Ra on cell proliferation, cell counts were done 24 h after plating equal numbers of cells. Cells from three icIL-1Ra-JWF2 clones showed significantly reduced growth rates compared with parental JWF2 cells. We subcutaneously injected five independent clones of icIL-1Ra-JWF2 cells into nude mice and measured the tumor doubling time by weekly measurements of tumor volume. IcIL-1Ra appeared to significantly slow the growth of tumors in vivo. Collectively these observations suggest that IL-1Ra has antiproliferative effects in murine skin carcinoma cells.

Difluoromethylornithine is effective as both a preventive and therapeutic agent against the development of UV carcinogenesis in SKH hairless mice.

Targeting specific events associated with tumor development represents a rational approach to chemoprevention as well as therapeutic intervention. In this study the ability of difluoromethylornithine (DFMO) to inhibit UV-induced skin carcinogenesis when administered before or after the appearance of tumors was examined. SKH hairless mice were irradiated 3 times per week with 90 mJ/cm(2); this dose was increased by 10% weekly to a maximum of 175 mJ/cm(2). Mice supplied 0.4% DFMO in the drinking water continuously throughout the experiment had an average of 2.0 tumors/mouse (72% incidence) at 30 weeks while controls had an average of 8.2 tumors/mouse (100% incidence). DFMO started after 12 weeks of UV, a time prior to tumor appearance, yielded 3.6 tumors and 100% incidence at 30 weeks. Starting DFMO at 22 weeks, when an average of 2.5 tumors were present, caused regression of tumors for several weeks, followed by a slight rebound. The final tumor number at 30 weeks was 3.0 (96% incidence). Thus, DFMO has strong chemopreventive efficacy, as well as therapeutic activity, against UV-induced skin tumors. Histological and proliferative markers support this conclusion.

Activators of peroxisome proliferator-activated receptor-alpha partially inhibit mouse skin tumor promotion.

Several recent reports have suggested that peroxisome proliferator-activated receptors (PPARs) may be involved in the development of neoplasias in different tissue types. The present study was undertaken to determine whether PPARs play a role in skin physiology and tumorigenesis. In an initiation-promotion study, SENCAR mice treated topically with the PPARalpha ligands conjugated linoleic acid and 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (Wy-14643) exhibited an approximately 30% lower skin tumor yield compared with mice treated with vehicle. The PPARgamma and PPARdelta activators troglitazone and bezafibrate, respectively, exerted little, if any, inhibitory activity. PPARalpha was detected in normal and hyperplastic skin and in papillomas and carcinomas by immunohistochemistry. In addition, PPARalpha, PPARdelta/PPARbeta, and PPARgamma protein levels were analyzed by immunoblotting in normal epidermis and papillomas. Surprisingly, the levels of all three isoforms were increased significantly in tumors as opposed to normal epidermis. In primary keratinocyte cultures, protein levels of PPARalpha and, to a lesser extent, PPARgamma were markedly increased when the cells were induced to differentiate with high-calcium (0.12 mM) conditions. In addition, we observed that Wy-14643 enhanced transcriptional activity of a peroxisome proliferator-response element-driven promoter in a mouse keratinocyte cell line. These results demonstrate that keratinocytes express functional PPARalpha, that PPARalpha may play a role in differentiation, and that ligands for PPARalpha are moderately protective against skin tumor promotion. We conclude that selective PPARalpha ligands may exert their protective role against skin tumor promotion by ligand activation of PPARalpha.

8S-lipoxygenase products activate peroxisome proliferator-activated receptor alpha and induce differentiation in murine keratinocytes.

To determine the function and mechanism of action of the 8S-lipoxygenase (8-LOX) product of arachidonic acid, 8S-hydroxyeicosatetraenoic acid (8S-HETE), which is normally synthesized only after irritation of the epidermis, transgenic mice with 8-LOX targeted to keratinocytes through the use of a loricrin promoter were generated. Histological analyses showed that the skin, tongue, and stomach of transgenic mice are highly differentiated, and immunoblotting and immunohistochemistries of skin showed higher levels of keratin-1 expression compared with wild-type mice. The labeling index, however, of the transgenic epidermis was twice that of the wild-type epidermis. Furthermore, 8S-HETE treatment of wild-type primary keratinocytes induced keratin-1 expression. Peroxisome proliferator activated receptor alpha (PPARalpha) was identified as a crucial component of keratin-1 induction through transient transfection with expression vectors for PPARalpha, PPARgamma, and a dominant-negative PPAR, as well as through the use of known PPAR agonists. From these studies, it is concluded that 8S-HETE plays an important role in keratinocyte differentiation and that at least some of its effects are mediated by PPARalpha.

15-LOX-1: a novel molecular target of nonsteroidal anti-inflammatory drug-induced apoptosis in colorectal cancer cells.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAIDs) appear to act via induction of apoptosis-programmed cell death-as potential colorectal cancer chemopreventive agents. NSAIDs can alter the production of different metabolites of polyunsaturated fatty acids (linoleic and arachidonic acids) through effects on lipoxygenases (LOXs) and cyclooxygenases. 15-LOX-1 is the main enzyme for metabolizing colonic linoleic acid to 13-S-hydroxyoctadecadienoic acid (13-S-HODE), which induces apoptosis. In human colorectal cancers, the expression of this enzyme is reduced. NSAIDs can increase 15-LOX enzymatic activity in normal leukocytes, but their effects on 15-LOX in neoplastic cells have been unknown. We tested the hypothesis that NSAIDs induce apoptosis in colorectal cancer cells by increasing the protein expression and enzymatic activity of 15-LOX-1. METHODS: We assessed 15-LOX-1 protein expression and enzymatic activity, 13-S-HODE levels, and 15-LOX-1 inhibition in association with cellular growth inhibition and apoptosis induced by NSAIDs (primarily sulindac and NS-398) in two colorectal cancer cell lines (RKO and HT-29). All P values are two-sided. RESULTS: Sulindac and NS-398 progressively increased 15-LOX-1 protein expression in RKO cells (at 24, 48, and 72 hours) in association with subsequent growth inhibition and apoptosis. Increased 13-S-HODE levels and the formation of 15-hydroxyeicosatetraenoic acid on incubation of the cells with the substrate arachidonic acid confirmed the enzymatic activity of 15-LOX-1. Inhibition of 15-LOX-1 in RKO cells by treatment with caffeic acid blocked NS-398-induced 13-S-HODE production, cellular growth inhibition, and apoptosis (P =. 007, P<.0001, and P<.0001, respectively); growth inhibition and apoptosis were restored by adding exogenous 13-S-HODE (P<.0001 for each) but not its parent compound, linoleic acid (P = 1.0 for each). Similar results occurred with other NSAIDs and in HT-29 cells. CONCLUSIONS: These data identify 15-LOX-1 as a novel molecular target of NSAIDs for inducing apoptosis in colorectal carcinogenesis.

Prostaglandin E(2) regulation of cyclooxygenase expression in keratinocytes is mediated via cyclic nucleotide-linked prostaglandin receptors.

Inflammatory responses are thought to be mediated in part by the prostaglandins derived from arachidonic acid (AA) by the action of prostaglandin H synthase, also referred to as cyclooxygenase (COX). The mitogen-inducible isoform, COX-2, is over-expressed in numerous chronic inflammatory disease conditions and in neoplasms from both human and experimental animal models. COX-1 expression, on the other hand, has been referred to as constitutive or non-inducible. In this study, we present evidence demonstrating autoregulation of prostaglandin (PG) production by the PGs themselves and their precursor, AA. We observed that AA and PGs induced COX-2, as well as COX-1, expression in cultured murine keratinocytes approximately 3 h after treatment. In primary keratinocytes transiently transfected with a full-length COX-2 promoter linked to a luciferase reporter gene, we observed enhanced transcription by AA, PGE(2), and the other prostaglandins. Forskolin, a known activator of adenylate cyclase, and dibutryl-cAMP, a cAMP analog, induced COX-1 and COX-2 mRNA, suggesting that cAMP is a second messenger for COX expression. SQ 22536, an adenylate cyclase inhibitor, inhibited COX-2 mRNA induction by PGE(2) in a dose-dependent manner suggesting that PGE(2)-induced expression may be through one of the cAMP-linked PGE(2) receptors. The results of this study demonstrate that both COX-1 and COX-2 are inducible. Further, both COX isoforms can be up-regulated by their products, the PGs, and this autoregulation probably occurs via prostaglandin receptors linked to a cAMP signal transduction pathway.