Adhesion and migration of tumor cells on and through the vascular endothelium are critical steps of the metastatic invasion. We investigated the roles of E-selectin and of stress-activated protein kinase-2 (SAPK2/p38) in modulating endothelial adhesion and transendothelial migration of HT-29 colon carcinoma cells. Tumor necrosis factor ␣ (TNF␣) strongly increased the expression of E-selectin in human umbilical vein endothelial cells (HUVEC). This effect was independent of the activation of SAPK2/p38 induced by TNF␣. Adhesion of HT-29 cells on a monolayer of HUVEC pretreated with TNF␣ was dependent on E-selectin expression but was independent of SAPK2/p38 activity of both HUVEC and tumor cells. The adhesion of HT-29 cells to E-selectin-expressing HUVEC led to the activation of SAPK2/p38 in the tumor cells as reflected by the increased phosphorylation of the actin-polymerizing factor HSP27 by mitogenactivated protein kinase 2/3, a direct target of SAPK2/ p38. Moreover, a recombinant E-selectin/Fc chimera quickly increased the activation of SAPK2/p38 in HT-29 cells. Blocking the increased activity of SAPK2/p38 of HT-29 cells by SB203580 or by expressing a dominant negative form of SAPK2/p38 inhibited their transendothelial migration. Similarly, HeLa cells stably expressing a kinase-inactive mutant of SAPK2/p38 showed a decreased capacity to cross a layer of HUVEC. Overall, our results suggest that the regulation of transendothelial migration of tumor cells involves two essential steps as follows: adhesion to the endothelium through adhesion molecules, such as E-selectin, and increased motogenic potential through adhesion-mediated activation of the SAPK2/p38 pathway.Circulating tumor cells attach to adhesive endothelial molecules, and these interactions are pivotal during the metastatic process. E-selectin, whose expression is induced by cytokines and growth factors released by tumor cells, promotes the endothelial adhesion of tumor cells from various origins, and this correlates with metastatic dissemination of tumor cells, e.g. to liver, lung, and bones (1-4). The ability of colon tumor cell clones to bind E-selectin on endothelial cells is even directly proportional to their metastatic potential (5). Moreover, inhibiting the expression of E-selectin with drugs such as cimetidine prevents metastasis (6). Metastatic colonization also correlates with the expression of other types of endothelial adhesion molecules such as P-selectin and ICAM 1 (7-12). Furthermore, the metastatic potential is associated with the circulating levels of soluble endothelial adhesion molecules shed by activated endothelial cells of cancer patients (13-17). The increased metastatic potential associated with adhesion of tumor cells to the endothelium might result from two distinctive processes as follows: local intravascular proliferation of the attached tumor cells or extravasation of these cells following their transendothelial migration into the sub-vascular tissues (18,19). In both cases, the underlying biochemical mechanisms remain ill-...

Xi, Lei, Mohiuddin Taher, Chang Yin, Fadi Salloum, and Rakesh C. Kukreja. Cobalt chloride induces delayed cardiac preconditioning in mice through selective activation of HIF-1␣ and AP-1 and iNOS signaling. Am J Physiol Heart Circ Physiol 287: H2369 -H2375, 2004. First published July 29, 2004 doi:10.1152/ajpheart. 00422.2004.-Acute systemic hypoxia induces delayed cardioprotection against ischemia (I)-reperfusion (R) injury via inducible nitric oxide synthase (iNOS)-dependent mechanism. Because CoCl2 is known to elicit hypoxia-like responses, we hypothesized that this chemical would mimic the delayed preconditioning effect in the heart. Adult male mice were pretreated with CoCl2 or saline. The hearts were isolated 24 h later and subjected to 20 min of global I and 30 min of R in Langendorff mode. Myocardial infarct size (% of risk area; mean Ϯ SE, n ϭ 6 -8/group) was reduced in mice pretreated with 30 mg/kg CoCl2 (16.1 Ϯ 3.1% vs. 27.6 Ϯ 3.3% with saline control; P Ͻ 0.05) without compromising postischemic cardiac function. Higher doses of CoCl2 failed to induce similar protection. Electrophoretic mobility gel shift assay demonstrated significant enhancement in DNA binding activity of hypoxia-inducible factor 1␣ (HIF-1␣) and activator protein 1 (AP-1) in nuclear extracts from CoCl2-treated hearts. Activation of HIF-1␣ and AP-1 was evident at 30 min and sustained for the next 4 h after CoCl2 injection. In contrast, CoCl2-induced protection was independent of NF-B activation because no DNA binding or p65 translocation was observed in nuclear extracts. Also, CoCl2-induced cardioprotection was preserved in p50 subunit NF-B-knockout (KO) mice (11.1 Ϯ 3.0% vs. 25.1 Ϯ 5.0% in saline-treated p50-KO mice; P Ͻ 0.05). The infarct-limiting effect of CoCl2 was absent in iNOS-KO mice (20.9 Ϯ 3.0%). We conclude that in vivo administration of CoCl2 preconditions the heart against I/R injury. The delayed protective effect of CoCl2 is achieved through a distinctive signaling mechanism involving HIF-1␣, AP-1, and iNOS but independent of NF-B activation.ischemia-reperfusion injury; myocardial infarction; hypoxia; nitric oxide synthase; transcription factors INCESSANT OR INTERMITTENT chronic systemic hypoxia can elicit a complex adaptive response that leads to enhanced tolerance to myocardial ischemia-reperfusion (I/R) injury in immature or adult mammals (2,3,20,28). The myocardial endogenous adaptation to hypoxia leading to resistance against subsequent lethal injuries appears to be an inherent part of the so-called "late phase of preconditioning" (5), which may have great promise for eventual clinical applications. Most recently, we reported a similar protective effect by acute exposure to systemic hypoxia in adult mice (36), where the adverse effects of chronic hypoxia, such as pulmonary hypertension and right ventricular hypertrophy, can be effectively avoided. In these studies, inducible nitric oxide synthase (iNOS) played a pivotal role in triggering as well as mediating the hypoxia-induced late cardioprotection (36). Cai et al...

Abstract-We investigated the role of stress-activated p38 MAP kinase (p38/SAPK-2) signaling in delayed preconditioning of the heart. Adult male out-bred ICR mice were treated with p38 activator, anisomycin (0.1 mg/kg IP), or vehicle (5% DMSO). Twenty-four hours later, hearts were perfused in Langendorff mode and subjected to 30 minutes of ischemia and 30 minutes of reperfusion. Improvement in postischemic recovery of end-diastolic pressure and reduction in infarct size was observed, which was abolished by SB203580, a specific p38 inhibitor, and pyrrolidinediethyldithiocarbamate (PDTC), the NF-B inhibitor, but not by PD 98059, a specific inhibitor for MEK1 or 2. Key Words: anisomycin Ⅲ p38 mitogen-activated protein kinase Ⅲ nuclear factor-B Ⅲ nitric oxide Ⅲ ischemia B rief ischemia before a second sustained ischemia decreases myocardial infarction. 1 This cardioprotective phenomenon, known as ischemic preconditioning (IPC), has been classified into two temporally distinct phases: an early or acute phase that wanes within 2 to 4 hours, and a late phase of IPC that becomes manifest 24 to 72 hours later. 2,3 Recent studies suggest an important role of MAP kinase family, particularly p38, in the early phase of IPC, 4 but the results have been controversial. 5 It has been shown that p38 and MAPKAP kinase-2 are strongly activated by ischemia in the perfused rat heart. 6 Direct activation of p38 by anisomycin mimicked IPC in the hearts, 7,8 myocytes, 9 and hepatocytes against hypoxic insults. 10 However, the role of p38 in the late preconditioning has not been investigated thoroughly. Dana et al 11 showed that late preconditioning induced by transient activation of adenosine A 1 receptor with 2-chloro-N 6 -cyclopentyladenosine (CCPA) was accompanied by significant rise in p38 activity. We further demonstrated that CCPA-induced delayed protection was abolished by p38 inhibitor SB203580 in the mouse heart, 12 suggesting an essential role of p38 in protection. Also, recently we showed that heat stress-induced delayed protection was mediated by MAP kinases. 13 However, we should consider the fact that IPC or possibly heat shock may trigger a number of additional signaling pathways, including activation of G-proteincoupled receptors, protein kinase C, and MAP kinases such as p44/42, p46/54 (JNK 1/2), and MAPKAP kinase-2. 6,14 -17 These signaling molecules can potentially induce the synthesis of effector protein(s) of cardioprotection either individually or in concert. Therefore, it is difficult to elucidate the direct role of a specific MAP kinase such as p38 in cardioprotection using an IPC model. Accordingly, a desirable approach would be to use an agent/drug that selectively targets the signaling molecule of interest. Furthermore, the downstream signaling mechanism(s) by which p38 phosphorylation transduces the stress signal at the level of transcription or posttranslation leading to the development of late preconditioning is not fully understood. Recent studies suggest that IPC triggered activation and translocation of...

Background: Vital pulp therapy preserves and maintains the integrity and the health of dental pulp tissue that has been injured by trauma, caries or restorative procedures. The enhancement of cells viability and formation of reparative dentine and new blood vessels are vital determinants of the success of direct pulp capping. Therefore, the aims of this study was to evaluate and compare the in vitro osteogenic, odontogenic and angiogenic effects of mineral trioxide aggregate (MTA), calcium hydroxide [Ca(OH) 2 ], Biodentine and Emdogain on dental pulp stem cells (DPSCs) and examine the effects of the tested materials on cell viability. Methods: DPSCs were treated with MTA, Ca(OH) 2 , Biodentine or Emdogain. Untreated cells were used as control. The cell viability was measured by MTT assay on day 3. Real-Time PCR with SYBR green was used to quantify the gene expression levels of osteogenic markers (alkaline phosphatase and osteopontin), odontogenic marker (dentin sialophosphoprotein) and angiogenic factor (vascular endothelial growth factor) on day 7 and day 14. Results: All capping materials showed variable cytotoxicity against DPSCs (77% for Emdogain, 53% for MTA, 26% for Biodentine and 16% for Ca(OH) 2 compared to control (P value < 0.0001). Osteopontin (OPN) and dentin sialophosphoprotein (DSPP) gene expression was increased by all four materials. However, alkaline phosphatase (ALP) was upregulated by all materials except Emdogain. Vascular endothelial growth factor (VEGF) expression was upregulated by all four tested materials except Ca(OH) 2. Conclusions: Our results suggest MTA, Biodentine and Emdogain exhibit similar attributes and may score better than Ca(OH) 2. Emdogain could be a promising alternative to MTA and Biodentine in enhancing pulp repair capacity following dental pulp injury. However, further future research is required to assess the clinical outcomes and compare it with the in vitro findings.

We have examined the role of stress-activated p38 MAP kinase in regulating human immunodeficiency virus (HIV) gene expression in response to ultraviolet light (UV). We found that UV activated p38 in HeLa cells harboring stably integrated copies of an HIVcat plasmid to levels similar to those obtained by hyperosmotic shock. However, hyperosmotic shock resulted in one order of magnitude smaller increase in CAT activity than treatment with UV. The specific p38 inhibitor SB203580 significantly decreased (>80%) UV activation of HIV gene expression whereas PD98059, a specific MEK-1 inhibitor did not, suggesting that p38 is specifically involved in the HIV UV response and little to no contribution is provided by MEK-1 and the p42/p44 MAP kinase pathway. Whereas increased binding of NF-kappaB to an oligonucleotide spanning the HIV enhancer was observed after UV, as expected, this binding was not affected by SB203580. Furthermore, UV activation of HIV gene expression in cells having the cat reporter gene under control of an HIV promoter deleted of the enhancer (-69/+80) produced results indistinguishable from those using HIVcat/HeLa cells with an intact HIV promoter (-485/+80), suggesting that SB203580 acts through the basal transcription machinery. Northern blot analysis of steady-state RNA from HIVcat/HeLa cells revealed an almost complete inhibition of UV activation with SB203580 at the RNA level. Similarly, the UV response was almost completely obliterated at the CAT and RNA levels in HIVcat/HeLa cells stably transfected with a plasmid expressing a kinase-inactive mutant of p38 (isoform alpha), without affecting NF-kappaB activation, providing strong genetic evidence that p38, at least the alpha isoform, is necessary for UV activation of HIV gene expression and that NF-kappaB activation alone is insufficient. These results firmly establish p38 MAP kinase as a key modulator of HIV gene expression in response to UV that acts independently of NF-kappaB.

BackgroundUltrasonography (US) is being recognized as a traditional way of the diagnosis of various thyroid disorders, and this will help in detecting the thyroid tumors in early stage. Thyroid nodules are common and usually benign; steps to diagnose malignancy should include a careful clinical evaluation, laboratory tests, a thyroid US exam and a fine-needle aspiration (FNA) biopsy.MethodsA total of 173 registered cases were used for analysis in this study. Diagnosis was made following US-guided FNA cytology (FNAC) and histopathological diagnosis; clinicopathological and demographic data of all such patients were obtained and analyzed for the present study. For statistical analysis, Statistical Package of Social Sciences v.22 (SPSS) was used.ResultsIn the current study, 87.3% of patients were female, and 12.7% were male. The mean age of the patients was 43.35 years, 86.4% were Saudi nationals and there was no significant difference between age groups. Overall, the distribution of lesions in all age groups was 41.6% in the right lobe, 9.3% lesions were adenomatous, 71.1% were colloid, and 10.4% were lymphocytic. The final diagnosis of thyroid lesions was confirmed after histopathological examinations. Out of 173 cases, 12.6% (20 cases) of male patients and 87.4% (139 cases) of female patients had benign lesions, respectively. Only one male case was malignant, and seven cases were malignant in female group. Eighty percent of males and 77.7% females have colloid nodules, and 15% of males and 9.3% of females have adenomatous nodules. Four cases were non-diagnostic, one case was atypia in females, and one case was suspicious of malignancy in a male.ConclusionsMost thyroid lesions in this study population were benign, while papillary carcinoma was the most common malignancy encountered. There was a marked female predominance in all types of thyroid diseases. The most common age group affected is 30 - 39 years. In Saudi Arabia, growing prevalence of thyroid cancer may be due to the increased screening using sensitive imaging in clinical practice, and ultrasonography is the most accurate and cost-effective method for detecting thyroid lesions.

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Adenovirus expressing herpes simplex virus-thymidine kinase (HSV-TK) sensitizes internal rat glioma cells to radiation in combination with acyclovir (ACV). However, relatively high concentrations of ACV (Ͼ10 M) are required to obtain significant radiosensitization. Serum levels rarely reach more than the lower micromolar range, preventing the full use of this genetic approach to radiosensitize cells in vivo. To better use the lower concentrations of ACV available in sera, we constructed an adenovirus expressing a mutant HSV-TK (HSV-TK (75)) isolated for its ϳ20 times greater sensitivity to ACV than wild-type (wt) HSV-TK. We demonstrate that rat RT2 glioma cells infected with adenovirus AdCMV-TK(75) and exposed to either ACV or ganciclovir become more sensitive to lower concentrations (1-3 M) of the drugs compared with cells infected with AdCMV-TK(wt), which expresses wt HSV-TK. Most importantly, the RT2 cells become more sensitive to low doses (2-4 Gy) of 60 Co radiation than cells infected with an adenovirus expressing wt HSV-TK. This sensitization is accompanied by an increased rate of apoptosis. In summary, we show that infection of rat glioma cells with an adenovirus expressing a mutant HSV-TK sensitizes the cells to low doses of radiation after exposure to ACV at lower concentrations than those required for wt HSV-TK. This finding suggests that this mutant adenovirus may improve the in vivo efficacy of HSV-TK-based cancer gene therapy approaches. Cancer Gene Therapy (2000) 7, 879 -884