�����ƽ��

Interactions between developmental signaling pathways govern the formation and function of stem cells. Prostaglandin (PG) E2 regulates vertebrate hematopoietic stem cells (HSC). Similarly, the Wnt signaling pathway controls HSC self-renewal and bone marrow repopulation. Here, we show that wnt reporter activity in zebrafish HSCs is responsive to PGE2 modulation, demonstrating a direct interaction in vivo. Inhibition of PGE2 synthesis blocked wnt-induced alterations in HSC formation. PGE2 modified the wnt signaling cascade at the level of beta-catenin degradation through cAMP/PKA-mediated stabilizing phosphorylation events. The PGE2/Wnt interaction regulated murine stem and progenitor populations in vitro in hematopoietic ES cell assays and in vivo following transplantation. The relationship between PGE2 and Wnt was also conserved during regeneration of other organ systems. Our work provides in vivo evidence that Wnt activation in stem cells requires PGE2, and suggests the PGE2/Wnt interaction is a master regulator of vertebrate regeneration and recovery.

The Smoothened receptor (Smo) mediates hedgehog (Hh) signaling critical for development, cell growth, and migration, as well as stem cell maintenance. Aberrant Hh signaling pathway activation has been implicated in a variety of cancers, and small-molecule antagonists of Smo have entered human clinical trials for the treatment of cancer. Here, we report the biochemical characterization of allosteric interactions of agonists and antagonists for Smo. Binding of two radioligands, [(3)H]3-chloro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)-phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.3) (agonist) and [(3)H]cyclopamine (antagonist), was characterized using human Smo expressed in human embryonic kidney 293F membranes. We observed full displacement of [(3)H]cyclopamine by all Smo agonist and antagonist ligands examined. N-[(1E)-(3,5-Dimethyl-1-phenyl-1H-pyrazol-4-yl)methylidene]-4-(phenylmethyl)-1-piperazinamine (SANT-1), an antagonist, did not fully inhibit the binding of [(3)H]SAG-1.3. In a functional cell-based beta-lactamase reporter gene assay, SANT-1 and N-[3-(1H-benzimidazol-2-yl)-4-chlorophenyl]-3,4,5-tris(ethyloxy)-benzamide (SANT-2) fully inhibited 3-chloro-4,7-difluoro-N-[trans-4-(methylamino)cyclohexyl]-N-{\{}[3-(4-pyridinyl)phenyl]methyl{\}}-1-benzothiophene-2-carboxamide (SAG-1.5)-induced Hh pathway activation. Detailed Schild-type" radioligand binding analysis with [(3)H]SAG-1.3 revealed that two structurally distinct Smoothened receptor antagonists SANT-1 and SANT-2 bound in a manner consistent with that of allosteric modulation. Our mechanism of action characterization of radioligand binding to Smo combined with functional data provides a better understanding of small-molecule interactions with Smo and their influence on the Hh pathway."

Recurrent/metastatic head and neck cancer remains a devastating disease with insufficient treatment options. We investigated the MET receptor tyrosine kinase as a novel target for the treatment of head and neck squamous cell carcinoma (HNSCC). MET/phosphorylated MET and HGF expression was analyzed in 121 tissues (HNSCC/normal) by immunohistochemistry, and in 20 HNSCC cell lines by immunoblotting. The effects of MET inhibition using small interfering RNA/two small-molecule inhibitors (SU11274/PF-2341066) on signaling, migration, viability, and angiogenesis were determined. The complete MET gene was sequenced in 66 head and neck cancer tissue samples and eight cell lines. MET gene copy number was determined in 14 cell lines and 23 tumor tissues. Drug combinations of SU11274 with cisplatin or erlotinib were tested in SCC35/HN5 cell lines. Eighty-four percent of the HNSCC samples showed MET overexpression, whereas 18 of 20 HNSCC cell lines (90%) expressed MET. HGF overexpression was present in 45% of HNSCC. MET inhibition with SU11274/PF-2341066 abrogated MET signaling, cell viability, motility/migration in vitro, and tumor angiogenesis in vivo. Mutational analysis of 66 tumor tissues and 8 cell lines identified novel mutations in the semaphorin (T230M/E168D/N375S), juxtamembrane (T1010I/R988C), and tyrosine kinase (T1275I/V1333I) domains (incidence: 13.5%). Increased MET gene copy number was present with textgreater10 copies in 3 of 23 (13%) tumor tissues. A greater-than-additive inhibition of cell growth was observed when combining a MET inhibitor with cisplatin or erlotinib and synergy may be mediated via erbB3/AKT signaling. MET is functionally important in HNSCC with prominent overexpression, increased gene copy number, and mutations. MET inhibition abrogated MET functions, including proliferation, migration/motility, and angiogenesis. MET is a promising, novel target for HNSCC and combination approaches with cisplatin or EGFR inhibitors should be explored.

Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy, although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore, in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells, cetuximab concentration, and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets, respectively, in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore, supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level, cetuximab concentration, and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P textless 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a, and secreted significantly (P textless 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab mediated clinical responses. Cellular and secreted immune profiles and FcgammaR genotypes from patients' lymphocytes may provide clinically useful biomarkers of immune activation in cetuximab treated patients.

Adult hematopoietic stem cells (HSCs) are routinely used to reconstitute hematopoiesis after myeloablation; however, transplantation efficacy and multilineage reconstitution can be limited by inadequate HSC number, or poor homing, engraftment, or self-renewal. Here we report that mouse and human HSCs express prostaglandin E2 (PGE2) receptors, and that short-term ex vivo exposure of HSCs to PGE2 enhances their homing, survival, and proliferation, resulting in increased long-term repopulating cell (LTRC) and competitive repopulating unit (CRU) frequency. HSCs pulsed with PGE2 are more competitive, as determined by head-to-head comparison in a competitive transplantation model. Enhanced HSC frequency and competitive advantage is stable and maintained upon serial transplantation, with full multilineage reconstitution. PGE2 increases HSC CXCR4 mRNA and surface expression, enhances their migration to SDF-1 in vitro and homing to bone marrow in vivo, and stimulates HSC entry into and progression through cell cycle. In addition, PGE2 enhances HSC survival, associated with an increase in Survivin mRNA and protein expression and reduction in intracellular active caspase-3. Our results define novel mechanisms of action whereby PGE2 enhances HSC function and supports a strategy to use PGE2 to facilitate hematopoietic transplantation.

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.

Splitomicin is derived from beta-naphthol and is an inhibitor of Silent Information Regulator 2 (SIR2). Its naphthoic moiety might be responsible for its inhibitory effects on platelets. The major goal of our study was to examine possible mechanisms of action of splitomicin on platelet aggregation in order to promote development of a novel anti-platelet aggregation therapy for cardiovascular and cerebrovascular diseases. To study the inhibitory effects of splitomicin on platelet aggregation, we used washed human platelets, and monitored platelet aggregation and ATP release induced by thrombin (0.1 U/ml), collagen (2 microg/ml), arachidonic acid (AA) (0.5 mM), U46619 (2 microM) or ADP (10 microM). Splitomicin inhibited platelet aggregation induced by thrombin, collagen, AA and U46619 with a concentration dependent manner. Splitomicin increased cAMP and this effect was enhanced when splitomicin (150 microM) was combined with PGE1 (0.5 microM). It did not further increase cAMP when combined with IBMX. This data indicated that splitomicin increases cAMP by inhibiting activity of phosphodiestease. In addition, splitomicin (300 microM) attenuated intracellular Ca(++) mobilization, and production of thromboxane B2 (TXB2) in platelets that was induced by thrombin, collagen, AA or U46619. The inhibitory mechanism of splitomicin on platelet aggregation may increase cyclic AMP levels via inhibition of cyclic AMP phosphodiesterase activity and subsequent inhibition of intracellular Ca(++) mobilization, TXB2 formation and ATP release.

Endothelial progenitor cells (EPCs) play a critical role in postnatal and tumor vasculogenesis. Vascular endothelial growth inhibitor (VEGI; TNFSF15) has been shown to inhibit endothelial cell proliferation by inducing apoptosis. We report here that VEGI inhibits the differentiation of EPCs from mouse bone marrow-derived Sca1(+) mononuclear cells. Analysis of EPC markers indicates a significant decline of the expression of endothelial cell markers, but not stem cell markers, on VEGI-treated cells. Consistently, the VEGI-treated cells exhibit a decreased capability to adhere, migrate, and form capillary-like structures on Matrigel. In addition, VEGI induces apoptosis of differentiated EPCs but not early-stage EPCs. When treated with VEGI, an increase of phospho-Erk and a decrease of phospho-Akt are detected in early-stage EPCs, whereas activation of nuclear factor-kappaB, jun N-terminal kinase, and caspase-3 is seen in differentiated EPCs. Furthermore, VEGI-induced apoptosis of differentiated EPC is, at least partly, mediated by death receptor-3 (DR3), which is detected on differentiated EPC only. VEGI-induced apoptosis signals can be inhibited by neutralizing antibodies against DR3 or recombinant extracellular domain of DR3. These findings indicate that VEGI may participate in the modulation of postnatal vasculogenesis by inhibiting EPC differentiation.

A primary goal of cancer immunotherapy is to improve the naturally occurring, but weak, immune response to tumors. Ineffective responses to cancer vaccines may be caused, in part, by low numbers of self-reactive lymphocytes surviving negative selection. Here, we estimated the frequency of CD8(+) T cells recognizing a self-antigen to be textless0.0001% ( approximately 1 in 1 million CD8(+) T cells), which is so low as to preclude a strong immune response in some mice. Supplementing this repertoire with naive antigen-specific cells increased vaccine-elicited tumor immunity and autoimmunity, but a threshold was reached whereby the transfer of increased numbers of antigen-specific cells impaired functional benefit, most likely because of intraclonal competition in the irradiated host. We show that cells primed at precursor frequencies below this competitive threshold proliferate more, acquire polyfunctionality, and eradicate tumors more effectively. This work demonstrates the functional relevance of CD8(+) T cell precursor frequency to tumor immunity and autoimmunity. Transferring optimized numbers of naive tumor-specific T cells, followed by in vivo activation, is a new approach that can be applied to human cancer immunotherapy. Further, precursor frequency as an isolated variable can be exploited to augment efficacy of clinical vaccine strategies designed to activate any antigen-specific CD8(+) T cells.

OBJECTIVE: T lymphocytes play an important role in systemic sclerosis (SSc), a connective tissue disease characterized by inflammation, fibrosis, and vascular damage. While their precise role and antigen specificity are unclear, T cell-derived cytokines likely contribute to the induction of fibrosis. The aim of this study was to establish the role of cytokine dysregulation by T cells in the pathogenesis of SSc. METHODS: To identify relationships between a specific cytokine, T cell subset, and the disease course, we studied a large cohort of patients with diffuse cutaneous SSc (dcSSc) or limited cutaneous SSc (lcSSc). Using Luminex analysis and intracellular cytokine staining, we analyzed the intrinsic ability of CD4+ and CD8+ T cell subsets to produce cytokines following in vitro activation. RESULTS: High levels of the profibrotic type 2 cytokine interleukin-13 (IL-13) were produced following activation of peripheral blood effector CD8+ T cells from SSc patients as compared with normal controls or with patients with rheumatoid arthritis. In contrast, CD4+ T cells showed a lower and more variable level of IL-13 production. This abnormality correlated with the extent of fibrosis and was more pronounced in dcSSc patients than in lcSSc patients. CONCLUSION: Dysregulated IL-13 production by effector CD8+ T cells is important in the pathogenesis of SSc and is critical in the predisposition to more severe forms of cutaneous disease. Our study is the first to identify a specific T cell phenotype that correlates with disease severity in SSc and can be used as a marker of immune dysfunction in SSc and as a novel therapeutic target.

Although the concept that cancers originate from stem cells (SC) is becoming scientifically accepted, mechanisms by which SC contribute to tumor initiation and progression are largely unknown. For colorectal cancer (CRC), investigation of this problem has been hindered by a paucity of specific markers for identification and isolation of SC from normal and malignant colon. Accordingly, aldehyde dehydrogenase 1 (ALDH1) was investigated as a possible marker for identifying colonic SC and for tracking them during cancer progression. Immunostaining showed that ALDH1(+) cells are sparse and limited to the normal crypt bottom, where SCs reside. During progression from normal epithelium to mutant (APC) epithelium to adenoma, ALDH1(+) cells increased in number and became distributed farther up the crypt. CD133(+) and CD44(+) cells, which are more numerous and broadly distributed in normal crypts, showed similar changes during tumorigenesis. Flow cytometric isolation of cancer cells based on enzymatic activity of ALDH (Aldefluor assay) and implantation of these cells in nonobese diabetic-severe combined immunodeficient mice (a) generated xenograft tumors (Aldefluor(-) cells did not), (b) generated them after implanting as few as 25 cells, and (c) generated them dose dependently. Further isolation of cancer cells using a second marker (CD44(+) or CD133(+) serially) only modestly increased enrichment based on tumor-initiating ability. Thus, ALDH1 seems to be a specific marker for identifying, isolating, and tracking human colonic SC during CRC development. These findings also support our original hypothesis, derived previously from mathematical modeling of crypt dynamics, that progressive colonic SC overpopulation occurs during colon tumorigenesis and drives CRC development.