Treatment of newborn G6pc(-/-) mice with bone marrow-derived myelomonocytes induces liver repair.

BACKGROUND & AIMS: Several studies have shown that bone marrow-derived committed myelomonocytic cells can repopulate diseased livers by fusing with host hepatocytes and can restore normal liver function. These data suggest that myelomonocyte transplantation could be a promising approach for targeted and well-tolerated cell therapy aimed at liver regeneration. We sought to determine whether bone marrow-derived myelomonocytic cells could be effective for liver reconstitution in newborn mice knock-out for glucose-6-phosphatase-α. METHODS: Bone marrow-derived myelomonocytic cells obtained from adult wild type mice were transplanted in newborn knock-out mice. Tissues of control and treated mice were frozen for histochemical analysis, or paraffin-embedded and stained with hematoxylin and eosin for histological examination or analyzed by immunohistochemistry or fluorescent in situ hybridization. RESULTS: Histological sections of livers of treated knock-out mice revealed areas of regenerating tissue consisting of hepatocytes of normal appearance and partial recovery of normal architecture as early as 1 week after myelomonocytic cells transplant. FISH analysis with X and Y chromosome paints indicated fusion between infused cells and host hepatocytes. Glucose-6-phosphatase activity was detected in treated mice with improved profiles of liver functional parameters. CONCLUSIONS: Our data indicate that bone marrow-derived myelomonocytic cell transplant may represent an effective way to achieve liver reconstitution of highly degenerated livers in newborn animals.

Aminoflavone, a ligand of the aryl hydrocarbon receptor, inhibits HIF-1alpha expression in an AhR-independent fashion.

Aminoflavone (AF), the active component of a novel anticancer agent (AFP464) in phase I clinical trials, is a ligand of the aryl hydrocarbon receptor (AhR). AhR dimerizes with HIF-1beta/AhR, which is shared with HIF-1alpha, a transcription factor critical for the response of cells to oxygen deprivation. To address whether pharmacologic activation of the AhR pathway might be a potential mechanism for inhibition of HIF-1, we tested the effects of AF on HIF-1 expression. AF inhibited HIF-1alpha transcriptional activity and protein accumulation in MCF-7 cells. However, inhibition of HIF-1alpha by AF was independent from a functional AhR pathway. Indeed, AF inhibited HIF-1alpha expression in Ah(R100) cells, in which the AhR pathway is functionally impaired, yet did not induce cytotoxicity, providing evidence that these effects are mediated by distinct signaling pathways. Moreover, AF was inactive in MDA-MB-231 cells, yet inhibited HIF-1alpha in MDA-MB-231 cells transfected with the SULT1A1 gene. AF inhibited HIF-1alpha mRNA expression by approximately 50%. Notably, actinomycin-D completely abrogated the ability of AF to downregulate HIF-1alpha mRNA, indicating that active transcription was required for the inhibition of HIF-1alpha expression. Finally, AF inhibited HIF-1alpha protein accumulation and the expression of HIF-1 target genes in MCF-7 xenografts. These results show that AF inhibits HIF-1alpha in an AhR-independent fashion, and they unveil additional activities of AF that may be relevant for its further clinical development.

A biology-driven approach identifies the hypoxia gene signature as a predictor of the outcome of neuroblastoma patients.

BACKGROUND: Hypoxia is a condition of low oxygen tension occurring in the tumor microenvironment and it is related to poor prognosis in human cancer. To examine the relationship between hypoxia and neuroblastoma, we generated and tested an in vitro derived hypoxia gene signature for its ability to predict patients' outcome. RESULTS: We obtained the gene expression profile of 11 hypoxic neuroblastoma cell lines and we derived a robust 62 probesets signature (NB-hypo) taking advantage of the strong discriminating power of the l1-l2 feature selection technique combined with the analysis of differential gene expression. We profiled gene expression of the tumors of 88 neuroblastoma patients and divided them according to the NB-hypo expression values by K-means clustering. The NB-hypo successfully stratifies the neuroblastoma patients into good and poor prognosis groups. Multivariate Cox analysis revealed that the NB-hypo is a significant independent predictor after controlling for commonly used risk factors including the amplification of MYCN oncogene. NB-hypo increases the resolution of the MYCN stratification by dividing patients with MYCN not amplified tumors in good and poor outcome suggesting that hypoxia is associated with the aggressiveness of neuroblastoma tumor independently from MYCN amplification. CONCLUSIONS: Our results demonstrate that the NB-hypo is a novel and independent prognostic factor for neuroblastoma and support the view that hypoxia is negatively correlated with tumors' outcome. We show the power of the biology-driven approach in defining hypoxia as a critical molecular program in neuroblastoma and the potential for improvement in the current criteria for risk stratification.

The hypoxic synovial environment regulates expression of vascular endothelial growth factor and osteopontin in juvenile idiopathic arthritis.

OBJECTIVE: Synovial angiogenesis, a critical determinant of juvenile idiopathic arthritis (JIA) pathogenesis, is sustained by various mediators, including vascular endothelial growth factor (VEGF) and osteopontin (OPN). We characterized the contribution of the local hypoxic environment to VEGF and OPN production by monocytic cells recruited to the synovium in JIA. METHODS: Paired synovial fluid (SF) and peripheral blood (PB) samples were collected from 20 patients with JIA. Mononuclear cells (MC) were isolated, and monocytic cells were purified by adherence, maintained in a hypoxic environment, or subjected to reoxygenation. VEGF and OPN protein concentrations were tested in SF, plasma, and culture supernatants by ELISA, and mRNA expression was assessed in freshly purified and cultured cells by reverse transcriptase-polymerase chain reaction. Synovial tissue was obtained at synovectomy from 4 patients with JIA, and analyzed by immunohistochemistry for VEGF, OPN, CD68, and hypoxia-inducible factor-1alpha (HIF-1alpha). RESULTS: VEGF mRNA expression was increased in SFMC and SF monocytic cells compared to matched PBMC and PB monocytic cells or SF lymphocytes, correlating with significantly higher protein levels in SF relative to plasma samples. Accordingly, OPN mRNA expression in SF monocytic cells was associated with significant increase of SF protein. Immunohistochemistry revealed the presence of both factors in synovial tissues at the level of the lining and sublining layers, which colocalized with intense CD68 and HIF-1alpha staining, suggesting production by hypoxic synovial monocytic cells. VEGF and OPN expression was abrogated upon SF monocytic cell reoxygenation and maintained by exposure to prolonged hypoxia. CONCLUSION: Hypoxic synovial monocytic cells are a likely source of VEGF and OPN in JIA. These data point to a role for hypoxia in the perpetuation of synovitis in JIA.

Monocytes and dendritic cells in a hypoxic environment: Spotlights on chemotaxis and migration.

A common denominator of several pathological conditions, such as solid tumors and inflammatory lesions, is represented by low partial oxygen pressure (pO(2))(2). Mononuclear phagocytes are recruited in large numbers as primary monocytes from the circulation to diseased tissues, where they accumulate within ischemic/hypoxic sites terminally differentiating into inflammatory and tumor-associated macrophages or myeloid dendritic cells (DCs). Thus, mononuclear phagocyte responses that ensue at pathological sites begin in the setting of reduced pO(2). In the last years, extensive work from several groups has been carried out to characterize hypoxia-mediated changes in mononuclear phagocyte gene expression and functional properties under different pathologic situations, demonstrating that oxygen availability is a critical regulator of their functional behavior. However, the majority of reports are focused on the characterization of differentiated macrophages, in particular tumor-infiltrating macrophages (TAM), whereas limited evidence is available for what concerns the responses of peripheral blood monocytes or DCs to the local hypoxic environment. This brief review provides an overview of the phenotypic and functional changes triggered by hypoxia in primary monocytes and DCs. A major focus is given to the chemotactic activity and migratory behavior of these cells when exposed to levels of hypoxia similar to those present in ischemic tissues. Specifically, we discuss the influence of the local hypoxic microenvironment on the expression profile of genes involved in cell motility/migration. Experimental evidence demonstrating that hypoxia modulates in primary monocytes the expression of a selected cluster of chemokine genes with a characteristic dichotomy resulting in the up-regulation of those active on neutrophils and the inhibition of those predominantly active on monocytes, macrophages, T lymphocytes, NK cells, basophils and/or DCs is reported. We also review the findings suggestive of a negative regulatory role of hypoxia on monocyte migration, which is exerted through several alternative or complementary mechanisms and results in monocyte "trapping" within ischemic/hypoxic sites of diseased tissues. Furthermore, we summarize data relative to the ability of hypoxia to differentially regulate in immature DCs (iDCs) the expression profile of genes coding for chemokines and chemokine receptors, the former being down-regulated and the latter up-regulated, thus promoting the switch from a proinflammatory to a migratory phenotype of iDCs by, respectively, reducing their capacity to recruit other inflammatory leukocytes and increasing their sensitivity to chemoattractants. Similarities and differences between the gene expression pattern induced by hypoxia in primary monocytes and that reported in differentiated macrophages are also outlined in this review, to attempt to establish which gene clusters representative of the hypoxic transcriptome of mononuclear phagocytes are specific for a certain stage of differentiation. In particular, we discuss the partial overlap existing among mononuclear phagocytes at various differentiation stages in the expression of a cluster of hypoxia-responsive genes coding for regulators of angiogenesis, proinflammatory cytokines/receptors, and inflammatory mediators and implicated in tissue neo-vascularization and cell activation. Finally, we review studies on the transcription pathways underlying hypoxia-regulated gene expression in monocytic lineage cells, which support a major role for the hypoxia-inducible factor-1 (HIF-1)/hypoxia responsive element (HRE) pathway in monocyte extravasation and migration to hypoxic sites and in the activation of monocyte/macrophage proinflammatory and immunoregulatory responses by hypoxia both in vitro and in vivo. Recent experimental evidence suggesting the requirement of additional transcription factors, such as nuclear factor-kappaB (NF-kappaB), Ets-1, CCAAT/enhancer binding protein-alpha/beta (C/EBPalpha/beta), activator-protein-1 (AP-1), and early growth response-1 (Egr-1), for hypoxic regulation of gene transcription in primary human monocytes and differentiated macrophages and indicative of the existence of both a positive and a negative O(2)-driven HIF-1-dependent feedback regulatory mechanism of hypoxia transcriptional response in primary monocytes, are also reported.

Human dendritic cells differentiated in hypoxia down-modulate antigen uptake and change their chemokine expression profile.

Dendritic cells (DCs) are the most potent antigen-presenting cells and fine-tune the immune response. We have investigated hypoxia's effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H-iDCs) significantly fail to capture antigens through down-modulation of the RhoA/Ezrin-Radixin-Moesin pathway and the expression of CD206. Moreover, H-iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.

Topotecan inhibits vascular endothelial growth factor production and angiogenic activity induced by hypoxia in human neuroblastoma by targeting hypoxia-inducible factor-1alpha and -2alpha.

Neuroblastoma produce angiogenic peptides, and the extent of angiogenesis correlates with tumor progression and poor clinical outcome. Hence, angiogenic factor inhibition represents an important therapeutic option. One of the major drives to tumor angiogenesis is hypoxia, a decrease in oxygen tension that characterizes the tumor microenvironment. We investigated the effects of the topoisomerase I inhibitor, topotecan, on vascular endothelial growth factor (VEGF) induction by hypoxia in advanced-stage human neuroblastoma cells. Topotecan counteracted hypoxic induction of VEGF and decreased angiogenic activity of conditioned medium from hypoxic cultures in vivo in the chick chorioallantoic membrane. Promoter-driven reporter studies showed the role of both hypoxia-inducible factor (HIF)-1alpha and -2alpha in VEGF transcription activation by hypoxia, because (a) overexpression of either protein by cotransfection with expression vectors resulted in VEGF promoter transactivation, which was abrogated by mutation in the HIF-binding site, and (b) targeted knockdown of HIF-1alpha/2alpha by RNA interference inhibited hypoxia-stimulated VEGF transcriptional activity and protein secretion. Topotecan-inhibitory effects on VEGF induction by hypoxia were mediated through suppression of both HIF-1alpha and HIF-2alpha protein accumulation and transactivation properties, which was specific and required ongoing RNA transcription. A similar pattern of results was obtained in cells treated with the hypoxia-mimetic agent, desferrioxamine. These data provide the first evidence that topotecan is a potent inhibitor of HIF-1alpha and HIF-2alpha subunits in hypoxic neuroblastoma cells, leading to decreased VEGF expression and angiogenic activity. An important clinical implication of these findings is that therapies targeted to the HIF pathway have the potential to inhibit neuroblastoma angiogenesis and growth.

Hypoxic synovial environment and expression of macrophage inflammatory protein 3gamma/CCL20 in juvenile idiopathic arthritis.

OBJECTIVE: Synovial inflammation is a major determinant of juvenile idiopathic arthritis (JIA) pathogenesis and is mediated by local chemokine secretion. Monocytic cells are an important source of chemokines. The purpose of this study was to investigate expression of CCL20, a macrophage inflammatory protein, in synovial fluid (SF) and SF-derived monocytic cells from JIA patients and its regulation by hypoxia, a common feature of the inflamed synovial environment. METHODS: Mononuclear cells and monocytic cells were isolated from paired SF and peripheral blood (PB) samples from JIA patients and were maintained in a hypoxic environment or subjected to reoxygenation. CCL20 concentrations in SF, PB, and culture supernatants were measured by enzyme-linked immunosorbent assay. CCL20 expression was assessed in both freshly purified and cultured cells by reverse transcriptase-polymerase chain reaction and immunocytochemistry. Hypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha were detected in the synovial tissue and cells of JIA patients by immunohistochemistry and Western blotting. RESULTS: CCL20 concentrations were significantly higher in SF compared with PB samples (P < 0.0001). SF mononuclear cells, but not matched PB mononuclear cells, constitutively expressed CCL20 messenger RNA. The SF monocytic cell fraction produced higher amounts of CCL20 than did SF lymphocytes, and CCL20 expression was associated with HIF positivity. Reoxygenation abrogated HIF and CCL20 expression, which was maintained in SF monocytic cells exposed to prolonged hypoxia. CONCLUSION: CCL20 is released into the SF of JIA patients, and SF monocytic cells are a major source of this chemokine. The hypoxic synovial microenvironment may directly contribute to the persistent inflammation associated with JIA by increasing CCL20 production by SF monocytic cells, thus representing a potential therapeutic target.

Transcriptome of hypoxic immature dendritic cells: modulation of chemokine/receptor expression.

Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process.

Hypoxia transcriptionally induces macrophage-inflammatory protein-3alpha/CCL-20 in primary human mononuclear phagocytes through nuclear factor (NF)-kappaB.

Hypoxia, a condition of low oxygen tension, occurring in many pathological processes, modifies the mononuclear phagocyte transcriptional profile. Here, we demonstrate hypoxic up-regulation of the CCL20 chemokine in primary human monocytes (Mn) and macrophages. mRNA induction was paralleled by protein secretion and dependent on gene transcription activation. Functional studies of the CCL20 promoter using a series of 5'-deleted and mutated reporter constructs demonstrated the requirement for the NF-kappaB-binding site located at position -92/-82 for gene transactivation by hypoxia, as 1) transcription was abrogated by a 3-bp mutation of the NF-kappaB motif; 2) three copies of the wild-type NF-kappaB-binding site conferred hypoxia responsiveness to a minimal heterologous promoter; and 3) hypoxia increased specific NF-kappaB binding to this sequence. Furthermore, we provide evidence of the specific role of a single NF-kappaB family member, p50, in mediating CCL20 gene transcription in hypoxic Mn. p50 homodimers were the only detectable NF-kappaB complexes binding the cognate kappaB site on the CCL20 promoter upon hypoxia exposure, and NF-kappaBp50 knockdown by lentiviral-mediated short hairpin RNA interference resulted in complete binding inhibition. NF-kappaBp50 overexpression in transient cotransfection studies promoted CCL20 gene transactivation, which was abrogated by mutation of the -92/-82 kappaB site. Moreover, nuclear expression of the other NF-kappaB family members was inhibited in hypoxic Mn. In conclusion, this study characterizes a previously unrecognized role for hypoxia as a transcriptional inducer of CCL20 in human mononuclear phagocytes and highlights the importance of the NF-kappaB pathway in mediating this response, with potential implications for inflammatory disease and cancer pathogenesis.

Hypoxia inhibits Moloney murine leukemia virus expression in activated macrophages.

Hypoxia, a local decrease in oxygen tension, occurring in many pathological processes, modifies macrophage (Mphi) gene expression and function. Here, we provide the first evidence that hypoxia inhibits transgene expression driven by the Moloney murine leukemia virus-long terminal repeats (MoMLV-LTR) in IFN-gamma-activated Mphi. Hypoxia silenced the expression of several MoMLV-LTR-driven genes, including v-myc, enhanced green fluorescence protein, and env, and was effective in different mouse Mphi cell lines and on distinct MoMLV backbone-based viruses. Down-regulation of MoMLV mRNA occurred at the transcriptional level and was associated with decreased retrovirus production, as determined by titration experiments, suggesting that hypoxia may control MoMLV retroviral spread through the suppression of LTR activity. In contrast, genes driven by the CMV or the SV40 promoter were up-regulated or unchanged by hypoxia, indicating a selective inhibitory activity on the MoMLV promoter. It is interesting that hypoxia was ineffective in suppressing MoMLV-LTR-controlled gene expression in T or fibroblast cell lines, suggesting a Mphi lineage-selective action. Finally, we found that MoMLV-mediated gene expression in Mphi was also inhibited by picolinic acid, a tryptophan catabolite with hypoxia-like activity and Mphi-activating properties, suggesting a pathophysiological role of this molecule in viral resistance and its possible use as an antiviral agent.

Hypoxia modifies the transcriptome of primary human monocytes: modulation of novel immune-related genes and identification of CC-chemokine ligand 20 as a new hypoxia-inducible gene.

Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize the molecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profile induced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the gene expression pattern were detected following 16 h exposure to 1% O(2), with 536 and 677 sequences showing at least a 1.5-fold increase and decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differences of selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whose responsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation of a significant cluster of genes with immunological relevance. These included scavenger receptors (CD163, STAB1, C1qR1, MSR1, MARCO, TLR7), immunoregulatory, costimulatory, and adhesion molecules (CD32, CD64, CD69, CD89, CMRF-35H, ITGB5, LAIR1, LIR9), chemokines/cytokines and receptors (CCL23, CCL15, CCL8, CCR1, CCR2, RDC1, IL-23A, IL-6ST). Furthermore, we provided conclusive evidence of hypoxic induction of CCL20, a chemoattractant for immature dendritic cells, activated/memory T lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mechanism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis of chronic inflammatory diseases.

Constitutively active Cdc42 mutant confers growth disadvantage in cell transformation.

The Rho family small GTPase Cdc42 is critical for diverse cellular functions including the regulation of actin organization, cell polarity, intracellular membrane trafficking, transcription, cell cycle progression and cell transformation. Like other members of the Rho family, Cdc42 cycles between the GTP-bound, active state, and the inactive, GDP-bound state under tight regulation, and it is believed that the GTP bound form of Cdc42 represents the active signaling module in eliciting effector activation and cellular responses. The constitutively active mutant, V12, derived from the analogous mutations found in oncogenic Ras that are GTPase-defective, and a "fast-cycling" self-activating mutant, F28, of Cdc42, have been widely in use to study the cellular effects of Cdc42. Here we report that the constitutively active V12 mutant of Cdc42, when stably expressed in cells, could behave in a dominant negative fashion in inhibiting cell proliferation while the F28 mutant was growth stimulatory. The V12 mutant failed to transform NIH3T3 cells while F28 potently stimulated anchorage-independent growth. The growth inhibitory effect of the V12 mutant correlated with activation of JNK2 and suppression of the cyclin D1 and NF-kappaB expressions that were instead upregulated by the F28 mutant. Furthermore, the V12 mutant could suppress, whereas the F28 mutant potentiated or had no effect on, a wide variety of oncogene-induced cell transformation, including that by the Dbl family GEFs Dbl, Vav and Lbc and the oncogenic Ras, ErbB-2, PDGF B or Raf. These results raise the possibility that over-saturation or constitutive activation of Cdc42 signal may negatively impact on cell proliferation and that both the activation and deactivation steps, or the complete GTPase cycle, of Cdc42 is required for proper function.

Induction of apoptosis by flavopiridol in human neuroblastoma cells is enhanced under hypoxia and associated with N-myc proto-oncogene down-regulation.

PURPOSE: Neuroblastoma is the most common extracranial solid tumor of children that arises from the sympathetic nervous system. Survival rates for neuroblastoma patients is low despite intensive therapeutic intervention, and the identification of new effective drugs remains a primary goal. The cyclin-dependent kinase inhibitor, flavopiridol, has demonstrated growth-inhibitory and cytotoxic activity against various tumor types. Our aim was to investigate flavopiridol effects on advanced-stage, N-myc proto-oncogene (MYCN)-amplified human neuroblastomas and the modulation of its activity by hypoxia, a critical determinant of tumor progression and a major challenge of therapy. EXPERIMENTAL DESIGN: Cell viability was monitored by 3-(4,5 dimethyl-2 thiazolyl)-2,5 diphenyl-2H tetrazolium bromide (MTT) and trypan blue dye exclusion assays; DNA synthesis was assessed with the bromodeoxyuridine pulse-labeling technique; apoptosis was studied by Giemsa staining, DNA fragmentation, terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling reaction, flow cytometric determination of hypodiploid DNA content, and evaluation of caspase activity and cytochrome c (CytC) release; MYCN expression was determined by Northern and Western blotting. RESULTS: Flavopiridol caused dose- and time-dependent decreases in neuroblastoma viability by inducing apoptosis, as confirmed by morphologic and biochemical criteria. Cell death was preceded by DNA synthesis inhibition and G1-G2 arrest, reversed by the pancaspase inhibitor, zVAD-fmk, and associated with caspase-3 and -2 activation and CytC increase. Moreover, flavopiridol strongly down-regulated MYCN mRNA and protein expression. Exposure to hypoxia enhanced both the extent of apoptosis and flavopiridol effects on CytC, caspase 3, and MYCN. CONCLUSIONS: These results indicate that flavopiridol has growth-inhibitory and apoptotic activity against advanced-stage neuroblastomas in vitro and is worthy of further investigation for the treatment of this disease.

Hypoxia inhibits the expression of the CCR5 chemokine receptor in macrophages.

Hypoxia, a decrease in oxygen tension occurring in pathological tissues, has a profound effect on macrophage functions. Here, we provide the first evidence that hypoxia inhibits CCR5 chemokine receptor expression in mouse macrophages. CCR5 was constitutively expressed in macrophages and upregulated by IFNgamma. Hypoxia downregulated both constitutive and IFNgamma-induced CCR5 mRNA and protein. Reoxygenation of hypoxic cells reverted CCR5 inhibition. CCR5 upregulation by IL-10, LPS, and IL-4 was also antagonized by hypoxia. CCR5 inhibition may be a way to retain/concentrate recruited macrophages at hypoxic sites or a feedback mechanism to control the autocrine activation of macrophages which produce CCR5 ligands.

Hypoxia selectively inhibits monocyte chemoattractant protein-1 production by macrophages.

Hypoxia, a local decrease in oxygen tension occurring in inflammatory and tumor lesions, modulates gene expression in macrophages. Because macrophages are important chemokine producers, we investigated the regulatory effects of hypoxia on macrophage-derived chemokines. We demonstrated that hypoxia inhibits the production of the macrophage and T lymphocyte chemotactic and activating factor, monocyte chemoattractant protein-1 (MCP-1). Exposure of mouse macrophages to low oxygen tension resulted in the down-regulation of constitutive MCP-1 mRNA expression and protein secretion. Hypoxia inhibitory effects were selective for MCP-1 because the chemokines macrophage inflammatory protein-1beta (MIP-1beta), RANTES, IFN-gamma-inducible protein-10, and MIP-2 were not affected, and MIP-1alpha was induced. Hypoxia also inhibited, in a time-dependent fashion, MCP-1 up-regulation by IFN-gamma and LPS. Moreover, the inhibitory action of hypoxia was exerted on human monocytic cells. MCP-1 down-regulation was associated with inhibition of gene transcription and mRNA destabilization, suggesting a dual molecular mechanism of control. Finally, we found that the triptophan catabolite picolinic acid and the iron chelator desferrioxamine, which mimic hypoxia in the induction of gene expression, differentially regulated the expression of MCP-1. This study characterizes a novel property of hypoxia as a selective inhibitor of MCP-1 production induced by different stimuli in macrophages and demonstrates that down-regulation of gene expression by hypoxia can be controlled at both transcriptional and posttranscriptional levels. Inhibition of MCP-1 may represent a negative regulatory mechanism to control macrophage-mediated leukocyte recruitment in pathological tissues.