Microparticle conferred microRNA profiles--implications in the transfer and dominance of cancer traits.

BACKGROUND: Microparticles (MPs) are membrane vesicles which are released from normal and malignant cells following a process of budding and detachment from donor cells. MPs contain surface antigens, proteins and genetic material and serve as vectors of intercellular communication. MPs comprise the major source of systemic RNA including microRNA (miRNA), the aberrant expression of which appears to be associated with stage, progression and spread of many cancers. Our previous study showed that MPs carry both transcripts and miRNAs associated with the acquisition of multidrug resistance in cancer. RESULTS: Herein, we expand on our previous finding and demonstrate that MPs carry the transcripts of the membrane vesiculation machinery (floppase and scramblase) as well as nucleic acids encoding the enzymes essential for microRNA biogenesis (Drosha, Dicer and Argonaute). We also demonstrate using microarray miRNA profiling analysis, the selective packaging of miRNAs (miR-1228*, miR-1246, miR-1308, miR-149*, miR-455-3p, miR-638 and miR-923) within the MP cargo upon release from the donor cells. CONCLUSIONS: These miRNAs are present in both haematological and non-haematological cancer cells and are involved in pathways implicated in cancer pathogenesis, membrane vesiculation and cascades regulated by ABC transporters. Our recent findings reinforce our earlier reports that MP transfer 're-templates' recipient cells so as to reflect donor cell traits. We now demonstrate that this process is likely to occur via a process of selective packaging of nucleic acid species, including regulatory nucleic acids upon MP vesiculation. These findings have significant implications in understanding the cellular basis governing the intercellular acquisition and dominance of deleterious traits in cancers.

Microparticle-associated nucleic acids mediate trait dominance in cancer.

Drug resistance is a major cause of cancer treatment failure, with multidrug resistance (MDR) being the most serious, whereby cancer cells display cross-resistance to structurally and functionally unrelated drugs. MDR is caused by overexpression of the efflux transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1). These transporters act to maintain sublethal intracellular drug concentrations within the cancer cell, making the population treatment unresponsive. Recently, we discovered a novel nongenetic basis to MDR whereby microparticles (MPs) transfer P-gp intercellularly from MDR donor cells to drug-sensitive recipient cells. MPs isolated from MDR leukemia and breast cancer cells were cocultured with their drug-sensitive counterparts. P-gp transfer was assessed by direct immunolabeling, and acquired transcripts and regulatory microRNAs by quantitative real-time PCR. We show that MDR MPs incorporate nucleic acids; MPs change recipient cells' transcriptional environment to reflect donor MDR phenotype, and distinct pathways exist among cancers of different origin that may be dependent on donor cells' ABCB1 overexpression. We demonstrate that this pathway exists for both hematological and nonhematological malignancies. By conferring MDR and "retemplating" the transcriptional landscape of recipient cells, MPs provide a novel pathway, having implications in the dissemination and acquisition of deleterious traits in clinical oncology.

Differential microRNA expression in experimental cerebral and noncerebral malaria.

MicroRNAs (miRNAs) are posttranscriptional regulatory molecules that have been implicated in the regulation of immune responses, but their role in the immune response to Plasmodium infection is unknown. We studied the expression of selected miRNAs following infection of CBA mice with Plasmodium berghei ANKA (PbA), which causes cerebral malaria (CM), or Plasmodium berghei K173 (PbK), which causes severe malaria but without cerebral complications, termed non-CM. The differential expression profiles of selected miRNAs (let-7i, miR-27a, miR-150, miR-126, miR-210, and miR-155) were analyzed in mouse brain and heart tissue by quantitative reverse transcription-PCR (qRT-PCR). We identified three miRNAs that were differentially expressed in the brain of PbA-infected CBA mice: let7i, miR-27a, and miR-150. In contrast, no miRNA changes were detected in the heart, an organ with no known pathology during acute malaria. To investigate the involvement of let-7i, miR-27a, and miR-150 in CM-resistant mice, we assessed the expression levels in gamma interferon knockout (IFN-γ(-/-)) mice on a C57BL/6 genetic background. The expression of let-7i, miR-27a, and miR-150 was unchanged in both wild-type (WT) and IFN-γ(-/-) mice following infection. Overexpression of these three miRNAs during PbA, but not PbK, infection in WT mice may be critical for the triggering of the neurological syndrome via regulation of their potential downstream targets. Our data suggest that in the CBA mouse at least, miRNA may have a regulatory role in the pathogenesis of severe malaria.

Human immunodeficiency virus infection alters tumor necrosis factor alpha production via Toll-like receptor-dependent pathways in alveolar macrophages and U1 cells.

Human immunodeficiency virus (HIV)-positive persons are predisposed to pulmonary infections, even after receiving effective highly active antiretroviral therapy. The reasons for this are unclear but may involve changes in innate immune function. HIV type 1 infection of macrophages impairs effector functions, including cytokine production. We observed decreased constitutive tumor necrosis factor alpha (TNF-alpha) concentrations and increased soluble tumor necrosis factor receptor type II (sTNFRII) in bronchoalveolar lavage fluid samples from HIV-positive subjects compared to healthy controls. Moreover, net proinflammatory TNF-alpha activity, as measured by the TNF-alpha/sTNFRII ratio, decreased as HIV-related disease progressed, as manifested by decreasing CD4 cell count and increasing HIV RNA (viral load). Since TNF-alpha is an important component of the innate immune system and is produced upon activation of Toll-like receptor (TLR) pathways, we hypothesized that the mechanism associated with deficient TNF-alpha production in the lung involved altered TLR expression or a deficit in the TLR signaling cascade. We found decreased Toll-like receptor 1 (TLR1) and TLR4 surface expression in HIV-infected U1 monocytic cells compared to the uninfected parental U937 cell line and decreased TLR message in alveolar macrophages (AMs) from HIV-positive subjects. In addition, stimulation with TLR1/2 ligand (Pam(3)Cys) or TLR4 ligand (lipopolysaccharide) resulted in decreased intracellular phosphorylated extracellular signal-regulated kinase and subsequent decreased transcription and expression of TNF-alpha in U1 cells compared to U937 cells. AMs from HIV-positive subjects also showed decreased TNF-alpha production in response to these TLR2 and TLR4 ligands. We postulate that HIV infection alters expression of TLRs with subsequent changes in mitogen-activated protein kinase signaling and cytokine production that ultimately leads to deficiencies of innate immune responses that predispose HIV-positive subjects to infection.

Tumor necrosis factor alpha leads to increased cell surface expression of CXCR4 in SK-N-MC cells.

Both host and viral factors play an important role in the pathogenesis of human immunodeficiency virus (HIV)-associated bran injury. In this study, the authors examined the interactions between tumor necrosis factor (TNF)-alpha, CXCR4, the alpha chemokine receptor, and three HIV isolates, including the T-tropic viruses, HIV-1(MN) and HIV-1(IIIB), and the dual tropic virus, HIV-1(89.6). The authors show by flow cytometry that treatment of differentiated SK-N-MC cells with TNF-alpha induces a significant increase in the cell surface expression of CXCR4 in a time- and dose-dependent manner. The effect is partly regulated at the level of transcription. To assess the biological significance of this finding, we show that TNF-alpha potentiates the ability of the above mentioned HIV isolates to induce neuronal apoptosis and that the effect is significantly reduced by pretreating cells with monoclonal antibodies to either CXCR4 and TNF-alpha. Together these results suggest that TNF-alpha may render neuronal cells vulnerable to the apoptotic effects of HIV by increasing the cell surface expression of CXCR4 and thus identify another mechanism by which TNF-alpha contributes to the pathogenesis of HIV-associated brain injury.

Stimulation of peroxisome proliferator-activated receptors alpha and gamma blocks HIV-1 replication and TNFalpha production in acutely infected primary blood cells, chronically infected U1 cells, and alveolar macrophages from HIV-infected subjects.

Metabolic disorders in HIV-infected patients, especially those receiving highly active antiretroviral therapy (HAART) regimens containing protease inhibitors, are associated with insulin resistance. These metabolic disorders include fat redistribution, diabetes, and hypertriglyceridemia. Thiazolidinediones (TZDs) are used to treat patients with diabetes secondary to insulin resistance, and TZDs are being studied in HAART-related metabolic disorders. We studied the effects of TZDs (peroxisome proliferator-activated receptor-gamma [PPARgamma] agonists) and a PPARalpha agonist on HIV replication and TNFalpha production in peripheral blood mononuclear cells (PBMCs) acutely infected with HIV-1, in a chronically infected monoblastoid cell line (U1) and in alveolar macrophages (AMs) from HIV-infected subjects and uninfected controls. Rosiglitazone, ciglitazone, troglitazone, and PgJ (PPARgamma agonists) as well as fenofibrate (PPARalpha agonist) inhibited HIV replication in both PBMCs and U1 cells. These agents also inhibited TNFalpha production, but the magnitude of TNFalpha inhibition was not directly correlated with the quantitative decreases in HIV replication. In AMs, ciglitazone, rosiglitazone, and troglitazone reduced TNFalpha production. We hypothesize that alterations in mitogen-activated protein kinase signaling pathways have contemporaneous and interrelated effects on HIV replication, cytokine production, and lipid metabolism. Modulation of these pathways using PPAR agonists may improve the metabolic alterations during HAART in conjunction with desirable decreases in HIV replication and TNFalpha production.