Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the conjugation of long chain fatty acid and cholesterol to form cholesteryl esters. It is an integral membrane protein located in the endoplasmic reticulum. Experiments performed in intact mammalian cells have shown that the rate of cholesteryl ester synthesis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL) or oxygenated sterols such as 25-hydroxycholesterol to the growth medium. However, the molecular mechanism(s) by which sterol(s) stimulate the ACAT activity remains to be elucidated. Recently, our laboratory reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268, 20747-20755). In the current study, we report the expression of human ACAT cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity. Infecting these cells with recombinant virus containing ACAT cDNA caused these cells to express high levels of ACAT protein and high levels of ACAT activity when assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. The combination of high level of ACAT protein expression and the low level of cellular cholesterol content in the infected cells have provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this system, we have shown that cholesterol itself can serve as an ACAT activator in vitro, in addition to its role as an ACAT substrate. The current work provides the experimental basis to hypothesize that, inside mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT.

BackgroundKinesin family member 2a (KIF2A), a type of motor protein found in eukaryotic cells, is associated with development and progression of various human cancers. The role of KIF2A during breast cancer tumorigenesis and progression was studied.MethodsImmunohistochemical staining, real time RT-PCR and western blot were used to examine the expression of KIF2A in cancer tissues and adjacent normal tissues from breast cancer patients. Patients’ survival in relation to KIF2A expression was estimated using the Kaplan–Meier survival and multivariate analysis. Breast cancer cell line, MDA-MB-231 was used to study the proliferation, migration and invasion of cells following KIF2A-siRNA transfection.ResultsThe expression of KIF2A in cancer tissues was higher than that in normal adjacent tissues from the same patient (P < 0.05). KIF2A expression in cancer tissue with lymph node metastasis and HER2 positive cancer were higher than that in cancer tissue without (P < 0.05). A negative correlation was found between KIF2A expression levels in breast cancer and the survival time of breast cancer patients (P < 0.05). In addition, multivariate analysis indicated that KIF2A was an independent prognostic for outcome in breast cancer (OR: 16.55, 95% CI: 2.216-123.631, P = 0.006). The proliferation, migration and invasion of cancer cells in vitro were suppressed by KIF2A gene silencing (P < 0.05).ConclusionsKIF2A may play an important role in breast cancer progression and is potentially a novel predictive and prognostic marker for breast cancer.

Together, our results indicated a possible role of Th22 pure Th17 cells and Th17 cells in RA, and blockade of the interleukin-22 may be a reasonable therapeutic strategy for RA.

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SummaryIt is well recognized that tissue microenvironments are involved in regulating the development and function of dendritic cells (DC). Oxygen supply, which varies in different tissues, has been accepted as an important microenvironmental factor in regulating the biological functions of several immune cells and as being involved in tumour progression and metastasis. However, little is known about the effect of hypoxia on the biological functions of DC and the effect of these hypoxia-conditioned DC on tumour metastasis. In this study, we analysed the transcriptional profiles of human monocyte-derived immature DC (imDC) and mature DC (mDC) cultured under normoxia and hypoxia by microarray, and found a body of potential targets regulating the functions of DC during hypoxia. In addition, the phagocytic ability of hypoxic imDC markedly decreased compared with that of normoxic imDC. Importantly, hypoxic DC poorly induced the proliferation of allogeneic T cells, but polarized allogeneic CD4 + naive T cells into a T helper type 2 (Th2) response.Moreover, hypoxic DC secreted large amounts of osteopontin, which were responsible for the enhanced migration of tumour cells. Therefore, our study provides new insights into the biological functions of DC under hypoxic conditions and one of mechanisms underlying tumour immune escape during hypoxia.Keywords: cancer; dendritic cells; hypoxia; T helper type 2Please cite this article in press as: Yang M. et al. Hypoxia skews dendritic cells to a T helper type 2-stimulating phenotype and promotes tumour cell migration by dendritic cell-derived osteopontin, Immunology (2009)

BackgroundNotch1 is a potent regulator known to play an oncogenic role in many malignancies including T-cell acute lymphoblastic leukemia (T-ALL). Tumor hypoxia and increased hypoxia-inducible factor-1α (HIF-1α) activity can act as major stimuli for tumor aggressiveness and progression. Although hypoxia-mediated activation of the Notch1 pathway plays an important role in tumor cell survival and invasiveness, the interaction between HIF-1α and Notch1 has not yet been identified in T-ALL. This study was designed to investigate whether hypoxia activates Notch1 signalling through HIF-1α stabilization and to determine the contribution of hypoxia and HIF-1α to proliferation, invasion and chemoresistance in T-ALL.MethodsT-ALL cell lines (Jurkat, Sup-T1) transfected with HIF-1α or Notch1 small interference RNA (siRNA) were incubated in normoxic or hypoxic conditions. Their potential for proliferation and invasion was measured by WST-8 and transwell assays. Flow cytometry was used to detect apoptosis and assess cell cycle regulation. Expression and regulation of components of the HIF-1α and Notch1 pathways and of genes related to proliferation, invasion and apoptosis were assessed by quantitative real-time PCR or Western blot.ResultsHypoxia potentiated Notch1 signalling via stabilization and activation of the transcription factor HIF-1α. Hypoxia/HIF-1α-activated Notch1 signalling altered expression of cell cycle regulatory proteins and accelerated cell proliferation. Hypoxia-induced Notch1 activation increased the expression of matrix metalloproteinase-2 (MMP2) and MMP9, which increased invasiveness. Of greater clinical significance, knockdown of Notch1 prevented the protective effect of hypoxia/HIF-1α against dexamethasone-induced apoptosis. This sensitization correlated with losing the effect of hypoxia/HIF-1α on Bcl-2 and Bcl-xL expression.ConclusionsNotch1 signalling is required for hypoxia/HIF-1α-induced proliferation, invasion and chemoresistance in T-ALL. Pharmacological inhibitors of HIF-1α or Notch1 signalling may be attractive interventions for T-ALL treatment.

Our data indicate that glioma-derived CD73 contributes to local adenosine-mediated immunosuppression in synergy with CD39 from infiltrating CD4(+)CD39(+) T lymphocytes, which could become a potential therapeutic target for treatment of malignant glioma and other immunosuppressive diseases.

Hypoxia is a common characteristic of many pathological and physiological conditions that can markedly change cellular metabolism and cause the accumulation of extracellular adenosine. Recent studies have shown that adenosine can modulate the function of certain immune cell types through binding with different adenosine receptors. Our previous studies have shown that hypoxia has an effect on the biological activity of dendritic cells (DCs) by inducing their differentiation towards a Th2 polarising phenotype. However, the mechanisms underlying this suppression remain unclear. In this study, we have demonstrated that hypoxic mDCs predominantly express adenosine receptor A2b. The A2b receptor antagonist MRS1754 was able to increase the production of IL-12p70 and TNF-a by hypoxic mDCs and elevate the amount of Th1 cytokine IFN-c production in a mDCs-T-cell co-culture system. We also found that the effect of hypoxia on IL-12p70 production was mediated via increased intracellular cAMP levels through the up-regulation of A2b adenosine receptor and the preferential expression of adenosine A2b receptors in hypoxic mDCs was HIF-1a dependent. Therefore, the hypoxic mDCs could provide a useful tool for researching the function of A2bR in human DCs. Our results offer new insights into understanding the molecular mechanisms underlying the biological activities of DCs in local-tissue hypoxic microenvironments.