Silencing of carbonic anhydrase I enhances the malignant potential of exosomes secreted by prostatic tumour cells

We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock‐down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as ‘enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.     

Sera of patients with spontaneous tumour regression and elevated anti‐CA I autoantibodies change the gene expression of ECM proteins

Spontaneous tumour regression after high‐dose therapy and autologous stem cell transplantation is associated with the aplastic anaemia‐like syndrome and the presence of polyclonal autoantibodies against carbonic anhydrase I (CA I). When tumour cells were grown in vitro in the presence of patients’ sera positive for anti‐CA I autoantibodies, their morphological pattern was altered. These changes were accompanied by modifications in the gene expression profile. We observed downregulation of genes of the basal lamina assembly (collagen type IV alpha 4, the laminin subunit gamma 2), the extracellular matrix (collagen type I alpha 1), the cytoskeleton (keratin 14 type I), the collagen triple helix repeat containing 1 and the proto‐oncogene WNT7B. On the other hand, the expression of the CA 1 gene was increased in the tumour cells. It was also noticed that the presence of anti‐CA I autoantibodies did not impair tumour cell proliferation and cell viability in vitro. These findings were observed only in the presence of patients’ sera positive for anti‐CA I autoantibodies.     

Effects of different sources of copper on Ctr1, ATP7A,ATP7B,MT and DMT1 protein and gene expression in Caco-2 cells

Copper sulfate (CuSO₄), micron copper oxide (micron CuO) and nano copper oxide (nano CuO) at different concentrations were, respectively, added to culture media containing Caco-2 cells and their effects on Ctr1, ATP7A/7B, MT and DMT1 gene expression and protein expression were investigated and compared. The results showed that nano CuO promoted mRNA expression of Ctr1 in Caco-2 cells, and the difference was significant compared with micron CuO and CuSO₄. Nano CuO was more effective in promoting the expression of Ctr1 protein than CuSO₄ and micron CuO at the same concentration. Nano CuO at a concentration of 62.5 μM increased the mRNA expression levels of ATP7A and ATP7B, and the difference was significant compared with CuSO₄. The addition of CuSO₄ and nano CuO to the culture media promoted the expression of ATP7B proteins. CuSO₄ at a concentration of 125 μM increased the mRNA expression level of MT in Caco-2 cells, and the difference was significant compared with nano CuO and micron CuO. Nano CuO at a concentration of 62.5 μM inhibited the mRNA expression of DMT1, and the difference was significant compared with CuSO₄ and micron CuO. Thus, the effects of CuSO₄, micron CuO and nano CuO on the expression of copper transport proteins and the genes encoding these proteins differed considerably. Nano CuO has a different uptake and transport mechanism in Caco-2 cells to those of CuSO₄ and micron CuO.     

Regulation of extracellular matrix proteins by transforming growth factor beta1 in cultured pulmonary endothelial cells

Transforming growth factor beta-1 (TGF-beta1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-beta1 in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-beta may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24 h in either the absence or in the presence of TGF-beta1 (0-20 ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5 ng/ml. Northern blot hybridization demonstrated that TGF-beta1 (5 ng/ml) treatment of BPAE cells caused an increase in steady-state levels     

Decreased hippocampal noradrenaline does not affect corticosterone release following electrical stimulation of CA1 pyramidal cells

Bipolar electrodes were implanted into the CA1 pyramidal cells of the dorsal hippocampus and the effect of electrical stimulation of these cells on corticosterone secretion was investigated in freely moving rats. Histology showed that the electrodes were positioned in close proximity to the CA1 pyramidal cells. Rats that were subjected to high intensity electrical stimulation (1, 10, and 100A) behaved differently when compared to their sham stimulated controls. They were more active and displayed wet dog shakes. Plasma corticosterone levels increased dose-dependently in rats subjected to different electrical stimulation intensities. Although prior treatment (24 hours) of rats with DSP₄ (60 mg/kg, i.p.) significantly reduced hippocampal noradrenaline content by 46%, it did not bring about any behavioural changes. DSP₄ treatment also had no effect on electrically stimulated corticosterone release. These data suggested that stimulation of CA1 pyramidal cells may lead to increased corticosterone release and that a decrease in hippocampal noradrenaline concentration was unable to alter this corticosterone response.     

Differential involvement of TGF-beta1 in mediating the motogenic effects of TSP-1 on endothelial cells, fibroblasts and oral tumour cells

The role of TSP-1 in tumour growth and angiogenesis remains controversial, with both stimulatory and inhibitory roles proposed. The effects of TSP-1 on the migration of endothelial cells, fibroblast and oral tumour cell lines were examined using the transmembrane assay. TSP-1 induced a bi-phasic effect on human and bovine endothelial cells: stimulation at low concentrations (0.1–10 µg/ml) and inhibition at high concentrations (25–100 µg/ml). FGF-2-stimulated endothelial cell migration was either further stimulated or inhibited by TSP-1, following the same bi-phasic dose response as in the absence of FGF-2. In contrast, TSP-1 stimulated the migration of human fibroblast and oral tumour cells in a dose dependent manner; a plateau was reached with 5–25 µg/ml and no inhibitory effect was observed. These effects were partly neutralised by antibodies to αvβ3 integrin. TGF-β1 (0.1–200ng/ml tested) mimicked the effects of TSP-1 on cell migration. Function-neutralising antibodies to TGF-β1 completely abolished both the stimulatory and inhibitory effects of TSP-1 on endothelial migration, but had no effect on TSP-1-stimulated migration of fibroblast and oral tumour cells. The effects of TGF-β1 were not affected by antibodies to TSP-1. These results indicate that the effects of TSP-1 on endothelial cell migration are mediated by TGF-β1, whereas the effects on fibroblast and tumour cell migration are TGF-β1-independent.     

Loss of PTEN expression does not contribute to PDK-1 activity and PKC activation-loop phosphorylation in Jurkat leukaemic T cells

Unopposed PI3-kinase activity and 3'-phosphoinositide production in Jurkat T cells, due to a mutation in the PTEN tumour suppressor protein, results in deregulation of PH domain-containing proteins including the serine/threonine kinase PKB/Akt. In Jurkat cells, PKB/Akt is constitutively active and phosphorylated at the activation-loop residue (Thr308). 3'-phosphoinositide-dependent protein kinase-1 (PDK-1), an enzyme that also contains a PH domain, is thought to catalyse Thr308 phosphorylation of PKB/Akt in addition to other kinase families such as PKC isoforms. It is unknown however if the loss of PTEN in Jurkat cells also results in unregulated PDK-1 activity and whether such loss impacts on activation-loop phosphorylation of other putative PDK-1 substrates such as PKC. In this study we have addressed if loss of PTEN in Jurkat T cells affects PDK-1 catalytic activity and intracellular localisation. We demonstrate that reducing the level of 3'-phosphoinositides in Jurkat cells with pharmacological inhibitors of PI3-kinase or expression of PTEN does not affect PDK-1 activity, Ser241 phosphorylation or intracellular localisation. In support of this finding, we show that the levels of PKC activation-loop phosphorylation are unaffected by reductions in the levels of 3'-phosphoinositides. Instead, the dephosphorylation that occurs on PKB/Akt at Thr308 following reductions in 3'-phosphoinositides is dependent on PP2A-like phosphatase activity. Our finding that PDK-1 functions independently of 3'-phosphoinositides in T cells is also confirmed by studies in HuT-78 T cells, a PTEN-expressing cell line with undetectable levels of 3'-phosphoinositides. We conclude therefore that loss of PTEN expression in Jurkat T cells does not impact on the PDK-1/PKC pathway and that only a subset of kinases, such as PKB/Akt, are perturbed as a consequence PTEN loss.     

The localization of hnRNP A2/B1 in nuclear matrix and the aberrant expression during the RA-induced differentiation of human neuroblastoma SK-N-SH cells

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is involved in the synthesis of RNA. Its expression is up-regulated in many tumor cell lines. In this study, we investigated the distribution of hnRNP A2/B1 in the nuclear matrix, including its co-localization with expression products of related genes. Results from 2-DE PAGE and MS showed that hnRNP A2/B1 is involved with components of nuclear matrix proteins of SK-N-SH cells, and that its expression level is down-regulated after retinoic acid (RA) treatment. Protein immunoblotting results further confirm the existence of hnRNP A2/B1 in the nuclear matrix, as well as its down-regulation after RA treatment. Immunofluorescence microscopy observation showed that hnRNP A2/B1 localized in nuclear matrix of SK-N-SH cells and its distribution regions were altered after RA treatment. Laser scanning confocal microscopy observation showed that hnRNP A2/B1 co-localized with c-Myc, c-Fos, P53, and Rb in SK-N-SH cells. The co-localized region was altered as a result of RA treatment. Our data proved that hnRNP A2/B1 is a nuclear matrix protein and can be up-regulated in human neuroblastoma. The expression and distribution of hnRNP A2/B1 can affect the differentiation of SK-N-SH cells, as well as its co-localization with related oncogenes and tumor suppressor genes.     

Silencing of the Pink1 gene expression by conditional RNAi does not induce dopaminergic neuron death in mice

Transgenic RNAi, an alternative to the gene knockout approach, can induce hypomorphic phenotypes that resemble those of the gene knockout in mice. Conditional transgenic RNAi is an attractive choice of method for reverse genetics in vivo because it can achieve temporal and spatial silencing of targeted genes. Pol III promoters such as U6 are widely used to drive the expression of RNAi transgenes in animals. Tested in transgenic mice, a Cre-loxP inducible U6 promoter drove the broad expression of an shRNA against the Pink1 gene whose loss-of-functional mutations cause one form of familial Parkinson's disease. The expression of the shRNA was tightly regulated and, when induced, silenced the Pink1 gene product by more than 95% in mouse brain. However, these mice did not develop dopaminergic neurodegeneration, suggesting that silencing of the Pink1 gene expression from embryo in mice is insufficient to cause similar biochemical or morphological changes that are observed in Parkinson's disease. The results demonstrate that silencing of the PINK1 gene does not induce a reliable mouse model for Parkinson's disease, but that technically the inducible U6 promoter is useful for conditional RNAi in vivo.     

Discoidin domain of Del1 protein contributes to its deposition in the extracellular matrix

The extracellular matrix (ECM) acts as a critical factor during morphogenesis. Because the organization of the ECM directly influences the structure of tissues and organs, a determination of the way that ECM organization is regulated should help to clarify morphogenesis. We have analyzed the assembly of Del1, an ECM protein produced by endothelial cells in embryos, in the ECM. Del1 consists of three epidermal growth factor repeats (E1–E3) at its N-terminus and two discoidin domains (C1, C2) at its C-terminus. Experiments with various deletion mutants of Del1 have revealed that fragments containing the C-terminus of C1, which has a lectin-like structure, direct deposition in the ECM. The efficiency of deposition varies according to the presence of other domains in Del1. A fragment containing E3 and C1 has the strongest deposition activity, whereas fragments containing C2, which is highly homologous to C1, have low deposition activity. Digestion of ECM with hyaluronidase from bovine testis releases Del1 from the ECM, suggesting that glycosaminoglycans are involved in the deposition of Del1. In vivo gene transfer experiments have shown that fusion with the deposition domain of Del1 dramatically alters the distribution of exogenous proteins in mice. Thus, the extent of Del1 deposition may modify the organization of the ECM.     

Enhancing effect of taurine on CYP7A1 mRNA expression in Hep G2 cells

Summary. Taurine has been reported to enhance cholesterol 7α-hydroxylase (CYP7A1) mRNA expression in animal models. However, no in vitro studies of this effect have been reported. The Hep G2 human hepatoma cell line has been recognized as a good model for studying the regulation of human CYP7A1. This work characterizes the effects of taurine on CYP7A1 mRNA levels of Hep G2 cells in a dose- and time-dependent manner. In the dose-dependent experiment, Hep G2 cells were treated with 0, 2, 10 or 20 mM taurine in the presence or absence of cholesterol 0.2 mM for 48 h. In the time-dependent experiment, Hep G2 cells were treated with 0 or 20 mM taurine for 4, 24 and 48 h with and without cholesterol 0.2 mM. Our data revealed that taurine showed time- and dose-response effects on CYP7A1 mRNA levels in Hep G2 cells. However, glycine – a structural analogue of taurine – did not have an effect on CYP7A1 gene expression. These results show that, in agreement to previous studies on animal models, taurine induces the mRNA levels of CYP7A1 in Hep G2 cells, which could enhance cholesterol conversion into bile acids. Also, Hep G2 cell line may be an appropriate model to study the effects of taurine on human cholesterol metabolism.     

利用CRISPRi技术沉默MRP1基因增强A549/DDP细胞对顺铂的敏感性*

目的: 采用CRISPRi技术沉默人肺癌A549/DDP细胞MRP1基因表达,观察细胞对顺铂敏感性的变化。方法: 采用生物信息学软件分析MRP1启动子序列,设计合成3对sgRNA干扰片段,定向克隆到pSPgRNA载体中,构建靶向MRP1的干扰表达载体,分别与dCas9表达载体共转染A549/DDP细胞。实验共分为5组,分别为A549/DDP细胞组,Scrambled组,sgRNA-MRP1-1组,sgRNA-MRP1-2组和sgRNA-MRP1-3组,每组设置3个复孔,处理48 h后进行后续实验。通过qRT-PCR和Western blot检测MRP1 mRNA和蛋白表达水平,MTT法检测细胞对药物的敏感性,激光共聚焦显微镜下观察细胞的形态变化。结果: 成功构建了sgRNA-MRP1-1、sgRNA-MRP1-2和sgRNA-MRP1-3 3种干扰载体,分别与dCas9表达载体共转染A549/DDP细胞后,均能显著降低细胞MRP1基因表达(P<0.01),其中sgRNA-MRP1-2干扰效果最好,MRP1 mRNA水平降低了72%,蛋白水平降低了53%。将sgRNA-MRP1-2转染细胞后,细胞对顺铂的敏感性显著增加,IC₅₀值由74.26±3.71降低至34.29±2.51,细胞形态由梭形变为椭圆形,染色质高度凝聚、边缘化,产生凋亡小体。结论: 成功构建3种靶向MRP1的干扰表达载体,均能有效沉默A549/DDP细胞中MRP1基因表达,可增强细胞对顺铂的敏感性。     

The expression levels of the translational factors eEF1A 1/2 correlate with cell growth but not apoptosis in hepatocellular carcinoma cell lines with different differentiation grade

Despite the involvement of the elongation factors eEF1A (eEF1A1 and eEF1A2) in the development of different cancers no information is available on their possible contribution to the biology of hepatocellular carcinoma (HCC). We investigated the expression of both forms of eEF1A in HepG2 and JHH6 cell lines considered to be a good in vitro model of HCC at different stage of differentiation. Our data indicate that the mRNA amount of eEF1A1 is increased in both cell lines as compared to normal liver tissue, but eEF1A2 mRNA level is markedly increased only in JHH6. Moreover, the less differentiated cell line JHH6 displays higher EEF1A1 and EEF1A2 mRNAs levels and an higher nuclear-enriched/cytoplasm ratio of EEF1A protein compared to the better differentiated HepG2 cell line. Over-expression depends only partially on gene amplification. The more abundant mRNA levels and the higher nuclear-enriched/cytoplasm ratio of eEF1A in JHH6 neither correlate with apoptosis resistance nor with proliferation rate in sub-confluent cells. However, in confluent cells, a clear tendency to maintain JHH6 into the cell cycle was observed. In conclusion, we document the increased mRNA levels of EEF1A genes in HCC cell lines compared to normal liver. Additionally, we show the increased nuclear-enriched/cytoplasmic protein ratio of eEF1A and the marked raise of the expression of both eEF1A forms in JHH6 compared to HepG2, suggesting the possibility that eEF1A forms might become a relevant markers related to HCC tumor phenotype.     

Regulation of arginase I activity and expression by both PD-1 and CTLA-4 on the myeloid-derived suppressor cells

An elevated number of Gr-1⁺CD11b⁺ myeloid-derived suppression cells (MDSCs) has been described in mice and human bearing tumor and associated with immune suppression. Arginase I production by MDSCs in the tumor environment may be a central mechanism for immunosuppression and tumor evasion. In this study and before, we found that Gr-1⁺CD11b⁺ MDSCs from ascites and spleen of mice bearing ovarian 18D carcinoma express a high level of PD-1, CTLA-4, B7-H1 and CD80 while other co-stimulatory molecules, namely CD40, B7-DC and CD86 are not detected. Further studies showed that PD-1 and CTLA-4 on the Gr-1⁺CD11b⁺ MDSCs regulated the activity and expression of arginase I. The blockage and silencing of PD-1, CTLA-4 or both PD-1 and CTLA4 molecules could significantly reduce arginase I activity and expression induced with tumor-associated factor. Similar results were also observed while their ligands B7-H1 and/or CD80 were blocked or silenced. Furthermore, CD80 deficiency also decreased the arginase I expression and activity. Antibody blockade or silencing of PD-1, CTLA-4 or both reduced the suppressive potential of PD-1+CTLA-4+MDSCs. Blockade of PD-1, CTLA-4 or both also slowed tumor growth and improved the survival rate of tumor-bearing mice. Thus, there may exist a coinhibitory and costimulatory molecules-based immuno-regulating wet among MDSCs. This research was supported by Nankai University grant, NSFC grant “30771967”, “985” grant,The Ministry of Science and Technology grant “2006AA020502”“06C26211200695”, Tianjin Grant “07JCZDJC03300” and “06ZHCXSH04800”.     

F-actin does not modulate the initial steps of the protein kinase C activation process in living nerve cells

Actin is a major substrate for protein kinase C (PKC) and PKC is considered a modulator of the actin network. In addition in vitro studies (Biochemistry 39 (2000) 271) have suggested that all PKC isoforms bind to actin during the process of activation of the enzyme. To test the physiological significance of such a coupling we used living PC12 cells and primary cultures of cerebellar granule cells. When PC12 cells were treated with either latrunculin B, which impairs actin polymerization, or phalloidin, which stabilizes actin filaments, we observed a significant reduction of the [Ca2+]i response revealed by Fura-2 fluorescence, while the PKC conformational changes followed by Fim-1 fluorescence were unaffected. The responses induced either by cell depolarization or muscarinic receptor activation were similarly affected by the toxin treatment of PC12 cells. In cerebellar granule cells the [Ca2+]i response induced by KCl depolarization was increased by latrunculin treatment, whereas no effect was observed on the PKC response. Latrunculin had no effect on the NMDA-induced responses in these cells. Finally we also show that the response induced by a long-lasting depolarization, which mimics stimulation leading to neuronal plasticity, was not significantly altered by latrunculin or phalloidin treatment of the cells. These results suggest that the actin network is not involved in the initial steps of the PKC activation process in living nerve cells.