Expression of mdm-2 oncoprotein in the primary and metastatic sites of mammary tumor (GI-101) implanted athymic nude mice

The expression of mdm-2 oncoprotein (p90) was determined in a human breast tumor xenograft line (GI-101) that was derived from a 57 year old female cancer patient with recurrent, infiltrating ductal adenocarcinoma (Stage IIIa, T3N2MX). Immunoprecipitation coupled western blot analysis of the primary tumors that have been obtained from xenograft implanted athymic nude mice, using mdm-2 (Ab-1) mouse monoclonal antibody, primarily revealed high level expression of a 90 kD full length mdm-2 protein. In the GI-101 tumor the level of full length mdm-2 (p90) protein expression increased with the increase in the size of the tumor (100 to 2,000 mm(3)) and a maximum expression was detected in 2,000 mm(3) size tumors. In addition to the expression in the primary site, a significantly high level expression of mdm-2 protein (p90) was detected in the lung and liver tissues also, which are the known metastatic sites for GI-101 xenograft tumors. However, the level of mdm-2 protein expression was undetectable in the lung and liver tissues obtained from control mice. A cell line (GI-101A) derived from the GI-101 xenograft tumor also showed a high level expression of mdm-2 protein after several generations of cell passage. When the GI-101A cells were treated with DES (Diethylstilbestrol) the mdm-2 protein expression increased after 10 min treatment and reached a peak level at 40 min. Interestingly, DES (10 and 20 microM) treatment increased the total cell number also after 96 hr treatment compared to the non-treated cells. It appears that mdm-2 (p90) may have a significant role in supporting the tumor cell growth as well as the metastatic process of the GI-101A cells.     

Specific association of increased vascular endothelial growth factor expression and its receptors with macrophage differentiation of HL-60 leukemia cells

We investigated the gene expression profiles of vascular endothelial growth factor (VEGF) and its receptors in HL-60 leukemia cells. In the VEGF family, both mRNA and protein expression of VEGF-C were up-regulated in phorbol myristate acetate (PMA)-differentiated HL-60 cells. We detected two bands of ∼31 and ∼60 kDa in cell lysates, and the higher expression of ∼31 kDa band was further increased after stimulation with tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS). A ∼31 kDa VEGF-C protein was also detected in conditioned media from PMA-differentiated HL-60 cells after LPS stimulation. The mRNA expression of VEGFR-1, VEGFR-2, and neuropilin-1 (NRP-1) was markedly up-regulated in PMA-differentiated HL-60 cells, corresponding to the results from VEGF binding studies, in which VEGF binding activity was increased in PMA-differentiated HL-60 cells. These did not occur in dimethylsulfoxide (DMSO)-differentiated HL-60 cells. The expression of VEGF-C and VEGF receptors is regulated specifically in HL-60 cells during macrophage differentiation.     

TGFβ1参与视黄酸诱导的HL-60细胞分化

为研究内源性TGF-β１对全反式视黄酸（ATRA）作用HL60细胞的影响,应用定量RT-PCR和ELISA方法,研究ATRA诱导HL-60细胞分化过程中TGF-β１ 表达的变化.并构建TGF-β１ RNA干扰表达质粒,抑制HL-60细胞内源性TGF-β１表达,进而研究ATRA诱导内源性TGF-β１表达下降的HL-60细胞分化的情况．结果发现,ATRA诱导HL-60细胞分化过程中,TGF-β１ 表达明显增高．得到4个针对不同靶位点的RNA干扰表达质粒.其中,针对起始编码区的质粒转染48 h后对HL-60细胞的TGF-β１蛋白抑制率为73~2％．内源性TGF-β１表达下降后,ATRA作用的HL-60细胞NBT还原试验的光密度值降低,CD33抗原阳性的细胞比例较对照组升高,CD11b抗原阳性的细胞比例较对照组降低.表明内源性TGF-β１表达下降后,ATRA诱导HL-60细胞分化的作用有所减弱,提示内源性TGF-β１在ATRA诱导HL-60细胞分化中起一定的作用．     

下调TSG101基因对人急性髓性白血病细胞的调节作用

构建肿瘤易感基因101(TSG101)基因的小干扰RNA载体并将其转导入HL-60细胞,获得稳定转染的阳性克隆后,应用RT-PCR和Western印迹进行鉴定;MTT法和流式细胞仪检测细胞转染前后生长速度和细胞周期的变化;DNA梯带(ladder)法和流式细胞仪检测细胞对顺铂诱导的凋亡的变化;Western印迹检测耐药相关蛋白P-gp和MRP的表达变化。结果表明,成功构建了TSG101的小干扰RNA载体;经蛋白质水平检测证实成功建立了稳定的TSG101低表达的白血病细胞模型;转染TSG101小干扰RNA后的细胞生长速度显著减慢,出现G1期阻滞;对顺铂的敏感性明显增强,形成DNA条带,且低表达P-gp。因此,下调TSG101基因能抑制HL-60细胞生长,增加细胞对化疗药物的敏感性,并提示该基因具有进行白血病基因治疗的可行性。     

Effect of systemic lupus erythematosus (SLE) treatment drugs on GI-101A breast tumor cell growth

The effect of systemic lupus erythematosus (SLE) treatment drugs on PKC (protein kinase C) activity and cell growth was studied using GI-101A breast tumor cells. Both hydroxychloroquine (HCQ) and prednisone treatments significantly increased the PKC activity in GI-101A cells after 60 min. Treatment of cells with a combination of HCQ/prednisone also produced the highest increase in PKC activity following 60 min incubation. When the GI-101A cells were treated with the same drugs, HCQ (10 ng/ml) prednisone (10 ng/ml) and HCQ/prednisone combination (10 ng/ml of each), for 72 hr the total PKC activity in the cells was significantly elevated and consequently the GI-101A cell growth was stimulated. As a result of drug induced cell growth stimulation the total number of cells in the treatment groups increased significantly compared to the non-treated controls. Interestingly HCQ and prednisone treatment induced cell growths were completely blocked by PKC specific inhibitor chelerythrine (50 microM). Our results suggest that HCQ and prednisone treatment can induce GI-101A cell growth via activating PKC.     

Reactive oxygen species induce expression of vascular endothelial growth factor in chondrocytes and human articular cartilage explants

Vascular endothelial growth factor (VEGF) promotes cartilage-degrading pathways, and there is evidence for the involvement of reactive oxygen species (ROS) in cartilage degeneration. However, a relationship between ROS and VEGF has not been reported. Here, we investigate whether the expression of VEGF is modulated by ROS. Aspirates of synovial fluid from patients with osteoarthritis (OA) were examined for intra-articular VEGF using ELISA. Immortalized C28/I2 chondrocytes and human knee cartilage explants were exposed to phorbol myristate acetate (PMA; 0-20 microg/ml), which is a ROS inducer, or 3-morpholino-sydnonimine hydrochloride (SIN-1; 0-20 microM), which is a ROS donor. The levels of VEGF protein and nitric oxide (NO) production were determined in the medium supernatant, using ELISA and Griess reagent, respectively. Gene expression of VEGF-121 and VEGF-165 was determined by splice variant RT-PCR. Expression of VEGF and VEGF receptors (VEGFR-1 and VEGFR-2) was quantified by real-time RT-PCR. Synovial fluid from OA patients revealed markedly elevated levels of VEGF. Common RT-PCR revealed that the splice variants were present in both immortalized chondrocytes and cartilage discs. In immortalized chondrocytes, stimulation with PMA or SIN-1 caused increases in the levels of VEGF, VEGFR-1 and VEGFR-2 mRNA expression. Cartilage explants produced similar results, but VEGFR-1 was only detectable after stimulation with SIN-1. Stimulation with PMA or SIN-1 resulted in a dose-dependent upregulation of the VEGF protein (as determined using ELISA) and an increase in the level of NO in the medium. Our findings indicate ROS-mediated induction of VEGF and VEGF receptors in chondrocytes and cartilage explants. These results demonstrate a relationship between ROS and VEGF as multiplex mediators in articular cartilage degeneration.     

Expression of LH receptor mRNA splice variants in bovine granulosa cells: changes with follicle size and regulation by FSH in vitro

In cattle, most evidence suggests that granulosa cells express LH receptors (LHR) after (or as) the follicle becomes dominant, however there is some suggestion that granulosa cells from smaller pre-dominant follicles may express several LHR mRNA splice variants. The objective of this study was to measure LHR expression in bovine follicles of defined size and steroidogenic ability, and in granulosa cells from small follicles (<6 mm diameter) undergoing differentiation in vitro. Semiquantitative RT-PCR demonstrated that LHR mRNA was undetectable in granulosa cells of follicles <7 mm diameter (nondominant follicles), and increased with follicle diameter in follicles >7 mm diameter. Splice variants with deletions of exon 10 and part of exon 11 were detected as previously described, and we detected a novel splice variant with a deletion of exon 3. Cultured granulosa cells contained LHR mRNA, but with significantly greater amounts of variants with deletions of exon 10 and/or exon 11 compared with cells from dominant follicles. FSH increased the abundance of some but not all LHR mRNA splice variants in cultured granulosa cells. The addition of LH to cultured cells did not increase progesterone secretion, despite the presence of LHR mRNA. Collectively, these data suggest that granulosa cells do not acquire functional LHR until follicle dominance occurs.     

雷帕霉素对低氧下HL-60细胞的低氧调控信号分子表达的影响

摘要目的：通过小分子化合物氯（CoCt2）模拟的低氧环境,分析低氧下及其雷帕霉素（RPM）作用下人急性髓细胞白血病细胞HL．60的低氧调控信号分子表达的变化;方法：常规方法复苏、传代、培养HL-60细胞,培养细胞进入对数生长期后用于实验。低氧模拟组、低氧雷帕霉素处理组、常氧雷帕霉素处理组分别用含2001xmol／LCoCl2、2001xmol／LCOCl2／20nmol／LRPM、20nmol／LRPM的1640培养基处理生长状态良好的细胞,对照组细胞用1640培养基培养,各组置培养箱以37℃、5％CO2培养,并于处理后24h、48h、72h收集细胞用于检测;采用实时荧光定量PCR方法检测低氧诱导因子（HIF-1α）、内皮细胞生长因于（VEGF）、雷帕霉素靶蛋白（mTOR）及GAPDH在转录水平的表达;结果：①与各时段对照组相比,低氧模拟组HIF-1α表达随时间逐渐增加,72h明显上调;与常氧雷帕霉素处理组各时段比较,低氧雷帕霉素处理组HIF-1α表达早期（24h）相对下调,后期相对上调;②．与对照组比较,各处理组mTOR表达均下调,低氧雷帕霉素处理组在早期（24h）下调显著;与常氧雷帕霉素处理组比较,低氧雷帕霉素处理组mTOR各时段的表达均相对下调;③与对照组各时段相比,低氧模拟组VEGF的表达在早期显著上调,但后期呈下调;常氧雷帕霉素处理组各时段VEGF的表达下调,与其比较,低氧雷帕霉素处理组各时段均呈相对下调。结论：常氧和低氧下RPM作用HL-60细胞后VEGF、mTOR的mRNA均表达下调,RPM可在低氧环境下增强了这种下调表达作用。     

Cell-specific changes in expression of mRNAs encoding splice variants of aplysia cell adhesion molecule accompany long-term synaptic plasticity

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long-term facilitation (LTF) of SN synapses evoked by serotonin (5-HT) or in the motor neuron L7 during the early phases of long-term depression (LTD) of SN synapses evoked by Phe-Met-Arg-Phe-amide (FMRFa). We used single cell RT-PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5-HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI-linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5-HT evoked changes that exaggerated cell-specific differences in isoform expression. FMRFa evoked changes that reduced cell-specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell-specific regulation of splice variant expression may contribute to different forms of long-term synaptic plasticity.     

Regional expression of the splice variants of Kv4.3 in rat brain and effects of C-terminus deletion on expressed K+ currents

In situ hybridization and RT-PCR analyses have revealed that, among three Kv4.3 splice variants (a, b, and c) with distinct C-terminal cytoplasmic domains, the mRNA for Kv4.3a is abundant in cerebral cortex, cerebellum, olfactory bulb, and medulla oblongata, whereas the mRNA for Kv4.3c is localized mainly to hippocampus. Three new distinct splice variants of Kv4.3 (Kv4.3d, e and f), which consist of 601, 635, and 628 amino acids, respectively, and have distinct C-terminal cytoplasmic domains, were isolated from rat brain by RT-PCR. Kv4.3b, d, e and f were expressed at much lower levels in brain. Mutagenesis which removed 149 amino acids in C-terminal domain of Kv4.3a significantly slowed its rate of recovery from inactivation as measured in heterologous expression in HEK293 cells. Surprisingly, however, neither the rate of inactivation nor voltage dependence of the activation and inactivation were changed.     

Aldosterone upregulates vascular endothelial growth factor expression in mouse cortical collecting duct epithelial cells through classic mineralocorticoid receptor

Accumulating evidence shows that aldosterone plays an important role in the pathogenesis of renal fibrosis but its mechanism has not been completely defined. Recently, exogenous administration of aldosterone significantly alleviated ischemic states in a model of femoral artery ligated rats, accompanied by an obvious enhancement of VEGF upregulation. We hypothesized that aldosterone may also regulate the expression of VEGF in the kidney. To confirm this, cultured cortical collecting duct epithelial cells (M-1 cell line) were incubated with aldosterone and control media, respectively. The pathway by which aldosterone regulates VEGF expression was tested by the administration of spironolactone, a specific mineralocorticoid receptor (MR) antagonist. VEGF expression was detected by immunofluorescence staining, ELISA, Western blot and RT-PCR. Aldosterone induced an elevation of VEGF excretion in a time- and dose-dependent manner. Western blotting showed a 1.4-fold elevation in cytosolic VEGF expression following aldosterone (10(-8) M) incubation for 48 h (p<0.01). After aldosterone (10(-7) M) incubation for 48 h, the mRNA level of VEGF164 and VEGF120 showed 1.8- and 1.7-fold increases, respectively (p<0.01). This upregulation was almost completely blocked by spironolactone as shown both by mRNA levels and cytosolic protein levels. In addition, the mRNA of aldosterone receptor was detected in M-1 cells. We demonstrated for the first time that aldosterone induced VEGF expression in M-1 cells, an effect mediated by classic mineralocorticoid receptor. This finding provides experimental evidence for the local non-hemodynamic action of aldosterone.