Specific induction of PAG608 in cranial and spinal motor neurons of L-DOPA-treated parkinsonian rats

We identified p53-activated gene 608 (PAG608) as a specifically induced gene in striatal tissue of L-DOPA (100 mg/kg)-injected hemi-parkinsonian rats using differential display assay. In the present study, we further examined morphological distribution of PAG608 in the central nervous system of L-DOPA-treated hemi-parkinsonian rats. PAG608 expression was markedly induced in fibers and neuronal cells of the lateral globus pallidus and reticular thalamic nucleus adjacent to internal capsule, specifically in the parkinsonian side of L-DOPA-treated models. The protein was also constitutively expressed in motor neurons specifically in either side of the pontine nucleus and motor nuclei of trigeminal and facial nerves. Furthermore, L-DOPA-induced PAG608 expression on motor neurons in the contralateral side of the ventral horn of the spinal cord and the lateral corticospinal tract without cell loss. The specific induction of PAG608 6–48 h after L-DOPA injection in the extrapyramidal tracts, pyramidal tracts and corresponding lower motor neurons of the spinal cords suggests its involvement in molecular events in stimulated motor neurons. Taken together with the constitutive expression of PAG608 in the motor nuclei of cranial nerves, PAG608 may be a useful marker of stressed or activated lower motor neurons.     

转染肿瘤坏死因子-α及MDR1反义RNA逆转乳腺癌多药耐药的研究

目的：转染肿瘤坏死因子-α(TNF-α)cDNA和多药耐药基因(MDR1)的反义RNA到乳腺癌耐药细胞株MCF-7/ADR中进行表达，并观察它们在乳腺癌耐药逆转中的作用。 方法：应用RT-PCR和DNA重组技术构建反义绿色荧光蛋白pEGFP-MDR1融合蛋白表达载体和红色荧光蛋白pDsRed2-TNF-α融合蛋白表达载体，分别和同时导入乳腺癌耐药细胞株MCF-7/ADR中进行表达，检测转染前后细胞的生长曲线、细胞凋亡程度、MDR1-mRNA和P糖蛋白（P-gp）表达情况及对ADR敏感性的变化。 结果：转染后的细胞生长明显减慢，凋亡率显著增加，MDR1-mRNA和P糖蛋白（P-gp）表达明显降低，对ADR的耐药性明显下降，敏感性增加。 结论：联合运用不同的逆转耐药机制，将TNF-α cDNA及MDR1反义RNA分别或同时导入乳腺癌耐药细胞中，能有效达到逆转耐药的目的。     

Commissural NTS lesions enhance the pressor response to central cholinergic and adrenergic activation

The clinical side-effects of increased cyclooxygenase (COX) activity induced by pathologic conditions have raised concerns recently. However, a better understanding of the mechanisms underlying the subsequent neurotoxicity requires knowledge of pathways downstream of COX, especially prostaglandin E2 (PGE2) and its receptors. Therefore, this study was performed to investigate the effects of PGE2 receptor 1 (EP1) activity on neuronal cell death resulting from hypoxia/reoxygenation (Hyp). As cyclinD1 activity has been shown to regulate neuronal apoptosis as well, the role of cyclinD1 was investigated, as well. Cortical neural cells isolated from fetal Wistar rats were cultured for 12 d and exposed to Hyp conditions to establish an in vitro Hyp model. To determine the effects of EP1 activity on Hyp-induced neurotoxicity, cells were treated with 17-phenyl trinor-PGE2 (17-pt), a synthetic EP1 agonist, or sc-51089, an EP1 antagonist, then exposed to hypoxic conditions for 3 h and reoxygenated for 21 h. Following Hyp, cell viability was quantified by MTT assays, and apoptosis was assessed by flow cytometry. Protein expression levels of caspase-3 and cyclinD1 were examined by Western blot analysis. Treatment of cultured cortical neurons with 17-pt significantly decreased the survival rate of Hyp-treated neurons (p < 0.05), while treatment with sc-51089 increased the survival rate. Treatment with 17-pt also led to increased expression levels of caspase-3, further supporting a role for EP1 in the observed neurotoxicity. However, cyclinD1 expression levels were unchanged following treatment with either 17-pt or sc-51089. Therefore, EP1 may play an important role in Hyp-induced neuronal apoptosis, but this neurotoxic activity is unlikely to involve cyclinD1.     

Altered EphA5 mRNA expression in rat brain with a single methamphetamine treatment

Methamphetamine is a potent and indirect dopaminergic agonist which can cause chronic brain dysfunctions including drug abuse, drug dependence and drug-induced psychosis. Methamphetamine is known to trigger molecular mechanisms involved in associative learning and memory, and thereby alter patterns of synaptic connectivity. The persistent risk of relapse in methamphetamine abuse, dependence and psychosis may be caused by such alterations in synaptic connectivity. EphA5 receptors constitute large families of tyrosine kinase receptor and are expressed almost exclusively in the nervous system, especially in the limbic structures. Recent studies suggest EphA5 to be important in the topographic projection, development, and plasticity of limbic structures, and to be involved in dopaminergic neurotransmission. We used in situ hybridization to examine whether methamphetamine alters EphA5 mRNA expression in the brains of adult male Wister rats. EphA5 mRNA was widely distributed in the medial frontal cortex, cingulate cortex, piriform cortex, hippocampus, habenular nucleus and amygdala. Compared to baseline expression at 0 h, EphA5 mRNA was significantly decreased (by 20%) in the medial frontal cortex at 24 h, significantly increased (by 30%) in the amygdala at 9 and 24 h, significantly but transiently decreased (by 30%) in the habenular nucleus at 1 h after a single injection of methamphetamine. Methamphetamine did not change EphA5 mRNA expression in the cingulate cortex, piriform cortex or hippocampus. Our results that methamphetamine altered EphA5 mRNA expression in rat brain suggest methamphetamine could affect patterns of synaptic connectivity, which might be responsible for methamphetamine-induced chronic brain dysfunctions.     

反义表皮生长因子受体cDNA转染抑制胶质母细胞瘤生长的机制研究

目的 探讨反义表皮生长因子受体(EGFR)cDNA转染抑制胶质母细胞瘤细胞株U87MG生长的机制。方法 反义EGFR cDNA转染胶质母细胞瘤细胞株U87MG后,筛选出低表达EGFR的细胞株;通过观察肿瘤细胞的形态和Western蛋白印迹免疫化学方法检测胶质纤维酸性蛋白(GFAP)的表达,来研究反义,EGFR cDNA转染对胶质母细胞瘤分化的影响;通过流式细胞仪进行细胞周期分析及免疫组织化学方法检测细胞周期调控蛋白p53、Rb、p16和CDK4等,以研究反义EGFR cDNA转染对细胞周期的影响及机制;用telomeric repeat amplification protocol(TRAP分析)检测肿瘤细胞的端粒酶活性。结果 反义EGFR cDNA转染胶质母细胞瘤U87MG后,肿瘤细胞的突起延长,细胞的GFAP表达增高;肿瘤细胞的G0／G1期细胞百分率明显增高,而S期细胞百分率明显减少;肿瘤细胞的野生型,p53蛋白表达明显增高,而Rb、p16和CDK4等的蛋白表达水平未发生明显改变;肿瘤细胞的端粒酶活性明显降低。结论 反义EGFR cDNA转染可以通过诱导胶质母细胞瘤细胞分化、诱导野生型p53的表达、G1细胞周期阻滞及抑制端粒酶活性等机制而抑制肿瘤细胞生长,而且这些作用机制之间是相互作用、相互协调而共同发挥抑制肿瘤细胞生长作用的。     

Immunohistochemical localization of AMPA-type glutamate receptor subunits in the nucleus of the Edinger-Westphal in embryonic chick

The ischemic damage in the hippocampal CA1 region following transient forebrain ischemia, delayed neuronal death, is a typical apoptotic response, but the underlying mechanisms are not fully understood. We have reported that mild hyperthermia (38 °C) accelerates DNA fragmentation of the gerbil CA1 pyramidal neurons following transient forebrain ischemia. Recently, we reported that galectin-3, a β-galactosidase-binding lectin, is spatio-temporally expressed only by activated microglial cells located within CA1 region following transient forebrain ischemia in gerbils. Furthermore, expression of galectin-3 and Iba-1 (a specific microglial cell marker) are strongly reduced by hypothermia during ischemic insult. To further elucidate the effect of hyperthermia on the expression of galectin-3 by micloglia in delayed neuronal death, we examined immunohistochemical expression of galectin-3 and Iba-1, in situ terminal dUTP-biotin nick end labeling of DNA fragmentation (for determination of cell death) and hematoxylin and eosin staining (for morphological observation). We observed that between 37 °C and 39 °C, there was a temperature-dependent enhancement of galectin-3 expression in microglial cells in the CA1 region following transient ischemia. Apoptotic DNA fragmentation, detected by TUNEL staining, was observed in CA1 region in normothermia. This TUNEL staining was enhanced by hyperthermia at 37.5 °C and 38 °C, but not at 39 °C. Ischemia-induced neuronal degeneration in CA1 region in gerbil hippocampus subjected to hyperthermia (37.5 °C, 38 °C and 39 °C) observed by HE staining is similar to that in normothermic gerbils. These findings imply that galectin-3 expression in microglia may influence the survival of CA1 pyramidal neurons in cases such as hyperthermia-related neuronal injury.     

反义VEGF165基因转染对人神经母细胞瘤生物学行为的影响

目的探讨反义VEGF165基因转染在抑制肿瘤血管生成和肿瘤生长中的作用。方法构建正义和反义VEGF165真核表达载体,用脂质体转染人神经母细胞瘤细胞SH-SY5Y,G418筛选稳定表达细胞克隆。应用RT-PCR证实外源性反义VEGF mRNA的表达,免疫细胞化学和EUSA检测VEGF蛋白的表达水平,MTT法检测肿瘤细胞体外生长情况,并接种于裸鼠皮下,观察体内肿瘤增殖能力。结果RT-PCR证实转染反义VEGF的细胞中有外源性反义VEGF mRNA的表达,免疫细胞化学和EUSA显示VEGF蛋白表达明显降低,MTT实验显示转染前后细胞体外生长速度基本一致。而转染反义VEGF的肿瘤细胞裸鼠体内生长速度明显减慢。结论反义VEGF基因转染能够有效抑制SH-SY5Y细胞内源性VEGF蛋白的表达,抑制裸鼠体内肿瘤的生长。     

AFP增强型四元复合体介导N-ras反义RNA抑制HepG2.2.15细胞成瘤性的研究

目的　观察AFP增强型四元复合体介导的N ras反义RNA转移系统对HBV转基因肝癌细胞系HepG2 .2 .15致瘤性的体内外抑制作用。方法　构建含有N ras反义RNA的AFP增强型四元复合体 ,体外瞬时转染HBV转基因HepG2 .2 .15细胞 ,流式细胞术检测转染前后N ras蛋白表达水平、细胞凋亡率及细胞周期的变化 ,同时建立稳定表达N ras反义RNA的肝癌细胞系HepG2 .2 .15 /as ras,绘制转染前后生长曲线。体内抑瘤试验分别以HepG2 .2 .15或HepG2 .2 .15 /as ras细胞制备荷瘤裸鼠模型 ,比较二者成瘤率。HepG2 .2 .15成瘤组局部瘤内注射N ras反义RNA四元复合体 ,研究其对肿瘤的抑制作用。结果　AFP增强型四元复合体介导N ras反义RNA体外瞬时转染HepG2 .2 .15细胞可显著降低胞内N ras蛋白的表达水平 (P <0 .0 5 ) ,使细胞生长停滞于G0 /G1期 ,且可诱导细胞凋亡。体内抑瘤实验显示 ,HepG2 .2 .15 /as ras细胞注射组成瘤率 ( 4 0 % )显著低于HepG2 .2 .15细胞注射组( 10 0 % )。瘤内注射N ras反义RNA四元复合体可使肿瘤体积明显缩小。结论　AFP增强型四元复合体介导的N ras反义RNA转移系统可降低HBV转基因肝癌细胞HepG2 .2 .15N ras蛋白的表达水平 ,逆转其恶性行为 ,体内外均可有效抑制肿瘤的生长增殖     

Hepatitis C virus protein expression induces apoptosis in HepG2 cells

The mechanisms of hepatocyte death and the events that lead to a high rate of chronic liver disease in patients infected with hepatitis C virus are not known. We established a HCV replication system in HepG2 cell culture and utilized this model to address the effect of HCV proteins on HepG2 cell growth and viability. After transfection of HepG2 cells with full-length RNA, a truncated RNA, or an antisense RNA, cell proliferation and cell viability were analyzed by thymidine uptake and the trypan blue exclusion method, respectively. Full-length RNA transfected HepG2 cells showed a decrease in cell proliferation and viability compared to cells transfected with HCV truncated RNA and antisense RNA control. A subset of cells expressing HCV proteins underwent apoptosis as documented by morphological studies, ultrastructural analysis, cell cycle analysis by flow cytometry, terminal transferase enzyme mediated end labeling of DNA, and DNA laddering. This study suggests that expression of HCV proteins can lead to cell death by apoptosis, which may be an important event in the pathogenesis of chronic hepatitis C virus infection in humans.     

Broad spectrum neuroprotection profile of phosphodiesterase inhibitors as related to modulation of cell-cycle elements and caspase-3 activation

Cellular injury can involve the aberrant stimulation of cell cycle proteins in part through activation of phosphodiesterases (PDEs) and downstream expression of cell-cycle components such as cyclin D1. In mature non-proliferating cells activation of the cell cycle can lead to the induction of programmed cell death. In the present study, we investigated the in vitro neuroprotective efficacy and mechanism of action of vinpocetine (PDE1 inhibitor), trequinsin (PDE3 inhibitor), and rolipram (PDE4 inhibitor) in four mechanistically-distinct models of injury to primary rat cortical neurons as related to cell cycle regulation and apoptosis. Cellular injury was induced by hypoxia/hypoglycemia, veratridine (10 μM), staurosporine (1 μM), or glutamate (100 μM), resulting in average neuronal cell death rates of 43–48% as determined by MTT assay. Treatment with each PDE inhibitor (PDEI) resulted in a similar concentration-dependent neuroprotection profile with maximal effective concentrations of 5–10 μM (55–77% neuroprotection) in all four neurotoxicity models. Direct cytotoxicity due to PDE inhibition alone was not observed at concentrations below 100 μM. Further studies indicated that PDEIs can suppress the excitotoxic upregulation of cyclin D1 similar to the effects of flavopiridol, a cyclin-dependent kinase inhibitor, including suppression of pro-apoptotic capase-3 activity. Overall, these data indicate that PDEIs are broad-spectrum neuroprotective agents acting through modulation of cell cycle elements and may offer a novel mode of therapy against acute injury to the brain.     

Protective effects of zinc chelation in traumatic brain injury correlate with upregulation of neuroprotective genes in rat brain

Chelation of excessive neuronal zinc ameliorates zinc neurotoxicity and reduces subsequent neuronal injury. To clarify the molecular mechanisms of this neuroprotective effect, we used a focused cDNA array of stress-response genes with zinc chelation (calcium EDTA) in our rat model of fluid percussion brain injury at 2 h, 24 h, and 7 days after injury. In parallel experiments, we compared neuronal cell death in TUNEL-stained brain sections in traumatized rats with and without calcium EDTA treatment. Zinc chelation induced the expression of several neuroprotective genes; neuroprotective gene expression correlated with substantially decreased numbers of TUNEL-positive cells.     

Ethanol-induced oxidative stress is mediated by p38 MAPK pathway in mouse hippocampal cells

It has been known that ethanol causes neuronal cell death through oxidative stress. Ethanol itself and reactive oxygen species (ROS) produced by ethanol modulate intracellular signaling pathways including mitogen-activated protein kinase (MAPK) cascades. This study was conducted to examine the impact of ethanol on MAPK signaling in HT22 cells. Ethanol (100 and 400 mM) caused activation of ERK, p38 MAPK, and JNK. ERK activation occurred in early time and p38 MAPK activation was evident when ERK activation was diminished. Specific inhibitor of p38 MAPK (SB203580) protected HT22 cells against ethanol, which was accompanied by an inhibition of ROS accumulation. However, inhibitors of ERK (U0126) and JNK (SP600125) had no effects on ethanol-induced neuronal cell death when they are treated with ethanol for 24 h. These results suggest that p38 MAPK may have important roles in ROS accumulation during ethanol-induced oxidative stress in HT22 cells.     

端粒酶反义腺病毒载体对乳腺癌细胞MCF-7端粒酶的抑制作用

端粒酶ＲＮＡ的反义地人乳腺癌细胞系ＭＣＦ－７细胞端粒酶活性的影响。方法用重组腺病毒转移并表达端粒酶ＲＮＡ的反义ｃＤＮＡ,采用基因重组腺脂质体共转当闰酶反义重组病毒,用Ｓｏｕｔｈｅｒｎ杂交鉴定病毒的整合功能,用ＴＲＡＰ－ 染法检测端粒酶活性。结果ＭＣＦ－７细胞是恶性乳腺癌的典型细胞系。对对照组ＭＣＦ－７、ＭＣＦ－７、ｖＡｄ－ＡＡＶ细胞相比,反义病毒感染后的细胞是恶性乳腺癌的典型细胞系。与对照组ＭＣＦ     

Lithium and oxidative stress lessons from the MPTP model of Parkinson's disease

Lithium has been successfully employed therapeutically for treatment of bipolar depressive illness; however, its mechanism of action is poorly understood. Recently, it has been demonstrated by us that lithium can prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) dopaminergic neurotoxicity in mice. From analyzing the pattern of protection in various parameters, we suggest that lithium protects against MPTP-induced depletion of striatal dopamine (DA) by preventing free radical-induced inactivation of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. Possible neuroprotective effect of lithium against H₂O₂-induced cell death was assessed in human neuroblastoma; SH-SY5Y cell line. Pretreatment with LiCl (2 mM and 4 mM) for 7 days protected against H₂O₂ neurotoxicity in a dose-dependent manner. However, this protection could not be achieved through short-term incubation with LiCl. In agreement; we found that lithium lacks immediate antioxidant activity using the in vitro lipid peroxidation essay indicating that not acute but chronic treatment with lithium allows cells to deal better with oxidative stress.     

Homocysteine induced SH-SY5Y apoptosis through activation of NADPH oxidase in U251MG cells

Epidemiological studies have indicated a correlation between homocysteinemia and dementia, including Alzheimer's disease. However, the mechanism by which homocysteine (Hcy) induces neuronal cell death remains unknown. We found that micromolar concentrations of Hcy induced neuroblastoma SH-SY5Y cell death only when co-cultured with glioblastoma U251MG cells. In this culture system, cysteine had no effect on SH-SY5Y cell death. There was an increase in TUNEL-positive cells and loss of mitochondrial membrane potential following treatment with 100 μM Hcy. Addition of conditioned medium prepared from U251MG cells in the presence of 100 μM Hcy also reduced SH-SY5Y cell viability, while this effect was prevented when using conditioned medium from U251MG cells exposed to 100 μM Hcy + apocynin, a specific NADPH oxidase inhibitor. Following exposure to 100 μM Hcy in U251MG cells, expression of Rac1, a compartment of NADPH oxidase, was translocated to the plasma membrane, and the active form of Rac1 was increased. There was no change in peroxide concentration in the medium of U251MG cells after addition of Hcy. Overall, these data suggest that Hcy stimulates Rac1 activation and NADPH oxidase, resulting in superoxide anion production that may induce SH-SY5Y cell apoptosis.     

Early induction of neuronal lipocalin-type prostaglandin D synthase after hypoxic-ischemic injury in developing brains

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is up-regulated in oligodendrocytes (OLs) in mouse models for genetic neurological disorders including globoid cell leukodystrophy (twitcher) and GM₁ and GM₂ gangliosidoses and in the brain of patients with multiple sclerosis. Since L-PGDS-deficient twitcher mice undergo extensive neuronal death, we concluded that L-PGDS functions protectively against neuronal degeneration. In this study, we investigated whether L-PGDS is also up-regulated in acute and massive brain injury resulting from neonatal hypoxic-ischemic encephalopathy (HIE). Analysis of brains from human neonates who had died from HIE disclosed that the surviving neurons in the infarcted lesions expressed L-PGDS. Mouse models for neonatal HIE were made on postnatal day (PND) 7. Global infarction in the ipsilateral hemisphere was evident at 24 h after reoxygenation in this model. Intense L-PGDS immunoreactivity was already observed at 10 min after reoxygenation in apparently normal neurons in the cortex, and thereafter, in neurons adjacent to the infarcted area. Quantitative RT-PCR revealed that the L-PGDS mRNA level of the infarcted hemisphere was 33-fold higher than that of the sham-operated mouse brains at 1 h after reoxygenation and that it decreased to the normal level by 24 h thereafter. Furthermore, in both human and mouse brains, many of L-PGDS-positive cells were also immunoreactive for p53; and some of these expressed cleaved caspase-3. The expression of L-PGDS in degenerating neurons implies that L-PGDS functions as an early stress protein to protect against neuronal death in the HIE brain.     

mfgl2凝血酶原酶反义载体的构建及其效应研究

目的：从基因转录水平抑制小鼠巨噬细胞系Raw264.7细胞表达mfgl2（mouse fibrinogen like protein 2/mfgl2 prothrominase,以下简称mfgl2）和其效应研究.方法：构建mfgl2反义载体（mfgl2-antisense-pcDNA3.0）并转染Raw264.7细胞,在IFN-γ刺激下,分别用RT-PCR、免疫组化、蛋白质印迹分析和mfgl2蛋白功能学检测方法（Procoagulant activity, PCA）检测mfgl2基因转录和蛋白表达水平及PCA的变化.结果：转染mfgl2-antisense-pcDNA3.0可显著抑制Raw264.7细胞mfgl2基因转录和蛋白表达并使其生物学活性显著下降,并且mfgl2表达水平与其PCA活性变化呈平行关系.结论：mfgl2-antisense-pcDNA3.0可有效抑制Raw264.7细胞表达mfgl2,同时其PCA也显著下降,表明Raw264.7细胞PCA与其表达mfgl2有关,此结果为探讨从基因水平治疗与fgl2高表达相关的疾病如重型乙型肝炎、同种移植排斥反应时脏器局部的微血栓形成、纤维素沉积、微循环障碍等病理学损伤寻找新的干预靶点提供了科学依据和手段.     

鞘糖脂微结构域1相关磷酸化蛋白基因的筛选及其对转移性前列腺癌细胞生物学行为的影响

目的 筛选肿瘤转移相关新基因,探讨鞘精脂微结构域1相关磷酸化蛋白基因(PAG1)转染对人前列腺癌细胞系PC-3M的高转移亚系PC-3M-1E8体外生物学行为的影响.方法 利用PC-3M高转移亚系PC-3M-1E8、低转移亚系PC-3M-284,人肺巨细胞痛细胞系(PG)高转移亚系PG-BE1和低转移亚系PG-LH7 cDNA制作4张基因芯片,筛选出PC-3M和PG高、低转移亚系共同差异表达基因.对在两个转移亚系共同表达下调的PAG1基因做进一步研究,采用即时定量PCR及Western blot验证PAG1在PC-3M细胞系中的表达.构建pcDNA3.0-PAG1真核表达载体,稳定转染PC-3M-1E8细胞.MTT比色实验及软琼脂集落形成实验检测肿瘤细胞体外增殖能力;流式细胞术检测肿瘤细胞周期及凋亡;Matrigel穿膜实验检测肿瘤细胞体外侵袭能力.结果 基因芯片初步筛选出PC-3M高、低转移亚系差异表达基因共327个,上调基因123个,下调基因204个.PG高、低转移亚系差异表达基因共281个,上调基因167个,下调基因114个.PC-3M与PG高转移亚系共同表达下调基因9个、上调基因8个.即时定量PCR及Western blot证实PAG1在PC-3M高转移亚系中表达低于低转移亚系.MTT比色及软琼脂集落形成实验显示转染pcDNA3.0-PAG1组细胞增殖速度明显低于转染空载体组和未转染组(均P<0.05).细胞周期检测转染pcDNA3.0-PAG1组比转染空载体组和未转染组处于G_0～G_1期的细胞百分数明显增加(P<0.05).转染pcDNA3.0-PAG1组与转染空载体组和未转染组相比凋亡细胞百分率无显著差异(P>0.05).体外穿膜侵袭实验结果表明转染pcDNA3.0-PAG1组比转染空载体组和未转染组穿膜细胞数目明显减少,分别为(35.1±4.9)、(127.6±6.6)和(135.0±5.0)个(P<0.05).结论 利用同一母系来源的高、低转移亚系制作基因芯片可以摒除转移无关基因的干扰,筛选出差异表达的肿瘤转移相关基因.PAG1基因稳定转染能抑制人前列腺癌高转移亚系PC-3M-1E8细胞的体外增殖能力和侵袭能力,PAG1基因可能是一个潜在的肿瘤增殖、侵袭和转移的抑制基因.     

Differential Gene Expression in Apoptosis: Identification of Ribosomal Protein 23K, a Cell Proliferation Inhibitor

Gene expression during the camptothecin-induced apoptotic death of human leukemic U937 cells and mouse T-cell hybridoma QW4.1 cells was studied by the mRNA differential display technique. Ten clones were confirmed to be differentially expressed, nine of which encoded novel sequences. One clone, U3.2, was induced approximately 10-fold in camptothecin-treated cells and was found to be identical to a highly basic 23-kDa human protein which contains basic leucine zipper-like motifs and has recently been identified as the human homologue of the rat ribosomal protein L13a. Northern blot analysis revealed a major mRNA of 0.9 kb and a minor mRNA of 1.3 kb. Overexpression of a full-length 23K cDNA, tagged with a FLAG sequence, in COS-7 cells revealed a predominantly nucleolar localization and the absence of any 23K protein from the cytoplasm. Subsequent transfection studies, using antisense phosphorothioate-modified oligonucleotides, revealed that inhibition of 23K expression results in an increased cell proliferation and greater sensitivity of U937 cells to the effects of camptothecin-induced cell death. Upregulation of 23K expression using a cDNA construct resulted in a decrease in cell proliferation and growth arrest, suggesting a role for 23K protein as a proliferation checkpoint following a cellular insult.