艰难梭菌A毒素的细胞致死活性研究

本文报道艰难梭菌Ａ毒素对４种培养细胞的细胞致死活性的探讨。４种培养细胞为Ｖｅｒｏ（非洲绿猴肾细胞）细胞、ＴＰＣ─１（人甲状腺肿瘤细胞）细胞、ＮＩＨ３Ｔ３细胞（小鼠成纤维细胞）及将ｒａｓ癌基因转基因于ＮＩＨ３Ｔ３细胞的ＮＩＨ３Ｔ３ｒａｓ细胞。应用台酚蓝排除能试验、噻唑蓝（ＭＴＳ）比色、细胞膜损害测定试验、荧光显微术观察细胞核的形态变化等测定Ａ毒素细胞致死活性。实验表明：４种培养细胞系对Ａ毒素细胞圆缩化作用的敏感性依次为ＮＩＨ３Ｔ３ｒａｓ,ＴＰＣ─１,Ｖｅｒｏ,ＮＩＨ３Ｔ３细胞。而对Ａ毒素细胞致死活性的敏感性依次为ＴＰＣ─１,ＮＩＨ３Ｔ３,Ｖｅｒｏ,ＮＩＨ３Ｔ３ｒａｓ细胞。从而得知Ａ毒素的细胞致死活性不但依细胞种类不同而不同,而且也不一定与Ａ毒素的细胞圆缩化作用有关。肿瘤细胞ＴＰＣ─１细胞对Ａ毒素致死活性有特殊敏感性。以上结果对探索抗癌新药的研制具有重要意义。     

转基因人肺腺癌细胞株的建立

以逆转录病毒ｐＬＸＳＬ为载体与人细胞介素２基因（ＩＬ－２）重组,用电穿孔技术将其重组子ｐＬＩＬ—２ＳＮ导入ＰＡ３１７细胞中,建立了逆转染病毒包装体系细胞ＰＡ３１７／ｐＬＩＬ—２ＳＮ。应用逆转录病毒包装体系细胞上清液去转染入肺腺癌细胞ＳＰＣ—Ａ１,经过２０天含Ｇ４１８培液的筛选式培养,历经了５０代的传代培养,获得了转白细胞介素２基因的人肺腺癌细胞株ＳＰＣ－Ａ１／ＩＬ－２。该细胞（ＳＰＣ－Ａ１／ＩＬ－２）经ＰＣＲ技术验证外源性目的基因（ＩＬ－２ｇｅｎｅ）导入细胞内,且证明该细胞能表达ＩＬ—２基因（有ＩＬ－２的分泌）。我们研究的目的是对肿瘤细胞的特异性抗原修饰、对肿瘤细胞进行处理。试图建立肿瘤疫苗。     

缺氧诱导因子1和红细胞生成素3‘—增强子调节内皮细胞？…

目的和方法：将红细胞生成素（ＥＰＯ）３＇－增强子野生片断及点突变片断借脂质体主人脐静脉内皮细胞株ＥＣＶ－３０４,用半定量ＲＴ－ＰＣＲ测定正常秘缺氧诱导因子－１（ＨＩＦ－１）诱导剂氧化钴（ＣｏＣｌ２）作用下培养６ｈ的细胞环氧合酶２（ＣＯＸ－２）和血栓素合酶（ＴＸＳ）的ｍＲＮＡ。结果：ＨＩＦ－１诱导剂ＣｏＣｌ２可放ＣＯＸ－２和ＴＸＳ基因转明显增强２,向细胞导入野生ＥＰＯ３＇增强子片断可阻断ＣｏＣｌ２诱     

神经节苷脂GM_3对人白血病J6－2细胞磷脂酰胆碱代谢的影响

神经节苷脂ＧＭ３诱导人单核样白血病Ｊ６－２细胞沿单核／巨噬细胞途径分化．在ＧＭ３诱导分化同时,Ｊ６－２细胞磷脂代谢发生了显著变化．采用（（３２）Ｐ）Ｐｉ、［ＧＨ３－３Ｈ］胆碱和［ＣＨ３－３Ｈ］ＳＡＭ参入实验对ＧＭ３影响Ｊ６－２细胞ＰＣ代谢的机制进行了初步的探讨．ＧＭ３促进［（３２）Ｐ］Ｐｉ参入Ｊ６－２细胞ＰＣ;抑制［ＣＨ３－３Ｈ］胆碱参入ＰＣ及ＰＣ合成的前体磷酸胆碱及ＣＤＰ－胆碱;ＧＭ３促进［ＣＨ３－３Ｈ］ＳＡＭ参入ＰＣ,但抑制［ＣＨ３－３Ｈ］ＳＡＭ参入ＰＣ合成的前体胆碱、磷酸胆碱和ＣＤＰ－胆碱．上述结果提示,ＧＭ３抑制Ｊ６－２细胞ＰＣ合成的ＣＤＰ－胆碱途径,促进ＰＣ合成的ＰＥ甲基化途径．     

HSV—tk／NAS系统基因治疗脑肿瘤中野生型腺病毒的检测及其安全性

在腺病毒介导的ＨＳＶ－ｔｋ／ＮＡＳ系统脑肿瘤基因治疗研究过程中,建立了野生型腺病毒（ＲＣＡ）的检测系统,主要包括腺病毒Ｅ１区的ＰＣＲ以及野生型病毒的细胞病理效应观察这两种方法,ＰＣＲ的灵敏度为１个２９３细胞所含有的Ｅ１片段数。对基因治疗脑肿瘤所采用的重组腺病毒ＡｄＴＫ及其感染的细胞进行ＲＣＡ的检测,没有发现ＲＣＡ。通过离体实验首次发现,重组腺病毒对脑肿瘤细胞的杀伤作用明显高于正常的脑胶质细胞;大鼠     

逆转录病毒介导的TNFa基因在胶质瘤细胞系（C6）中的表达及对肿瘤细胞恶性生长的影响

利用逆转录病毒载体ＬＸＳＮ构建了含有完整编码ＴＮＦｃＤＮＡ的重组逆转录病毒质粒ｐＬＸＳＮ－ｔｎｆ,用Ｌｉｐｏｆｅｃｔａｍｉｎｅ将重组质粒导入病毒包装细胞ｐＡ３１７,经Ｇ４１８筛选培养获得抗性克隆,用ＮＩＨ３Ｔ３细胞测定病毒滴度,获得滴度为５×１０５ＣＦＵ／ｍｌ的细胞克隆。利用病毒上清液感染大鼠胶质瘤细胞系Ｃ６,得到Ｇ４１８抗性克隆细胞Ｃ６ｐＬＸＳＮ－ｔｎｆ,经ＰＣＲ检测,ＴＮＦｃＤＮＡ完整地整合在细胞基因组中。测定Ｃ６ｐＬＸＳＮ－ｔｎｆ细胞上清中ＴＮＦ的生物活性,结果显示ＴＮＦ有相对稳定的表达（４８～１８０Ｕ／ｍｌ１０６ｃｅｌｌｓ／２４ｈ）。实验还显示经ＴＮＦ基因转导的Ｃ６ｐＬＸＳＮ－ｔｎｆ细胞生长速度较之亲本肿瘤细胞Ｃ６明显下降,基因修饰后的肿瘤细胞在Ｗｉｓｔａｒ大鼠体内形成肿瘤的能力明显受到抑制。进一步用超离心法浓缩病毒对胶质瘤移植模型进行了体内治疗研究。     

Bcl——2基因表达对TNF及OA诱发的细胞编程死亡的不同效应

用ＴＮＦ和ＯＡ（Ｏｋａｄａｉｃａｃｉｄ）诱发人神经母细胞瘤ＳＫ细胞死亡,并证明细胞死亡为编程死亡（ＰｒｏｇｒｍｍｅｄＣｅｌｌＤｅａｔｈ,简称ＰＣＤ）。将编码Ｂｃｌ－２全长蛋白的ｃＤＮＡ植入ＰＪＸ４１ｎｅｏ载体中,使其表达由ＨＣＭＶ病毒起动子控制。形成的顺义（ｐＢｃｌ－２－Ｓ）及反义（ｐＢｃｌ－２－ＡＳ）表达质粒经转染导入ＳＫ细胞中获得稳定转染子。Ｗｅｓｔｅｒｎ印迹表明顺义转染子表达大量的２６ｋｄＢｃｌ－２蛋白,而反义转染子则不表达。增强表达的Ｂｃｌ－２蛋白能抑制由ＴＮＦ引发的ＰＣＤ,但不影响ＯＡ引发的ＰＣＤ,从而证明了Ｂｃｌ－２基因产物抗细胞死亡效应的特异性。     

Ⅰ-A~k基因克隆转导及对肿瘤细胞成瘤的影响

采用ＲＴＰＣＲ方法合成小鼠ＭＨＣⅡ类分子ⅠＡｋ基因α和β链ｃＤＮＡ,插入逆转录病毒载体ｐＬＳＸＮ,构建ⅠＡｋα和ⅠＡｋβ表达载体,采用脂质体介导的重组质粒转移方法将ⅠＡｋα和ⅠＡｋβ基因导入ＥＬ４小鼠淋巴瘤细胞和Ｐ８１５小鼠肥大细胞瘤细胞,经流式细胞仪检测在细胞表面有ⅠＡｋ表达．将以上两种细胞注射到同源小鼠Ｃ５７ＢＬ／６（Ｈ２ｄ）皮下,观察到肿瘤产生后又消退,证明在肿瘤细胞中单独导入同种异型ＭＨＣⅡ类分子基因也能激活肿瘤的细胞免疫,为进一步开展肿瘤的基因治疗奠定了基础．     

乙肝病毒PreSl片段与乙肝表面抗原羧端的融合表达

我们利用聚合酶链反应法（ＰＣＲ）得到了编码乙肝病毒肝细胞受体结合位点ＰｒｅＳｌ（２１￣４７）的基因片段,并将它分别融合到Ｓ基因中相应于第１７５,１８８和２２３位氨基酸残基处。所得到的融合基因插入痘苗病毒表达载体ｐＧＪＰ－５后,在哺乳动物细胞ＣＶ－１中进行了暂时表达,对融合蛋白的表达、分泌和抗原性的研究表明,３种融合基因均能表达具有Ｓ和ＰｒｅＳｌ双重抗原性的融合蛋白,但融合位点对表达水平和分泌性质有     

中国人雄激素受体N端转示激活区的测序及突变检测

雄激素受体（ａｎｄｒｏｇｅｎ ｒｅｃｅｐｔｏｒ,ＡＲ）Ｎ端转录激活功能区（ＡＦ１）是ＡＲ发挥转录激活所必需的。用４对引物（Ａ３－Ａ４）ＰＣＲ扩增２０例中国正常男性ＡＲ的ＡＦ１,双链ＤＮＡ循环测序以确定正常中国人ＡＲ的ＡＦ１的核苷酸顺序。在此基础上,用ＰＣＲ－ＳＳＣＰ分析和双链ＤＮＡ循环测序法对２例雄激素抵抗征（ＡＩＳ）患者外周血白细胞和１５例前列腺癌（ＰＣ）患者癌组织中ＡＲ的ＡＦ１区进行突变检测。     

Evaluation of photodynamic treatment using aluminum phthalocyanine tetrasulfonate chloride as a photosensitizer: new approach

Photodynamic therapy (PDT) has been the subject of several clinical studies. Evidence to date suggests that direct cell death may involve apoptosis. T(24) cells (bladder cancer cells, ATCC-Nr. HTB-4) were subjected to PDT with aluminum phthalocyanine tetrasulfonate chloride (AlS(4)Pc-Cl) and red laser light at 670 nm. Morphological changes after PDT were visualized under confocal microscopy. Raman microspectroscopy is considered as one of the newly established methods used for the detection of cytochrome c as an apoptotic marker. Results showed that PDT treated T(24) cells seem to undergo apoptosis after irradiation with 3 J cm(-2). Cytochrome c could not be detected from cells incubated with AlS(4)Pc-Cl using Raman spectroscopy whereas AlS(4)Pc-Cl seems to interfere with the Raman spectrum of cytochrome c.     

Photodynamic modification of disulfonated aluminium phthalocyanine fluorescence in a macrophage cell line.

Disulfonated aluminium phthalocyanine (AlS(2)Pc) is used experimentally as a photosensitiser for both photodynamic therapy (PDT) and photochemical internalisation (PCI). In this study we have focused on modifications in intracellular photosensitiser localisation and fluorescence intensity in macrophages during and after photoirradiation. Since macrophages are highly abundant in tumour tissue and readily accumulate AlS(2)Pc both in vivo and in vitro, we investigated PDT-induced changes of AlS(2)Pc fluorescence in the murine macrophage cell line J774A.1 using CCD fluorescence imaging microscopy. The distinct intracellular localization disappeared upon red laser irradiation and was replaced by a uniform distribution accompanied by a transient fluorescence intensity increase using higher AlS(2)Pc concentrations, followed by photobleaching after further irradiation. A short period of irradiation was sufficient to induce the intracellular redistribution and intensity increase, which then continued in the dark without further laser irradiation. However in the absence of oxygen no fluorescence intensity increase or redistribution was observed. This finding favours the general assumption of photodynamic destruction of organelle membranes resulting in the observed redistribution of the phthalocyanine. No other long-lived fluorescent photoproducts were observed during irradiation. Under deoxygenated conditions slower photobleaching was observed, and photobleaching quantum yields were estimated under aerated and deoxygenated conditions. The participation of reactive oxygen intermediates (ROS) generated during irradiation was indicated by intracellular oxidation of 2',7'-dichlorodihydrofluorescein to the fluorescent 2',7'-dichlorofluorescein in macrophages. The oxygen dependence of these photomodification processes is relevant to the application of AlS(2)Pc to photochemical internalisation which relies on photosensitiser redistribution in cells upon light exposure.     

酸性森林土壤中Ali/(Ca+Mg)摩尔比值的分布特征及影响因素

研究了西南和华南酸性森林土壤中Al_i/(Ca+Mg)摩尔比值的分布特征与影响因素,用主成分分析(PCA)和偏最小二乘法(PLS)回归综合评价各种影响因素及其相对重要性. 2000～2002年连续3年的监测结果表明,大多数土壤水中该摩尔比值都小于临界值1.0,说明土壤铝释放还未对植被造成显著伤害.偏最小二乘法(PLS)回归显示,土壤铝饱和度(AlS)是影响A层土壤水中Al_i/(Ca+Mg)摩尔比值的首要因素;土壤铝饱和度愈高,土壤水中Al_i/(Ca+Mg)摩尔比值愈高.5个流域中,尽管流溪河流域酸沉降量偏低,但由于土壤铝饱和度较高,A层土壤水中Al_i/(Ca+Mg)摩尔比值高于其他流域.土壤水中无机铝(Al_i)浓度是影响深层(B₁、B₂、BC层)土壤水中Al_i/(Ca+Mg)摩尔比值的主导因素;土壤水中无机铝浓度愈高,Al_i/(Ca+Mg)摩尔比值愈高.各流域内摩尔比值沿土壤深度的变化与无机铝浓度的变化基本一致.可以认为,土壤铝饱和度是影响土壤水中Al_i/(Ca+Mg)摩尔比值区域性差异的主要因素,土壤水中无机铝浓度是影响Al_i/(Ca+Mg)摩尔比值纵向差异的主要因素.     

Synthesis,degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3

Murine TIS7 and TIS21 cDNAs were cloned from phorbol ester-induced Swiss 3T3 cells. The cognate rat cDNAs. PC4 and PC3, were cloned from nerve growth factor (NGF)-treated PC12 pheochromocytoma cells. The TIS7/PC4 and TIS21/PC3 primary response genes are rapidly and transiently induced in response to serum, phorbol esters, and polypeptide growth factors in quiescent Swiss 3T3 cells and by NGF and other ligands in PC12 cells. In both 3T3 and PC12 cells the appearance of the TIS21/PC3 message precedes that of TIS7/PC4 message following ligand stimulation, suggesting that the TIS21/PC3 protein is likely to be synthesized more rapidly than the TIS7/PC4 protein. Using antisera prepared against recombinant TIS21 and TIS7 proteins, we find that the TIS21/PC3 protein is, indeed, synthesized more rapidly than the TIS7/PC4 protein following stimulation in both 3T3 and PC12 cells. In addition, “pulse-chase” experiments demonstrate that the TIS21/PC3 protein is degraded much more rapidly than the TIS7/PC4 protein. The sequences of the predicted PC3 and PC4 proteins have lead to the speculation that these two proteins may both be secreted from cells following stimulation. The PC4 protein is reported to have some sequence similarity to interferons. The TIS21/PC3 protein contains a presumptive leader sequence. Using our antisera to the recombinant proteins, however, we cannot detect secretion of radiolabelled TIS7/PC4 or TIS21/PC3 protein. Immunohistochemical and subcellular fractionation experiments suggest that the TIS7 protein is a membrane associated, non-nuclear intracellular protein. The TIS21 protein, in contrast, is' a non-nuclear, soluble intracellular protein. © 1994 Wiley-Liss, Inc.     

Prohormone convertase 1/3 is essential for processing of the glucose-dependent insulinotropic polypeptide precursor

The physiology of the incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), and their role in type 2 diabetes currently attract great interest. Recently we reported an essential role for prohormone convertase (PC) 1/3 in the cleavage of intestinal proglucagon, resulting in formation of GLP-1, as demonstrated in PC1/3-deficient mice. However, little is known about the endoproteolytic processing of the GIP precursor. This study investigates the processing of proGIP in PC1/3 and PC2 null mice and in cell lines using adenovirus-mediated overexpression. Supporting a role for PC1/3 in proGIP processing, we found co-localization of GIP and PC1/3 but not PC2 in intestinal sections by immunohistochemistry, and analysis of intestinal extracts from PC1/3-deficient animals demonstrated severely impaired processing to GIP, whereas processing to GIP was unaltered in PC2-deficient mice. Accordingly, overexpression of preproGIP in the neuroendocrine AtT-20 cell line that expresses high levels of endogenous PC1/3 and negligible levels of PC2 resulted in production of GIP. Similar results were obtained after co-expression of preproGIP and PC1/3 in GH4 cells that express no PC2 and only low levels of PC1/3. In addition, studies in GH4 cells and the alpha-TC1.9 cell line, expressing PC2 but not PC1/3, indicate that PC2 can mediate processing to GIP but also to other fragments not found in intestinal extracts. Taken together, our data indicate that PC1/3 is essential and sufficient for the production of the intestinal incretin hormone GIP, whereas PC2, although capable of cleaving proGIP, does not participate in intestinal proGIP processing and is not found in intestinal GIP-expressing cells.     

Immunosuppressant FK506 induces sustained activation of MAP kinase and promotes neurite outgrowth in PC12 mutant cells incapable of differentiating

During the continuous culturing of neural PC12 cells, a drug hypersensitive PC12 mutant cell line (PC12m3) was obtained, which demonstrated high neurite outgrowth when stimulated by various drugs. When the immunosuppressant drug FK506 and nerve growth factor (NGF) were introduced to the PC12m3 cells, the frequency of neurite outgrowth increased approximately 40-fold for NGF alone. However, the effect of FK506 on neuritogenesis in PC12 parental and drug insensitive PC12m1 mutant cells was much lower than in PC12m3 cells. The sustained activation of mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth of PC12 cells. Interestingly, the drug hypersensitive PC12m3 cells exhibited the sustained activation of MAP kinase with FK506 in comparison to low or no activities in PC12 parental or drug insensitive PC12m1 cells. These results indicate that PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.     

Disruption of proprotein convertase 1/3 (PC1/3) expression in mice causes innate immune defects and uncontrolled cytokine secretion

The proprotein convertase 1/3 is expressed in the regulated secretory pathway of neural and endocrine cells. Its major function is in the post-translational processing and activation of precursor proteins. The PC1/3 knock-out (KO) mouse model has allowed us to elucidate its physiological functions in studies focused primarily on neuroendocrine tissues. However, PC1/3 is also expressed in cells of the immune system, mainly in macrophages. The present study explores the effects of innate immune challenge in the PC1/3 KO mouse. PC1/3 KO mice have an enlarged spleen with marked disorganization of the marginal zone and red pulp. Immunohistochemical studies using various markers demonstrate a depletion of dendritic cells in PC1/3 KO spleens. When challenged with lipopolysaccharide, PC1/3 KO mice are more susceptible to septic shock than wild-type controls or other PC KO mice, such as PC2 and PC7 null mice. Plasma levels of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) were very significantly elevated in PC1/3 KO mice, consistent with a hypercytokinemia, i.e. indicative of a major systemic uncontrolled inflammatory response or cytokine storm. Peritoneal macrophages isolated from PC1/3 KO mice also demonstrate elevated cytokine secretion when treated with LPS. Electron micrographs show morphological features indicating a prolonged activation of these cells following LPS stimulation. We also present evidence that the proinflammatory T(h)1 pathway is dominant in the PC1/3 KO mouse model. We conclude that aside from its important role in neuroendocrine functions PC1/3 also has an important role in the regulation of the innate immune system, most likely through the regulation of cytokine secretion in macrophages.     

Proprotein Convertase 1/3 (PC1/3) in the Rat Alveolar Macrophage Cell Line NR8383: Localization,Trafficking and Effects on Cytokine Secretion

The proprotein convertase 1/3 (PC1/3) is an important post-translational processing enzyme for the activation of precursor proteins within the regulated secretory pathway. Well characterized for its role in the neural and endocrine systems, we recently reported an unconventional role of PC1/3 as a modulator of the Toll-like receptor innate immune response. There are only a few reports that have studied PC1/3 expression in macrophages, and more investigation is needed to better characterize its function. These studies would greatly benefit from model cell lines. Our study aims to identify and characterize PC1/3 in a relevant model macrophage cell line and to determine the links between PC1/3 and innate immune cellular responses. We describe the rat alveolar cell line, NR8383, as expressing PC1/3 and the most common Toll-like receptors. In NR8383 cells, PC1/3 is localized at the Trans-Golgi network and traffics to lysosome related vesicles upon lipopolysaccharide stimulation. Moreover, we report the co-localization of PC1/3 and Toll-like receptor 4 upon lipopolysaccharide stimulation. Down regulation of PC1/3 by shRNA produce a similar phenotype in NR8383 to what we previously reported in isolated peritoneal macrophages. PC1/3 shRNA induced changes in the cellular organization and expression of the specific trafficking regulator RAB GTPase. As a consequence, NR8383 down-regulated for PC1/3, present an abnormal cytokine secretion profile. We conclude that the NR8383 cell line represents a good model to study PC1/3 in macrophages and we present PC1/3 as an important regulator of vesicle trafficking and secretion in macrophages.     

Combining Paclitaxel with ABT-263 Has a Synergistic Effect on Paclitaxel Resistant Prostate Cancer Cells

We assessed the capability of paclitaxel, one of the taxanes, to induce death in two prostate cancer lines, LNCaP and PC3. Paclitaxel drove an apoptotic pathway in LNCaP, but not in PC3 cells, in response to G2/M arrest. An examination of the levels of anti-apoptotic proteins revealed that Bcl-xl was much higher in PC3 cells than in LNCaP cells and Bcl2 could be detected only in PC3 cells, not in LNCaP cells. Knocking down Bcl-xl enhanced paclitaxel-induced apoptosis in LNCaP cells, while we were unable to knock down Bcl-xl efficiently in PC3 cells. Significantly, a comparison of ABT-263, a specific inhibitor of Bcl2 and Bcl-xl, with ABT-199, a Bcl2 selective inhibitor, disclosed that only ABT-263, not ABT-199, could induce apoptosis in LNCaP and PC3 cells. The results indicate that Bcl-xl has a protective role against paclitaxel-induced apoptosis in LNCaP and PC3 cells, and its overexpression causes the paclitaxel resistance seen in PC3 cells. Interestingly, combined paclitaxel with ABT-263 to treat LNCaP and PC3 cells demonstrated synergistic apoptosis activation, indicating that ABT-263 could enhance paclitaxel-induced apoptosis in LNCaP cells and overcome Bcl-xl overexpression to trigger paclitaxel-induced apoptosis in PC3 cells. We also observed that the activation of apoptosis in LNCaP cells was more efficient than in PC3 cells in response to paclitaxel plus ABT-263 or to ABT-263 alone, suggesting that the apoptosis pathway in PC3 cells might have further differences from that in LNCaP cells even after Bcl-xl overexpression is accounted for.     

Regulation of alpha3 nicotinic acetylcholine receptor subunit mRNA levels by nerve growth factor and cyclic AMP in PC12 cells

To investigate the effects of nerve growth factor (NGF) and cyclic AMP (cAMP) on the level of the nicotinic acetylcholine receptor subunit alpha3 mRNA, we used PC12h cells, PC12 cells expressing dominant-negative Ras protein, and the parental PC12 cells. PC12h cells have NGF-responsive tyrosine hydroxylase activity. Expression of dominant-negative Ras protein prevents the signaling through the Ras-mitogen-activated protein kinase cascade. The morphological changes of the parental PC12 cells in response to NGF and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPTcAMP), a cell-penetrating cAMP analogue, were similar to those of PC12h cells. NGF up-regulated the alpha3 mRNA level in PC12h cells and down-regulated the alpha3 mRNA level in the parental PC12 cells. Expression of dominant-negative Ras protein and an inhibitor of mitogen-activated protein kinase kinase inhibited the effects of NGF on alpha3 mRNA level. CPTcAMP down-regulated the alpha3 mRNA level in all three PC12 cell lines. An inhibitor of protein kinase A inhibited the CPTcAMP-induced down-regulation of alpha3 mRNA. The alpha3 mRNA down-regulation required prolonged treatment with CPTcAMP even after cAMP response element binding protein phosphorylation was decreased. Membrane depolarization with high K+ had no effect on the alpha3 mRNA level in PC12h cells. Based on these results, we propose that at least two unknown effectors regulate alpha3 mRNA levels in PC12 cells.