Gene silencing efficiency of shRNA expression vectors targeting Cx43 in vitro

Our previous studies showed that there were close relationships between connexin 43 (Cx43) and acupoints and meridians. In order to further investigate the effect of Cx43 in acupuncture treatment, RNA interference technique was used to construct small hairpin RNA (shRNA) expression vectors targeting Cx43 and identify the efficiency of RNA interference in NIH/3T3 cell lines for further use in vivo. Aiming directly at the two targets of Cx43 mRNA sequence of the rat and mouse homology region, we synthesized two pairs of complementary oligonucleotide strands in vitro. Double strands were formed after annealing, and then inserted into Pgenesil-1 plasmid expression vector. After identification by enzyme cutting and sequencing, the recombinant plasmids named P-Cx43-shRNA (1), P-Cx43-shRNA (2) and P-con-shRNA were transfected into the NIH/3T3 cells. Immunofluorescence and Western blot assays were used to detect the protein level of Cx43 after being screened by G418. The results of enzyme cutting and sequencing showed that we successfully constructed two shRNA expression vectors targeting Cx43, and a control expression vector for rat and mouse. Also, the Cx43 protein level was decreased by 73.5% (P<0.01) and 10.8%, accordingly. The Cx43 protein level was not influenced by the transfection of P-con-shRNA. The outcomes demonstrate that the plasmid P-Cx43-shRNA (1) can specifically silence better the expression of Cx43 in NIH/3T3 cells, which offers an experimental evidence for further in vivo investigation.     

Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As(+3)- and MMA(+3)-induced apoptosis through inhibition of telomerase activity via JNK activation

The effects of six arsenic compounds including As(+3), MMA(+3), DMA(+3), As(+5), MMA(+5), and DMA(+5) on the viability of NIH3T3 cells were examined. As(+3) and MMA(+3), but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As(+3) and MMA(+3) were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As(+3) and MMA(+3) treatments. An increase in the intracellular peroxide level was examined in As(+3)- and MMA(+3)-treated NIH3T3 cells, and As(+3)- and MMA(+3)-induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As(+3)- and MMA(+3)-induced cytotoxicity. Suppression of JNKs significantly inhibited As(+3)- and MMA(+3)-induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As(+3)- and MMA(+3)-induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As(+3) or MMA(+3). These data provide the first evidence to indicate that apoptosis induced by As(+3) and MMA(+3) is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved.     

Cadmium-induced alterations of connexin expression in the promotion stage of in vitro two-stage transformation

During the multistage carcinogenesis, functions of several key genes involved in the cell cycle control and cell-cell communication can be damaged. Gap junction intercellular communication (GJIC) is known to transfer small, water-soluble molecules through intercellular channels composed of proteins called connexins (Cxs). Therefore, aberrant expression of Cx may be one of the critical factors for the clonal expansion of initiated cells during the two-stage transformation. We already improved the classical in vitro two-stage transformation method using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as an initiator and cadmium as a promoter on Balb/3T3 A31 cells, and reconfirmed the promotional effect of cadmium with this method (Fang, M.Z., Cho, M.H., Lee, H.W., 2001. Improvement of in vitro two-stage transformation assay and detection of the promotional effect of cadmium, Toxicol. In Vitro (in press). In this study, precise roles of Cd on Cx expression in normal Balb/3T3 A31 and during the promotion stage of the in vitro two-stage transformation were elucidated. For this purpose, the Cx43, Cx32 and Cx26 protein levels, Cx43 and Cx26 mRNA levels and the cellular distribution location of Cx43 protein were determined. Normal Balb/3T3 cells expressed Cx43 and Cx32, but not Cx26. After a short-term treatment of cadmium on normal cells, phosphorylation of Cx43 protein increased and Cx32 protein level decreased. However, during the promotion stage of the in vitro two-stage transformation, transformed cells treated with cadmium for long periods expressed Cx43 and Cx32 highly, similar to the level of normal Balb/3T3 cells, compared to the nontransformed cells. Moreover, Cx43 of the transformed cells was distributed mostly in the perinuclear region rather than the intercellular membrane. These data suggest that cadmium may inhibit the GJIC by increasing the phosphorylation of Cx43 and decreasing the expression of Cx32 in the normal Balb/3T3 A31 cells. Our results also suggest that these changes are not associated with the cell transformation; transformed cells may reexpress Cx43 and Cx32 similar to the normal cells, though Cx43 protein is distributed aberrantly during the transformation process. Further studies are needed to clarify the relationship between transformation and posttranslational modification of the Cx proteins.     

小鼠干扰素调节因子3启动子质粒的构建及其启动子活性分析

目的构建包含小鼠干扰素调节因子3(mIRF-3)基因启动子的质粒,并评价其启动子活性。方法以小鼠全血细胞总DNA为模板,PCR扩增mIRF-3目的片段,亚克隆此片段至pGL3-basic荧光素酶报告基因的多克隆位点,构建含mIRF-3启动子的重组报告质粒pmIRF-3。将pmIRF-3、pGL3-basic和pGL3-control质粒分别转染小鼠胚胎成纤维细胞NIH3T3后,行荧光素酶活性检测,计算相对活性单位(RLU)。生物信息学分析转录因子结合位点。结果成功构建了重组报告质粒pmIRF-3。与pGL3-basic组相比,pmIRF-3、pGL3-control组RLU明显增加(0.443±0.113vs.18.907±3.335、25.704±5.850)(P<0.01)。mIRF-3启动子区域内存在AML-1a、E2F、MZF1、CdxA、OCT-x等多个转录因子结合位点。结论 mIRF-3转录起始位点附近1479bp序列在NIH3T3细胞中具有较强的启动子活性。     

Effects of heptachlor epoxide on components of various signal transduction pathways important in tumor promotion in mouse hepatoma cells. Determination of the most sensitive tumor promoter related effect induced by heptachlor epoxide

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE; 0, 0.1, 1, 10, and 50 μM) on several cellular tumor promoter-sensitive parameters were studied in mouse 1c1c7 hepatoma cells in an effort to identify the most sensitive biomarker for OC promoter exposure and the critical pathway and target of OC promoters. The levels of Ca²⁺ in the endoplasmic reticulum (ER) store, connexin43 (Cx43), PLCγ₁, nPKC, and AP-1 DNA binding in nucleus were studied to screen for effects induced by submicromolar HE levels. While all the parameters tested elicited effects, particulate PLCγ₁ and AP-1 DNA binding were found to be the most sensitive parameters affected by HE on both dose and temporal bases. Their levels were increased with 10- to 100-fold lower HE concentrations than were required to affect nPKC or Cx43. Further, with the lower HE dosages, particulate PLCγ₁ and nuclear AP-1 were positively modulated by HE after 1 h versus 3 or 72 h for nPKC and Cx43. Ca²⁺ store depletion was probably the third most sensitive parameter, after AP-1 and PLCγ₁. These results suggest the tyrosine kinase growth factor receptor pathway is the probable critical pathway for HE-induce tumor promotion with the critical target most likely being upstream of PLCγ₁ and AP-1. This work also demonstates that upon exposure to a tumor promoter such as HE, many hepatocellular effects or changes result, suggesting that a cellular-program shift occurs similar to that described by the resistant hepatocyte model after exposure to a carcinogen or enzyme inducer.     

A new role for extracellular Ca2+ in gap-junction remodeling: studies in humans and rats

We wanted to elucidate whether extracellular calcium may regulate the expression of the cardiac gap-junction proteins connexin 40 and connexin43. In the free wall of the left atria of 126 cardiac surgery patients with either sinus rhythm (SR) or chronic atrial fibrillation (AF), we determined the expression of the cardiac gap-junction proteins Cx43 and Cx40 by Western blot and immunohistology. For deeper investigation, we incubated cultured neonatal rat cardiomyocytes at 2 or 4 mM Ca⁺⁺ for 24 h and determined intercellular coupling, Cx40, Cx43 protein and mRNA expression, protein trafficking and sensitivity to verapamil (10–100 nM), cyclosporin A (1 μM),and BMS605401 (100 nM), a specific inhibitor of Ca²⁺-sensing receptor (CaSR). We found in patients that both Cx are up-regulated in AF in the left atrium (by 100–200%). Interestingly, Cx40 was mainly up-regulated, if total serum calcium was ≥2.2 mM, while Cx43 was independent from extracellular [Ca⁺⁺]. In cultured cells, 4 mM Ca⁺⁺-exposure lead to up-regulation of Cx40, but not Cx43. We found enhanced Cx40 in the plasma membrane and reduced Cx40 in the Golgi apparatus. The membrane Cx40 up-regulation resulted in enhanced gap-junction intercellular coupling with a shift in the Boltzmann fit of voltage-dependent inactivation indicating a higher contribution of Cx40 as revealed by dual whole cell voltage clamp experiments. BMS605401 could prevent all Ca²⁺-induced changes. Moreover, cyclosporin A completely abolished the Ca²⁺-induced changes, while verapamil was ineffective. We conclude that extracellular calcium (24 h exposure) seems to up-regulate Cx40 but not Cx43.     

氯化镧通过细胞周期机制促进NIH 3T3小鼠胚胎成纤维细胞增殖(英文)

镧离子(La3+)对NIH 3T3小鼠胚胎成纤维细胞的增殖有重要影响,但其机理尚需深入研究。在本研究中,我们运用增殖分析、周期阻滞-释放和BrdU追踪等方法详细分析了La3+对NIH 3T3细胞增殖及细胞周期动力学的影响。结果显示,La3+处理12小时即有显著促增殖作用,在48小时促增殖效应的EC50为2.4μM。La3+促NIH 3T3细胞增殖的作用也在BrdU掺入实验中得到了验证。La3+推动更多细胞跨过G1/S周期检查点进入S期,但不改变细胞周期的时间长度。免疫荧光染色发现,La3+处理增加了两个关键细胞周期调控因子cyclin D1和c-Myc的蛋白水平和细胞核定位。本研究显示La3+的促增殖作用与报道的可能引起肾源性系统性纤维化疾病的Gd3+非常相似,并提供了镧系元素细胞生物学活性的新信息。     

PKC及缝隙连接蛋白Cx43改变在吗啡和舒芬太尼预处理缺氧复氧损伤心肌中的作用

目的：探讨蛋白激酶C（PKC）信号通路及心肌缝隙连接蛋白Cx43改变在阿片药物预处理中的作用。方法：原代心肌细胞分离培养。将培养5 d的心肌细胞分成8组。正常对照组（C组）不给予任何处理,建立培养乳鼠心肌细胞缺氧/复氧损伤模型（I/R组）,吗啡组（MF组）加入吗啡至终浓度0.3 μmol·L^-1,舒芬太尼组（SF组）加入舒芬太尼至终浓度0.000 3 μmol·L^-1,PKC激动剂PMA+吗啡组（PMA+MF组）加入PMA至终浓度0.02 μmol·L^-1,再进行吗啡预处理;PKC抑制剂Rottlerin+吗啡组（ROT+MF组）加入Rottlerin至终浓度5 μmol·L^-1,再进行吗啡预处理;PKC激动剂PMA+舒芬太尼组（PMA+SF组）加入PMA至终浓度0.02 μmol·L^-1,再进行舒芬太尼预处理;PKC抑制剂Rottlerin+舒芬太尼组（ROT+SF组）加入Rottlerin至终浓度5 μmol·L^-1,再进行舒芬太尼预处理。各组做上述相应处理后取材检测细胞存活率。免疫荧光共聚焦技术检测缝隙连接蛋白Connexin 43（Cx43）的平均光密度（AOD）,用Western-blot检测细胞Cx43总蛋白及其磷酸化水平（P-Cx43）的表达量。结果：与C组比较,其余各组心肌细胞存活率、Cx43 AOD值、心肌细胞Cx43总蛋白表达及P-Cx43表达降低（P<0.05）;与I/R组比较,MF组、SF组、ROT+MF组、ROT+SF组、PMA+MF组、PMA+SF组心肌细胞存活率、Cx43 AOD值、Cx43总蛋白和P-Cx43表达增高（P<0.05）;与MF组比较,ROT+MF组心肌细胞存活率、Cx43 AOD值、Cx43总蛋白及P-Cx43表达降低（P<0.05）,SF组心肌细胞存活率、Cx43 AOD值和Cx43总蛋白降低（P<0.05）,而P-Cx43表达增高（P<0.05）,PMA+MF组心肌细胞存活率、Cx43 AOD值、Cx43总蛋白和P-Cx43表达均增高（P<0.05）;ROT+SF组较SF组心肌细胞Cx43 AOD值、Cx43总蛋白和P-Cx43表达低（P<0.05）,而PMA+SF组较SF组心肌细胞存活率、心肌细胞Cx43 AOD值、Cx43总蛋白和P-Cx43表达高（P<0.05）。结论：吗啡和舒芬太尼预处理可减轻心肌细胞缺氧/复氧损伤,且吗啡对心肌细胞的保护作用较舒芬太尼强,这种心肌细胞保护作用可能与PKC信号通路的激活有关。     

Silver nanoparticles up-regulate Connexin43 expression and increase gap junctional intercellular communication in human lung adenocarcinoma cell line A549

Abstract

Silver nanoparticles (Ag NPs) are increasingly being used in wound dressings, medical settings, and various household products due to their unique properties and antimicrobial activity. Despite the widespread use of Ag NP products, the molecular mechanisms underlying the biological effects of Ag NPs remain unclear. Gap junctional intercellular communication (GJIC), formed by the connexin protein family, plays a critical role in the maintenance of tissue and organ homeostasis. This study was undertaken to investigate the effects of well characterized, PVP-coated Ag NPs (69 ± 3 nm) and silver nitrate on GJIC and connexin43 (Cx43) expression in human lung adenocarcinoma cell line A549. Our results showed that Ag NPs increased GJIC in A549 cells as assayed by dye transfer method. Western blot analysis showed that incubation of cells with Ag NPs significantly increased the expression of Cx43 protein. In addition, Ag NPs up-regulated expression of Cx43 mRNA in a dose-dependent manner. Silver nitrate failed to increase GJIC and the expression of Cx43 protein. It, however, increased Cx43 mRNA expression in A549 cells. Taken together, our results provide the first evidence that Ag NPs induced the increase of GJIC activity in A549 cells through up-regulation of Cx43 protein, suggesting that Cx43 and GJIC may be one of the targets for Ag NPs biological effects.     

(-)-Epicatechin effects in rat liver epithelial cells: stimulation of gap junctional communication and counteraction of its loss due to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate

Gap junctional intercellular communication (GJIC) is a direct signaling pathway for neighboring cells. Disturbances in GJIC are suggested to play a role in carcinogenesis and may be involved in cardiac arrhythmia. Tumor promoters like 12-O-tetradecanoylphorbol-13-acetate (TPA) are capable of inhibiting GJIC, whereas GJIC is stimulated by several micronutrients like genistein, retinoids or carotenoids. (-)-Epicatechin (4-40 microM), a major flavonoid in cocoa and green tea, exhibited stimulatory effects on GJIC in WB-F344 rat liver epithelial cells after 24-72hr of incubation; no change was observed after 90 min. However, treatment of cells for 90 min with TPA (5 or 10nM) led to complete loss of GJIC, whereas 40% loss was found with 1nM. These inhibitory effects of TPA were largely suppressed when (-)-epicatechin or genistein (40 microM) were present during the incubation. In communicating WB-F344 cells, most of the major gap junction protein connexin43 (Cx43) was located in the plasma membrane. When the cells were exposed to TPA, considerably less protein was found in the membrane. Such a delocalization of Cx43 proteins was not observed when TPA was coincubated with the flavonoids, (-)-epicatechin or genistein. It is concluded that TPA affects Cx43 trafficking between cellular compartments, and that this effect is counteracted by (-)-epicatechin or genistein.     

Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells

1. We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS-1 fibroblasts and human HeLa epithelial cells. 2. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within approximately 1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS-1 cells selected to express the rat alpha1 Na+/K+-ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1-10 microM) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous alpha1 subunits possess relatively high affinity for the glycoside (Ki approximately 0.3 vs approximately 100 microM) Ouabain-induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+-ATPase isoenzymes expressed in these different cell lines. 3. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 4. In separate experiments, the effects of ouabain on real-time trafficking of connexin (Cx) protein were monitored by time-lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43-green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of Cx40 and Cx43 evaluated in A7r5 cells by immunochemical and Western blot analysis. 5. Ouabain (1 mM) depressed vesicular trafficking of Cx43-GFP after approximately 1 h, and caused a time-dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+-ATPase into a general signal transducer that regulates downstream protein synthesis.     

Rictor/mTORC2 involves mitochondrial function in ES cells derived cardiomyocytes via mitochondrial Connexin 43

Rictor is a key component of the mammalian target of rapamycin complex 2 (mTORC2) and is required for Akt phosphorylation (Ser473). Our previous study shows that knockdown of Rictor prevents cardiomyocyte differentiation from mouse embryonic stem (ES) cells and induces abnormal electrophysiology of ES cell-derived cardiomyocytes (ESC-CMs). Besides, knockdown of Rictor causes down-expression of connexin 43 (Cx43), the predominant gap junction protein, that is located in both the sarcolemma and mitochondria in cardiomyocytes. Mitochondrial Cx43 (mtCx43) plays a crucial role in mitochondrial function. In this study, we used the model of cardiomyocyte differentiation from mouse ES cells to elucidate the mechanisms for the mitochondrial damage in ESC-CMs after knockdown of Rictor. We showed swollen and ruptured mitochondria were observed after knockdown of Rictor under transmission electron microscope. ATP production and mitochondrial transmembrane potential were significantly decreased in Rictor-knockdown cells. Furthermore, knockdown of Rictor inhibited the activities of mitochondrial respiratory chain complex. The above-mentioned changes were linked to inhibiting the translocation of Cx43 into mitochondria by knockdown of Rictor. We revealed that knockdown of Rictor inactivated the mTOR/Akt signalling pathway and subsequently decreased HDAC6 expression, resulted in Hsp90 hyper-acetylation caused by HDAC6 inhibition, thus, inhibited the formation of Hsp90-Cx43-TOM20 complex. In conclusion, the mitochondrial Cx43 participates in shRNA-Rictor-induced mitochondrial function damage in the ESC-CMs.     

以siRNA表达载体稳定抑制缝隙连接蛋白43表达的睾丸间质细胞和睾丸支持细胞系的建立

目的构建Cx43-siRNA真核表达载体,获得连接蛋白43(connexin43,Cx43)被长期稳定抑制的睾丸间质细胞(TM3细胞)系和睾丸支持细胞(TM4细胞)系,为研究Cx43及其形成的细胞缝隙连接(gap junction,GJ)在睾丸组织中的作用提供有用模型。方法设计合成3对针对Cx43的短发夹样siRNA的DNA模板序列,定向连接到siRNA真核表达载体pSilencerTM2.1-U6neo上,通过测序鉴定后以脂质体法瞬时转染睾丸支持细胞,以Western blot方法检测Cx43蛋白表达水平,筛选出最有效的干扰序列,再将之分别转染睾丸间质细胞和睾丸支持细胞,G418筛选出能稳定表达siRNA的细胞系,以"Parachute"荧光传递示踪法检测细胞缝隙连接功能。结果 Western blot结果显示,第3对干扰序列对Cx43表达抑制效果最佳,以表达该序列的质粒稳定转染的TM3细胞和TM4细胞上Cx43蛋白表达水平均明显降低;荧光传递示踪法检测表明,两种细胞系的GJ功能均被明显抑制。结论以Cx43-siRNA真核表达载体稳定转染的方法能长期干扰TM3和TM4细胞上Cx43的表达,并抑制由其形成的GJ功能。     

Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells

1. We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS-1 fibroblasts and human HeLa epithelial cells. 2. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within approximately 1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS-1 cells selected to express the rat alpha1 Na+/K+-ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1-10 microM) attenuated dye transfer in wild-type COS-1 and HeLa cells, whose endogenous alpha1 subunits possess relatively high affinity for the glycoside (Ki approximately 0.3 vs approximately 100 microM) Ouabain-induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+-ATPase isoenzymes expressed in these different cell lines. 3. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. 4. In separate experiments, the effects of ouabain on real-time trafficking of connexin protein were monitored by time-lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43-green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of connexins (Cx) 40 and 43 evaluated in A7r5 cells by immunochemical and Western blot analysis. 5. Ouabain (1 mM) depressed vesicular trafficking of Cx43-GFP after approximately 1 h, and caused a time-dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed approximately 90 min following washout of the glycoside. 6. We conclude that ouabain exerts biphasic effects on the intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+-ATPase into a general signal transducer that regulates downstream protein synthesis.     

Nickel compounds induce apoptosis in human bronchial epithelial Beas-2B cells by activation of c-Myc through ERK pathway

Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15% of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ion-induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells.