Mouse KL2 is a unique MTSE involved in chromosome-based spindle organization and regulated by multiple kinases during female meiosis

Microtubule-severing enzymes(MTSEs)play important roles in mitosis and meiosis of the primitive organisms.However,their roles in mammalian female meiosis,which accounts for over 80%of gamete-originated human reproductive diseases,remain unexplored.In the current study,we reported that katanin-like 2(KL2)was the only MTSE concentrating at chromosomes.Furthermore,the knockdown of KL2 significantly reduced the chromosome-based increase in the microtubule(MT)polymer,increased aberrant kinetochore-MT(K-MT)attachment,delayed meiosis,and severely affected normal fertility.We demonstrated that the inhibition of aurora B,a key kinase for correcting aberrant K-MT attachment,significantly eliminated KL2 expression from chromosomes.Additionally,KL2 interacted with phosphorylated eukaryotic elongation factor-2 kinase,and they competed for chromosome binding.Phosphorylated KL2 was also localized at spindle poles,with its phosphorylation regulated by extracellular signal-regulated kinase 1/2.In summary,the current study reveals a novel function of MTSEs in mammalian female meiosis and demonstrates that multiple kinases coordinate to regulate the levels of KL2 at chromosomes.

Vaccinia-related kinase 2 variants differentially affect breast cancer growth by regulating kinase activity

Genetic information is transcribed from genomic DNA to mRNA,which is then translated into threedimensional proteins.mRNAs can undergo various post-transcriptional modifications,including RNA editing that alters mRNA sequences,ultimately affecting protein function.In this study,RNA editing was identified at the 499th base(c.499)of human vaccinia-related kinase 2(VRK2).This RNA editing changes the amino acid in the catalytic domain of VRK2 from isoleucine(with adenine base)to valine(with guanine base).Isoleucine-containing VRK2 has higher kinase activity than the valine-containing VRK2,which leads to an increase in tumor cell proliferation.Earlier we reported that VRK2 directly interacts with dystrobrevin-binding protein(dysbindin)and results in reducing its stability.Herein,we demonstrate that isoleucine-containing VRK2 decreases the level of dysbindin than valinecontaining VRK2.Dysbindin interacts with cyclin D and thereby regulates its expression and function.The reduction in the level of dysbindin by isoleucine-containing VRK2 further enhances the cyclin D expression,resulting in increased tumor growth and reduction in survival rates.It has also been observed that in patient samples,VRK2 level was elevated in breast cancer tissue compared to normal breast tissue.Additionally,the isoleucine form of VRK2 exhibited a greater increase in breast cancer tissue.Therefore,it is concluded that VRK2,especially dependent on the 167th variant amino acid,can be one of the indexes of tumor progression and proliferation.

Detection of LAMA2 c.715C>G:p.R239G mutation in a newborn with raised creatine kinase: A case report

BACKGROUND We report a rare case of primary clinical presentation featuring elevated creatine kinase(CK)levels in a neonate,which is associated with the LAMA2 gene.In this case,a heterozygous mutation in exon5 of the LAMA2 gene,c.715C>G(resulting in a change of nucleotide number 715 in the coding region from cytosine to gua-nine),induced an amino acid alteration p.R239G(No.239)in the patient,repre-senting a missense mutation.This observation may be elucidated by the neonatal creatine monitoring mechanism,a phenomenon not previously reported.CASE SUMMARY We analysed the case of a neonate presenting solely with elevated CK levels who was eventually discharged after supportive treatment.The chief complaint was identification of increased CK levels for 15 d and higher CK values for 1 d.Ad-mission occurred at 18 d of age,and despite prolonged treatment with creatine and vitamin C,the elevated CK levels showed limited improvement.Whole exo-me sequencing revealed the presence of a c.715C>G mutation in LAMA2 in the newborn,correlating with a clinical phenotype.However,the available informa-tion offers insufficient evidence for clinical pathogenicity.CONCLUSION Mutations in LAMA2 are associated with the clinical phenotype of increased neonatal CK levels,for which no specific treatment exists.Whole genome sequen-cing facilitates early diagnosis.

Interleukin-1 receptor associated kinase 2 is a functional downstream regulator of complement factor D that controls mitochondrial fitness in diabetic cardiomyopathy

Diabetic cardiomyopathy is a disorder of the cardiac muscle that affects patients with diabetes.The exact mechanisms underlying diabetic cardiomyopathy are mostly unknown,but several factors have been implicated in the pathogenesis of the disease and its progression towards heart failure,including endothelial dysfunction,autonomic neuropathy,metabolic alterations,oxidative stress,and alterations in ion homeostasis,especially calcium transients[1].In Military Medical Research,Jiang et al.[2]sought to determine the functional role of complement factor D(Adipsin)in the pathophysiology of diabetic cardiomyopathy.

Crosslink among cyclin-dependent kinase 9,ATP binding cassette transporter G2 and Beclin 1 in colorectal cancer

Colorectal cancer(CRC)ranks third in the number of cancers mainly because of the inability to diagnose it at an early stage.The pathogenesis of CRC is complicated,which is the result of the complex interaction of multiple genetic and environmental factors.Currently,one of the main treatments for CRC is chemotherapy.But the primary cause of CRC treatment failure is drug resistance.The expression of cyclin-dependent kinase 9(CDK9)was correlated with elevated autophagy levels in colon cancer,and high expression of CDK9 indicates a poor prognosis in CRC.The incidence of autophagy and the expressions of Beclin 1 and ATP binding cassette transporter G2 are different in left and right colon cancer,and autophagy may be involved in the occurrence of chemotherapy resistance.In this article,the roles of CDK9,ATP binding cassette transporter G2 and Beclin 1 in CRC were elucidated,emphasizing the linkages among them and providing potential therapeutic targets of CRC.

Casein kinase 2 interacts with and phosphorylates ataxin-3

Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.

Cloning of the Full-length Gene for Tobacco Ethylene Receptor NTHK2 and Characterization of Its Kinase Domain

Previously the partial sequence of an ethylene receptor gene NTHK2 was isolated from tobacco (Nicotiana tabacum L. var. Xanthi) plants and it was wound and drought inducible. In the present study full-length cDNA of NTHK2 was cloned by 5'-RACE method. NTHK2 gene has 3 216 bp, with 509 bp of 5'-non-coding region and 427 lip of 3'-non-coding region, and encodes an ethylene-receptor homolog of 760, amino acids. NTHK2 protein has a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. In the putative histidine kinase domain, the histidine at the phosphorylation site was replaced by an asparagine. To study the biochemical property of NTHK2, its kinase domain was expressed as a fusion protein with glutathione S-transferase (GST) using yeast Schizzosaccharomyces pombe as an expression system. In vitro kinase assay showed that NTHK2 kinase domain can autophosphorylate in the presence of Mg2+, indicating that NTHK2 may function as a kinase. Further studies will elucidate the function of NTHK2 in plant.

bFGF激活tyrosine kinase／PI3K／PLCγ增加血管内皮细胞[Mg^2＋]i的研究

目的探讨碱性成纤维细胞生长因子(basic fibroblastgrowth factor,bFGF)对人脐带静脉内皮细胞(human umbilicalvein endothelial cells,HUVECs)内游离镁离子浓度([Mg2+]i)的调节机制研究。方法我们采用荧光指示剂mag-fura-2,运用PTi阳离子测定系统动态测HUVECs的[Mg2+]i。结果经酪氨酸激酶阻断剂(tyrphostin A23和genistein)、3-磷脂酰肌醇激酶阻断剂(wortmannin和LY294002)、磷脂酶Cγ阻断剂(U73122)预处理,能阻断bF-GF诱导的[Mg2+]i增加。但经磷脂酶Cγ阻断剂无活性的类似物(U73343)和丝裂原活化蛋白激酶阻断剂(SB202190和PD98059)预处理,不能阻断bFGF诱导的[Mg2+]i增加。结论bFGF通过酪氨酸激酶/3-磷脂酰肌醇激酶/磷脂酶Cγ信号传递途径使细胞内的Mg2+库释放Mg2+,从而增加HUVECs的[Mg2+]i。

NIMA related kinase 2 promotes gastric cancer cell proliferation via ERK/MAPK signaling

BACKGROUND NIMA related kinase 2(NEK2) is closely related to mitosis, and it is currently considered to be over-expressed frequently in many poorly prognostic cancers.However, the effect of the up-regulated NEK2 on cellular signaling in tumors,such as gastric cancer(GC), is con-fusing.AIM To determine the role of the up-regulation of NEK2 in GC.METHODS To investigate the pathological significance of NEK2 in GC, the expression pattern of NEK2 in GC was investigated based on the 'Oncomain' database and compared between 30 pairs of cancer samples and adjacent tissues. The coexpression of NEK2 and ERK in GC was analyzed using The Cancer Genome Atlas(TCGA) database and confirmed in clinical samples by quantitative realtime PCR(qRT-PCR), and the survival curve was also plotted. Western blot or qRT-PCR was used to analyze the effect of NEK2 on the phosphorylation levels of ERK and c-JUN in two GC cell lines(BGC823 and SGC7901) with NEK2 overexpression, and the expression of the downstream effector cyclin D1.Furthermore, CCK8, EdU incorporation assay, and flow cytometry were used to detect the proliferative ability of BGC823 and SGC7901 cells with stably silenced ERK.RESULTS NEK2 was significantly up-regulated in human GC tissues. ERK was significantly associated with NEK2 expression in human clinical specimens, and combined overexpression of NEK2 and ERK potentially forecasted a poor prognosis andsurvival in GC patients. NEK2 knockdown in GC cells inhibited ERK and c-JUN phosphory-lation and reduced the transcription of cyclin D1. More interestingly,NEK2 can rescue the inhibition of cellular viability, proliferation, and cell cycle progression due to ERK knockdown.CONCLUSION Our results indicate that NEK2 plays a carcinogenic role in the malignant proliferation of GC cells via the ERK/MAPK signaling, which may be important for treatment and improving patient survival.

Ca2+/calmodulin-dependent protein kinase II regulates colon cancer proliferation and migration via ERK1/2 and p38 pathways

AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.

Activation of extracellular signal-related kinases 1 and 2 in Sertoli cells in experimentally cryptorchid rhesus monkeys

Aim： To assess the spatiotemporal changes in the expression of extracellular signal-regulated kinases 1 and 2 （ERK1/ 2）, c-Jun N-terminal kinases （JNK） and p38 mitogen-activated protein kinases （MAPK） in response to heat stress in the cryptorchid testis, and to investigate a possible relation to Sertoli cell dedifferentiation. Methods： Immunohistochemistry and western blot were used to examine the expression and activation of ERK1/2, p38 and JNK in the cryptorchid testis at various stages after experimental cryptorchidism. Results： The abdominal temperature did not obviously change the total ERK1/2 expression but significantly activated phospho-ERK1/2 in the Sertoli cells of the cryptorchid testis. Heat stress increased total JNK expression in the Sertoli cells of the cryptorchid testis but did not activate phospho-JNK. Neither total p38 nor phospho-p38 was induced by heat stress in the Sertoli cells of the cryptorchid testis. Changes in the spatiotemporal expression of cytokeratin 18 （CK18）, a marker of immature or undifferentiated Sertoli cells, were induced in the cryptorchid testis in a pattern similar to the activation of ERK1/2. Condusion： The activation of ERK1/2 in the testis may be related to dedifferentiation of Sertoli cells under heat stress induced by experimental cryptorchidism.

Sphingosine kinase 1 is upregulated with lysophosphatidic acid receptor 2 in human colorectal cancer

AIM: To examine the expression of SphK1, an oncogenic kinase that produces sphingosine 1-phosphate (S1P), and its correlation with the expression of LPAR2, a major lysophosphatidic acid (LPA) receptor overexpressed in various cancers, in human colorectal cancer.METHODS: Real-time reverse-transcription polymerase chain reaction was used to measure the mRNA expression of SphK1, LPAR2, and the three major S1P receptors in 27 colorectal cancer samples and corresponding normal tissue samples. We also examined the correlation between the expression of SphK1 and LPAR2.RESULTS: Colorectal cancer tissue in 22 of 27 patients had higher levels of SphK1 mRNA than in normal tissue. In two-thirds of the samples, SphK1 mRNA expression was more than two-fold higher than in normal tissue. Consistent with previous reports, LPAR2 mRNA expression in 20 of 27 colorectal cancer tissue samples was higher compared to normal tissue samples. Expression profiles of all three major S1P receptors, S1PR1, S1PR2, and S1PR3, varied without any trend, with no significant difference in expression between cancer and normal tissues. A highly significant positive correlation was found between SphK1 and LPAR2 expression [Pearson’s correlation coefficient (r) = 0.784 and P < 0.01]. The mRNA levels of SphK1 and LPAR2 did not correlate with TNM stage.CONCLUSION: Our findings suggest that S1P and LPA may play important roles in the development of colorectal cancer via the upregulation of SphK1 and LPAR2, both of which could serve as new therapeutic targets in the treatment of colorectal cancer.

Sodium Butyrate Induces Apoptosis of Human Colon Cancer Cells by Modulating ERK and Sphingosine Kinase 2

Objective To investigate the role of extracellular signal-regulated kinase （ERK） in apoptosis of human colon cancer （HCT116） cells. Methods After the HCT116 cells were pretreated with specific ERK inhibitor （U0126） or specific siRNA and exposed to 10 mmol/L sodium butyrate （NaBT） for 24 h, their apoptosis was detected by flow cytometry, levels of SphK2 and ERK protein were measured by Western blot, and translocation of SphK2 was assayed by immunofluorescence microscopy. Results The U0126 and siRNAs specific for SphK2 blocked the export of SphK2 from nuclei to cytoplasm and increased the apoptosis of HCT116 cells following NaBT exposure. Over-expression of PKD decreased NaBT-induced apoptosis of HCT116 cells, which was reversed by U0126. Furthermore, transfection of HCT116 cells with constitutively activated PKD plasmids recovered the UO126-blocked export of SphK2. Conclusion ERK regulates the export of SphK2 and apoptosis of HCT116 cells by modulating PKD. Modulation of these molecules may help increase the sensitivity of colon cancer cells to the physiologic anti-colon cancer agent, NaBT.

Dexamethasone suppresses DU145 cell proliferation and cell cycle through inhibition of the extracellular signal-regulated kinase 1 /2 pathway and cyclin D1 expression

Aim： To determine the mechanisms of glucocorticoids in inhibiting advanced prostate cancer growth. Methods： The cell proliferation and cell cycle of prostate cancer DU145 cells following dexamethasone treatment were determined by proliferation assay and fluorescence-activated cell sorter. Western blot analysis was carried out to evaluate the effects of dexamethasone on phosphorylation of extracellular signal-regulated kinase （ERK）1/2 and expression of cyclin D1 in DU145 cells with or without glucocorticoid receptor （GR） antagonist RU486. Reverse transcription- polymerase chain reaction verified the expression of GR mRNA in DU145 cells. Results： Dexamethasone significantly inhibited DU 145 cell proliferation at the G0/G1 phase. Westem blot analysis showed a dramatic reduction of ERK1/2 activity and cyclin D1 expression in dexamethasone-treated cells. The decreased phosphorylation of ERK1/2 in dexamethasone-treated cells was attenuated by GR blockade. Additionally, the effects of dexamethasone in inhibiting cyclin D1 expression were altered by GR blockade. Conclusion： Dexamethasone suppresses DU145 cell proliferation and cell cycle, and the underlying mechanisms are through the inhibition of phosphorylation of ERK1/2 and cyclin D1 expression. The inhibition of ERK1/2 phosphorylation and cyclin D1 expression is attenuated by GR blockade, suggesting that GR regulates ERK1/2 and cyclin D1 pathways. These observations suggest that dexamethasone has a potential clinical application in prostate cancer therapy.

Caffeic acid phenethyl ester up-regulates antioxidant levels in hepatic stellate cell line T6 via an Nrf2-mediated mitogen activated protein kinases pathway

AIM To investigate the antioxidant effect of caffeic acid phenethyl ester (CAPE) in hepatic stellate cell-T6 (HSC-T6) cells cultured in vitro and the potential mechanisms. METHODS HSC-T6 cells were cultured in vitro and treated with various concentrations of CAPE for 24, 48 and 72 h, respectively. Cell proliferation was investigated using the MTT assay, and cell ultrastructural alterations were observed by transmission electron microscopy. Flow cytometry was employed to investigate the effects of CAPE on apoptosis and the levels of reactive oxygen species in HSC-T6 cells cultured in vitro. An enzyme immunoassay instrument was used to evaluate antioxidant enzyme expression. The effect on alpha-smooth muscle actin was shown using immunofluorescence. Gene and protein levels of Nrf2, related factors, and mitogen activated protein kinases (MAPKs), in HSC-T6 cells were detected using RT-PCR and Western blot, respectively. RESULTS CAPE inhibited the proliferation and activation of HSC-T6 cells cultured in vitro. CAPE increased the antioxidant levels and the translocation of Nrf2 from the cytoplasm to the nucleus in HSC-T6 cells. Moreover, the phosphorylation of MAPKs in cells decreased in response to CAPE. Interestingly, CAPE-induced oxidative stress in the cells was significantly attenuated by pretreatment with MAPKs inhibitors. CONCLUSION CAPE inhibits cell proliferation and up-regulates the antioxidant levels in HSC-T6 cells partly through the Nrf2-MAPKs signaling pathway.

Inhibition of DNA-dependent Protein Kinase Catalytic Subunit by Small Molecule Inhibitor NU7026 Sensitizes Human Leukemic K562 Cells to Benzene Metabolite-induced Apoptosis

Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.

Chronic stress causes protein kinase C epsilon-aldehyde dehydrogenase 2 signaling pathway perturbation in the rat hippocampus and prefrontal cortex,but not in the myocardium

Chronic stress is strongly associated with the occurrence and development of depression and cardiovascular disease.Stress can induce altered mitochondrial function and activation of apoptosis in the cardio-cerebral system.However,it is unknown whether the protein kinase C ε（PKCε）-aldehyde dehydrogenase 2（ALDH2） pathway is altered under chronic stress,and this study sought to address this question.A rat model of depression was established using a chronic unpredictable mild stress（CUMS） protocol.After experiencing CUMS for 4 weeks,the sucrose preference test and the forced swim test verified depressive-like behaviors.Enzyme linked immunosorbent assays showed that ALDH2 activity was decreased in the rat hippocampus and prefrontal cortex,but was not altered in the myocardium.Western blot assays demonstrated reduced levels of ALDH2 and PKCε,but increased levels of 4-hydroxy-2-nonenal（4 HNE） adducts.Caspase-3 expression did not obviously alter,but active forms of caspase-3 were increased in the hippocampus and prefrontal cortex.In the myocardium,expression of ALDH2,PKCε and 4 HNE adducts did not remarkably alter;while caspase-3 expression was reduced and the active forms of caspase-3 were upregulated.Pearson＇s correlation test demonstrated that expression of 4 HNE adducts was positively correlated with levels of the active forms of caspase-3 in the hippocampus and prefrontal cortex,but not in the myocardium.In conclusion,chronic stress can damage the PKCε-ALDH2 signaling pathway in the hippocampus and prefrontal cortex,but not in the myocardium.Moreover,4 HNE is associated with active forms of caspase-3 in the hippocampus and prefrontal cortex.

Signal transducer and activator of transcription 3 promotes the Warburg effect possibly by inducing pyruvate kinase M2 phosphorylation in liver precancerous lesions

BACKGROUND Study shows that signal transducer and activator of transcription 3(STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2(PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats.AIM To investigate the mechanism of the Warburg effect in liver precancerous lesions in rats.METHODS A model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with Nmethyl-N'-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen(PCNA), STAT3,and PKM2 were examined by Western blot and immunofluorescence.RESULTS We found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liverprecancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression,PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells.Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2.CONCLUSION The Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.

Tolerance of neurite outgrowth to Rho kinase inhibitors decreased by cyclooxygenase-2 inhibitor

In this study, PC12 Adh cells and Neuro-2a cells were treated with Rho-associated kinase inhibitors （Y27632 and Fasudil）, a cyclooxygenase-1 selective inhibitor （SC560）, and a cyclooxygenase-2 inhibitor （NS398）. We found that these cells became tolerant to Rho-associated kinase inhibitors, as neurite outgrowth induced by these inhibitors diminished following more than 3 days of exposure in either cell line. The proteins cyclooxygenase-2 and cytosolic prostaglandin E synthetase were upregulated at day 3. NS398 decreased the tolerance to neurite outgrowth induction in both cell lines, whereas SC560 had almost no effect. These findings indicate that cells become tolerant to neurite outgrowth induced by Rho-associated kinase inhibitors, this is at least partly associated with upregulation of proteins involved in the cyclooxygenase-2 pathway, and cyclooxygenases-2 inhibition prevents this tolerance.

Synthesis of 5-substituted benzyl-2,4-diamino pyrimidine derivatives as c-Fms kinase inhibitors

A serials of novel 5-substituted benzyl-2,4-diamino pyrimidine derivatives have been synthesized and evaluated as inhibitors of c-Fms kinase by the standard MTT method.The results showed that compound 15,5-[3-methoxy-4-（pyridine-3-yl）benzyl]-2,4-diamino pyrimidine,had an IC50 of 1.45μmol/L in inhibiting the proliferation of M-CSF-dependent myeloid leukemia cells in mice （NFS-60）,which was similar with GW2580,a selective inhibitor of c-Fms kinase.