1.Expression of positive and negative regulators of cell cycle during wound healing.
Xudong ZHU ; Yanfei DI ; Chengxiang HU ; Zhengguo WANG
Chinese Medical Journal 2002;115(3):326-330
OBJECTIVETo detect the expression of cell cycle positive regulators cyclin D(1), cyclin E, CDK(2), CDK(4) and negative regulators p21(cip1), p27(kip1), p16(ink4a) and p15(ink4b) during wound healing in rats.
METHODSOpen wounds of full-thickness skin, diameter 1.8 cm, on rat backs were used as the wound model. Wound tissues were harvested on postwounding days 3, 5, 7, 9, 11, 14, 21 and 30. Ki67 expression in granulation tissue was detected by immunohistochemical assay. The patterns of the expression of cyclin D(1), cyclin E, CDK(2), CDK(4) and p21(cip1), p27(kip1), p16(ink4a), p15(ink4b) were detected by Western blot.
RESULTSCell proliferation in granulation tissue took place predominantly within the first week after injury, with the proliferation peak occurring at postwounding day 5. There were no dramatic variations in the expression of cyclin D(1), CDK(2) and CDK(4) during wound healing. Up-regulated cyclin E was maintained from day 3 to 11 after injury, and then was down-regulated. No expression of p16(ink4a) and p15(ink4b) was found. p21(cip1) was expressed only from day 7 to 14, with peak expression observed on day 9. Constitutive p27(kip1) was expressed throughout wound healing with low levels in the proliferating period of day 3 to 5 and with increased levels in the post-mitotic and remodeling stage. The expression of p21(cip1) and p27(kip1) showed an inverse gradient to that of Ki67.
CONCLUSIONp21(cip1) and p27(kip1) play a supervising role in preventing the hyperproliferative tendency in tissue repair.
Animals ; Cell Cycle ; physiology ; Cell Cycle Proteins ; biosynthesis ; physiology ; Cell Division ; physiology ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Cyclin-Dependent Kinases ; antagonists & inhibitors ; biosynthesis ; Cyclins ; biosynthesis ; Male ; Rats ; Rats, Wistar ; Skin ; cytology ; metabolism ; Tumor Suppressor Proteins ; biosynthesis ; physiology ; Wound Healing
2.Expression of NDRG2 is related to tumor progression and survival of gastric cancer patients through Fas-mediated cell death.
Seung Chul CHOI ; Suk Ran YOON ; Yuk Pheel PARK ; Eun Young SONG ; Jae Wha KIM ; Woo Ho KIM ; Young YANG ; Jong Seok LIM ; Hee Gu LEE
Experimental & Molecular Medicine 2007;39(6):705-714
Although N-myc downstream regulated gene 2 (NDRG2) has been known to be a tumor suppressor gene, the function of this gene has not been elucidated. In the present study, we investigated the expression and function of NDRG2 in human gastric cancer. Among seven gastric cancer and two non-cancer cell lines, only two gastric cancer cell lines, SNU-16 and SNU-620, expressed NDRG2, which was detected in the cytoplasm. Interestingly, NDRG2 was highly expressed in normal gastric tissues, but gastric cancer patients were divided into NDRG2-positive and -negative groups. The survival rate of NDRG2-negative patients was lower than that of NDRG2-positive patients. We confirmed that the loss of NDRG2 expression was a significant and independent prognostic indicator in gastric carcinomas by multivariate analysis. To investigate the role of NDRG2 in gastric cancer cells, we generated a NDRG2-silenced gastric cancer cell line, which stably expresses NDRG2 siRNA. NDRG2-silenced SNU-620 cells exhibited slightly increased proliferation and cisplatin resistance. In addition, inhibition of NDRG2 decreased Fas expression and Fas-mediated cell death. Taken together, these data suggest that inactivation of NDRG2 may elicit resistance against anticancer drug and Fas-mediated cell death. Furthermore, case studies of gastric cancer patients indicate that NDRG2 expression may be involved in tumor progression and overall survival of the patients.
Apoptosis/*physiology
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Cell Line, Tumor
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Down-Regulation
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Fas Ligand Protein/*physiology
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Gene Expression Regulation, Neoplastic
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Humans
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Stomach Neoplasms/metabolism/*mortality/pathology
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Tumor Markers, Biological/*metabolism
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Tumor Suppressor Proteins/biosynthesis/genetics/immunology/*metabolism
3.Inhibitory effect of tumor suppressor PTEN on cell growth of endometrial carcinoma.
Xiao-yun WAN ; Yi-fu SHI ; Juan-qing LI
Chinese Journal of Oncology 2004;26(5):275-278
OBJECTIVETo evaluate the inhibitory effect of tumor suppressor PTEN on cell growth of endometrial carcinoma.
METHODSThe exogenous wild PTEN cDNA via an adenoviral vector (Ad-PTEN) was introduced into Ishikawa cells. The expression of PTEN protein was detected by Western blot. The growth of Ishikawa cells was evaluated by trypan blue exclusion method and MTT.
RESULTSThe expression of PTEN protein was induced on day 1, and greatly increasing on day 3 - 5 after Ad-PTEN infection. The expression of PTEN significantly inhibited the growth of Ishikawa cells, and also significantly inhibited the growth of Ishikawa cells induced by IGF-II.
CONCLUSIONAdenovirus-mediated introduction of exogenous PTEN into human endometrial carcinoma cells can induce growth suppression. PTEN gene may be a novel therapeutic agent for endometrial carcinoma.
Adenoviridae ; genetics ; Cell Proliferation ; Endometrial Neoplasms ; metabolism ; pathology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Insulin-Like Growth Factor II ; pharmacology ; PTEN Phosphohydrolase ; Phosphoric Monoester Hydrolases ; biosynthesis ; genetics ; physiology ; Recombination, Genetic ; Transfection ; Tumor Suppressor Proteins ; biosynthesis ; genetics ; physiology
5.Construction of deltaNp63 specific small hairpin RNA expressing plasmid and its role in bladder cancer--a preliminary study.
Yun-feng HE ; Xiao-hou WU ; Chun-li LUO ; Dai-yin TIAN ; Liang-suo ZHANG ; Fei GAO
Chinese Journal of Oncology 2006;28(11):820-825
OBJECTIVETo construct deltaNp63 specific small hairpin RNA (shRNA) expressing plasmid,to examine its inhibitory effect to the expression of deltaNp63 protein and mRNA in transitional cell carcinoma of the bladder (TCCB) , its effect on TCCB cells cycle and proliferation.
METHODSDeltaNp63 specific oligonucleotides were designed and synthesized. These oligonucleotides were annealed to form double strand DNA fragments and this fragment was cloned into Pgenesil-1 plasmid. The recombinant deltaNp63-shRNA expression construct was confirmed by using Pst I + Sal I double digestion and by sequencing. Fluorescence staining was used to confirm the success of transfection in TCCB cells under the fluorescence microscope. The inhibitory effect of deltaNp63-shRNA construct was examined with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical staining assay. The cell cycle of TCCB cells was assayed by flow cytometry (FCM). The cellular proliferation of TCCB cells was assayed by tetrazolium bromide (MTT) colorimetry.
RESULTSThe deltaNp63-shRNA expression plasmid was successfully constructed and transfected into TCCB cells. It can effectively reduce the expression of deltaNp63 protein and mRNA. The reduction rate of deltaNp63 mRNA was 63.0%, and the G0/G1 ratio was increased and S phase was decreased in transfected TCCB cells. The cellular proliferation was also lower in transfected 5637 cells in comparrison with that of non-transfected TCCB cells.
CONCLUSIONA deltaNp63-shRNA expression plasmid, constructed from Pgenesil-1 plasmid, can successfully be transfected into TCCB cells and can effectively inhibit the expression of deltaNp63 protein and mRNA. It also can take part in regulation of the cell cycling and inhibit the cellular proliferation of TCCB cells.
Carcinoma, Transitional Cell ; genetics ; metabolism ; pathology ; Cell Cycle ; genetics ; physiology ; Cell Line, Tumor ; Cell Proliferation ; DNA-Binding Proteins ; biosynthesis ; genetics ; physiology ; Humans ; Immunohistochemistry ; Microscopy, Fluorescence ; Plasmids ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Trans-Activators ; biosynthesis ; genetics ; physiology ; Transcription Factors ; Transfection ; Tumor Suppressor Proteins ; biosynthesis ; genetics ; physiology ; Urinary Bladder Neoplasms ; genetics ; metabolism ; pathology
6.Induction of HSF1 expression and sporadic colorectal cancer.
Yong-ming FANG ; Qi DONG ; Hui CEN ; Xiao-ping TANG ; Shu ZHENG
Journal of Zhejiang University. Medical sciences 2004;33(5):390-394
OBJECTIVETo explore the activation of the signal transduction pathways related with the carcinogenesis of sporadic colon cancers.
METHODSA gene microarray monitoring activation of 8 signal transduction pathways (PathwayFinder GEArray) was used to screen the differentially expressed genes between colorectal cancer and normal colon tissue. The differentially expressed genes were further analyzed by RT-PCR, using RNA extracted from cancer tissue and matched normal colon mucosa of 35 patients with colorectal cancer.
RESULTSThe expression of hsf1, hsf27 and inos was increased in colon cancer compared with normal colon mucosa using PathwayFinder GEArray. The RT-PCR results showed that the expression of hsf1 was detected in 86% of patients(30/35)and the expression of inos detected in 63% patients(22/35).
CONCLUSIONHsf1 induces heat shock stress signaling pathway, which might play a role in the carcinogenesis of sporadic colorectal cancer.
Colorectal Neoplasms ; genetics ; metabolism ; DNA-Binding Proteins ; biosynthesis ; genetics ; Gene Expression Regulation, Neoplastic ; Heat Shock Transcription Factors ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; physiology ; Transcription Factors ; Tumor Suppressor Proteins ; biosynthesis ; genetics
7.Impaired Delta NP63 Expression is Associated with Poor Tumor Development in Transitional Cell Carcinoma of the Bladder.
Yunfeng HE ; Xiaohou WU ; Wei TANG ; Daiyin TIAN ; Chunli LUO ; Zhikang YIN ; Hu DU
Journal of Korean Medical Science 2008;23(5):825-832
The oncogenic isoform of the p63 protein, delta NP63, plays an important role in the pathogenesis of many epithelial carcinomas, and emerging evidences suggest that delta NP63 is a promising drug target. However, the functions of delta NP63 in transitional cell carcinoma of bladder (TCCB) are poorly defined. In this study, a delta NP63 shRNA expression vector was transfected into TCCB cell line 5637 and cell cycling, cell proliferation and protein expression were assessed by flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-dimethyl tetrazolium bromide (MTT) assay, and immunohistochemistry, respectively. The delta NP63 shRNA expression vector was also injected into 5637 cell xenograft tumors in nude mice, and tumor size was measured, tumor tissue morphology was assessed by immunohistopathology and transmission electron microscopy. In the in vitro study, delta NP63 shRNA transfection caused successful delta NP63 gene silencing and resulted in significant arrest of cell cycling and cellular proliferation (p<0.05) as well as cyclin D1 expression. In the nude mouse xenograft model, delta NP63 shRNA greatly inhibited tumor growth, induced tumor cell apoptosis (p<0.05) and resulted in cyclin D1 downregulation. Our data suggest that delta NP63 may play an oncogenic role in TCCB progression through promoting cell survival and proliferation. Intratumoral administration of delta NP63-specific shRNA suppressed tumor delta NP63 expression and cellular proliferation while promoted tumor cellular apoptosis, and therefore inhibited tumor growth and improved survival of xenograft-bearing mice, which was not accompanied by significant signs of systemic toxicity.
Animals
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Carcinoma, Transitional Cell/*genetics/metabolism
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Cell Line, Tumor
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Cell Proliferation
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Cyclin D1/biosynthesis
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Disease Progression
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Female
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Humans
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Mice
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Mice, Nude
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Microscopy, Electron, Transmission
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Models, Biological
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Neoplasm Transplantation
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Trans-Activators/*biosynthesis/*physiology
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Tumor Suppressor Proteins/*biosynthesis/*physiology
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Urinary Bladder Neoplasms/*genetics/metabolism
8.Experimental study on apoptosis of leukemia cell line NB4 transfected with WT1 gene.
Hui-Ling SHEN ; Zi-Xing CHEN ; Wei WANG ; Jian-Nong CEN ; Shao-Yan HU ; Ye ZHAO
Journal of Experimental Hematology 2005;13(6):989-995
In order to study the potential effects of exogenous WT1 gene isoform on apoptosis in leukemia cell line NB4 and its possible molecular mechanisms, the eukaryotic expression recombinant vector (pCB6(+)/WTA) containing full-length human WT1 isoform (WTA: -17aa/-KTS) cDNA and the vacant vector-alone were introduced into the leukemia cell line NB4 respectively by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Binding of Annexin V were tested by flow cytometry and agarose gel electrophoresis to verify whether exogenous WTA could induce apoptosis of NB4 cells. Expressions of p21, p53, bcl-2, bcl-XL and c-myc genes were determined by semi-quantitative RT-PCR after introducing recombinant vectors into the NB4 cells. The results showed that in exposure to As(2)O(3) at 0.8 micromol/L for 48 hours, the NB4/WTA cells exhibited the morphological hallmarks of apoptosis, the marked DNA ladder shown by gel electrophoresis, and the enhanced apoptosis rate marked by Annexin V. RT-PCR showed an increase in p21 and c-myc genes expression, a decrease in bcl-2 and a relative constant expression of p53, bcl-XL in NB4/WTA cells. It is concluded that the introduction and expression of exogenous WTA gene can lead to apoptosis of NB4/WTA cells by down-regulating the Bcl-2 gene expression and up-regulating the p21 and c-myc genes expression.
Apoptosis
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genetics
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physiology
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Blotting, Western
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Cell Line, Tumor
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Cyclin-Dependent Kinase Inhibitor p21
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genetics
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Gene Expression Regulation, Neoplastic
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Humans
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Leukemia
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genetics
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metabolism
;
pathology
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Proto-Oncogene Proteins c-bcl-2
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genetics
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Proto-Oncogene Proteins c-myc
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genetics
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RNA, Messenger
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biosynthesis
;
genetics
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Reverse Transcriptase Polymerase Chain Reaction
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Transfection
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Tumor Suppressor Protein p53
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genetics
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WT1 Proteins
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genetics
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metabolism
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physiology
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bcl-X Protein
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genetics
9.The relationship of expression of bcl-2, p53, and proliferating cell nuclear antigen (PCNA) to cell proliferation and apoptosis in renal cell carcinoma.
Zhaohui ZHU ; Shian XING ; Ping CHENG ; Guosheng LI ; Yu YANG ; Fuqing ZENG ; Gongcheng LU
Journal of Huazhong University of Science and Technology (Medical Sciences) 2004;24(4):354-357
To investigate the relationship of bcl-2, p53, proliferating cell nuclear antigen (PCNA) to cell proliferation, apoptosis and pathological parameters, the patterns of cell growth and turnover in renal cell carcinoma (RCC), formalin-fixed and paraffin-embedded tissue blocks from 34 patients with RCC were examined. Cell proliferation activity was detected by PCNA immunostaining and the proliferation index (PI) was expressed as a percentage of the PCNA-positive cells in the tumor cells. Apoptosis was detected by terminal deoxy- nucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), and the apoptotic index (AI) was expressed as a percentage of the TUNEL-positive cells in the tumor cells. Expressions of bcl-2 and p53 were assessed immunohistochemically. Our results showed that the PI ranged from 6.0% to 24.0% (median 12.3%) and the AI from 2.0% to 8.0% (median 5.4%) in RCC. The expression of the bcl-2 protein was demonstrated in 15 cases (44.1%); the expression of the p53 protein, however, was seen in only 3 case. bcl-2 positivity was not associated with PI or AI or any pathological parameters. There were close associations between PI and tumor grade and stage, and a significant relationship between AI and the tumor grade of RCC, Our study suggests that bcl-2 positivity was not associated with PI or AI or any pathological parameters. There are close associations between PI and AI and tumor grade and stage of RCC. Active cell proliferation may be accompanied by frequent apoptosis in RCC.
Adult
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Aged
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Apoptosis
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physiology
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Carcinoma, Renal Cell
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metabolism
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pathology
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Cell Division
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Female
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Humans
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Kidney Neoplasms
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metabolism
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pathology
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Male
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Middle Aged
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Proliferating Cell Nuclear Antigen
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biosynthesis
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genetics
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Proto-Oncogene Proteins c-bcl-2
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biosynthesis
;
genetics
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Tumor Cells, Cultured
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Tumor Suppressor Protein p53
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biosynthesis
;
genetics
10.AMPK interacts with DSCAM and plays an important role in netrin-1 induced neurite outgrowth.
Kun ZHU ; Xiaoping CHEN ; Jianghong LIU ; Haihong YE ; Li ZHU ; Jane Y WU
Protein & Cell 2013;4(2):155-161
Down syndrome cell adhesion molecule (DSCAM) acts as a netrin-1 receptor and mediates attractive response of axons to netrin-1 in neural development. However, the signaling mechanisms of netrin-DSCAM remain unclear. Here we report that AMP-activated protein kinase (AMPK) interacts with DSCAM through its γ subunit, but does not interact with DCC (deleted in colorectal cancer), another major receptor for netrin-1. Netrin-treatment of cultured cortical neurons leads to increased phosphorylation of AMPK. Both AMPK mutant with dominant-negative effect and AMPK inhibitor can significantly suppress netrin-1 induced neurite outgrowth. Together, these findings demonstrate that AMPK interacts with DSCAM and plays an important role in netrin-1 induced neurite outgrowth. Our study uncovers a previously unknown component, AMPK, in netrin-DSCAM signaling pathway.
AMP-Activated Protein Kinases
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antagonists & inhibitors
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genetics
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metabolism
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Animals
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Cell Adhesion Molecules
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genetics
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metabolism
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Cells, Cultured
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HEK293 Cells
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Humans
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Mice
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Nerve Growth Factors
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pharmacology
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Netrin-1
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Neurites
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physiology
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Neurons
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cytology
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drug effects
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metabolism
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Phosphorylation
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Protein Binding
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Protein Kinase Inhibitors
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pharmacology
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RNA Interference
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RNA, Small Interfering
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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Signal Transduction
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drug effects
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Transfection
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Tumor Suppressor Proteins
;
pharmacology