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4.
J Fish Biol ; 99(2): 373-383, 2021 Aug.
Article in English | MEDLINE | ID: mdl-33715166

ABSTRACT

The genus Microphysogobio was established by Mori [Mori, T. (1934). The fresh water fishes of Jehol. In Report of the first scientific expedition to Manchoukuo. 1: pp. 1-61] based on a single specimen (Microphysogobio hsinglungshanensis) collected in the Luanhe River basin, Xinglong County, Hebei Province, China. Because the genus characteristics were derived from its type species, M. hsinglungshanensis, the detailed description is essential. In addition, to distinguish M. hsinglungshanensis and Microphysogobio chinssuensis, the description based on holotype and more specimens is needed. M. hsinglungshanensis can be distinguished from all other congeners by the following combination of characters: mouth shallow arc shaped and inferior; medial pad on lower lip inverted trapezoid and usually grooved; barbel short, 6.9%-14.3% in head length; lateral-line scales 38-39; ventral region between pectoral-fin origin and pelvic-fin origin scaleless; scales above lateral line 4-4.5; predorsal scales 10-11; vertebrae 4 + 32 - 34; caudal-fin membrane with two or three rows of irregular black spots. The characteristics of this genus were redefined based on M. hsinglungshanensis in this study.


Subject(s)
Cyprinidae , Cypriniformes , Animals , China , Head , Rivers
5.
Eur J Pharmacol ; 588(1): 1-8, 2008 Jun 24.
Article in English | MEDLINE | ID: mdl-18448095

ABSTRACT

Artesunate, a remarkable antimalarial agent, also reveals profound cytotoxic activity. In the present investigation, we compared the anticancer effects of artesunate on three colorectal cancer cell lines and analyzed the relationship between drug sensitivity and malignant phenotype of the tumor cells. The findings are as follows: poorly-differentiated was colorectal cancer cell line CLY showing nuclear beta-catenin accumulation and loss of E-cadherin; moderately-differentiated were Lovo cells with cytoplasmic distribution of the two proteins; and well-differentiated were HT-29 cells with membranous localization of them. Also, both in vitro and in vivo, poorly- or moderately-differentiated CLY and Lovo were more susceptible to artesunate treatment than well-differentiated HT-29. Furthermore, the sensitive response of CLY and Lovo to artesunate was associated with membranous translocation of beta-catenin and increased expression of E-cadherin, which indicated the inhibition of hyperactive Wnt signaling pathway and the reversion of the epithelial to mesenchymal transition, respectively. Due to the vital roles of Wnt pathway and the epithelial to mesenchymal transition in tumor differentiation, we thought artesunate could promote the re-differentiation and apoptosis of colorectal cancer cells by inhibition of hyperactive Wnt pathway and reversion of the epithelial to mesenchymal transition.


Subject(s)
Adenocarcinoma/drug therapy , Adenocarcinoma/metabolism , Antineoplastic Agents , Artemisinins/pharmacology , Cadherins/biosynthesis , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , beta Catenin/biosynthesis , Animals , Apoptosis/drug effects , Artesunate , Blotting, Western , Cell Cycle/drug effects , Cell Differentiation/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Female , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Middle Aged , Neoplasm Proteins/biosynthesis , Subcellular Fractions/drug effects
6.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 24(1): 16-9, 2008 Jan.
Article in Chinese | MEDLINE | ID: mdl-18177610

ABSTRACT

AIM: Investigate the molecular mechanism of regulating survivin expression and related signal transduction pathway, molecular cascade reaction and biological effects in activated PBMC. METHODS: The expression of survivin and related proteins were detected by Western blot in PBMC stimulated by PHA and rhIL-2 with or without JAK inhibitor-AG490 treatment, and FCM was performed to analyze cell cycle and cell division. RESULTS: Our results indicated that molecular and cellular reactions in PBMC activated by PHA and rhIL-2 were dependent on time series. At first, the phosphorylation of Stat3 and Stat5 were observed, then, protein levels of CyclinD3 and CyclinE increased, and the stimulated PBMC began to enter to S phage with survivin protein expression was initiated, which at last resulted in cell division with dramatically increasing expression of survivin protein. AG490 could significantly inhibit all these reactions but had no effect on the expressions of the cell cycle inhibitor-P21 and anti-apoptosis protein-Bcl-2. CONCLUSION: The expression of survivin in stimulated PBMC was dependent on the primarily activated JAK-STAT pathway, which upregulated CyclinD3 and CyclinE protein levels, initiated the cell cycle progression, and induced cell cycle-dependent survivin expression, and so survivin was involved in cell division and cell proliferation.


Subject(s)
Cell Cycle/drug effects , Cell Survival/genetics , Microtubule-Associated Proteins/metabolism , Cell Adhesion Molecules , Cell Cycle/physiology , Cell Proliferation/drug effects , Gene Expression , Humans , Inhibitor of Apoptosis Proteins , Microtubule-Associated Proteins/genetics , Protozoan Proteins , STAT3 Transcription Factor , Signal Transduction , Survivin , Tyrphostins/pharmacology
7.
Int J Cancer ; 121(6): 1360-5, 2007 Sep 15.
Article in English | MEDLINE | ID: mdl-17520675

ABSTRACT

Artesunate (ART), a remarkable antimalarial agent, also inhibited the growth of human colorectal carcinoma. As determined by MTT assay, flow cytometry analysis on apoptosis and indirect immunofluorescence analysis on the proliferation-associated marker Ki67, ART suppressed the proliferation and promoted the apoptosis of colorectal cancer cells in a dose-dependent manner. Furthermore, immunofluorescence analysis on beta-catenin and RT-PCR analysis on Wnt/beta-catenin target genes demonstrated ART translocated beta-catenin from nucleus to adherent junctions of membrane and reduced transcription mediated by beta-catenin. These results suggested the anticancer activity of ART correlated with the inhibition of hyperactive Wnt/beta-catenin signaling pathway. In vivo, ART significantly slowed the growth of colorectal tumor xenografts. Bioluminescent imaging also revealed that ART decreased the physiological activity of tumor xenografts and delayed spontaneous liver metastasis. These antitumor effects were related to the membranous translocation of beta-catenin and the inhibition of the unrestricted activation of Wnt/beta-catenin pathway, which was confirmed by the immunohistochemical staining of tumor tissues. These results and the known low toxicity are clues that ART might be a promising candidate drug for the treatment of colorectal carcinoma.


Subject(s)
Adenocarcinoma/metabolism , Antineoplastic Agents/pharmacology , Artemisinins/pharmacology , Colorectal Neoplasms/metabolism , Sesquiterpenes/pharmacology , Animals , Apoptosis/drug effects , Artesunate , Blotting, Western , Cell Proliferation/drug effects , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression/drug effects , Humans , Immunohistochemistry , Male , Mice , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Wnt Proteins/drug effects , Wnt Proteins/metabolism , Xenograft Model Antitumor Assays , beta Catenin/drug effects , beta Catenin/metabolism
8.
Oncol Rep ; 17(4): 835-40, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17342324

ABSTRACT

Colorectal cancer (CRC) with liver metastasis is a fatal disease with rapid progression and poor patient outcome. However, the molecular mechanism involved in liver metastasis of CRC remains essentially unknown, largely because of the presence of few available CRC cell lines with liver metastasis origin and spontaneous metastatic potentials in nude mice. In this study, we established a novel metastatic CRC cell line, CLY, derived from liver metastasis of a 64-year-old Chinese CRC patient. The cell line was characterized by morphology, growth kinetics, tumorigenicity, spontaneous liver metastatic potential, and cytogenetics. Immunofluorescence analysis of beta-catenin and E-cadherin and methylation-specific PCR (MSP) of the E-cadherin gene (CDH1) promoter were also used to compare CLY with conventional CRC cell lines (HT-29 and Lovo). CLY exhibited compact and polygonal-shaped epithelial cells in vitro and its population doubling time was 29.5 h. Subcutaneous transplantation of CLY into nude mice resulted in subcutaneous tumor formation and spontaneous liver metastasis in all of 10 nude mice. Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 60. In immunofluorescence analysis, CLY exhibited a low expression but high restricted nuclear localization of beta-catenin and a silenced expression of E-cadherin, which may be induced by hypermethylation of the E-cadherin gene (CDH1) promoter and differed from conventional CRC cell lines. Therefore, CLY is an ideal cell model for further exploring the metastatic mechanisms of CRC and testing new therapeutic reagents for CRC with liver metastasis.


Subject(s)
Cell Line, Tumor , Colorectal Neoplasms/pathology , Liver Neoplasms/secondary , Models, Biological , Animals , Cadherins/analysis , Cadherins/genetics , Cadherins/metabolism , Colorectal Neoplasms/chemistry , Colorectal Neoplasms/genetics , DNA Methylation , Humans , Karyotyping , Liver Neoplasms/chemistry , Liver Neoplasms/genetics , Mice , Neoplasm Transplantation , beta Catenin/analysis , beta Catenin/genetics , beta Catenin/metabolism
9.
Ai Zheng ; 25(5): 533-7, 2006 May.
Article in Chinese | MEDLINE | ID: mdl-16687069

ABSTRACT

BACKGROUND & OBJECTIVE: Proteasome inhibitor, which can induce apoptosis in various tumor cells, is a kind of potential antitumor drug. This study was to identify the proteins involved in G(2)/M arrest of leukemia cell line HL-60 exposed to proteasome inhibitor MG132 by proteomic techniques. METHODS: Flow cytometry was used to examine cell cycle of HL-60 cells exposed to 2.5 micromol/L MG132. Nuclear extracts of HL-60 cells were prepared, and the purity was detected by light microscopy and Western blot, and the differentially expressed protein spots were determined by two-dimensional gel electrophoresis and identified with MALDI-TOF-TOF/MS. RESULTS: There was a distinct G(2)/M phase arrest before the apoptosis of HL-60 cells induced by 2.5 micromol/L MG132. Twenty-three differentially expressed protein spots were found between MG132-treated and control HL-60 cells; 8 nuclear proteins were identified by MALDI-TOF-TOF/MS analysis. CONCLUSIONS: The detected proteins, such as eIF5A and splicing factor, may be involved in regulation of G(2)/M arrest of HL-60 cells. These findings will be helpful for revealing molecular mechanisms of proteasome inhibitor-induced G(2)/M phase arrest and apoptosis of leukemia cell line.


Subject(s)
Cell Cycle/drug effects , Leupeptins/pharmacology , Nuclear Proteins/analysis , Proteomics/methods , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Electrophoresis, Gel, Two-Dimensional , G2 Phase/drug effects , HL-60 Cells , Heterogeneous-Nuclear Ribonucleoproteins/analysis , Humans , Peptide Initiation Factors/analysis , RNA-Binding Proteins/analysis , Serine-Arginine Splicing Factors , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Eukaryotic Translation Initiation Factor 5A
10.
Mol Biotechnol ; 32(3): 197-204, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16632886

ABSTRACT

The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18-25 nucleotides) with important roles in the regulation of gene expression. Although a large number of miRNAs have been identified in a variety of eukaryotic systems, the function of the vast majority of these molecules remains unknown. To study the functions of miRNAs, it is crucial to determine their spatial and temporal expression patterns. Although there are some existing methods that can analyze the expression of miRNAs, it is not an easy task for routine gene-expression studies. In this study, we have established a simple method to detect the expression of mature miRNAs. Total RNA was polyadenylated by poly(A) polymerase, and then cDNA was synthesized by a specific reverse transcriptase (RT) primer and reverse transcriptase using the poly(A)-tailed total RNA as templates. The expression of several mature miRNAs was assayed by this method. The expression profile of two miRNAs, determined by the polymerase chain reaction (PCR) assay, was identical to that determined by Northern blotting. All these data show that the poly(A)-tailed RT-PCR is a convenient method to detect the expression of miRNAs.


Subject(s)
Gene Expression Profiling/methods , MicroRNAs/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Blotting, Northern , Cell Line , Cell Line, Tumor , HeLa Cells , Humans , MicroRNAs/metabolism , Poly A/genetics , Reproducibility of Results , Sequence Analysis, DNA
11.
Yi Chuan ; 28(1): 11-6, 2006 Jan.
Article in Chinese | MEDLINE | ID: mdl-16469709

ABSTRACT

To learn the variation in the gene for UGT1A1 enzyme, the genetic mechanism in a Chinese Han nationality family suffered from Gilbert's syndrome was studied. At first, genomic DNA from peripheral blood of the sufferer in this family was used for amplifying all of the five exons of the UGT1A1 gene by PCR, and then direct sequencing of the PCR product was applied to analyze gene mutation. The results showed that there existed a G-->A homozygous transition at nucleotide 211 leading the substitution of arginine for glycine at position 71 of corresponding protein product (G71R) and a T-->G homozygous transition at nucleotide 1456 leading the substitution of aspartic acid for tyrosine at position 486 of corresponding protein product (Y486D). No mutation was detected in promoter region and the splicing junction sites. The relevant mutation sites of the other family members were sequenced and identified to be heterozygous in the two above-mentioned mutation sites and in the TA repeat mutation in the promoter region. Furthermore, fresh blood samples were collected from all of the members to detect the serum bilirubin levels to determine the sufferer. The result was consistent with the mutation analysis. It could thus be inferred that this family was caused by mutation in the open reading frame of the gene UGT1A1.


Subject(s)
Gilbert Disease/genetics , Glucuronosyltransferase/genetics , Asian People/genetics , Bilirubin/blood , Child , Exons/genetics , Female , Gilbert Disease/blood , Humans , Male , Mutation , Pedigree , Polymerase Chain Reaction , Promoter Regions, Genetic/genetics
12.
Ai Zheng ; 24(5): 525-30, 2005 May.
Article in Chinese | MEDLINE | ID: mdl-15890091

ABSTRACT

BACKGROUND & OBJECTIVE: The expression of Survivin in cancer cells highly correlates with that of human telomerase reverse transcriptase (hTERT). Both of them are ideal targets for cancer gene therapy. This study aimed to clarify if they regulate each other in cancer cells. METHODS: The expressions of Survivin and hTERT in HeLa S3 cells were inhibited by antisense oligonucleotide respectively. Activity of telomerase was detected by telomerase repeat amplification (TRAP) assay. Protein and mRNA levels of Survivin were analyzed by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) respectively. Proliferation of HeLa S3 cells was analyzed by MTT assay. RESULTS: Inhibiting the expression of Survivin in HeLa S3 cells had no effects on telomerase activity. Inhibiting the expression of hTERT by antisense oligonucleotide No.14 decreased protein level of Survivin, which was negatively correlated with the concentration of No.14 (200-1 000 nmol/L), but didn't change mRNA level of survivin. The decrease of Survivin level was inhibited by proteasome inhibitor lactacystin and MG132. Furthermore, simultaneous inhibition of hTERT and survivin co-efficiently inhibited proliferation of HeLa S3 cells. CONCLUSION: Inhibiting the expression of hTERT in HeLa S3 cells promotes ubiquitin-proteasome degradation of Survivin.


Subject(s)
Acetylcysteine/analogs & derivatives , Cysteine Proteinase Inhibitors/pharmacology , DNA-Binding Proteins/metabolism , Microtubule-Associated Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Oligonucleotides, Antisense/pharmacology , Telomerase/metabolism , Acetylcysteine/pharmacology , Cell Proliferation , HeLa Cells , Humans , Inhibitor of Apoptosis Proteins , Leupeptins/pharmacology , Microtubule-Associated Proteins/genetics , Neoplasm Proteins/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Survivin
13.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 21(3): 330-3, 2005 May.
Article in Chinese | MEDLINE | ID: mdl-15862152

ABSTRACT

AIM: To prepare and characterize anti-human beta-Netrin antibodies. METHODS: B cell dominant epitopes of human beta-Netrin C-terminal 114 amino acid sequences were predicated by the GoldKey software. One of the epitopes was synthesized and coupled with bovine serum album (BSA) by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The BALB/c mice were immunized with the coupled protein. The splenocytes of immunized mice were fused with Sp2/0 cells by routine method and the hybridomas were selected in HAT medium. The hybridoma cells secreting specific antibody were detected by ELISA and cloned by limiting dilution. The titer specificity, and Ig subclass of anti-beta-Netrin mAbs were characterized by ELISA, Western blot and immunocytochemical staining. In addition, New Zealand rabbits were immunized with the coupled protein to prepare polyclonal antibody against beta-Netrin. The specificity of the antiserum was verified by Western blot. RESULTS: A 16-mer peptide NH2-FRGKRTLYPES-WTDRG-COOH was the dominant epitope of the B cells. Synthesized peptide coupled with BSA was used as the immunogen to immunize BALB/c mice. Three hybridoma cell lines that stably secrete specific mAbs were obtained. The result of immunocytochemical staining showed that prepared mAb specifically recognize the antigen in the neuronal cells. The polyclonal antibody against beta-Netrin had high specificity. Western blot analysis showed that the antiserum bind with the prokaryotically expressed beta-Netrin specifically. CONCLUSION: Using the synthesized peptides as hapten, we have prepared epitope-specific mAbs and pAb against beta-Netrin successfully.


Subject(s)
Antibodies/immunology , Epitopes/analysis , Membrane Proteins/chemistry , Membrane Proteins/immunology , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/immunology , Amino Acid Sequence , Animals , Antibodies/analysis , Antibody Specificity , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Epitopes/immunology , Female , Gene Expression Regulation , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Netrins , Neurons/metabolism , Peptides/chemical synthesis , Peptides/chemistry , Peptides/immunology , Protein Structure, Tertiary , Rats
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 13(2): 174-81, 2005 Apr.
Article in Chinese | MEDLINE | ID: mdl-15854272

ABSTRACT

To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.


Subject(s)
Apoptosis/drug effects , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , Hydroxamic Acids/pharmacology , Blotting, Western , Cell Cycle/drug effects , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Flow Cytometry , Histone Deacetylase Inhibitors , Humans , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology
15.
Ai Zheng ; 23(10): 1144-8, 2004 Oct.
Article in Chinese | MEDLINE | ID: mdl-15473924

ABSTRACT

BACKGROUND & OBJECTIVE: Proteasome inhibitor is a kind of potential anti-tumor drug,it can induce apoptosis in various tumor cells. This study was designed to investigate the molecular mechanism of apoptosis and G(2)/M arrest in leukemia cell line HL-60 induced by proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO). METHODS: Apoptosis in HL-60 cells was observed under fluorescent microscope, flow cytometry and immunoblot were used to analyze cell apoptosis, cell cycle arrest, and the mechanisms. RESULTS: MG132 (2 micromol/L)induced apoptosis in HL-60 cells after 24-h treatment. Meanwhile, HL-60 cells were arrested at G(2)/M phase before apoptosis after induced by MG132. The percentage of G(2)/M phase in MG132-treated HL-60 cells at 12 h was 63.42+/-2.02,while that in untreated cells was 7.29+/-3.01 (P< 0.01). The percentage of apoptosis in MG132-treated HL-60 cells at 24 h was 16.67+/-1.48, while untreated cells had no death (P< 0.01). Compared to the treatment with MG132 only, caffeine (2 mmol/L) exposure can reduce G(2)/M arrest and apoptosis in MG132-treated HL-60 cells. Expression of cyclin-dependent kinase inhibitor p21waf/cip1 up-regulated after treated with MG132 for 3 h, but no p53 or p27 detected. CONCLUSIONS: Proteasome inhibitor MG132 can induce G2/M arrest before the apoptosis appeared in HL-60 cells. The obvious up-regulation of p21 indicated that it is p21(waf/cip1), but not p53 or p53-related proteins,that involved in the regulation of G(2)/M arrest and subsequent apoptosis induced by MG132 in HL-60 cells.


Subject(s)
Apoptosis/drug effects , Cysteine Proteinase Inhibitors/pharmacology , G2 Phase/drug effects , Leupeptins/pharmacology , Antineoplastic Agents/pharmacology , Cell Cycle Proteins/metabolism , Cell Division/drug effects , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21 , HL-60 Cells , Humans , K562 Cells , Leukemia, T-Cell/pathology , Up-Regulation
16.
Ai Zheng ; 23(5): 487-92, 2004 May.
Article in Chinese | MEDLINE | ID: mdl-15142440

ABSTRACT

BACKGROUND & OBJECTIVE: Papillary thyroid carcinoma is characterized by RET (rearranged during transfection)/PTC (papillary thyroid carcinoma) rearrangements. However, the function of RET/PTC in carcino- genesis is not well understood. This study was designed to investigate the interaction between DNA double-strand break sensor ATM (mutated in ataxia telangiectasia) kinase and PTC1, a rearranged form of proto-oncogene ret, to explore the role of ret rearrangements in carcinogenesis. METHODS: RET TK phosphorylation was determined by in vitro kinase assay using the immunoprecipitation with anti-ATM antibody as kinase and the immunoprecipitation of HA-tagged TK as substrate. The location of ATM-LZPR in COS7 cells coexpressed with PTC1 was investigated by protein extracts of cytoplasm and nucleus. The phosphorylation level of p53, was determined by Western blot analysis with the antibody against phosphorylated p53, and the cell cycle was determined by flow cytometry when PTC1 overexpressed. RESULTS: ATM directly phosphorylated TK domain of PTC1 in vitro kinase assay. ATM-LZPR was located in both cell cytoplasm and nucleus when PTC1 was not expressed, however, co-overexpression of PTC1 and ATM-LZPR made the latter locate only in the cytoplasm. In addition, overexpression of PTC1 inhibited the phosphorylation level of p53 by ATM and caused G(1)/S phase arrest of cell cycle. CONCLUSIONS: PTC1 may remain ATM kinase in cytoplasm and inhibit the phosphorylation of p53 by ATM. PTC1, a rearrangement form of ret, may result in the disorder of cell damage repair and cell cycle checkpoint and destroy cell homeostasis.


Subject(s)
Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Oncogene Proteins, Fusion/metabolism , Oncogene Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , COS Cells , Cell Cycle Proteins/genetics , Cell Cycle Proteins/pharmacology , Cell Nucleus/metabolism , Chlorocebus aethiops , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/pharmacology , G1 Phase , HeLa Cells , Humans , Leucine Zippers/genetics , Oncogene Proteins/genetics , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/pharmacology , Protein-Tyrosine Kinases , Proto-Oncogene Mas , Proto-Oncogene Proteins c-ret , Receptor Protein-Tyrosine Kinases/genetics , S Phase , Transfection , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/pharmacology
17.
Sheng Wu Gong Cheng Xue Bao ; 20(1): 30-3, 2004 Jan.
Article in Chinese | MEDLINE | ID: mdl-16108485

ABSTRACT

Human telomeric repeat binding factor 1(TRF1) contains one Myb-type DNA-binding repeat and an amino-terminal acidic domain. It can bind to the duplex array of TTAGGG repeats at chromosome ends and is shown to be important in preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Interestingly, the double strand DNA breaks sensor ATM interacts with and phosphorylates Pin2/TRF1 and inhibits its function after DNA damage. Are there some proteins else that can interact with TRF1 and influence its function? In order to analysis the interaction between TRF1 and other proteins, we must prepare the antiserum that can recognize the endogenous TRF1 of cell lysates. TRF1 cDNA was amplified using cDNA Library of HeLa cell by PCR and cloned into pUCm-T vector. Sequence analysis reveals identity to the GenBank report. The TRF1 cDNA was subcloned into expression vector pET-28c(+) and expressed in E. coli as a fusion protein of 65 kD. The recombinant TRF1 can express in the supernatant (about 12.3% in total protein) on the induction of 0.5 mmol/L IPTG at 37 degrees C for 3 hours. Western-blot analysis showed the recombinant protein can react with TRF1 polyclonal antibody sc-6165 (from Santa Cruz Company). His6-TRF1 was purified by Ni(2+) -NTA resin affinity chromatography made by ourselves and showed to be homogeneity in SDS-PAGE. Rabbits were immunized for four times to prepare polyclonal antibody. The unpurified antiserum can recognize the overexpressed TRF1 with myc-tag and the endogenous Pin2/TRF1 of cell lysate by Western-blot at 1:1000 dilution. At 1:400 dilution, the antiserum can interact with endogenous TRF1 by Immunofluorescence cell staining analysis. The endogenous TRF1 in different cell lines, such as HepG2, 803, MCF7 and HeLa, locates in the nucleus. The soluble expression TRF1 and preparation of its antibody lay the foundation to study it further.


Subject(s)
Antibodies/immunology , Escherichia coli/genetics , Recombinant Fusion Proteins/biosynthesis , Telomeric Repeat Binding Protein 1/genetics , Animals , Cloning, Molecular , HeLa Cells , Humans , Immune Sera/immunology , Rabbits , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Telomeric Repeat Binding Protein 1/immunology
18.
Ai Zheng ; 22(6): 575-8, 2003 Jun.
Article in Chinese | MEDLINE | ID: mdl-12948403

ABSTRACT

BACKGROUND & OBJECTIVE: Specific gene expression plays an important role in cancer gene therapy. Construction of specific expression vector is the basis of cancer gene therapy. This study was conducted to explore the expression specificity of osm (oncostatin M) gene driven by human telomerase reverse transcriptase (hTERT) gene promoter in tumor cells with telomerase activity and to investigate the growth inhibitory capability of expression of osm gene on telomerase-positive tumor cells. METHODS: The authors constructed the expression vector of osm gene afforded by the hTERT promoter and investigated its effect on tumors in vitro using reverse transcription polymerase chain reaction (RT-PCR), transient transfection, and MTT method. RESULTS: Expression of extrinsic osm gene driven by hTERT gene promoter was detected in HepG2 cell with telomerase activity, and not detected in human embryonic lung fibroblast (HEL) cell without telomerase activity. After transfection of phTERT-osm, the proliferation of HepG2, HeLa, Glc, and A549 cells showed significant inhibitory effect, and the inhibitory rate was 12.4-46%. No inhibitory effect appeared in HEL cell. CONCLUSION: The expression of osm gene under the control of hTERT gene promoter can restrict toxic effect to telomerase-positive tumor cells, and alleviate the toxic effect on normal cells without telomerase activity.


Subject(s)
Genetic Therapy , Neoplasms/therapy , Peptides/genetics , Telomerase/genetics , Cell Division , DNA-Binding Proteins , Humans , Neoplasms/pathology , Oncostatin M , Promoter Regions, Genetic , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Simian virus 40/genetics
19.
Article in English | MEDLINE | ID: mdl-12040397

ABSTRACT

To study the function of survivin in cancer cells, survivin cDNA was amplified from HeLa cells by RT-PCR. Then the coding fragment was subcloned into an inducible eukaryotic expression plasmid pHC in reverse direction.The obtained plasmid pHSC was transfected into HeLa cells by Lipofectamine. An inducible cell line which can express antisense RNA of survivin with 2 mmol/L Zn(2 ) was obtained by the selection with 800 nmol/L G418 solution. The expression of antisense RNA of survivin inhibited the expression of survivin protein. Inhibition of survivin expression suppressed the proliferation of HeLa cells and its cell cycle. And it also sensitized HeLa cells to chemotherapeutics.

20.
Article in English | MEDLINE | ID: mdl-12050815

ABSTRACT

A novel protein named apoptin induces a p53-independent, Bcl-2 insensitive type apoptosis in various human tumor cells but not in normal cells. Apoptin is considered to be a promising candidate for safe and effective anti-tumortherapy. Moreover, apoptin may be important for fundamental studies on molecularbasis of apoptosis and cancer. Apoptin gene was subcloned into prokaryotic expression vector pPROEXHT and pBV220, respectively. The apoptin protein was obtained from pPROEXHT-apoptin expression system and not from pBV220-apoptin the former contains a 6xhistidine affinity tag for ease of purification. Expressed protein was purified by chromatography on a co-chelated affinity column and was renatured by dialysis. The renatured apoptin was able to induce purified Hela nuclei apoptosis in vitro even without the participation of the cytoplasm, showing that apoptin expressed in E.coli was active to induce apoptosis.

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