The aim of this study is to establish stable transfected cell lines which could produce SPAG4L protein labeled with Myc and His tags in vitro. The open reading frame (ORF) of human SPAG4L was amplified by PCR and the fragments were cloned into eukaryotic expression vector pcDNA3.1/myc-His(-)A. The recombined plasmids of pcDNA3.1/myc-His(-)A/SPAG4L were verified by sequencing and digestion with enzymes. Then, the recombined plasmids were introduced into HeLa cells and screened by G418. Western blotting was performed to detect the expression of SPAG4L and its tags in stable transfected cell lines. SPAG4L and its tags were expressed in the stable cell lines transfected with pcDNA3.1/myc-His(-)A/SPAG4L, but not in the control group. Further study showed that SPAG4L colocalized with the endoplasmic reticulum(ER) marker PDI by immunofluorescence. The stable transfected cell lines established in this study will provide a powerful tool for further studies such as co-immunoprecipitation and pull-down.
Carrier Proteins ; biosynthesis ; genetics ; Cloning, Molecular ; Female ; Genetic Vectors ; genetics ; HeLa Cells ; Histidine ; genetics ; Humans ; Male ; Proto-Oncogene Proteins c-myc ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Transfection

In order to construct a eukaryotic expression vector of bovine c-myc gene, the coding sequence (CDS) of c-myc gene was amplified from bovine primordial genital ridges by RT-PCR. The CDS was subcloned into pMD19-T vector, and then inserted into vector pIRES2-AcGFP1-Nuc. After confirmed by restriction enzyme digestion and sequencing, the recombined plasmid was transfected into skin fibroblast cells. RT-PCR and Western Blotting were used to detect the expression of c-myc mRNA and protein, respectively. The results show that the complete CDS of c-myc gene was cloned from fetal bovine primordial genital ridges. The eukaryotic expression vector of bovine c-myc gene was constructed and efficiently expressed in the skin fibroblast cells. The present study will lay a good foundation for further study of c-myc gene function and bovine induced pluripotent stem cells from somatic cells by defined factors.
Animals ; Cattle ; Cloning, Molecular ; Fetus ; cytology ; Fibroblasts ; cytology ; Genetic Vectors ; genetics ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Skin ; cytology ; Transfection

The expression of a soluble protein on cell surface is often desirable for study of a functional protein, wide application of a protein or investigation of protein-protein interaction. The expression of a soluble protein on the surface of a cell is often achieved by genetically linking a protein to the extra-cellular fragment of a transmembrane partner. In this study, the myc epitope was linked with N terminal of transmembrane proteins either A2TM or deltaLNGFR amplified by overlapping PCR. The plasmids expressing fusion protein were transfected into 293FT cells and the expression of target proteins was evaluated by fluorescent microscope, flow cytometry and Western blotting. The results of flow cytometry revealed that both A2TM and deltaLNGFR were expressed on the cell surface, but A2TM could only be detected with high copy number. Western blotting showed that the expression level of deltaLNGFR was very high and protein was heavily glycosylated, by contrast the expression of A2TM was hardly detected. The results indicate that glycosylated deltaLNGFR is a good candidate partner for the expression of a soluble protein on the cell surface.
Apoptosis Regulatory Proteins ; biosynthesis ; genetics ; Cell Membrane ; metabolism ; Genes, MHC Class II ; genetics ; HLA-A2 Antigen ; biosynthesis ; genetics ; Humans ; Membrane Fusion Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism

OBJECTIVETo investigate the protective effects of ginkgolides on glucose deprivation-induced apoptosis in PC12 cells and the mechanism underlying the protective effect.

METHODPC12 cells were treated under glucose deprivation, and the proliferation was determined by tetrazolium (MTT) assay. Furthermore, the mRNA levels of Bcl-2, Bax, c-myc were measured by Fluorescence Quantitative PCR (FQ-PCR).

RESULTGinkgolides could markedly inhibit the injury of glucose deprivation on the PC12 cells and increase the cell proliferation compared with the model groups (P <0.01). Ginkgolides could up-regulate Bcl-2 and down-regulate Bax and c-myc at 12 h, respectively. There were no significant differences in the Bcl-2 and Bax levels in both groups at 24 h, and ginkgolides only reduced the elevation of c-myc from 4. 32-fold to 2. 87-fold at this time.

CONCLUSIONGinkgolides are able to protect the injured PC12 cells against cell apoptosis. During the early period of glucose deprivation, Bcl-2, Bax and c-myc were regulated to inhibit cell apoptosis by ginkgolides. After that, ginkgolides seems inhibit the apoptosis through attenuating the elevation of c-myc.

Animals ; Apoptosis ; drug effects ; genetics ; Apoptosis Regulatory Proteins ; genetics ; Cell Proliferation ; drug effects ; Fluorescence ; Gene Expression Regulation ; drug effects ; Ginkgolides ; pharmacology ; Glucose ; deficiency ; pharmacology ; PC12 Cells ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; bcl-2-Associated X Protein ; genetics

OBJECTIVETo investigate the expressions of human telomerase reverse transcriptase (h-TERT), c-myc, and proliferating cell nuclear antigen (PCNA) in chronic viral hepatitis (CVH), liver cirrhosis and primary hepatocellular carcinoma (HCC) and understand their possible role in liver carcinogenesis.

METHODSTotally 157 liver disease specimens were collected, including 56 CVH, 52 liver cirrhosis and 49 primary HCC specimens. In situ hybridization was performed on these specimens to examine the expressions of h-TRET and c-myc mRNA, and immunohistochemistry carried out for PCNA detection, with the cell apoptosis detected with in situ ending labeling.

RESULTSIn the CVH, liver cirrhosis and primary HCC specimens, h-TERT expression was detected at the frequencies of 11/56 (19.6%), 43/52 (82.7%) and 44/47 (93.6%), c-myc expression at 7/56 (12.5%), 21/52 (40.4%) and 26/47 (55.3%), with apoptotic index of (27.3-/+4.7)%, (16.5-/+2.6)% and (8.7-/+1.3)% and PCNA expression rate of (17.1-/+2.9)%, (49.3-/+7.8)% and (62.5-/+9.1)%, respectively. Correlations among h-TERT, c-myc, and PCNA expressions and the apoptotic index were not found in the examined tissues (P>0.05).

CONCLUSIONLiver carcinogenesis may involve increased h-TERT, c-myc, and PCNA expressions and suppressed cell apoptosis.

Adult ; Apoptosis ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Cell Transformation, Neoplastic ; Female ; Hepatitis B, Chronic ; genetics ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Liver Cirrhosis ; genetics ; metabolism ; pathology ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Proliferating Cell Nuclear Antigen ; biosynthesis ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; genetics ; metabolism ; Telomerase ; genetics

OBJECTIVETo study the function of c-myc and K-ras in tumorigenesis of ovarian cancer.

METHODSK-ras and/or c-myc cDNAs were introduced into mouse ovarian surface epithelium cells (MOSE) using recombinant Moloney retroviral vectors. The resulting MOSE cells were studied by cell proliferation assays, the ability to form colonies in soft agarose, matrigel invasion assays and tumorigenicity assays in nude mice.

RESULTSK-ras and c-myc can be easily delivered to the normal MOSE cells by recombinant retroviruses. mRNA and protein of the target genes can be detected by RT-PCR and Western blot. Cell proliferation assays showed that MOSE-Ras cells and MOSE-RM cells (MOSE-Ras/Myc) grew more rapidly than parental cells (MOSE) and MOSE-Myc cells (P <0.01). In addtition, MOSE-RM cells grew more rapidly than MOSE-Ras cells (P <0. 05). Cell colony formation assays showed that while MOSE-Ras and MOSE-RM cells can form colonies in soft-agarose, the MOSE-Myc and MOSE cells did not. Matrigel invasion assays showed that MOSE-Ras and MOSE-RM cells have invasion ability, but not MOSE-Myc ascites and the control MOSE cells. Xenograft experiments showed that MOSE-Ras and MOSE-RM cells were able to form tumors in nude mice following intraperitoneal injection. Tumors were not observed in animals injected with either MOSE-Myc or MOSE cells.

CONCLUSIONThe recombinant Moloney retroviral system is a highly efficient and convenient method for introducing and expressing foreign genes in murine surface epithelial cell cultures. In this model, expression of K-ras alone is sufficient to generate tumorigenic MOSE, however expression of c-myc in conjunction with K-ras results in cells with a higher index of malignancy. Based on the assays described in this report, expression of c-myc alone can not transform MOSE cultures although it does play a role in cooperation with K-ras.

Animals ; Blotting, Western ; Cell Movement ; Cell Proliferation ; Cell Transformation, Neoplastic ; Cells, Cultured ; Epithelial Cells ; cytology ; metabolism ; Female ; Immunohistochemistry ; Mice ; Mice, Nude ; Mice, Transgenic ; Neoplasms, Experimental ; genetics ; metabolism ; pathology ; Oncogene Protein p21(ras) ; biosynthesis ; genetics ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Ovary ; cytology ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; Retroviridae ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

A 15-mer phosphorothioate antisense oligonucleotide (ASON) complementary to the translation start region of the C-myc oncogene mRNA was labeled with 131I or 125I and the labelled compound was linked to the vasoactive intestinal peptide (VIP) to be bound covalently to a polylysine chain so as to deliver oligonucleotide into tumor cells. The effect of the VIP as carrier on cell uptake of ASON in tissue culture was evaluated in a human colon adenocarcinoma HT29 cell line. The efficacy of VIP-131-ASON on cell growth was evaluated using the MTT assay. Expression of c-myc-encoded protein was measured by flow cytometry. Sense and nosense control Oligonucleotides with VIP carrier were used as control. The results showed that VIP competed effectively with VIP-125I-ASON to bind the HT29 cells. Cell uptake was increased 3-4 fold using the VIP carrier compared to the same dosage of naked DNA. HT29 cells treated with VIP-131I-ASON complexes exhibited 4-fold lower proliferation than those treated with 13I-ASON and 6-fold lower proliferation than those treated with radioiodinated Sense and nosense DNA. Cancer protein expression of HT29 cells treated with VIP-131I-ASON was decreased 2-fold compare with that in 131I-ASON treated cell. The use of a VIP carrier greatly increased 131I-ASON cellular uptake and inhibition of cell proliferation and C-myc cancer protein expressing in HT29 cell by radioiodinated antisense Oligonucleotides.
Adenocarcinoma ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; Drug Carriers ; Humans ; Iodine Radioisotopes ; Isotope Labeling ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Vasoactive Intestinal Peptide

Carcinoma, Hepatocellular ; pathology ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; pathology ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Transfection ; Tumor Cells, Cultured ; Xeroderma Pigmentosum ; genetics

Aldehydes ; pharmacology ; Animals ; Anthracenes ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Hepatocytes ; cytology ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Rats

OBJECTIVE: To study the apoptosis of retina and the expression of c-myc protein in form-deprivation myopia. METHODS: Two-day-old chickens were sutured with right eyelid for 4, 8 and 12 weeks. After measurement of refracation, the eyeballs were observed by light microscope and taken photos. Retinal apoptotic cells were measured by TUNEL staining and flow cytometry. C-myc protein were examined by immunohistochemistry and flow cytometry. RESULTS: Lacquer crack lesions were found in sutured eyes at 12 weeks. Apoptotic cells were observed in retinal outer and inner nuclear layer of the sutured eyes at 12 weeks and obvious peak of apoptosis was observed in sutured eyes at 12 weeks. The expression of c-myc protein was significantly more than control eyes at 8 and 12 weeks. CONCLUSION The apoptosis of retinal was present in form-deprivation myopia with the degeneration of retina. C-myc protein plays an important role in retinal apoptosis of myopia.
Animals ; Animals, Newborn ; Apoptosis ; physiology ; Chickens ; Flow Cytometry ; Myopia ; metabolism ; pathology ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Random Allocation ; Retina ; metabolism ; pathology