OBJECTIVE: To analyze the expression and location of coding protein of UBAP1 gene and to understand the relationship between the expression pattern of the protein and cell carcinogenesis. METHODS: Bioinformatics was used to analyze the protein character to provide an available clue of subsequent research. The codon frame cDNA was amplified by PCR, and subcloned into enhance green fluorescence protein (EGFP) of pEGFP-C2. The recombinant plasmid was transfected into HNE1 cells. The expression of coding protein was observed by fluorescence microscopy. RESULTS: The expressed GFP-fusion protein generated striking green fluorescence in the cytoplasm in HNE1 cells. EGFP/UBAP1 was expressed and existed mainly in the nuclear, especially accumulated on the nuclear envelope. CONCLUSION The expression difference in HNE1 might be related to the carcinogenesis of NPC.
Base Sequence ; Carrier Proteins ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Molecular Sequence Data ; Nasopharyngeal Neoplasms ; metabolism ; Neoplasm Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo study the expressions of survivin, PTEN and their relationships with tissue grade and pathology stage in prostatic carcinoma (PCa).

METHODSThe immunohistological staining was used to evaluated the expressions of survivin protein and PTEN in 43 case of prostatic carcinoma (PCa) and 5 cases of benign prostatic hyperplasia (BPH).

RESULTSThe positive rate of survivin protein was 81.40%. Expression of survivin protein in 5 case of BPH was negative. The positive rate of PTEN was 30.23%, and the higher the grade and the clinical stage of tumors were, the lower the expression of PTEN was, PTEN of 5 case of BPH was positive.

CONCLUSIONThe positively correlation was found between the abnormal expressions of survivin protein, PTEN and the biological behavior of prostatic carcinoma (PCa). Detection of survivin combined with PTEN is valuable for diagnosing PCa, and evaluating malignancy extent and prognosis.

Aged ; Humans ; Immunohistochemistry ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; biosynthesis ; Middle Aged ; Neoplasm Proteins ; biosynthesis ; PTEN Phosphohydrolase ; biosynthesis ; Prognosis ; Prostatic Neoplasms ; metabolism ; pathology

OBJECTIVETo construct the recombinant plasmid pcDNA3.0-RGC32 and evaluate the effect of the response gene to complement-32 (RGC32) on cell cytoskeleton in vitro.

METHODSThe full-length cDNA of RGC32 was obtained by RT-PCR and inserted into the eukaryotic expression vector pcDNA3.0 to generate the recombinant plasmid pcDNA3.0-RGC32. After transfection of the recombinant plasmid into SW480 cells, the expression of RGC32 in the cells was detected by Western blotting. The cytoskeleton of SW480 cells was visualized before and after the transfection, and the changes in the cell migration ability was assessed by wound-healing assay.

RESULTSThe recombinant plasmid pcDNA3.0-RGC32 was successfully constructed. The expression of RGC32 was significantly increased in SW480 cells after transfection with pcDNA3.0-RGC32. Before the transfection, the microfilaments of SW480 cells were few and short without obvious polarity, but after the transfection, the microfilaments were increased and elongated with also an obvious polarity, and the invasive structures of lamellae and lamellipodia occurred. The migration ability of the cells was enhanced after transfection with pcDNA3.0-RGC32.

CONCLUSIONOverexpression of RGC32 can cause the reorganization of cytoskeleton and promotes the cell migration, which can be an important mechanism of RGC32 in promoting cancer metastasis.

Cell Cycle Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; Cell Movement ; Colorectal Neoplasms ; genetics ; metabolism ; pathology ; Cytoskeleton ; chemistry ; metabolism ; Genetic Vectors ; Humans ; Muscle Proteins ; biosynthesis ; genetics ; Neoplasm Metastasis ; genetics ; Nerve Tissue Proteins ; biosynthesis ; genetics ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

AIMTo characterize the coexpression of survivin, an inhibitor of apoptosis (IAF), and human telomerase reverse transcriptase (hTERT) in human testes with varying spermatogenic function.

METHODSTranscript levels of survivin mRNA and hTERT mRNA were determined in normal testes (n=11) and testes with defective spermatogenesis (n=28) using real-time reverse-transcription polymerase chain reaction (RT-PCR). The histological work-up was performed according to a modified Johnsen score.

RESULTSExpressions of both survivin and hTERT were highest at median levels of 96.8 and 709 in normal spermatogenesis and dropped to 53.3 and 534 in testes with postmeiotic spermatogenic arrest (n=10). In severe spermatogenic failure (n=18), survivin expression was lacking in most specimens (n=16), whereas at least low levels of testicular hTERT expression were largely detectable with a normalized expression of 73 in premeiotic spermatogenic arrest (n=7) and 45 in patients with Sertoli cell-only syndrome (SCOS) (n=3). Both survivin and hTERT expressions increased with a progressing Johnsen score (P for trend=0.001).

CONCLUSIONAlthough both survivin and hTERT are correlated with spermatogenic function, they show different expression patterns in testes of infertile patients. These findings substantiate results from studies in the rodent testis suggesting a predominant expression of survivin in meiotically dividing germ cells.

Biopsy ; DNA-Binding Proteins ; biosynthesis ; Gene Expression ; physiology ; Humans ; Infertility, Male ; metabolism ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; biosynthesis ; Neoplasm Proteins ; biosynthesis ; Spermatogenesis ; physiology ; Telomerase ; biosynthesis ; Testis ; metabolism

OBJECTIVETo investigate the expression of cyclooxygenase-2 (COX-2), human mut-l homologue 1 (hMLH1) and human mut-s homologue 2 (hMSH2) proteins in human paired gastric carcinoma (GC) and adjacent normal mucosa, and analyze their relationship with microsatellite instability (MSI).

METHODSThe protein expressions were examined by western blotting. Five MSI loci were assessed by PCR.

RESULTSIn 30 surgically excised GC tissues, the overexpression rate of COX-2, the low expression rate of hMLH1 and hMSH2 were 66.7%, 40% and 33.3%, respectively. Significant differences were found when compared with those of adjacent normal mucosa (P < 0.05). MSI was detected in 13 GC. The number of MSI-H (MSI-High, > or = 2 loci), MSI-L (MSI-Low, only one locus), and MSS (microsatellite stable) were 9, 4 and 17, respectively. The number of low expression rates of COX-2, hMLH1 and hMSH2 in MSI-H were 6, 8 and 5, respectively. There were significant differences compared to that of MSS (P < 0.05).

CONCLUSIONThe results suggest that microsatellite instability pathway is probably involved in the carcinogenesis of gastric carcinoma, which is frequently accompanied by low expression of hMLH1 and hMSH2, and may be also by low expression of COX-2.

Adaptor Proteins, Signal Transducing ; biosynthesis ; genetics ; Cyclooxygenase 2 ; biosynthesis ; genetics ; Humans ; Microsatellite Repeats ; genetics ; MutL Protein Homolog 1 ; MutL Proteins ; Neoplasm Proteins ; biosynthesis ; genetics ; Nuclear Proteins ; biosynthesis ; genetics ; Stomach Neoplasms ; genetics ; metabolism

OBJECTIVE: To explore the down-expression mechanism of MYETS1 gene in multiple myeloma cell lines ARH-77 or KM3, and express MYETS1 gene in prokaryotic express system. METHODS: The region of chromosome 13q14.3 in ARH-77 and KM3 was detected by FISH. MYETS1 gene was amplified by RT-PCR and cloned into prokaryotic expression vector pGEX-4T. RESULTS: Positive consequence was acquired in 13q14.3 where MYETS1 located by FISH in ARH- 77 and KM3 cell lines. Bioinformatics indicated highly sequence homology between MYETS1 and LECT1, but excluded the homology of open reading frame between MYETS1 and that of LECT1 by RT-PCR. Myets1 protein was expressed and harvested successfully. CONCLUSION The region of chromosome 13q14.3 ,where MYETS1 gene located, was not defected in ARH-77 and KM3 cell lines. Down-expression of MYETS1 might be regulated by other mechanisms in multiple myeloma cell lines.
Cell Line, Tumor ; Chromosomes, Human, Pair 13 ; genetics ; Gene Deletion ; Genetic Vectors ; genetics ; Humans ; Intercellular Signaling Peptides and Proteins ; biosynthesis ; genetics ; Membrane Proteins ; biosynthesis ; genetics ; Multiple Myeloma ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

ATR-Fc is a fusion protein consisting of extracellular domain of human anthrax toxin receptor (ATR) and a fragment (hinge, CH2, and CH3 domains) of the Fc of human IgG1. The aim of ATR-Fc expression is to get an antibody-like molecule binding to protective antigen (PA), a component of anthrax toxins, this fusion protein may compete with cell surface receptor for PA binding, and block the transport of lethal factor (LF) and edema factor (EF) into cells, thereby act as an antitoxin to prevent and treat anthrax infection. A DNA fragment encoding N-terminal amino acids 1-227 of ATR and human IgG1 Fc was inserted into the Hind III and Not I sites of pcDNA3.1 to generate the eukaryotic vector pcDNA3.1/ATR-Fc for expression of ATR-Fc fusion protein. Using lipofectine-mediated gene transfer technique, pcDNA3.1/ATR-Fc was transfected into CHO-K1 cells. After selected with G418, a recombinant CHO cell line, ATR-Fc-1D5, whose expression level was about 10 - 15 microg/(10(6) cells x d), was established. The recombinant protein expressed by the ATR-Fc-1D5 cells was purified with protein A chromatography. The experimental results demonstrated a direct and specific interaction between ATR-Fc and PA assessed by ELISA.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Receptors, Cell Surface ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

Recently, the immunotherapy has been highlighted among cancer treatments. Cancer-testis antigen (CTA) has been studied in a variety of solid tumors because of its specific expression in tumors, and testis, ovary and placenta tissues, but not in other normal tissues. In order to provide a new approach for multiple myeloma (MM) immunotherapy, we examined the CTA expression in MM cell lines, and primary myeloma cells in patients with MM. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of MAGE-C1/CT7, SSX1, SSX2 and SSX4 in MM cell lines of RPMI-8226 and U266, and bone marrow (BM) cells of 25 MM patients and 18 healthy volunteers. The results showed that the 4 CTAs were expressed in RPMI-8226 and U266 cell lines. The positive expression rate of MAGE-C1/CT7, SSX1, SSX2 and SSX4 in the BM cells of 25 MM patients was 28% (7/25), 80% (20/25), 40% (10/25) and 68% (17/25), respectively. In contrast, the expression of any member of the CTAs was not detected in BM cells of 18 healthy volunteers. The expression of two or more CTAs was detected in 80% (20/25) MM patients, and that of at least one CTA in 88% (22/25). The mRNA expression levels of SSX1 and SSX4 were significantly higher in patients with MM at stage III than in those at stage I and II (P<0.05). No statistically significant differences were observed in the mRNA expression levels of MAGE-C1/CT7 and SSX2 in further stratified analyses by age, gender, MM types and percentage of MM cells in BM (P>0.05). In conclusion, our present study showed that MAGE-C1/CT7, SSX1, SSX2 and SSX4 were co-expressed in MM cell lines and the primary myeloma cells in MM patients, but not expressed in BM cells of healthy subjects. The mRNA levels of SSX1 and SSX4 are associated with MM clinical stage. This work may provide a new insight into MM immunotherapy in the future.
Adult ; Aged ; Antigens, Neoplasm ; biosynthesis ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Multiple Myeloma ; genetics ; pathology ; Neoplasm Proteins ; biosynthesis ; Neoplasm Staging ; Repressor Proteins ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

Survivin, a new member of the inhibitor of apoptosis protein (IAP) family, expressed in the most human cancers but not in terminally differentiated adult tissues, so survivin may be a target for tumor therapy. In addition, some scholars found that survivin expression is associated with the resistance in chemotherapy. To explore the relationship between survivin and drug-resistance, the alteration of survivin mRNA and protein of HL-60 cells treated with daunomycin (DNR), mitoxantrone (MIT) and arsenic trioxide (As2O3) was investigated, the expressions of survivin mRNA and survivin protein were detected on the first and third day by RT-PCR and Western blot, respectively. The results showed that survivin mRNA levels all decreased after the first day of treatment with drugs. It was decreased by 10% in DNR group, 40% (P < 0.01) in MIT group, and 25% (P < 0.01) in As2O3 group in comparison with control cells. In the third day, the survivin mRNA treated with DNR was up-regulated by 20% (P < 0.05), compared with the first day, and MIT was up-regulated by 65% (P < 0.01), but As2O3 was still down-regulated by 32% (P < 0.01). In Western blot, survivin protein level increased 14% after treated with DNR for three days, compared with the control cells, and 11% in MIT, but decreased by 82% in As2O3. It is concluded that after treatment with chemotherapeutic drugs, the survivin level descended at first day and then ascended obviously. This phenomenon may be associated with the resistance in chemotherapy for leukemia. On the other hand, As2O3 shows a different mechanism that may play a significant role to reverse resistance.
Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Daunorubicin ; pharmacology ; Drug Resistance, Neoplasm ; drug effects ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Mitoxantrone ; pharmacology ; Neoplasm Proteins ; biosynthesis ; genetics ; Oxides ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics

To explore the change of sensitivity to chemotherapy of antisense RNA targeting survivin on hepatocarcinoma carcinoma cells in vitro. Survivin mRNA structure region was amplified by RT-PCR and inserted inversely into eukaryotic expression vector pcDNA3. The antisense expression plasmid pcDNA3/survivin was transfected into HepG2 with lipofectAMINE 2000 (LF2000), with low concentration of 5-fluorouracil (5-Fu) added. Survivin protein was detected by Western-blot, the growth activity was measured by MTT, and apoptosis was detected by Flow Cytometry 12 h, 24 h, 48 h after transfection. The activity of caspase-3 was found by quantitative assay 48 h after transfection. The construction of antisense RNA vector pcDNA3/survivin was verified by restricted endonuclease digestion and nucleotide sequencing. Compared with normal group, 5-Fu and antisense survivin group, the cells growth inhibition, apoptosis index, and caspase-3 activity were increased in antisense survivin transfected + 5-Fu group. The threshold of apoptosis was decreased after survivin was silenced, and the sensitivity to chemotherapy was increased. These findings suggest the existence of a potential new target for gene therapy.
Antigens, Neoplasm ; biosynthesis ; genetics ; Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; Fluorouracil ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; Liver Neoplasms ; metabolism ; pathology ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; Plasmids ; RNA, Messenger ; biosynthesis ; genetics ; Transfection