Mouse L1210 leukemia cell line is widely used as a model in the study of tumorigenesis, as well as the efficacy of chemotherapeutic drugs; however, like other suspension cell lines, the mouse L1210 cell line has lowest transfection efficiency, that many barriers exist to study about the structure, function, as well as metabolism in leukemia cells. This study was aimed to obtain higher transfection efficiency of L1210 cell line to facilitate scientific research. The transfection efficiencies of nucleofector and liposome in L1210 leukemia cells were detected by converted fluorescence microscopy and flow cytometry using EGFP (enhance green fluorescent protein); cell viability was observed by trypan blue exclusion test. The results showed that the transfection efficiency of nucleofector primarily through reporter gene pEGFP by Amaxa Nucleofector(TM) nuclear transfer apparatus was significantly higher than lipofectamine 2000 transfection, furthermore, in the same cell density (2 × 10(6)/ml) and plasmid content (10 µg), the transfection efficiency of nuclear transfer apparatus default mode A-20 was higher than that of other modes (S-18, T-20). Its survival rate was up to 50.5% after 24 hours. Cell viability of liposome transfection reached to 88% after 24 hours, but the transfection efficiency was lower (< 1%). It is concluded that the nuclear transfer apparatus A-20 transfected L1210 can reach higher transfection efficiency up to 61.6%, which is significantly higher than that of lipofectamine transfection. The survival rate is up to 50.5% well meeting the needs of scientific research. Higher transfection efficiency is helpful for in-depth research about the morphology, functions and pathogenesis in leukemia model L1210, and provides more searching space for the treatment of leukemia diseases.
Animals ; Cell Line, Tumor ; Cell Nucleus ; genetics ; Cell Survival ; genetics ; Genes, Reporter ; Green Fluorescent Proteins ; genetics ; Liposomes ; Mice ; Transfection ; methods

In order to assess whether gene transfection could be mediated by ultrasound in association with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irradiated by ultrasound at 1 MHz, 0.4-2.0 W/cm(2) and 50% duty cycle with plasmid encoding enhanced green fluorescent protein (EGFP) as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal transfection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm(2) for 30 s. The transfection efficacy in ulstrasound+P85 group was three times higher than in single ultrasound group [(17.63+/-1.07)% vs (5.57+/-0.56)%, P<0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound+P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm(2) for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.
Cell Survival/genetics ; Green Fluorescent Proteins/genetics ; Hep G2 Cells ; Poloxalene/*pharmacology ; Transfection ; Ultrasonics

In order to study the physicochemical characteristics of cytosolic PrP (CytoPrP) and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K (PK) digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.
Cell Line, Tumor ; Cell Survival ; Cytosol ; chemistry ; Endopeptidase K ; pharmacology ; Humans ; Prions ; genetics ; physiology ; Transfection

OBJECTIVEThe present study was conducted to understand the effects of PrP in different octapeptide repeats on proliferation of HeLa cells.

METHODS AND RESULTSMutant PrPs with octapeptide repeat insertion were transiently expressed in HeLa cells and their results of MTT assay showed stronger cytotoxic effect on the proliferation of cells than wild-type PrP. Annexin V/PI assay also demonstrated that the expression of mutant PrPs was much easier to induce apoptosis than wild-type in HeLa cells. The percentage of both early and late stage apoptosis in mutant groups were significantly higher than that of wild type.

CONCLUSIONThese data suggest that the expression of mutant PrPs associated with familial CJD is much easier to induce apoptosis in cultured cells than expression of wild type PrP.

Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Proliferation ; Cell Survival ; genetics ; physiology ; Colorimetry ; HeLa Cells ; Humans ; Mutation ; Oligopeptides ; genetics ; Plasmids ; genetics ; Prion Proteins ; Prions ; genetics ; metabolism ; physiology ; Transfection

OBJECTIVETo investigate the feasibility of life span extension of transformed human embryonic tendon cells (THETC) by reconstitution of the telomerase activity.

METHODSTHETC were transfected by pGRN145 plasmid containing the human telomerase reverse transcriptase (hTERT) cNDA in vitro by molecular cloning technique. The biological characteristics of transfected cells were detected and compared by morphological observation, plate cloning efficiency, soft agar culture, growth curve of cells cultured in different conditions, immunohistochemistry, telomerase activity assay by telomeric repeat amplification protocol (TRAP).

RESULTSThe THETC transfected by pGRN145 plasmid (telT) could express the telomerase activity with extension of life span. The telT maintained the original characteristics of temperature-dependant and serum-dependant, as well as secretion of type I collagen normally and without tendency of malignant transformation.

CONCLUSIONSThe life span of THETC can be prolonged by reconstitution of telomerase activity, which provides the novel experimental methods to establish the standard cells line.

Cell Line ; Cell Survival ; Embryo, Mammalian ; Humans ; Plasmids ; genetics ; RNA-Directed DNA Polymerase ; Telomerase ; genetics ; metabolism ; Tendons ; cytology ; enzymology ; Transfection

BACKGROUNDAstrocyte elevated gene-1 (AEG-1), primarily identified as a late response gene induced by HIV-1 infection, plays multiple roles in the process of oncogenesis. This novel gene has been demonstrated to be involved in the several potent carcinogenic pathways, including PI3K/Akt pathway, nuclear factor (NF)-κB pathway, and Wnt/κ-catenin pathway. Although the function of AEG-1 has been intensively investigated in recent years, the molecular mechanism underlying its oncogenic role is largely unknown. The aim of this research was to explore the potential function of AEG-1 in breast cancer development and progression.

METHODSAEG-1 was ectopically overexpressed in breast cancer MCF-7 cells and its biological effects on the proliferation and invasion of MCF-7 cells were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and invasion assays. The expression of HER2/neu, a crucial oncogene involving in breast cancer carcinogenesis, was also determined.

RESULTSOverexpression of the AEG-1 promoted the proliferation and invasion ability of breast cancer cells, and upregulated the expression of HER2/neu, a crucial oncogene involving in breast cancer carcinogenesis.

CONCLUSIONAEG-1 might facilitate the proliferation and invasion of breast cancer cells by upregulating HER2/neu expression, which provides a potential target for breast cancer therapy.

Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; Cell Adhesion Molecules ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; genetics ; physiology ; Humans ; Neoplasm Invasiveness ; genetics ; Real-Time Polymerase Chain Reaction ; Receptor, ErbB-2 ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology

BACKGROUNDCalreticulin (CRT) is major Ca 2+ -binding chaperone mainly resident in the endoplasmic reticulum (ER) lumen. Recently, it has been shown that non-ER CRT regulates a wide array of cellular responses. We previously found that CRT was up-regulated during hypoxia/reoxygenation (H/R) and this study was aimed to investigate whether CRT nuclear translocation aggravates ER stress (ERS)-associated apoptosis during H/R injury in neonatal rat cardiomyocytes.

METHODSApoptosis rate and lactate dehydrogenase (LDH) leakage in culture medium were measured as indices of cell injury. Immunofluorescence staining showed the morphological changes of ER and intracellular translocation of CRT. Western blotting or reverse transcription polymerase chain reaction was used to detect the expression of target molecules.

RESULTSCompared with control, H/R increased apoptosis rate and LDH activity. The ER became condensed and bubbled, and CRT translocated to the nucleus. Western blotting showed up-regulation of CRT, Nrf2, activating transcription factor 4 (ATF4), CHOP and caspase-12 expression after H/R. Exogenous CRT overexpression induced by plasmid transfection before H/R increased cell apoptosis, LDH leakage, ER disorder, CRT nuclear translocation and the expression of ERS-associated molecules. However, administration of the ERS inhibitor, taurine, or CRT siRNA alleviated cell injury, ER disorder, and inhibited ERS-associated apoptosis.

CONCLUSIONSOur results indicated that during H/R stress, CRT translocation increases cell apoptosis and LDH leakage, aggravates ER disorder, up-regulates expression of nuclear transcription factors, Nrf2 and ATF4, and activates ERS-associated apoptosis.

Animals ; Apoptosis ; genetics ; physiology ; Calreticulin ; genetics ; metabolism ; Cell Hypoxia ; genetics ; physiology ; Cell Survival ; genetics ; physiology ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Myocytes, Cardiac ; cytology ; metabolism ; RNA Interference ; Rats

OBJECTIVETo investigate the effect of a sequence-specific small interfering RNA (siRNA) in suppressing survivin expression and cell proliferation and inducing apoptosis of PC-2 cells.

METHODSThe plasmid expression vector of siRNA targeted against survivin was constructed and transfected into PC-2 cells with Lipofectamine 2000. The changes of survivin expression were detected by semi-quantitative RT-PCR and immunohistochemical SP methods. The effect of siRNA in suppressing the proliferation of PC-2 cells was detected by MTT assay, and its role in inducing PC-2 cell apoptosis evaluated by flow cytometry.

RESULTSThe sequence-specific siRNA effectively suppressed survivin expression at both mRNA and protein levels with inhibition rate of 81.25% at mRNA level and 74.24% at protein level. Survivin expression suppression significantly inhibited the proliferation of PC-2 cells, and at 24 and 48 h after cell seeding, the proliferation inhibition rate was 28.00% and 33.38% respectively; 24, 48 h after the transfection, apoptosis occurred in 8.46% and 7.53% of the cells, respectively.

CONCLUSIONSThe plasmid expression vector for the siRNA against survivin constructed in the study can effectively and specifically suppress survivin expression in PC-2 cells, and blocking survivin expression suppresses PC-2 cell proliferation and induces cell apoptosis. siRNA targeted against survivin has a potential value in gene therapy for pancreatic cancer.

Apoptosis ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Pancreatic Neoplasms ; genetics ; pathology ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVETo construct a recombinant retrovirus vector expressing small interfering RNA (siRNA) targeting human telomerase reverse transcriptase (hTERT), and assess its effect on proliferation and apoptosis of human hepatocellular carcinoma cells.

METHODSThe sequence of the siRNA targeting hTERT, U6 promoter and EGFP gene were amplified by PCR and inserted into the mammalian retroviral expression vector pLXSN to construct the recombinant retroviral vector pLXSN-EGFP-U6-siTERT. The vector was then used to infect human hepatocellular carcinoma cell HepG2. The telomerase activity of the infected cells was detected by telomerase repeat amplification protocol-silver staining, and the cell apoptosis was examined using flow cytometry. The inhibition rate of HepG2 cell proliferation was analyzed by MTT assay.

RESULTSSequence analysis and restriction enzyme digestion showed confirmed successful construction of the recombinant expression vector pLXSN-EGFP-U6-siTERT. The telomerase activity of the infected HepG2 cells was reduced by 23.84%, 58.03% and 85.01% at 24, 48 and 72 h after the infection, respectively (P<0.05). The cell apoptosis rate of the infected cells was 29.05% at 24 h after the infection. The cell proliferation was markedly inhibited by the infection with the vector in comparison to that of the control group.

CONCLUSIONhTERT siRNA can effectively silence hTERT gene and suppress the telomerase activity and proliferation of HepG2 cells.

Apoptosis ; Carcinoma, Hepatocellular ; enzymology ; genetics ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Genetic Vectors ; genetics ; Humans ; Liver Neoplasms ; enzymology ; genetics ; pathology ; RNA Interference ; RNA, Small Interfering ; genetics ; Retroviridae ; genetics ; Telomerase ; genetics ; metabolism ; Transfection

OBJECTIVETo establish an immortalized ameloblastoma cell line.

METHODSThe primary cultured ameloblastoma cells were transfected with pRSV-Tag using Transfect AMINE kit. Tansfected cells were passaged to pass through crisis period and immortalize.

RESULTSCultured ameloblastoma cells were composed predominantly of closely packed small polygonal cells with epithelial morphology. They had limited life-span of 51 days in vitro. The small polygonal cells were eventually replaced by large flattened cells and subsequently became senescent and dead. On the other side, those tumor cells transfected with SV40Tag could live for a longer time. The majority of them died in crisis period while the survived cells from crisis period gained the ability to proliferate. There was no morphological change in TAM-1 compared with original cultured cells. A cell clone was harvested which was alive and keeping on proliferating after having been subcultured for 25 times. It was named TAM-1. The epithelial origin of TAM-1 was confirmed by strong immunoreactivity for cytokeratin in contrast to negative vimentin expression. It was detected that SV40Tag had been transfected into TAM-1 genesome and expressed continuously by PCR and RT-PCR.

CONCLUSIONSTAM-1 is immortalized ameloblastoma cell line in vitro.

Ameloblastoma ; genetics ; metabolism ; pathology ; Antigens, Polyomavirus Transforming ; genetics ; Cell Division ; genetics ; Cell Line, Transformed ; Cell Survival ; genetics ; Female ; Humans ; Immunohistochemistry ; Jaw Neoplasms ; genetics ; metabolism ; pathology ; Keratins ; analysis ; Plasmids ; genetics ; Time Factors ; Transfection ; Tumor Cells, Cultured ; Vimentin ; analysis