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PubMed2003

[Feasibility of real-time quantitative PCR in assessing the efficiency of gene transfection in vivo].

To explore feasibility of real-time quantitative PCR in assessing efficiency of gene transfection, clonal PCR was employed to analyze efficiency of retroviral-mediated neo gene transfection in primary myoblast, simultaneous real-time PCR were performed for estimation of transfection efficiency; for measuring integrated gene copy number per cell, linear amplification mediated-PCR (LAM-PCR) and retroviral 5'LTR integration analysis also were used. The results showed that: (1) the data from clonal PCR are similar as that from real-time PCR in low efficiency of transfection (< 36%); but in high efficiency of transfection, it is significantly differentiation between clonal PCR and real-time PCR. (2) One copy of transduced gene per cell was observed in retroviral-mediated gene transfection in primary myoblast. It is concluded that real-time PCR can be used to estimate gene transfer vector in vivo, but it is not available for assessing gene transfection in vitro, because high efficiency of transfection could be obtained in most of gene transfection in vitro.

Animals↗
PubMed2003

[Transfection of the nm23-H1 gene into BcaCD885 cell line inhibits the potential of invasion, adhesion and mobility].

OBJECTIVE: To transfect nm23-H1 into the BcaCD885 cell lines in order to get safe high-efficiency and low-toxicity, and to find out whether nm23-H1 could affect the invasion and metastases ability of BcaCD885 cell lines. METHODS: Lipofect was used to transfect nm23-H1 into BcaCD885 cell lines; immunohistochemistry was used to detect the difference expression of nm23-H1 between transfected and non-transfected cell lines; then transwell-room and wash way were used to detect the difference of invasion and metastases ability between transfected and non-transfected cell lines. RESULTS: PCMV-NEO-BAM system gave the stability expression of nm23-H1; there was significant different NDPKA expression between transfected and non-transfected BcaCD885 cell lines; the invasion and metastases ability of transfected BcaCD885 cell lines decreased obviously. CONCLUSION: nm23-H1 can inhibit the metastases of BcaCD885 cell lines significantly.

Cell Adhesion↗
PubMed1992

Neuromedin B receptors retain functional expression when transfected into BALB 3T3 fibroblasts: analysis of binding, kinetics, stoichiometry, modulation by guanine nucleotide-binding proteins, and signal transduction and comparison with natively expressed receptors.

The receptor that interacts with the mammalian bombesin-related peptide neuromedin B (NMB) is ubiquitous in the gastrointestinal tract and central nervous system. However, little is known regarding its cellular mechanisms of action. This receptor has been recently cloned, sequenced, and stably transfected into BALB 3T3 fibroblasts, permitting detailed study of the pharmacology and coupled biological activities of this receptor. In the present study, we compare the ability of transfected receptors to alter cell function with that of receptors natively expressed in small numbers by the rat glioblastoma cell line C6. NMB inhibited binding of 125I-[D-Tyro]NMB with high affinity in transfected cells (Ki = 3.08 +/- 0.14 nM) and in C6 cells (Ki = 1.90 +/- 1.10 nM), whereas the bombesin-related agonists gastrin-releasing peptide (GRP) and [D-Phe6, D-Ala11, Leu14]bombesin(6-16) (GRP analogue) had 100- and 300-fold lower affinities, respectively, for NMB receptors in either cell type. For both cell systems, maximal binding was observed between 5 and 15 min at 22 degrees. Both cell types internalized NMB at similar rates, with > 70% of bound ligand being internalized by 60 min at 22 degrees. The nonhydrolyzable guanosine analogue guanosine 5'-(beta,gamma-imido)triphosphate was equipotent in causing a decrease in binding of 125I-[D-Tyro]NMB due to decreased receptor affinity in both cell types, without a change in receptor number, demonstrating that the NMB receptor remained coupled to a guanine nucleotide-binding protein in both native and transfected cells. In both cell systems, NMB increased inositol monophosphate, inositol bisphosphate, and inositol trisphosphate in a time-dependent fashion. Inositol phosphates were increased in a dose-dependent fashion, with similar half-maximal values being obtained for NMB in both cell types (transfected, 1.01 +/- 0.09 nM; C6, 2.09 +/- 0.15 nM) and for the GRP analogue (transfected, 1855 +/- 140 nM; C6, 2129 +/- 250 nM). NMB mobilized intracellular Ca2+ in both cell systems, and the dose-response curves were superimposible (EC50 for transfected, 0.10 +/- 0.08 nM; C6, 0.11 +/- 0.02 nM). These data demonstrate that activation of the receptor for NMB stimulates phospholipase C and increases intracellular Ca2+. These results also demonstrate that transfected and native NMB receptors behave similarly, suggesting that the transfected cell line will be useful in future studies investigating ligand-receptor interactions, as well as in molecular biological studies of the structure-function relationship of the receptor.

3T3 Cells↗
PubMed2004

[Effect of fas gene transfection on tumourigenicity and proliferation of transplanted tumor of oral squamous cell carcinoma cell line Tca8113].

OBJECTIVE: To investigate the effect of fas gene transfection and monoclonal anti-fas antibody on tumorigenicity and proliferation of transplanted tumor of Tca8113 cell. METHODS: Plasmid including fas gene was transfected into Tca8113 cell by lipofectamine kit. Some transfected cells were treated by monoclonal anti-fas antibody after 48 hours since transfection. Untransfected cell (control), fas-tansfected cell and fas-transfected cell treated with antibody were transplanted to nude mice subcutaneously. Growth of transplanted tumor was observed and recorded regularly. Animals were sacrificed and tumor samples were harvested at the end of experiment. Fas expression in each neoplasm was assessed by RT-PCR. Apoptosis, proliferation and expression of fas protein in tumor tissue were measured by flow cytometry (FCM). RESULTS: Tumor occurred much later in fas-transfected group and fas-transfected plus antibody treated group. Growth arrest was found in them. RT-PCR and FCM suggested that fas-transfection up-regulated the expression of fas mRNA and protein, increased apoptosis index (AI). But no effect on proliferation index (PI) was observed. Monoclonal anti-fas antibody did not effect the expression of fas mRNA and protein, but increased AI and decreased PI. CONCLUSION: Fas-transfection suppressing tumorigenesis of Tca8113 cell transplanted in nude mice might be caused by up-regulation of expression of fas gene and enhancement of apoptosis. However, anti-fas antibody suppressing tumorigenesis might be associated with activation of apoptosis and repression of proliferation.

Animals↗
PubMed2004

Additive effect of steroids and cholesterol on the liposomal transfection of the breast cancer cell line T-47D.

The efficiency of gene transfer is many times higher in viral than in liposomal transfection. One reason for this is an insufficient intracellular transport of the exogenous DNA into the nucleus in lipofection. Using liposomal transfection techniques for gene therapy is safer than viral approaches, so it would be of great importance to find solutions for their enhancement. We found a 4.51-fold increase in liposomal transfection of T-47D breast cancer cells by the addition of progesterone and a 2.81-fold increase by the addition of cholesterol. The transfection efficiency was measured as the activity of the delivered reporter gene luciferase. The addition of progesterone and cholesterol in combination led to a further enhancement of the transfection efficiency up to 13.72-fold. This additive effect could also be seen when we combined cholesterol with other steroids, but not by the combination of different steroids. All of these steroids alone had also the potential to increase liposomal transfection. Therefore we suggest that steroids and cholesterol enhance liposomal transfection by different mechanisms. Both substances have been shown to shift the exogenous DNA from the cytosol to the nucleus. Steroids normally act through intracellular steroid receptors, which migrate into the nucleus upon activation. The transfected DNA might be co-transported to the nucleus together with the migration of the activated steroid receptors. Even if cholesterol causes also an intracellular shift of DNA to the nucleus, its impact on the fluidity of cellular membranes or on the stability of lipoplexes in serum containing media could be mainly responsible for its effect. If the steroid enhanced liposomal transfection is dependent on the presence of steroid receptors, a specifically-enhanced gene delivery for a subgroup of gynecological tumours expressing high levels of steroid receptors could be possible.

Breast Neoplasms↗
PubMed2003

[Establishment and identification of stable transfection of CHO and COS7 cells with TIF3cDNA].

OBJECTIVE: To establish and identify CHO and COS7 cell lines transfected with TIF3cDNA. METHODS: CHO and COS7 cell lines transfected with TIF3cDNA were established with plasmid (pcDNA3.1/V5-His-TOPO) as the vector, using calcium phosphate inductive transfection techniques and G418 cell selection protocols. Expression of the transfected gene was analyzed and identified using western blotting analysis. The non-transfected group and vector-transfected group were designed to compare with the target gene-transfected group. RESULTS: 3 out of 7 CHO cell lines transfected with TIF3cDNA expressed stably high levels of TIF3 encoded protein. Similarly, 2 out of 4 COS7 cell lines had significant over-expression of TIF3 encoded protein. Conclusion Three CHO and two COS7 cell lines transfected with TIF3cDNA have been successfully established. The cell lines are of great value for the future exploration of Cd compounds related oncogenens and for the studies of the other biological functions of the compounds.

Animals↗
PubMed2004

[Study of osteoblasts transfected with gfp in vitro and traced in vivo].

OBJECTIVE: To observe the osteoblasts transfected with green fluorescent protein (GFP)by adenovirus vector expressed in vitro and traced it in vivo in order to research the feasibility of GFP as a tracer of seeding cells for tissue engineering. METHODS: CFP were transfected into the osteoblasts which derived from adult human bone marrow stromal cell (hBMSc) by adenovirus vector after being packed in 293A cells. The nontransfected hBMSc was used as the control group. The osteoblasts in each group were observed under an inverted phase contrast microscope and fluorescence microscope. The expressive efficiency of GFP was examined by flow cytometry,and alkaline phosphatase (ALP) activities and osteocalcin (OCN) synthesis. After eight days of the transfection,the osteoblasts were implanted into the muscle of nude mouse thigh while the non-transfected osteoblasts were also implanted as a control. Four and eight weeks after the operation, the nude rats were killed and the continuous tissue sections were examined using fluorescence microscopy after adjacent sections were performed by immunohistochemistry or routine HE staining. RESULTS: The green fluorescence was shown the transfected osteoblasts which derived from bone marrow. The rate of positive expression was over 75%. After eight days of the transfection, the marker proteins of the surface of the osteoblasts showed extremely efficient expression of CD29 and CD44, but the CD34 expressed negative. Either ALP or OCN of the osteoblasts was no significant difference between the two groups (P > 0.05) four and eight days after the transfection. The GFP were obviously expressed in nude mouse both at four and eight weeks, meanwhile it did not harm on the morphology and function of the transfected osteoblasts whose immunohistochemistry examination showed positive reaction. CONCLUSIONS: GFP could be transfected osteoblasts effectively in vitro and traced in vivo in nude mouse. It may be an optimal tracer for living cells on tissue engineering research.

Adult↗
PubMed1992

HLA-B27 expression modulates gram-negative bacterial invasion into transfected L cells.

The mechanism by which HLA-B27 confers genetic susceptibility to the seronegative spondyloarthropathies ankylosing spondylitis, Reiter's syndrome, and reactive arthritis, is not well understood. The current concept of an extraarticular bacterial infection functioning as the triggering event in a genetically susceptible host suggests the possibility of direct microbial-MHC interaction. We have addressed the role of HLA-B27 in microbial-host cell interaction by examining invasion by putatively arthritogenic gram-negative bacteria. Target cells used were murine L cells transfected with HLA-B27, HLA-A3, HLA-A2, HLA B44, HLA B18, or pSV2neo vector alone. Relative to the pSV2neo control and the HLA-A3 transfectant, HLA-B27-transfected cells demonstrated a consistent decrease in invasion for each of the following pathogens: Salmonella typhimurium (45 +/- 2% decrease), Shigella sonnei (53 +/- 13% decrease), Shigella flexneri (45 +/- 5% decrease), and enteroinvasive Escherichia coli (57 +/- 8% decrease). This decrease was specific for the HLA B27-transfected L cells and was not observed in the other B allele transfectants. The decreased invasion in the HLA-B27 transfectants is not the result of either altered endogenous mouse class I expression as a result of human class I transfection or increased intracellular bacterial killing within the B27 transfectants. There was an inverse relationship between the amount of surface expression of HLA-B27, as measured by FACS, and the degree of invasion. Blocking of surface B27 Ag with anti-B27 mAb augmented bacterial invasion in the B27 transfectants. These studies demonstrate a novel bacterial-B27 interaction that may have relevance to the pathogenesis of B27-related arthritis.

Animals↗
PubMed2005

[Immunostimulatory effect of human peripheral blood dendritic cells transfected by adeno-associated virus containing Her2/neu gene].

OBJECTIVE: To observe the immunostimulatory effect of human peripheral blood dendritic cells (DCs) transfected by Her2/neu gene delivered by adeno-associated virus. METHODS: The mononuclear cells in healthy donors were isolated by Ficol-Hypaque density gradient separation and divided into transfection group and control group without transfection by the recombinant virus. The cells were initially cultured in RPMI 1640 medium supplemented with 10% AB human serum, followed by addition of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4 and tumor necrosis factor (TNF)-alpha into the medium. The surface markers of DC were detected by flow cytometry, allogeneic mixed lymphocyte reaction assayed by 3H-TdR incorporation and the specific killing activity of T cells evaluated by MTT assay. RESULTS: The expression rates of CD1a, CD86 and CD83 of the transfected DCs were 98.10%, 99.42%, and 84.59%, and those of the non-transfected DCs were 92.69%, 98.07%, and 82.72%, respectively, showing no obvious differences between the two groups. The rates of CD4 and CD80 expression were 61.02% and 97.61%, respectively, in the transfected DCs, significantly higher than those in the non-transfected cells (36.19% and 55.5%). Both groups of DCs stimulated a strong T cell proferation response. The transfected DCs were capable of inducing specific killing of the target tumor cells, with the highest killing rate of (39.7+/-7.2)%. CONCLUSION: The immunostimulatory effect of human peripheral blood DCs can be enhanced by Her2/neu gene transfection mediated by adeno-associated virus.

Cells, Cultured↗
PubMed2005

Barriers to productive transfection of trabecular meshwork cells.

PURPOSE: A critical function of trabecular meshwork cells is to degrade cellular debris, including DNA. We hypothesize that low transfection efficiencies of primary human trabecular meshwork (HTM) cell cultures with plasmid DNA are a function of retained capacity to efficiently degrade exogenous DNA in vitro. METHODS: To determine mechanisms responsible for low transfection efficiencies of cultured HTM cells, steps of DNA entry into cytoplasm and nucleus were characterized. Following synchronization with sequential serum starvation and serum reintroduction, the HTM cell cycle was characterized using 5-bromo-2-deoxyuridine incorporation into replicating DNA. HTM cells were transfected during S-phase with plasmid DNA encoding green fluorescence protein (GFP) or plasmid DNA conjugated with Cy3. In some experiments, cells were treated with a DNase I inhibitor, 100 nM aurintricarboxylic acid. Uptake of plasmid DNA was measured by intracellular fluorescence of Cy3 and productive transfection efficiency was measured by intracellular fluorescence of GFP. RESULTS: HTM cells enter S-phase between 18 and 20 h after synchronization. Plasmid DNA reached the cytosolic compartment in 95% of transfected cells, regardless of synchronization. Synchronization dramatically increased productive transfection efficiency in HTM cells, from 3.0 to 9.0%. DNase I inhibition increased productive transfection efficiency of HTM cells two fold. CONCLUSIONS: Cultured HTM cells have a lower transfection efficiency than other primary ocular cell cultures, likely due partially to cytoplasmic digestion of DNA. We suggest that the difficulties in transfecting cultured HTM cells may be related to the filter function of the cells in vivo where the cells must degrade exogenous DNA.

Adult↗
PubMed1992

Loss of transforming growth factor beta 1 (TGF-beta 1)-induced growth arrest and p34cdc2 regulation in ras-transfected epithelial cells.

Transforming growth factor beta 1 (TGF-beta 1) is a potent inhibitor of mink lung epithelial (CCL64) cell growth in culture. The fact that many transformed epithelial cells have escaped from negative growth control by TGF-beta 1 suggests that transfected epithelial cells may be an appropriate model for investigating the growth-inhibitory mechanism of TGF-beta 1. We transfected CCL64 cells with a mouse c-myc oncogene (pSVc-myc1), a mutated Harvey-ras (Ha-ras) oncogene, or a combination of both. The results indicate that cells transfected with c-myc alone exhibit normal morphology and maintain sensitivity to TGF-beta 1 growth arrest, but are unable to form colonies in soft agar in the presence or absence of TGF-beta 1. Cells transfected with Ha-ras, or co-transfected with c-myc, display a transformed morphology, grow spontaneously under anchorage-independent conditions and acquire a complete resistance to growth inhibition by TGF-beta 1. Affinity cross-linking of [125I]TGF-beta 1 to cell-surface receptors from these transfectants revealed that all three TGF-beta receptor types were present and no significant differences in [125I]TGF-beta 1 labeling of these receptors was observed. Since we have previously demonstrated that modulation of p34cdc2 kinase is a marker for TGF-beta 1 growth inhibition, we investigated p34cdc2 activity in the CCL64 transfected clones. The results show that in the control CCL64 cells and in the myc-transfected clones TGF-beta 1 regulation of p34cdc2 activity is maintained. In the ras- and ras + myc-transfected cells p34cdc2 phosphorylation and histone H1 kinase activity is significantly increased and regulation by TGF-beta 1 is lost.

Animals↗
PubMed2006

[In vitro gene transfection into rabbit articular chondrocytes mediated by recombinant adeno-associated virus vector].

OBJECTIVE: To investigate the in vitro efficiency of recombinant adeno-associated virus (rAAV2) vector-mediated gene transfection into the rabbit articular chondrocytes. METHODS: Articular chondrocytes were isolated from New Zealand rabbits, cultured, and transfected with rAAV2 containing enhanced green fluorescent protein (eGFP) gene at different values multiplicity of infection (MOI). Successful transfection of chondrocytes was confirmed by detection of recombinant enhanced green fluorescent protein using inverted fluorescence microscopy. The transfection efficiency was determined using the fluorescence-activated cell sorter. A maximum level of 4.76% was set as the background autofluorescence in live, uninfected chondrocytes. RESULTS: The percentage of eGFP-positive chondrocytes increased in a vector dose-dependent manner. When the MOI value was <1 x 10(5) v.g./cell the transfection rate was positively linearly correlated with the dose. However, the MOI > 10(5) v.g./cell did not markedly increase the transfection rate. The greatest population of eGFP-expressing cells was 97.7% and the highest mean fluorescence intensity was 17.4 7 days after transfection. EGFP gene expression remained high until 56 days after transfection. CONCLUSION: The in vitro efficiency of rAAV2 vector-mediated eGFP gene transfection into rabbit articular chondrocytes was high. The eGFP expression can be maintained for a rather long time. EGFP gene is an ideal tracer for chondrocytes transduction.

Animals↗
PubMed2006

[Influence of liposome induced gene transfection on the characteristics of human immature dendritic cells].

OBJECTIVE: To investigate the influence of liposome induced gene transfection on the phenotypic characteristics and immune function of human immature dedritic cells (imDC). METHODS: Monocytes were isolated from human cord blood, and they were differentiated into imDC by rhGM-CSF and rhIL-4 induction. Then the morphologic observation and immune phenotypic identification were performed in im-DCs. imDCs were divided into transfection (T, with liposome transfection of pEGFP vector) and control (C, without transfection) groups. The transfection rate and expression of cell maturation marker (CD83, CD86 and HLA-DR) were determined with flow cytometry, and the proliferation of non-sensitized T lymphocyte before and after transfection was determined with allogeneic mixed leukocyte reaction (MLR). RESULTS: im-DC derived from human cord blood cells had typical appearance and surface markers consistent to what reported in the literature. The expression rates of CD86, CD83 and LA-DR in T group were (12 +/- 6) %, (8.6 +/- 2.3) % and (71 +/- 7) %, respectively, which exhibited no difference compared with those in C group (13 +/- 6) %, (9.1 +/- 3.8) % and (72 +/- 8) %, (P > 0.05). MLR results indicated that there was no obvious change in the immune stimulation function of imDC after transfection (P > 0.05) , with stimulation index lower than 2. CONCLUSION: There is no change in maturation of imDC after liposome transfection, but the transfection efficiency needs to be elevated.

Antigens, CD↗
PubMed2006

[An experimental study on rat bone marrow mesenchymal stem cells transfected with dentin sialophosphoprotein gene in vitro].

OBJECTIVE: To evaluate the expression of dentin sialophosphoprotein (DSPP) in transfected rat bone marrow mesenchymal stem cells (BM-MSC) and the influence of the transfection. METHODS: Plasmid containing mice dentin DSPP was constructed by using the cytomegalovirus (CMV) promoter and then transfected the cultured BM-MSC by lipofectamine; The expression of Pax-9 and dentin matrix protein 1 (DMP1) gene of transfected BM-MSC were detected by RT-PCR. The expression of DSPP was examined by immunocytochemical staining, and the formation ratio of mineralized nodules of transfected BM-MSC was compared with untransfected ones after mineralized induction. RESULTS: The constructed pcDNA3.1(+)/mDSPP could produced 3.0 kb and 5.4 kb fragments, DSPP gene and Pax9 gene were expressed 24 h and 48 h respectively, after BM-MSC were transfected Pax-9 gene was exprssed, but DMP1 gene was not; Immunohistochemical staining showed that DSPP was positive in transfected BM-MSC; The formation ratio of mineralized nodules of transfected BM-MSC was higher than that of untransfected ones after mineralized induction. CONCLUSIONS: The expression of mice DSPP in BM-MSC by gene transfection can induce the expression of tooth development-associated gene Pax9 and enhance the formation of mineralized nodules, which suggests that DSPP gene might induce odontogenic differentiation of BM-MSC.

Animals↗
PubMed2006

Enhancing radiosensitivity of Hela cells by combining transfection of bcl-2 c-myc ASODNs.

Previous studies have detected bcl2 and c-myc upregulated in Hela cells. In this study, we investigated the effect on apoptosis of Hela cells by combining transfection of bcl2 and c-myc antisense oligonucleotides(ASODNs), and to explore the relationship between combining gene transfection and radiosensitivity of Hela cells, furthermore, to seek a new method to enhance the radiosensitivity of tumor cells. ASODNs of bcl2 and c-myc coated with cationic liposomes were designed and synthesized and transfected to Hela cells. The expressions of BCL2 and C-MYC were assessed by immunohistochemistry (IHC) staining and flow cytometry (FCM), and the induced-apoptosis phenomenon of Hela cells transfected with bcl2 ASODN or c-myc ASODN was observed by Giemsa Staining, DNA Ladder and FCM. Irradiation was performed on ASODNs transfected cells, then the radiosensitivity, biological reaction and cell proliferation of the transfected cells were investigated by cellular clone formation analysis, MTT assay and FCM. Both bcl2 and c-myc ASODNs could partially inhibit the growth of Hela cells by reducing the expression of bcl2 and c-myc (p < 0.05). The apoptosis rate of Hela cells treated with bcl2 ASODN was 6.60 +/- 0.70%, c-myc ASODN 10.29 +/- 0.66%, bcl2 + c-myc ASODNs 11.83 +/- 0.57 %, which were much higher than that of control 1.79 +/- 0.19%(p < 0.05). The sensitivity enhancement ratio(SER) of Hela cells transfected with bcl2 ASODN,c-myc ASODN and bcl2 + c-myc ASODNs was 1.20, 1.53 and 2.18 respectively. Combining transfection of bcl2 and c-myc ASODN could more effectively induce apoptosis of Hela cells, which subsequently enhanced radiosensitivity of Hela cells. Furthermore, combining transfection ASODNs of apoptosis-inhibiting genes or oncogenes followed radiotherapy to malignant tumor cells might be a new potential strategy.

Analysis of Variance↗
PubMed1990

Expression of human immunodeficiency virus 1 (HIV-1) envelope gene products transcribed from a heterologous promoter. Kinetics of HIV-1 envelope processing in transfected cells.

The expression of human immunodeficiency virus 1 (HIV-1) envelope glycoprotein products was studied in cells transfected with env gene constructs transcribed from an SV40 promoter. Gene constructs possessing the complete tat, rev (tat+ rev+) and env genes were transiently expressed in COS-1 cells as precursor SU-TM (gp160), SU.TM (gp120 x 41), and nucleolar rev protein. In addition, envelope glycoprotein was detected on the surface of those transfected COS-1 cells expressing abundant levels of env protein. Transfected constructs possessing a mutated tat translational initiation codon (tat-rev+) were expressed in COS-1 cells with at least a 10-fold increase in the level of envelope glycoprotein expression compared to the analogous constructs with an intact tat AUG codon (tat+ rev+). Mutation of the rev initiation codon (tat+ rev-) and (tat-rev-) resulted in no detectable expression of env products but expression of these proteins could be rescued by co-transfection of a cDNA encoding the rev gene. Subgenomic tat/rev transcripts were detected following transfection of all of the gene constructs indicating splicing of the env mRNAs transcribed from a heterologous promoter. Unspliced env transcripts were only detected in the cytoplasm of cells transfected with (rev+) constructs or with the (tat- rev-) construct in the presence of the rev cDNA supplied in trans. In contrast, unspliced transcripts were detected in the nuclear and total cellular RNA of all transfected cells. Expression of rev protein was localized to the nucleolus of transfected COS-1 cells. These results indicate that the export of unspliced env mRNA to the cytoplasm is facilitated by the expression of rev. Following env synthesis, the conversion of SU-TM (gp160) to SU.TM (gp120 x 41) was not quantitative. After a 20-h pulse-chase, only 40% of the SU-TM (gp160) present at the start of the chase period was subsequently accountable as mature SU (gp120), and approximately 30% of the detectable SU (gp120) was found in the culture medium of transfected COS-1 cells. The findings indicate that the surface expression of SU.TM (gp120 x 41) derived from heterologous gene transcripts is modulated (i) the co-expression of rev, (ii) the efficiency of proteolytic processing of SU-TM (gp160), and (iii) the degree of SU (gp120) shedding and/or secretion from the cell.

Animals↗
PubMed1989

Metastatic potential of cloned murine melanoma cells transfected with activated c-Ha-ras.

We sought to determine whether the transfection of tumorigenic but not metastatic cells with the activated c-Ha-ras oncogene was invariably associated with acquisition of the metastatic phenotype. Three clonally derived lines of the K-1735 murine melanoma, characterized as nonmetastatic or poorly metastatic, were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, that confers resistance to neomycin (pSV2neoEJ). Cells transfected with pSV2neo, a plasmid containing the neo gene, served as controls for the procedure of Polybrene-mediated transfection. All cell lines were injected into syngeneic C3H/HeN and into athymic mice, and the results were compared with those produced by highly metastatic K-1735 M-2 cells. Although the pSV2neoEJ-transfected cells produced more rapidly growing s.c. tumors than the control cell lines did, the incidence of spontaneous metastasis was not increased. Following i.v. inoculation, the c-Ha-ras transfectants were retained in lung vasculature in greater proportions than pSV2neo counterpart transfectants were. The c-Ha-ras transfectants also produced significantly more lung tumor colonies, which grew faster than the few lung tumor colonies in mice given injections of control melanoma cells. We concluded that transfection of the activated c-Ha-ras oncogene into nonmetastatic K-1735 melanoma cells leads to accelerated tumor growth in vivo and can confer the ability to form lung colonies after i.v. injection but not the ability to metastasize from a primary s.c. tumor.

Animals↗
PubMed1989

Enhanced efflux of [3H]vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene.

Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain [3H]vinblastine. Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in [3H]vinblastine accumulation, compared to their drug-sensitive counterparts. After ATP depletion, this difference in [3H]vinblastine accumulation between mdr1 transfectants and control cells effectively disappears. This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner. In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells. In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of [3H]vinblastine release. The observation that the mdr1 transfectants show a decreased [3H]vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner. However, attempts to establish competition for [3H]vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants.

Adenosine Triphosphate↗