scholarly journals Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product.

1993 ◽  
Vol 13 (2) ◽  
pp. 762-768 ◽  
Author(s):  
A M Chan ◽  
T P Fleming ◽  
E S McGovern ◽  
M Chedid ◽  
T Miki ◽  
...  
Keyword(s):  
Cdna Cloning ◽  
Cell Transformation ◽  
Human Tumor ◽  
Soft Agar ◽  
Gene Encoding ◽  
Serum Factors ◽  
Cell Library ◽  
G Alpha 12 ◽  
Cloned Cdna ◽  
Nih 3T3

Using an expression cDNA cloning approach, we examined human tumor cell lines for novel oncogenes that might evade detection by conventional techniques. We isolated a transforming sequence that was highly efficient in transforming NIH 3T3 mouse fibroblasts. DNA sequence analysis identified the gene as the human homolog of a recently cloned alpha subunit of mouse GTP-binding protein G alpha 12. NIH 3T3 cells transfected with G alpha 12 cDNA grew in soft agar and were tumorigenic in nude mice. There were no apparent mutations in the cloned cDNA in comparison with a G alpha 12 cDNA clone isolated from a normal human epithelial cell library, implying that overexpression alone was sufficient to cause NIH 3T3 cell transformation. The observed altered growth properties mediated by G alpha 12 showed a certain degree of dependency on serum factors, and its mitogenic potential was also potently inhibited by suramin treatment.

Expression cDNA cloning of a transforming gene encoding the wild-type G alpha 12 gene product

1993 ◽  
Vol 13 (2) ◽  
pp. 762-768
Author(s):  
A M Chan ◽  
T P Fleming ◽  
E S McGovern ◽  
M Chedid ◽  
T Miki ◽  
...  
Keyword(s):  
Cdna Cloning ◽  
Cell Transformation ◽  
Human Tumor ◽  
Soft Agar ◽  
Gene Encoding ◽  
Serum Factors ◽  
Cell Library ◽  
G Alpha 12 ◽  
Cloned Cdna ◽  
Nih 3T3

Using an expression cDNA cloning approach, we examined human tumor cell lines for novel oncogenes that might evade detection by conventional techniques. We isolated a transforming sequence that was highly efficient in transforming NIH 3T3 mouse fibroblasts. DNA sequence analysis identified the gene as the human homolog of a recently cloned alpha subunit of mouse GTP-binding protein G alpha 12. NIH 3T3 cells transfected with G alpha 12 cDNA grew in soft agar and were tumorigenic in nude mice. There were no apparent mutations in the cloned cDNA in comparison with a G alpha 12 cDNA clone isolated from a normal human epithelial cell library, implying that overexpression alone was sufficient to cause NIH 3T3 cell transformation. The observed altered growth properties mediated by G alpha 12 showed a certain degree of dependency on serum factors, and its mitogenic potential was also potently inhibited by suramin treatment.

scholarly journals NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype.

1994 ◽  
Vol 14 (1) ◽  
pp. 646-654 ◽  
Author(s):  
T Johansen ◽  
G Bjørkøy ◽  
A Overvatn ◽  
M T Diaz-Meco ◽  
T Traavik ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Constitutive Expression ◽  
Soft Agar ◽  
3T3 Cells ◽  
Gene Encoding ◽  
Stable Transfectants ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.

scholarly journals Suppression of vinculin expression by antisense transfection confers changes in cell morphology, motility, and anchorage-dependent growth of 3T3 cells

1993 ◽  
Vol 122 (6) ◽  
pp. 1285-1294 ◽  
Author(s):  
JL Rodríguez Fernández ◽  
B Geiger ◽  
D Salomon ◽  
A Ben-Ze'ev
Keyword(s):  
Cell Transformation ◽  
Soft Agar ◽  
Cell Junctions ◽  
Cell Periphery ◽  
3T3 Cells ◽  
Serum Factors ◽  
Growth Activation ◽  
Cdna Construct ◽  
Dependent Growth ◽  
In Cells

The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, is markedly modulated in cells during growth activation, differentiation, motility and cell transformation. The stimulation of quiescent cells by serum factors and the culturing of cells on highly adhesive matrices induce vinculin gene expression, whereas the transformation of fibroblast and epithelial cells often results in decreased vinculin expression (reviewed in Rodríguez Fernández, J. L., B. Geiger, D. Salomon, I. Sabanay, M. Zöller, and A. Ben-Ze'ev. 1992. J. Cell Biol. 119:427). To study the effect of reduced vinculin expression on cell behavior, 3T3 cells were transfected with an antisense vinculin cDNA construct, and clones displaying decreased vinculin levels down to 10-30% of control levels were isolated. These cells showed a round phenotype with smaller and fewer vinculin-positive plaques localized mostly at the cell periphery. In addition, they displayed an increased motility compared to controls, manifested by a faster closure of "wounds" introduced into the monolayer, and by the formation of longer phagokinetic tracks. Moreover, the antisense transfectants acquired a higher cloning efficiency and produced larger colonies in soft agar than the parental counterparts. The results demonstrate that the regulation of vinculin expression in cells can affect, in a major way, cell shape and motility, and that decreased vinculin expression can induce cellular changes reminiscent of those found in transformed cells.

NIH 3T3 cells stably transfected with the gene encoding phosphatidylcholine-hydrolyzing phospholipase C from Bacillus cereus acquire a transformed phenotype

1994 ◽  
Vol 14 (1) ◽  
pp. 646-654
Author(s):  
T Johansen ◽  
G Bjørkøy ◽  
A Overvatn ◽  
M T Diaz-Meco ◽  
T Traavik ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Constitutive Expression ◽  
Soft Agar ◽  
3T3 Cells ◽  
Gene Encoding ◽  
Stable Transfectants ◽  
Nih 3T3 ◽  
Hydrolysis Of ◽  
Nih 3T3 Cells

In order to determine whether chronic elevation of intracellular diacylglycerol levels generated by hydrolysis of phosphatidylcholine (PC) by PC-hydrolyzing phospholipase C (PC-PLC) is oncogenic, we generated stable transfectants of NIH 3T3 cells expressing the gene encoding PC-PLC from Bacillus cereus. We found that constitutive expression of this gene (plc) led to transformation of NIH 3T3 cells as evidenced by anchorage-independent growth in soft agar, formation of transformed foci in tissue culture, and loss of contact inhibition. The plc transfectants displayed increased intracellular levels of diacylglycerol and phosphocholine. Expression of B. cereus PC-PLC was confirmed by immunoperoxidase and immunofluorescence staining with an affinity-purified anti-PC-PLC antibody. The NIH 3T3 clones expressing plc induced DNA synthesis, progressed through the cell cycle in the absence of added mitogens, and showed significant growth in low-concentration serum. Transfection with an antisense plc expression vector led to a loss of PC-PLC expression accompanied by a complete reversion of the transformed phenotype, suggesting that plc expression was required for maintenance of the transformed state. Taken together, our results show that chronic stimulation of PC hydrolysis by an unregulated PC-PLC enzyme is oncogenic to NIH 3T3 cells.

Ultrastructure of two neoplasms in culture compared with original biopsies

1984 ◽  
Vol 42 ◽  
pp. 50-51
Author(s):  
Joseph M. Harb ◽  
James T. Casper ◽  
Vlcki Piaskowski
Keyword(s):  
Electron Microscopy ◽  
Tissue Culture ◽  
Biopsy Specimen ◽  
Cultured Cells ◽  
Light And Electron Microscopy ◽  
Human Tumor ◽  
Soft Agar ◽  
Human Tumor Cells ◽  
Morphological Distinction ◽  
Tumor Types

The application of tissue culture and the newer methodologies of direct cloning and colony formation of human tumor cells in soft agar hold promise as valuable modalities for a variety of diagnostic studies, which include morphological distinction between tumor types by electron microscopy (EM). We present here two cases in which cells in culture expressed distinct morphological features not apparent in the original biopsy specimen. Evaluation of the original biopsies by light and electron microscopy indicated both neoplasms to be undifferentiated sarcomas. Colonies of cells propagated in soft agar displayed features of rhabdomyoblasts in one case, and cultured cells of the second biopsy expressed features of Ewing's sarcoma.

cDNA cloning and chromosomal mapping of the gene encoding adenylate kinase 2 from Drosophila melanogaster

2000 ◽  
Vol 1490 (1-2) ◽  
pp. 109-114 ◽  
Author(s):  
Takafumi Noma ◽  
Ryutaro Murakami ◽  
Yasuhiro Yamashiro ◽  
Koichi Fujisawa ◽  
Sachie Inouye ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Cdna Cloning ◽  
Adenylate Kinase ◽  
Chromosomal Mapping ◽  
Gene Encoding

Effects of ouabain on NIH/3T3 cells transformed with retrovirai oncogenes and on human tumor cell lines

1987 ◽  
Vol 40 (5) ◽  
pp. 653-658 ◽  
Author(s):  
Pierosandro Tagliaferri ◽  
Kazuyoshi Yanagihara ◽  
Fortunate Ciardiello ◽  
Neil Talbot ◽  
Ursula Flatow ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Cell Lines ◽  
Human Tumor ◽  
Tumor Cell Lines ◽  
3T3 Cells ◽  
Human Tumor Cell ◽  
Human Tumor Cell Lines ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.

1983 ◽  
Vol 3 (2) ◽  
pp. 280-289 ◽  
Author(s):  
H Okayama ◽  
P Berg
Keyword(s):  
Cdna Cloning ◽  
Mammalian Cells ◽  
Human Fibroblasts ◽  
Simian Virus 40 ◽  
Plasmid Vector ◽  
Simian Virus ◽  
Cdna Clones ◽  
Alpha Globin ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Cloned Cdna

This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).

Posttranscriptional down-regulation of ras oncogene expression by inhibitors of cellular glutathione

1993 ◽  
Vol 13 (7) ◽  
pp. 4416-4422
Author(s):  
A C Miller ◽  
J Gafner ◽  
E P Clark ◽  
D Samid
Keyword(s):  
Transcriptional Activation ◽  
De Novo ◽  
Drug Effects ◽  
Human Tumor ◽  
Cell Phenotype ◽  
Similar Drug ◽  
Ras Family ◽  
Steady State Levels ◽  
Or Gene ◽  
Nih 3T3

Alterations in intracellular glutathione (GSH) content are known to affect intrinsic responses to ionizing radiation. More recently, it became apparent that radiation responses may depend also on the expression of specific oncogenes, including ras. These findings, suggesting a possible link between GSH and ras, led us to examine the effect of various GSH modulators on ras expression. Treatment of c-Ha-ras-transformed NIH 3T3 cells with L-buthionine S'R'-sulfoximine, dimethylfumarate, or N',N'-1,3-bis(trans-4-hydroxycyclohexyl)-N'-nitrosourea resulted in dose- and time-dependent reduction in ras mRNA steady-state levels followed by a decrease in ras-encoded p21 protein production. The effect on ras correlated with the extent of GSH decline, was common to different members of the ras family, and was independent of the mode of oncogene activation or cell phenotype. Indeed, similar drug effects were observed with murine cells in which overexpression of the c-Ha-ras proto-oncogene was due to transcriptional activation (PR4, nontumorigenic) or gene amplification (NIH 136, tumorigenic) and with malignant cells expressing a mutated Ha-ras (RS504). Moreover, N-ras, EJras, and Ki-ras in human tumor cells were similarly affected. Molecular analysis revealed a significant decrease in ras mRNA half-life in cells subjected to GSH inhibition, an effect that required de novo protein synthesis, but there was no change in the rate of gene transcription. These results indicate that pharmacological manipulation of cellular GSH content can down-regulate ras expression at the posttranscriptional level by destabilizing ras transcripts. The potential clinical implications are discussed.

The K-fgf/hst oncogene induces transformation through an autocrine mechanism that requires extracellular stimulation of the mitogenic pathway

1991 ◽  
Vol 11 (2) ◽  
pp. 1138-1145
Author(s):  
D Talarico ◽  
C Basilico
Keyword(s):  
Growth Factor ◽  
Receptor Activation ◽  
Soft Agar ◽  
Serum Free Medium ◽  
Free Medium ◽  
3T3 Cells ◽  
Serum Free ◽  
Nih 3T3 ◽  
Autocrine Mechanism ◽  
Nih 3T3 Cells

The K-fgf/hst oncogene encodes a secreted growth factor of the fibroblast growth factor (FGF) family. The ability of K-fgf-transformed cells to grow in soft agar and in serum-free medium is inhibited by anti-K-FGF neutralizing antibodies, consistent with an autocrine mechanism of transformation. The transformed properties of clones that express high levels of K-FGF are, however, only partially affected. To better define the autocrine mechanism of transformation by K-fgf and to determine whether receptor activation could occur intracellularly, we constructed two mutants of the K-fgf cDNA. Deletion of the sequences encoding the signal peptide suppressed K-fgf ability to induce foci in NIH 3T3 cells. A few morphologically transformed colonies were observed in cotransfection experiments, and they were found to express high levels of cytoplasmic K-FGF. However, their ability to grow in serum-free medium and in soft agar was inhibited by anti-K-FGF antibodies. Addition of a sequence encoding the KDEL endoplasmic reticulum and Golgi retention signal to the K-fgf cDNA led to accumulation of the growth factor in intracellular compartments. The ability of the KDEL mutant to induce foci in NIH 3T3 cells was much lower than that of the wild-type cDNA, and also in this case the transformed phenotype was reverted by anti-K-FGF antibodies. These and other findings indicate that the transformed phenotype of cells expressing a nonsecretory K-FGF is due to the extracellular activation of the receptor by the small amounts of growth factor that these cells still release. Thus, transformation by K-fgf appears to be due to an autocrine growth mechanisms that requires activation of the mitogenic pathway at the cell surface.

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