scholarly journals Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells.

1989 ◽  
Vol 9 (5) ◽  
pp. 2258-2263 ◽  
Author(s):  
N Kuzumaki ◽  
Y Ogiso ◽  
A Oda ◽  
H Fujita ◽  
H Suzuki ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Oncogenic Transformation ◽  
3T3 Cells ◽  
Cellular Mechanisms ◽  
Ras Gene ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.

scholarly journals Resistance to oncogenic transformation in revertant R1 of human ras-transformed NIH 3T3 cells

1989 ◽  
Vol 9 (5) ◽  
pp. 2258-2263
Author(s):  
N Kuzumaki ◽  
Y Ogiso ◽  
A Oda ◽  
H Fujita ◽  
H Suzuki ◽  
...  
Keyword(s):  
Simian Virus 40 ◽  
Soft Agar ◽  
Simian Virus ◽  
T Antigen ◽  
Oncogenic Transformation ◽  
3T3 Cells ◽  
Cellular Mechanisms ◽  
Ras Gene ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

A flat revertant, R1, was isolated from human activated c-Ha-ras-1 (hu-ac-Ha-ras) gene-transformed NIH 3T3 cells (EJ-NIH 3T3) treated with mutagens. R1 contained unchanged transfected hu-ac-Ha-ras DNA and expressed high levels of hu-ac-Ha-ras-specific mRNA and p21 protein. Transfection experiments revealed that NIH 3T3 cells could be transformed by DNA from R1 cells but R1 cells could not be retransformed by Kirsten sarcoma virus, DNA from EJ-NIH 3T3 cells, hu-ac-Ha-ras, v-src, v-mos, simian virus 40 large T antigen, or polyomavirus middle T antigen. Somatic cell hybridization studies showed that R1 was not retransformed by fusion with NIH 3T3 cells and suppressed anchorage independence of EJ-NIH 3T3 and hu-ac-Ha-ras gene-transformed rat W31 cells in soft agar. These results suggest that the reversion and resistance to several oncogenes in R1 is due not to cellular defects in the production of the transformed phenotype but rather to enhancement of cellular mechanisms that suppress oncogenic transformation.

Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus

1988 ◽  
Vol 8 (12) ◽  
pp. 5495-5503
Author(s):  
L Fischer-Fantuzzi ◽  
C Vesco
Keyword(s):  
Amino Acid ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
3T3 Cells ◽  
Rat Embryo ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 cells, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 cells) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 cells were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 cells, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among cells of the same population, as if the nuclear uptake of some molecules depended on individual cell states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.

scholarly journals Cell-dependent efficiency of reiterated nuclear signals in a mutant simian virus 40 oncoprotein targeted to the nucleus.

1988 ◽  
Vol 8 (12) ◽  
pp. 5495-5503 ◽  
Author(s):  
L Fischer-Fantuzzi ◽  
C Vesco
Keyword(s):  
Amino Acid ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
3T3 Cells ◽  
Rat Embryo ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We investigated the requisites for, and functional consequences of, the relocation to the nucleus of a transforming nonkaryophilic mutant of the simian virus 40 large T antigen (a natural deletion mutant lacking an internal large-T-antigen domain that includes the signal for nuclear transport). Synthetic oligonucleotides were used to obtain gene variants with one or more copies of the signal-specifying sequence inserted near the gene 3' end, in a region dispensable for the main large-T-antigen functions. The analysis of stable transfectant populations showed that mouse NIH 3T3 cells, rat embryo fibroblasts, and simian CS cells (a subclone of CV1 cells) differed considerably in their ability to localize some variant molecules into the nucleus. CS cells were always the most efficient, and NIH 3T3 cells were the least efficient. The nuclear localization improved either with reiteration of the signal or with a left-flank modification of the signal amino acid context. Three signals appeared to be necessary and sufficient, even in NIH 3T3 cells, to obtain a nuclear accumulation comparable to that of wild-type simian virus 40 large T antigen; other signal-cell combinations caused a large variability in subcellular localization among cells of the same population, as if the nuclear uptake of some molecules depended on individual cell states. The effect of the modified location on the competence of the protein to alter cell growth was examined by comparing the activity of variants containing either the normal signal or a signal with a mutation (corresponding to large-T-antigen amino acid 128) that prevented nuclear transport. It was found that the nuclear variant was slightly more active than the cytoplasmic variants in rat embryo fibroblasts and NIH 3T3 cells and was notably less active in CS cells.

scholarly journals Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts.

1994 ◽  
Vol 14 (7) ◽  
pp. 4398-4407 ◽  
Author(s):  
A S Alberts ◽  
M Montminy ◽  
S Shenolikar ◽  
J R Feramisco
Keyword(s):  
Transcriptional Activity ◽  
Expression Vector ◽  
Active Form ◽  
Simian Virus 40 ◽  
T Antigen ◽  
3T3 Cells ◽  
Phosphorylation State ◽  
Phosphorylated Creb ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We have examined the activity and phosphorylation state of the cyclic AMP (cAMP) response element binding factor (CREB) in intact NIH 3T3 cells following microinjection of expression plasmids encoding regulatory proteins of type 1 (PP1) and 2A (PP2A) serine/threonine-specific protein phosphatases. Changes in CREB phosphorylation in the injected cells were monitored by indirect immunofluorescence using an affinity-purified antiserum (Ab5322) which specifically recognizes CREB phosphorylated at Ser-133, and changes in transcriptional activity of CREB were monitored by expression of a reporter gene regulated by cAMP. cAMP-stimulated phosphorylation in NIH 3T3 cells is normally transient, and as expected, after stimulation of cells with cell-permeable cAMP analogs, the level of phosphorylated CREB was found to initially increase and then return to a basal level within 4 h. Microinjection of an expression vector encoding a constitutively active form of inhibitor 1 (I-1), a PP1-specific inhibitor, by itself resulted in an apparent increase in phosphorylated CREB in unstimulated cells. Moreover, injection of the I-1 vector resulted in the prolonged appearance of phosphorylated CREB in cells after cAMP stimulation. In contrast, injection of a plasmid encoding simian virus 40 small t antigen, which interacts with PP2A to inhibit its activity towards several phosphoprotein substrates, had no effect on the phosphorylation state of CREB in stimulated or unstimulated NIH 3T3 cells. Consistent with these results, injection of the I-1 expression vector activated expression from a coinjected CRE-lacZ reporter plasmid, indicating that the increased phosphorylation of CREB also activated its transcriptional activity. These results provide further evidence for a role of a PP1 as the primary protein (Ser/Thr) phosphatase regulating the dephosphorylation of Ser-133 and thereby limiting the transcriptional activity of CREB.

scholarly journals Isolation of cellular genes differentially expressed in mouse NIH 3T3 cells and a simian virus 40-transformed derivative: growth-specific expression of VL30 genes.

1985 ◽  
Vol 5 (10) ◽  
pp. 2590-2598 ◽  
Author(s):  
K Singh ◽  
S Saragosti ◽  
M Botchan
Keyword(s):  
Cell Lines ◽  
Simian Virus 40 ◽  
Mouse Cell ◽  
Simian Virus ◽  
Differentially Expressed ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Transformed Cell ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We constructed and screened a cDNA library made from simian virus 40 (SV40)-transformed NIH 3T3 cells, and we isolated cDNAs representing genes that are differentially expressed between the parental cell and its SV40-transformed derivative. We found only a small number of cDNAs representing such genes. Two isolated cDNA clones represented RNAs expressed at elevated levels in the transformed cell line in a manner relatively independent of growth conditions. The expression of two other cDNAs was growth specific because transformed cells and nonconfluent parental cells contained higher levels of the homologous RNAs than did confluent, contact-inhibited parental cells. Another cDNA was well expressed in confluent parental and confluent transformed cells, but not in nonconfluent cells. The expression of some of these cDNAs varied strikingly in different mouse cell lines. Thus the genotype or histories of different cell lines can also affect the expression of certain genes. Interestingly, the only cDNA isolated that was expressed exclusively in the transformed cell was from an SV40 message. We focused on a growth-specific cDNA which we show is derived from a mouse endogenous retrovirus-like family called VL30. We sequenced the 3' long terminal repeat (LTR) of this transcriptionally active VL30 gene. This LTR has good homology with other VL30 LTR sequences, but differences occur, particularly upstream of the VL30 promoter. We found that VL30 gene expression varied in different mouse cell lines such that C3H cell lines had very low levels of VL30 transcripts relative to NIH 3T3 cell lines. However, Southern analysis showed that both cell lines had about the same number of VL30 genes homologous to our probe and that the position of the majority of these genes was conserved. We discuss possible explanations for this difference in VL30 expression.

scholarly journals Mechanism of activation of the ret proto-oncogene by multiple endocrine neoplasia 2A mutations.

1995 ◽  
Vol 15 (3) ◽  
pp. 1613-1619 ◽  
Author(s):  
N Asai ◽  
T Iwashita ◽  
M Matsuyama ◽  
M Takahashi
Keyword(s):  
Cell Surface ◽  
Multiple Endocrine Neoplasia ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
3T3 Cells ◽  
Endocrine Neoplasia ◽  
Transforming Activity ◽  
Nih 3T3 ◽  
Men 2A ◽  
Nih 3T3 Cells

Transforming activity of the c-ret proto-oncogene with multiple endocrine neoplasia (MEN) 2A mutations was investigated by transfection of NIH 3T3 cells. Mutant c-ret genes driven by the simian virus 40 or cytomegalovirus promoter induced transformation with high efficiencies. The 170-kDa Ret protein present on the cell surface of transformed cells was highly phosphorylated on tyrosine and formed disulfide-linked homodimers. This result indicated that MEN 2A mutations induced ligand-independent dimerization of the c-Ret protein on the cell surface, leading to activation of its intrinsic tyrosine kinase. In addition to the MEN 2A mutations, we further introduced a mutation (lysine for asparaginic acid at codon 300 [D300K]) in a putative Ca(2+)-binding site of the cadherin-like domain. When c-ret cDNA with both MEN 2A and D300K mutations was transfected into NIH 3T3 cells, transforming activity drastically decreased. Western blot (immunoblot) analysis revealed that very little of the 170-kDa Ret protein with the D300K mutation was expressed in transfectants while expression of the 150-kDa Ret protein retained in the endoplasmic reticulum was not affected. This result also demonstrated that transport of the Ret protein to the plasma membrane is required for its transforming activity.

scholarly journals Transcriptional control of the mouse alpha 2(I) collagen gene: functional deletion analysis of the promoter and evidence for cell-specific expression.

1986 ◽  
Vol 6 (2) ◽  
pp. 347-354 ◽  
Author(s):  
A Schmidt ◽  
P Rossi ◽  
B de Crombrugghe
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Chimeric Gene ◽  
Collagen Gene ◽  
3T3 Cells ◽  
Base Pairs ◽  
Specific Expression ◽  
Nih 3T3 ◽  
Cat Gene ◽  
Nih 3T3 Cells

A chimeric gene was constructed in which sequences between 2,000 base pairs upstream of the start of transcription of the mouse alpha 2(I) collagen gene and 54 base pairs downstream of this site were fused to the chloramphenicol acetyltransferase (CAT) gene. We present evidence suggesting that this collagen gene segment is sufficient for cell-specific expression of the chimeric gene. Indeed, the levels of CAT activity in transient expression experiments were at least 10 times higher after transfection of NIH 3T3 cells than after transfection of a mouse myeloma cell line, whereas much less difference was found after transfection of these two cell types with pSV2-CAT, a plasmid in which the early simian virus 40 promoter is fused to the CAT gene. Several deletions were introduced in the same 5'-flanking segment of the alpha 2(I) collagen gene, and the effects of these deletions were examined after DNA transfection of the chimeric collagen-CAT gene into NIH 3T3 cells. At least two segments broadly located between -979 and -502 and between -346 and -104 are needed for optimal expression of the chimeric gene. These results were obtained both in transient expression experiments and by analysis of pools of NIH 3T3 cells that were stably transfected with the different mutants. In general, the effects of the deletions on the activity of the alpha 2(I) collagen promoter were analogous, whether the plasmids harbored the simian virus 40 enhancer sequence or not, although the overall levels of expression of the chimeric gene were increased when the recombinant plasmids contained this sequence.

Expression of a peptide inhibitor of protein phosphatase 1 increases phosphorylation and activity of CREB in NIH 3T3 fibroblasts

1994 ◽  
Vol 14 (7) ◽  
pp. 4398-4407
Author(s):  
A S Alberts ◽  
M Montminy ◽  
S Shenolikar ◽  
J R Feramisco
Keyword(s):  
Transcriptional Activity ◽  
Expression Vector ◽  
Active Form ◽  
Simian Virus 40 ◽  
T Antigen ◽  
3T3 Cells ◽  
Phosphorylation State ◽  
Phosphorylated Creb ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We have examined the activity and phosphorylation state of the cyclic AMP (cAMP) response element binding factor (CREB) in intact NIH 3T3 cells following microinjection of expression plasmids encoding regulatory proteins of type 1 (PP1) and 2A (PP2A) serine/threonine-specific protein phosphatases. Changes in CREB phosphorylation in the injected cells were monitored by indirect immunofluorescence using an affinity-purified antiserum (Ab5322) which specifically recognizes CREB phosphorylated at Ser-133, and changes in transcriptional activity of CREB were monitored by expression of a reporter gene regulated by cAMP. cAMP-stimulated phosphorylation in NIH 3T3 cells is normally transient, and as expected, after stimulation of cells with cell-permeable cAMP analogs, the level of phosphorylated CREB was found to initially increase and then return to a basal level within 4 h. Microinjection of an expression vector encoding a constitutively active form of inhibitor 1 (I-1), a PP1-specific inhibitor, by itself resulted in an apparent increase in phosphorylated CREB in unstimulated cells. Moreover, injection of the I-1 vector resulted in the prolonged appearance of phosphorylated CREB in cells after cAMP stimulation. In contrast, injection of a plasmid encoding simian virus 40 small t antigen, which interacts with PP2A to inhibit its activity towards several phosphoprotein substrates, had no effect on the phosphorylation state of CREB in stimulated or unstimulated NIH 3T3 cells. Consistent with these results, injection of the I-1 expression vector activated expression from a coinjected CRE-lacZ reporter plasmid, indicating that the increased phosphorylation of CREB also activated its transcriptional activity. These results provide further evidence for a role of a PP1 as the primary protein (Ser/Thr) phosphatase regulating the dephosphorylation of Ser-133 and thereby limiting the transcriptional activity of CREB.

scholarly journals Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells

1988 ◽  
Vol 8 (1) ◽  
pp. 461-465
Author(s):  
D W Meek ◽  
W Eckhart
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

We observed six major tryptic phosphopeptides in p53 from simian virus 40-transformed and normal NIH 3T3 cells. Analyses of the phosphopeptides indicated that serines 37, 310 and/or 312, 389 and one or more of serines 7, 9, 12, 18, and 23 were phosphorylated. Phosphorylation of serines 310 and/or 312 was twofold higher in the simian virus 40-transformed cells as compared with that in normal NIH 3T3 cells.

scholarly journals Transcriptional control of the mouse alpha 2(I) collagen gene: functional deletion analysis of the promoter and evidence for cell-specific expression

1986 ◽  
Vol 6 (2) ◽  
pp. 347-354
Author(s):  
A Schmidt ◽  
P Rossi ◽  
B de Crombrugghe
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Chimeric Gene ◽  
Collagen Gene ◽  
3T3 Cells ◽  
Base Pairs ◽  
Specific Expression ◽  
Nih 3T3 ◽  
Cat Gene ◽  
Nih 3T3 Cells

A chimeric gene was constructed in which sequences between 2,000 base pairs upstream of the start of transcription of the mouse alpha 2(I) collagen gene and 54 base pairs downstream of this site were fused to the chloramphenicol acetyltransferase (CAT) gene. We present evidence suggesting that this collagen gene segment is sufficient for cell-specific expression of the chimeric gene. Indeed, the levels of CAT activity in transient expression experiments were at least 10 times higher after transfection of NIH 3T3 cells than after transfection of a mouse myeloma cell line, whereas much less difference was found after transfection of these two cell types with pSV2-CAT, a plasmid in which the early simian virus 40 promoter is fused to the CAT gene. Several deletions were introduced in the same 5'-flanking segment of the alpha 2(I) collagen gene, and the effects of these deletions were examined after DNA transfection of the chimeric collagen-CAT gene into NIH 3T3 cells. At least two segments broadly located between -979 and -502 and between -346 and -104 are needed for optimal expression of the chimeric gene. These results were obtained both in transient expression experiments and by analysis of pools of NIH 3T3 cells that were stably transfected with the different mutants. In general, the effects of the deletions on the activity of the alpha 2(I) collagen promoter were analogous, whether the plasmids harbored the simian virus 40 enhancer sequence or not, although the overall levels of expression of the chimeric gene were increased when the recombinant plasmids contained this sequence.

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