scholarly journals A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

1986 ◽  
Vol 6 (5) ◽  
pp. 1843-1846 ◽  
Author(s):  
A G Papageorge ◽  
B M Willumsen ◽  
M Johnsen ◽  
H F Kung ◽  
D W Stacey ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
S Phase ◽  
3T3 Cells ◽  
Ras Gene ◽  
Mutant Proteins ◽  
Ras Protein ◽  
Ras Genes ◽  
Essential Function ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene

1986 ◽  
Vol 6 (5) ◽  
pp. 1843-1846
Author(s):  
A G Papageorge ◽  
B M Willumsen ◽  
M Johnsen ◽  
H F Kung ◽  
D W Stacey ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
S Phase ◽  
3T3 Cells ◽  
Ras Gene ◽  
Mutant Proteins ◽  
Ras Protein ◽  
Ras Genes ◽  
Essential Function ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.

scholarly journals Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

1988 ◽  
Vol 8 (8) ◽  
pp. 3235-3243 ◽  
Author(s):  
L A Feig ◽  
G M Cooper
Keyword(s):  
Cell Proliferation ◽  
Inhibitory Effect ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Protein ◽  
Mammalian Expression ◽  
Guanine Nucleotide Binding ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

scholarly journals Desensitization of prostaglandin F2α-stimulated inositol phosphate generation in NIH-3T3 fibroblasts transformed by overexpression of normal c-Ha-ras-1, c-Ki-ras-2 and c-N-ras genes

1990 ◽  
Vol 267 (3) ◽  
pp. 809-813 ◽  
Author(s):  
F M Black ◽  
M J O Wakelam
Keyword(s):  
Inositol Phosphate ◽  
Prostaglandin F2α ◽  
Exponential Growth Phase ◽  
Maximal Effect ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Genes ◽  
3T3 Fibroblasts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The stimulation of inositol phosphate generation in control and ras-gene-transformed NIH-3T3 cells by prostaglandin F2 alpha (PGF2 alpha) was investigated. Compared with the control cells, a desensitization of the response was observed in cells transformed by the overexpression of N-, Ha-, or Ki-ras genes. This desensitization was without effect upon the concentration causing half-maximal effect (EC50), dissociation constant (Kd) or number of PGF2 alpha receptors. Inhibition of PG synthesis was without effect upon desensitization, demonstrating that the effect was not agonist-induced. Desensitization could be induced in NIH-3T3 cells by culturing under conditions where the cells were all in the exponential growth phase, or by a 12 h exposure to a C-kinase-activating phorbol ester. These results suggest that desensitization of certain agonist-induced inositol phospholipid responses in ras-transformed cells is a consequence of increased cell proliferation and associated amplification in C-kinase activity and is an indirect consequence of transformation by ras.

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP

1988 ◽  
Vol 8 (8) ◽  
pp. 3235-3243 ◽  
Author(s):  
L A Feig ◽  
G M Cooper
Keyword(s):  
Cell Proliferation ◽  
Inhibitory Effect ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Protein ◽  
Mammalian Expression ◽  
Guanine Nucleotide Binding ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system

1988 ◽  
Vol 8 (2) ◽  
pp. 786-793
Author(s):  
J Leon ◽  
H Kamino ◽  
J J Steinberg ◽  
A Pellicer
Keyword(s):  
Animal Model ◽  
Model System ◽  
Skin Tumors ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Genes ◽  
Ras Activation ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Codon 61

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.

scholarly journals H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system.

1988 ◽  
Vol 8 (2) ◽  
pp. 786-793 ◽  
Author(s):  
J Leon ◽  
H Kamino ◽  
J J Steinberg ◽  
A Pellicer
Keyword(s):  
Animal Model ◽  
Model System ◽  
Skin Tumors ◽  
3T3 Cells ◽  
Ras Gene ◽  
Ras Genes ◽  
Ras Activation ◽  
Nih 3T3 ◽  
Nih 3T3 Cells ◽  
Codon 61

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.

Mutation of amino acids in pp60c-src that are phosphorylated by protein kinases C and A

1989 ◽  
Vol 9 (6) ◽  
pp. 2453-2463
Author(s):  
P Yaciuk ◽  
J K Choi ◽  
D Shalloway
Keyword(s):  
Protein Kinase ◽  
3T3 Cells ◽  
Triple Mutant ◽  
Dependent Protein Kinase ◽  
Mutant Proteins ◽  
Tetradecanoyl Phorbol Acetate ◽  
Dependent Protein ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The product of the c-src proto-oncogene, pp60c-src, is phosphorylated at Ser-17 by cyclic AMP-dependent protein kinase A and at Ser-12 by calcium-phospholipid-dependent protein kinase C (when stimulated by 12-O-tetradecanoyl phorbol acetate). We tested the effects of Ser----Ala and Ser----Glu mutations at these sites in pp60c-src and in pp60c-src(F527) (a mutant whose transforming activities are enhanced by Tyr-527----Phe mutation) by transfecting single-, double-, and triple-mutant src expression plasmids into NIH 3T3 cells. Tryptic phosphopeptide analyses of the mutant proteins confirmed prior biochemical identifications of the phosphorylation sites and showed that neither separate nor coordinate mutations at Ser-12 and Ser-17 affected Tyr-416, Tyr-527, or Ser-48 phosphorylation or prevented mitosis-specific phosphorylations of either pp60c-src or pp60c-src(F527). Ser-12 mutation did not affect phosphorylation of the Ser-17-containing peptide, but mutation of Ser-17 significantly increased phosphorylation at Ser-12. Specific kinase activities (both with and without in vivo 12-O-tetradecanoyl phorbol acetate treatment) and the abilities of pp60c-src and pp60c-src(F527) to induce foci, transformed morphologies, and anchorage-independent growth were unaffected by any of the serine mutations. Thus, pp60c-src transforming activity in NIH 3T3 cells is relatively insensitive to phosphorylation at these sites, but there is a suggestion that Ser-17 phosphorylation may have a subtle regulatory effect.

scholarly journals Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

1999 ◽  
Vol 19 (7) ◽  
pp. 4623-4632 ◽  
Author(s):  
Masahiro Hitomi ◽  
Dennis W. Stacey
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Time Lapse ◽  
S Phase ◽  
3T3 Cells ◽  
Cycle Progression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

scholarly journals CtIP Activates Its Own and Cyclin D1 Promoters via the E2F/RB Pathway during G1/S Progression

2006 ◽  
Vol 26 (8) ◽  
pp. 3124-3134 ◽  
Author(s):  
Feng Liu ◽  
Wen-Hwa Lee
Keyword(s):  
Cyclin D1 ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Mutant Form ◽  
3T3 Cells ◽  
Primary Mouse ◽  
Rb Pathway ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT Cell cycle progression from G1 to S phase is mainly controlled by E2F transcription factors and RB family proteins. Previously we showed that the presence of CtIP is essential for G1/S transition in primary mouse blastocysts, as well as in NIH 3T3 cells. However, how CtIP executes this function remains to be elucidated. Here we show that in NIH 3T3 cells the expression of CtIP is regulated by the E2F/RB pathway during late G1 and S phases. The presence of wild-type CtIP, but not the E157K mutant form, which failed to interact with RB, enhanced its own promoter activity. Chromatin immunoprecipitation analysis indicated that the recruitment of CtIP to its promoter occurs concomitantly with TFIIB, a component of the RNA polymerase II complex, and with dissociation of RB from the promoter during late G1 and G1/S transition. Similar positive regulation of cyclin D1 expression by CtIP was also observed. Consistently, cells expressing the CtIP(E157K) protein alone exhibited growth retardation, an increase in the G1 population, and a decrease in the S-phase population. Taken together, these results suggest that, contrary to the postulated universal corepressor role, CtIP activates a subset of E2F-responsive promoters by releasing RB-imposed repression and therefore promotes G1/S progression.

scholarly journals Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells.

1987 ◽  
Vol 7 (7) ◽  
pp. 2512-2520 ◽  
Author(s):  
R D Owen ◽  
M C Ostrowski
Keyword(s):  
Dna Sequences ◽  
Regulation Of Gene Expression ◽  
Hormone Treatment ◽  
Northern Blotting ◽  
3T3 Cells ◽  
Ras Gene ◽  
Cellular Genes ◽  
Conditional Expression ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-related RNA and retrovirus-related VL30 RNA. Run-on transcription in isolated nuclei shows that the increase in v-sis-related RNA is not dependent on transcription and therefore must arise by a post-transcriptional mechanism. The increase in VL30 expression is a transcriptional effect. Hormone treatment of normal NIH 3T3 cells has no effect on the expression of these DNA sequences. These results suggest that v-ras stimulation of autocrine factors may play a role in transformation of cells by this gene and also suggest a reverse genetic strategy to determine the nucleic acid sequences and cellular factors involved in the regulation of gene expression that is observed.

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