scholarly journals NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

1985 ◽  
Vol 5 (1) ◽  
pp. 259-262 ◽  
Author(s):  
U P Thorgeirsson ◽  
T Turpeenniemi-Hujanen ◽  
J E Williams ◽  
E H Westin ◽  
C A Heilman ◽  
...  
Keyword(s):  
Nude Mice ◽  
Dna Sequences ◽  
Immune Cell ◽  
Human Tumor ◽  
3T3 Cells ◽  
Metastatic Phenotype ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Tumor Dna ◽  
Nih 3T3 Cells

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice

1985 ◽  
Vol 5 (1) ◽  
pp. 259-262
Author(s):  
U P Thorgeirsson ◽  
T Turpeenniemi-Hujanen ◽  
J E Williams ◽  
E H Westin ◽  
C A Heilman ◽  
...  
Keyword(s):  
Nude Mice ◽  
Dna Sequences ◽  
Immune Cell ◽  
Human Tumor ◽  
3T3 Cells ◽  
Metastatic Phenotype ◽  
Ras Oncogenes ◽  
Nih 3T3 ◽  
Tumor Dna ◽  
Nih 3T3 Cells

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.

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 Identification of ErbB3-stimulated genes using modified representational difference analysis

1997 ◽  
Vol 323 (1) ◽  
pp. 113-118 ◽  
Author(s):  
Carl F. EDMAN ◽  
Sally A. PRIGENT ◽  
Andrea SCHIPPER ◽  
James R. FERAMISCO
Keyword(s):  
Dna Sequences ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Representational Difference Analysis ◽  
3T3 Cells ◽  
Difference Analysis ◽  
Total Rna ◽  
Egfr Family ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

The epidermal growth factor receptor (EGFR) family of tyrosine kinases is involved in the growth of normal and tumour cells. The specific contribution of each of the four family members to these processes remains unclear. In the present study we have used a PCR-based subtractive approach to identify differences in messages induced in response to activation of ErbB3 and EGFR. The approach described is a modification of the representational difference analysis technique adapted for analysis of cDNA, which we have modified to permit identification of differential gene expression using as little as 20 μg of total RNA as the starting material. The mRNA obtained from EGF-stimulated NIH-3T3 cells expressing chimaeric EGFR-ErbB3 receptors provided the tester amplicons (small PCR-amplified fragments) which were subtracted against driver amplicons derived from unstimulated NIH-3T3 cells expressing the EGFR-ErbB3 chimaera or EGF-stimulated NIH-3T3 cells overexpressing the EGFR. A total of 22 different clones were isolated, 90% of which showed increased expression in the tester amplicons. Six of these, corresponding to known DNA sequences, were selected for further Northern blot analysis against total RNA prepared from the starting cell lines. Of these, the gene encoding the protein dlk (or a closely related protein, Pref-1) was identified as being regulated by ErbB3 but not by the EGFR. Other genes appeared to be elevated by both ErbB3 and EGFR, including those encoding c-jun, Ret finger protein (RFP), neuroleukin and amyloid protein precursor. One gene product, TIS11, was identified as being regulated by EGFR but not by ErbB3.

scholarly journals Mutants of the RNA-dependent protein kinase (PKR) lacking double-stranded RNA binding domain I can act as transdominant inhibitors and induce malignant transformation.

1995 ◽  
Vol 15 (6) ◽  
pp. 3138-3146 ◽  
Author(s):  
G N Barber ◽  
M Wambach ◽  
S Thompson ◽  
R Jagus ◽  
M G Katze
Keyword(s):  
Malignant Transformation ◽  
Nude Mice ◽  
Rna Binding ◽  
Critical Role ◽  
Point Mutations ◽  
Dominant Negative ◽  
3T3 Cells ◽  
Double Stranded Rna ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

Recently we reported that introduction of catalytically inactive PKR molecules into NIH 3T3 cells causes malignant transformation and the development of tumors in nude mice. We have proposed that PKR may be a tumor suppressor gene possibly because of its translational inhibitory properties. We have now designed and characterized a number of PKR mutants encoding proteins that retain their catalytic competence but are mutated in their regulatory double-stranded RNA (dsRNA) binding domains (RBDs). RNA binding analysis revealed that PKR proteins either lacking or with point mutations in the first RBD (RBD-1) bound negligible amounts of dsRNA activator or adenovirus VAI RNA inhibitor. Despite the lack of binding, such variants remained functionally competent but were much less active than wild-type PKR. PKR variants completely lacking RBD-1 were largely unresponsive to dsRNA in activation assays but could be activated by heparin. To complement these studies, we evaluated the effects of point mutations in RBD-1 or the removal of either RBD-1 or RBD-2 on the proliferation rate of mouse 3T3 cells. We were unsuccessful at isolating stably transformed cells expressing RBD-1 point mutants or RBD-2-minus mutants. In contrast, NIH 3T3 cells, which constitutively expressed PKR proteins that lacked RBD-1, were selected. These cells displayed a transformed phenotype and caused tumors after inoculation in nude mice. Further, levels of endogenous eIF-2 alpha phosphorylation in RBD-1-minus cell lines were reduced, suggesting that such mutants act in a dominant negative manner to inhibit the function of endogenous PKR. These results emphasize the importance of RBD-1 in PKR control of cell growth and provide additional evidence for the critical role played by PKR in the regulation of malignant transformation.

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.

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.

scholarly journals Depletion of c-myc with specific antisense sequences reverses the transformed phenotype in ras oncogene-transformed NIH 3T3 cells.

1991 ◽  
Vol 11 (7) ◽  
pp. 3699-3710 ◽  
Author(s):  
M D Sklar ◽  
E Thompson ◽  
M J Welsh ◽  
M Liebert ◽  
J Harney ◽  
...  
Keyword(s):  
Nude Mice ◽  
Growth Rates ◽  
Monolayer Culture ◽  
Soft Agar ◽  
Ras Oncogene ◽  
Minimum Level ◽  
3T3 Cells ◽  
Sense Orientation ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ras oncogene-transformed NIH 3T3 cells expressing glucocorticoid-inducible antisense c-myc cDNA transcripts at levels sufficient to deplete c-myc protein lost their transformed morphology and the ability to grow in soft agar; their ability to form tumors in nude mice was also impaired. These changes were dependent on the continuous expression of the antisense sequences. No major effects on plating efficiencies, growth rates in monolayer culture, or immortalization were observed in the revertant cells, indicating that the observed effects were not a toxic consequence of c-myc protein depletion. Transfection with the same vector expressing c-myc in the sense orientation or other control vectors had no effect on transformation. These results suggest that a certain minimum level of expression of c-myc is required for the maintenance of ras transformation in NIH 3T3 cells.

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