Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “NIH 3T3 Cells”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1997

Interleukin-1beta-induced expression of protein kinase C (PKC)-delta and epsilon in NIH 3T3 cells.

NIH 3T3 cells express the alpha, delta, epsilon and zeta isoenzymes of protein kinase C(PKC). Following stimulation of cells (24 h) with the pro-inflammatory cytokine, interleukin 1beta (IL-1beta), we observed, by Western blotting, a dose-dependent effect on the levels of PKC-epsilon and delta, but not on alpha or zeta. Moreover, time course analysis revealed that the isoenzymes, PKC-delta and epsilon were induced by IL-1beta after 7 h. Again, no change in PKC-alpha or zeta levels after IL-1beta treatment were detected. Incubation with selective PKC inhibitor peptides blocked the PKC-alpha, delta, epsilon and zeta antibodies binding to their respective isoenzyme bands. We also observed that the addition of the tumour-promoting phorbol ester, Phorbol 12-myristate 13-acetate (PMA), downregulated PKC-alpha, delta and epsilon by 7 h in NIH 3T3 cells. PMA did not affect constitutively produced PKC-zeta protein levels even after 24-h treatment. In summary, these results demonstrate that IL-1beta induces protein synthesis of the Ca2+-independent PKC-delta and epsilon isoforms in NIH 3T3 cells. The differences observed here between PKC isoenzymes in response to IL-1beta suggest that each isoenzyme may have a unique role in the signal transduction pathways of IL-1beta and that such isoenzyme may have a unique role in the signal transduction pathways of IL-1beta and that such selective expression may influence the action of agents which require PKC for signal transduction acting in concert with IL-1.

3T3 Cells↗
PubMed1990

Physiological induction and reversal of focus formation and tumorigenicity in NIH 3T3 cells.

NIH 3T3 cells undergo morphological transformation in response to conditions of constrained growth, such as occur in low serum concentrations or at confluence. Transformation is expressed in a small fraction of the cells by the appearance of discrete foci of multiplying cells on a confluent monolayer of quiescent cells. We isolated and expanded cell populations from three dense and three light foci. Cells from each of these populations efficiently reproduced foci of the same morphotype when grown on a background of nontransformed NIH 3T3 cells. Using cultures derived from one of the dense foci (subline D/2), we found that the number of focus-forming units was stable and the cells remained tumorigenic when they were subjected to repeated thrice-weekly passage in 2% calf serum. However, equivalent passage in 10% calf serum eventually rendered the cells incapable of both focus production and tumor formation. The results show that the capacity to produce tumors as well as morphological transformation are produced as a response to physiological constraints of growth and/or metabolism in the absence of carcinogens and that both properties can be reversed by lifting the constraints. This behavior is typical of an adaptational response and, taken together with other supporting evidence, shows that tumorigenesis does not require conventional genetic alteration.

Animals↗
PubMed1985

Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells.

NIH 3T3 cells were transfected with plasmids containing Moloney murine leukemia virus long terminal repeats and either chicken c-src or v-src genes. In contrast with the effects observed after transfection with plasmids containing c-src and avian retrovirus or simian virus 40 promoter-enhancers (H. Hanafusa, H. Iba, T. Takeya, and F. R. Cross, p. 1-8, in G. F. Vande Woude, A. J. Levine, W. C. Topp, and J. D. Watson, ed., Cancer Cells, vol. 2, 1984; H. Iba, T. Takeya, F. R. Cross, T. Hanafusa, and H. Hanafusa, Proc. Natl. Acad. Sci. U.S.A. 81:4424-4428, 1984; R. C. Parker, R. Swanstrom, H. E. Varmus, and J. M. Bishop, p. 19-26, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984; R. C. Parker, H. E. Varmus, and J. M. Bishop, Cell 37:131-139, 1984; D. Shalloway, P. M. Coussens, and P. Yaciuk, p. 9-17, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984; D. Shalloway, P. M. Coussens, and P. Yaciuk, Proc. Natl. Acad. Sci. U.S.A. 81:7071-7075; and K. C. Wilhelmsen, W. G. Tarpley, and H. M. Temin, p. 303-308, in G. F. Vande Woude et al., ed., Cancer Cells, vol. 2, 1984), we found that both types of Moloney murine leukemia virus long terminal repeat-src expression plasmids induced focus formation, although c-src induced only 1% as many foci as v-src. The focus-selected c-src overexpressed cells had altered morphology and limited growth in soft agarose but were not tumorigenic in vivo. Cleveland digests, comparative in vitro kinase assays, secondary transfections, and immunoprecipitations indicated that focus formation was caused by rare transfection events that resulted in very high-level pp60c-src expression rather than by mutations of the transfected c-src genes. These results suggest that pp60v-src induced transformation is not a completely spurious activity which is unrelated to the function of pp60c-src but that it represents a perturbation of already existent molecular control processes involving pp60c-src.

Animals↗
PubMed1988

The mechanisms of ornithine decarboxylase deregulation in c-Ha-ras oncogene-transformed NIH 3T3 cells.

NIH 3T3 cells transformed with the human c-Ha-rasVal-12 oncogene showed markedly enhanced activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, as compared with their nontransformed counterparts. While in normal and in c-Ha-ras proto-oncogene-transfected cells stimulation with serum caused a transient induction of ODC, in cells transfected with the mutant c-Ha-ras oncogene the activity of ODC persisted at high levels for greatly extended periods of time. The amounts of immunoreactive ODC protein roughly paralleled the changes in the enzyme activity. The augmentation of ODC content by transformation could be largely, but not solely, accounted for by an enhanced accumulation of ODC mRNA. Nuclear run-off transcription assays demonstrated that in transformed cells the rate of transcription of the ODC gene was increased but to a much lower extent than the increase in the level of ODC mRNA. The turnover of ODC mRNA, as measured after actinomycin D treatment, was negligible in transformed cells for up to 8 h, whereas in normal cells the messenger content was initially decreased, by 40% within 4 h, and then remained constant. In normal cells, however, actinomycin D depressed the expression of ODC by more than 80%, while in transformed cells the activity of ODC was slightly superinduced, corresponding to the changes of ODC mRNA. These findings suggest that labile proteins may be involved in the regulation of both the stability and translatability of the ODC mRNA. Transformation led also to about 3-fold stabilization of ODC as determined by an exposure of the cells to cycloheximide. The results thus suggest ODC deregulation at multiple levels in the ras-oncogene-transformed cells.

Animals↗
PubMed1984

Overexpression of the c-src protein does not induce transformation of NIH 3T3 cells.

NIH 3T3 mouse cells were transfected with plasmids that induce efficient expression of either (i) the Rous sarcoma virus v-src gene, (ii) the chicken c-src gene, or (iii) a recombinant gene combining the 5' portion of c-src with the 3' end of v-src. Focus formation in tissue culture and formation of large colonies in soft agar did not occur in cells transfected with c-src. Cells transfected with c-src expression plasmids did not form foci but were isolated using a coselectable biological marker. They display morphological and substrate-independent growth characteristics intermediate between those of normal and v-src-transformed mouse cells, and lysates from these cells have enhanced in vitro tyrosine kinase activity. Transfection with the c-src-v-src recombinant induced focus formation with an efficiency similar to that obtained with a v-src expression plasmid. These results imply that v-src-induced transformation does not result just from overexpression of an essentially normal cellular protein but, at least in part, depends on the mutations distinguishing the cellular and viral proteins.

Amino Acid Sequence↗
PubMed2000

NIH 3T3 cells or engineered NIH 3T3 cells stably expressing GDNF can protect primary dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) shows potent and relatively specific protective effects on dopaminergic neurons. However, the size of the GDNF protein (MW 32-42 kDa) precludes the clinical use of GDNF via parenteral administration. It would thus be useful to have a cell line that stably secretes GDNF with full biological activities. The present study shows that NIH 3T3 cells express a considerable amount of GDNF. After co-culturing with primary E14-E16 midbrain neurons, such cells protected primary rat midbrain TH-immunopositive neurons from degeneration and MPP+ toxicity. In order to enhance endogenous GDNF expression, NIH 3T3 cells were stably transfected with GDNF cDNA with the Kozak sequence. The clones with the highest GDNF expression level were selected. The protective effects of engineered cells increased as the GDNF expression level increased. These cell lines may merit clinical investigation.

3T3 Cells↗
PubMed1989

Regulation of Na+-H+ exchange in normal NIH-3T3 cells and in NIH-3T3 cells expressing the ras oncogene.

Our laboratory and others have demonstrated that Na+-H+ exchange can be regulated by two different pathways; one that is mediated by an inositol trisphosphate-stimulated increase in intracellular calcium activity, and one that is mediated by an increase in protein kinase C activity. To determine whether one of these pathways is more important than the other, or whether one pathway is physiologically relevant, we employed normal NIH-3T3 cells (3T3 cells) and NIH-3T3 cells expressing the EJ human bladder ras oncogene (EJ cells). The EJ cells were chosen because they provide a genetic model that does not exhibit serum- or platelet-derived growth factor (PDGF)-stimulated inositol trisphosphate release or Ca2+ mobilization. It was found that serum- or PDGF-stimulated Na+-H+ exchange was more pronounced in EJ cells than in control 3T3 cells. As expected, serum- or PDGF-stimulated Na+-H+ exchange in 3T3 cells was inhibited by chelating intracellular Ca2+ with the intracellular Ca2+ chelator quin2, by the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and by the calmodulin antagonist trifluoperazine. In contrast, these agents did not inhibit serum- or PDGF-stimulated Na+-H+ exchange in EJ cells. Activators of protein kinase C (e.g., 1-oleoyl-2-acetylglycerol or biologically active phorbol esters) were found to stimulate Na+-H+ exchange in EJ cells to the same extent as serum. However, these agents were considerably less effective than serum in control 3T3 cells. Despite these findings, PDGF did not stimulate diacylglycerol levels in EJ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1991

Reduction in the calcium requirement for growth is correlated with intracellular calcium stores in normal and SV40-transformed NIH 3T3 cells.

Untransformed NIH 3T3 cells do not proliferate in media with reduced calcium, while SV40-transformed NIH 3T3 cells do. Intracellular calcium stores of untransformed cells were depleted to a higher extent than those of transformed cells under these conditions, which led to a decreased intracellular calcium transient in response to serum, compared to SV40-transformed cells. Furthermore, untransformed cells could be gradually adapted to proliferate in the low-calcium medium and, after adaptation, maintained their stores and serum response in low calcium media. Our experiments indicate that it is the ability of the cells to maintain adequate calcium stores in low calcium media that correlates with a full serum response and the ability to proliferate, rather than any differences reflected in alterations of resting calcium levels.

3T3 Cells↗
PubMed1996

Differential effects of protein kinase C activation on calcium storage and capacitative calcium entry in NIH 3T3 cells.

In NIH 3T3 cells, treatment with phorbol 12-myristate 13-acetate (PMA) reduced the release of Ca2+ by thapsigargin, but did not activate Ca2+ entry; Ca2+ influx was triggered after the residual pool was emptied by thapsigargin, and this Ca2+ influx was similar to that induced by thapsigargin in control cells. The effect of PMA was due to decreased Ca2+ storage because 1) Ca2+ release by ionomycin was similarly affected by PMA, and in both control and PMA-treated cells, ionomycin did not release Ca2+ following thapsigargin treatment; 2) PMA reduced 45Ca2+ accumulation; and 3) studies with Ca2+ indicator compartmentalized into the endoplasmic reticulum indicated that stored Ca2+ was reduced by PMA. Although PMA did not itself activate Ca2+ entry, PMA potentiated Ca2+ entry with low concentrations of cyclopiazonic acid. With a somewhat higher concentration of cyclopiazonic acid, PMA had no effect on calcium entry. Thus, protein kinase C has two apparent actions on calcium signaling in NIH 3T3 cells: 1) reduced intracellular Ca2+ storage capacity and 2) augmented calcium entry with submaximal intracellular Ca2+ pool depletion. These actions indicate a complex and potentially important role for the protein kinase C system in calcium homeostasis in this cell type.

3T3 Cells↗
PubMed2006

Lanthanum induces extracellular signal-regulated kinase phosphorylation through different mechanisms in HeLa cells and NIH 3T3 cells.

Lanthanum ion (La3+) was generally regarded as calcium antagonist and was used as calcium channel blocker. However, its potential biological effects on cells were poorly understood. In the present work, it was found that La3+ could induce rapid extracellular signal-regulated kinase (ERK) phosphorylation in both HeLa cells and NIH 3T3 cells, but different mechanisms were involved. At a concentration of 30 microM or higher, La3+ enters the cells and activates ERK through a mechanism involving calmodulin activation inside the cells, which is similar to the action of intracellular Ca2+. However, at lower concentration, free La3+ promoted ERK phosphorylation in NIH 3T3 cells outside the cells through an unknown La3+ sensing mechanism, while Ca2+ exerted much weaker effect. The present results suggested that the biological effects of La3+ on cells maybe involve mechanisms beyond calcium antagonist.

Animals↗
PubMed1989

Cyclic AMP can partially restore platelet-derived growth factor-stimulated prostaglandin E2 biosynthesis, and calcium mobilization in EJ-ras-transformed NIH-3T3 cells.

NIH-3T3 cells transformed by the EJ-ras oncogene display reduced platelet-derived growth factor (PDGF)-stimulated phospholipase C activity as measured by inositol 1,4,5-triphosphate (IP3) synthesis and Ca2+ mobilization. The lack of PDGF-stimulated Ca2+ mobilization in EJ-ras transformed cells is not due to a loss of IP3 sensitivity, because microinjected IP3 elevates intracellular Ca2+. Treatment of EJ-ras transformed cells with cholera toxin or 8-bromo-cyclic AMP, but not pertussis toxin or the beta-subunit of cholera toxin, results in a slight recovery of PDGF-stimulated IP3 synthesis, a marked increase in intracellular Ca2+ mobilization, and an almost complete recovery of prostaglandin E2 biosynthesis. These data suggest that EJ p21-mediated inhibition of PDGF-stimulated intracellular events can be partially and transiently reversed by cyclic AMP.

Animals↗
PubMed1990

Suppression of transformation by and growth adaptation to low concentrations of glutamine in NIH-3T3 cells.

NIH-3T3 cells, commonly used as targets for oncogene-mediated neoplastic transformation, undergo high rates of spontaneous transformation. When the glutamine concentration in the medium was reduced from 5 to 1 mM or less, the transformation rate was reduced. This effect was not dependent upon a reduction in the growth rate, which remained unaffected by reduction of glutamine even to 0.6 mM. Upon trypsinization and transfer to 5 mM glutamine-containing medium, cells exposed to 0.2 mM glutamine for as little as 4 days formed fewer foci than control cells exposed over a similar period to 5 mM glutamine. This indicates that short term changes in the supply of this polyfunctional metabolite have heritable consequences in later cell generations. If populations containing highly transformed cells were passaged weekly for 1-3 weeks in 0.2 mM glutamine, resultant populations were better adapted to grow in low-glutamine medium and formed fewer transformed foci upon re-transfer to 5 mM glutamine medium, suggesting that the transformed state is at least partially reversible. If similar cell populations were exposed to low-glutamine medium but were not passaged, growth adaptation occurred but there was no reduction in focus formation, indicating that maintenance of a moderate rate of cell division may be required in addition to the lowered glutamine for reversal of transformation. Transformed and non-transformed cells originating from foci and from nonfocal areas of the same culture dishes multiplied at the same reduced rate in 0.2 mM glutamine. This indicates that suppression of spontaneous transformation in low-glutamine medium was not the result of selecting pre-existing variants but was itself an adaptive response of the population.

Adaptation, Physiological↗
PubMed1995

Both v-Ha-Ras and v-Raf stimulate expression of the vascular endothelial growth factor in NIH 3T3 cells.

Stimulation of NIH 3T3 cells with platelet-derived growth factor (PDGF)-BB and 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances vascular endothelial growth factor (VEGF) gene expression. To address the question of whether Ras and Raf are involved in the induction of VEGF gene expression by PDGF and TPA, we examined the effects of both factors on NIH 3T3 cells stably transfected with v-Ha-ras or v-raf. In serum-starved NIH 3T3 cells, only low levels of mRNA expression can be detected, whereas both ras and raf transformed cell lines express enhanced levels of a 4.3-kilobase VEGF transcript. Stimulation with PDGF or TPA resulted in increased VEGF mRNA in all cell lines, with highest levels found in the transformed cells. Immunofluorescence studies confirmed that the elevated VEGF mRNA expression correlated with enhanced protein levels. Positive immunofluorescence signals could be detected in v-Ha-ras or v-raf transformed cell lines but not in unstimulated NIH 3T3 cells. VEGF from conditioned medium of v-raf transformed NIH 3T3 cells was partially purified by chromatography on heparin-Sepharose. Biological activity of this VEGF protein was demonstrated by competition with binding of recombinant 125I-VEGF165 to human umbilical vein endothelial cells and by its ability to stimulate proliferation of these cells.

3T3 Cells↗
PubMed1990

Enhanced expression of ganglioside GD3 in human and rat hepatocellular carcinoma cells and NIH 3T3 cells transfected with human tumor DNAs.

The gangliosides of human hepatoma biopsies, human hepatoma cell lines, and diethylnitrosamine-induced rat hepatomas were examined. These malignant tissues all expressed increased content of disialolactosylceramide (GD3) with respect to their normal counterparts. During the induction of rat hepatoma by diethylnitrosamine, an increase in GD3 levels appeared as early as 12 wk after initiation of diethylnitrosamine, concurrent with the appearance of precancerous hepatocytes. GD3 levels gradually increased to a peak of 4 times that of normal rat liver at 20 wk. CMP-NeuAc:GM3 sialyltransferase, the enzyme that synthesizes GD3 by transfer of sialic acid to GM3, also had tumor-associated elevation during the course of diethylnitrosamine-induction of rat hepatomas. To investigate the relationship of oncogene transformation and changes in ganglioside biosynthesis, NIH 3T3 cells transfected DNAs from human hepatoma or nasopharyngeal carcinoma were studied. The transfectants each expressed the same ganglioside composition, including a detectable level of GD3, as well as enhanced activity of CMP-NeuAc:GM3 sialyltransferase. A correlation between the tumor DNA transfection and the augmentation of GD3 in malignant cells is discussed. Because of the early appearance of GD3 in hepatoma and its possible relationship to oncogene activation, GD3 may be a potentially useful early tumor marker.

Animals↗
PubMed1986

Adenylate cyclase activity of NIH 3T3 cells morphologically transformed by ras genes.

The observed homology between G-proteins which regulate adenylate cyclase and ras proteins and the suggested role of ras in the regulation of adenylate cyclase in yeast prompted us to examine the regulation of adenylate cyclase in three cell lines: (i) NIH 3T3 cells, (ii) NIH 3T3 cells transformed by high levels of the normal rasH gene product and (iii) NIH 3T3 cells transformed by a mutated rasH gene product. We found that the regulation of adenylate cyclase by G-proteins is identical in the three cell lines, although the response of the transformed NIH 3T3 cells to agonists is strongly attenuated. Our data suggest that mammalian ras products do not interact directly with adenylate cyclase, although their increased expression may indirectly inhibit the interaction of adenylate cyclase stimulatory receptors with G-proteins.

Adenylyl Cyclases↗
PubMed1988

Some immunological properties of high and low tumorigenic cellular variants of c-H-ras transformed 3T3 cells.

NIH 3T3 cells transformed in vitro with the c-H-ras oncogene were subcloned. The resulting subclones were assayed for in vivo tumorigenicity in nude and in immunocompetent mice. The response of two high tumorigenic and two low tumorigenic clones to mediators of natural immunity was analyzed. The clones did not differ in sensitivity to NK cell-mediated lysis. However, compared to low tumorigenic clones, the high tumorigenic ones had a down-regulated expression of a membrane determinant recognized by a certain monoclonal naturally occurring antibody. The determinants recognized by other monoclonal naturally occurring antibodies available in the laboratory were equally expressed on the high and low tumorigenic clones. The high tumorigenic cells showed an increased resistance to cytotoxicity mediated by lymphotoxin. These results suggest that naturally occurring antibodies and lymphotoxin may participate in controlling the tumorigenicity of transformed cells. The high tumorigenic clones but not the low tumorigenic ones contained a novel 3.5-kb ras mRNA.

Animals↗
PubMed1991

Activation and cytotoxicity of 5'-deoxy-5-fluorouridine in c-H-ras transformed NIH 3T3 cells.

Transformation of NIH 3T3 cells with c-H-ras has been demonstrated to result in significantly increased activation of 5'-deoxy-5-fluorouridine and significantly increased cytotoxicity in vitro as compared to non-transformed NIH 3T3. FUra cytotoxicity appeared to be increased also in vitro upon transformation; the level of significance however was beyond that of accepted significance (0.05 less than P less than 0.01). Furthermore dFUrd proved to be less active in vivo in nude mice bearing v-fos transformed NIH 3T3 cells than in nude mice bearing c-H-ras transformed cells.

Animals↗