Hormonal regulation of expression of the oncogene v-src in mouse fibroblasts NIH 3T3

NIH 3T3 cells were transfected by plasmid containing v-src under control of hormone-regulated LTR MMTV (pMLsrc10). This plasmid caused the foci of morphologically transformed cells. The transformed cells induced rapidly growing tumours in nude mice. In the presence of dexamethasone the efficiency of NIH 3T3 cell transformation increased ten times, while tumourigenicity remained unchanged.     

Genetic transformation of somatic cells. X. Morphological transformation and changes in the growth characteristics of NIH 3T3 cells administered the oncogene v-myc

Transfection of the plasmid containing the cloned gag-myc part of retrovirus MC 29 into mouse NIH 3T3 cells induces focuses of morphological transformation. Isolated morphological transformants have a decreased dependence on serum growth factors, a higher saturation density in monolayer, and an increased cloning efficiency on the glass and in agar. Induced traits are stably inherited, and may constitute the direct consequence of stable maintenance and expression of transfected oncogens.     

Senescence-like changes induced by expression of p21Waf1/Cip1 in NIH3T3 cell line

P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/cip1involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidencefor a link between p21Waf1/cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/cip1 expression. Induc-tion of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features,such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed thatp21Waf1/Cip1-transduced NIH3T3 cells expressedβ-galactosidase activity at pH 6.0, which is known to bea marker of senescence. Our results suggest that p21Waf1/cipx can also induce senescence-like changes inmurine cells.     

Effects of bradykinin on cell volume and intracellular pH in NIH 3T3 fibroblasts expressing the ras oncogene.

BCECF fluorescence has been applied to determine intracellular pH (pHi) in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ras) and otherwise identical cells not expressing the oncogene (-ras). In +ras cells, pHi is significantly more alkaline (6.79 +/- 0.03 n = 12) than in -ras cells (6.64 +/- 0.02, n = 8). Bradykinin (100 nmol/l) leads to intracellular alkalinization in both +ras (to 6.96 +/- 0.04, n = 12) and -ras cells (to 6.85 +/- 0.02, n = 8). The effect of bradykinin is completely abolished in the presence of dimethylamiloride (100 mumol/l), which does not modify pHi in the absence of bradykinin. Similar to bradykinin, cell shrinkage by addition of 15 mmol/l NaCl to the extracellular fluid leads to intracellular alkalinization (by 0.08 +/- 0.01, n = 15). Cell volume is significantly greater in +ras cells (2.7 +/- 0.4 pl, n = 15) than in -ras cells (2.2 +/- 0.4 pl, n = 15). Bradykinin leads to cell shrinkage in both +ras cells (by 7 +/- 1%, n = 17) and -ras cells (by 5 +/- 1%, n = 15). The effect of bradykinin on cell volume can be reversed by the reduction of extracellular NaCl concentration by 15 mmol/l NaCl in +ras cells and by 7 mmol/l NaCl in -ras cells. This maneuver completely abolishes (in -ras cells) or blunts (in +ras cells) the alkalinizing effect of bradykinin. In conclusion, +ras cells are more alkaline than -ras cells. Bradykinin leads to further intracellular alkalinization by activation of the Na+/H(+)-exchanger, at least in part secondary to hormone-induced cell shrinkage.     

Identification of a PDGF-like mitoattractant produced by NIH/3T3 cells after transformation with SV40

It has previously been shown that fibroblastic cells transformed by SV40 exhibit a reduced requirement for PDGF for growth. In addition, NIH/3T3 cells lose both their chemotactic response to PDGF and specific cell surface binding of PDGF after transformation with SV40. We have now examined whether the SV40 transformed NIH/3T3 cells are producing a factor which acts similarly to PDGF. Our studies indicate that NIH/3T3 cells transformed with SV 40 produce a factor which shares many biological properties with PDGF. We were unable to detect this activity in conditioned media from nontransformed NIH/3T3 cells. The SV40/NIH/3T3 derived factor appears to possess both chemotactic and mitogenic activity for connective tissue cells but not endothelial or epithelial cells. Furthermore, in preliminary studies, this activity competes with 125I-PDGF for binding to smooth muscle cells. The biochemical properties of the SV40/NIH/3T3 derived factor are different from those of PDGF. The SV40 activity appears to reside in a heat labile acidic protein (pI less than 7.0) of MW less than 30,000 whereas PDGF is a heat stable basic protein (pI9.8) of 30,000 MW. Production of this factor may play a role in the decreased serum requirement for cell replication exhibited by SV40-transformed NIH/3T3 cells by supplying the cells with their own PDGF-like growth factor.     

NIH3T3 transforming gene not a general feature of atherosclerotic plaque DNA (atherosclerosis/oncogene/NIH3T3 transfection assay)

A recent study indicated that the DNA isolated from human coronary atherosclerotic lesions is capable of transforming NIH3T3 cells in culture. Using DNA isolated from rabbit aortic and human carotid atherosclerotic lesions, we failed to observe such transforming activity. Thus, NIH3T3 transforming activity does not appear to be a general feature of atherosclerotic lesions.     

Senescence-like changes induced by expression of p21(waf1/Cip1) in NIH3T3 cell line

P21(Waf1/Cip1) is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21(Waf1/Cip1) involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21(Waf1/Cip1) and cellular senescence. While in murine cells, the role of p21(Waf1/Cip1) is indefinite. We explored this issue using NIH3T3 cells with inducible p21(Waf1/Cip1) expression. Induction of p21(Waf1/Cip1) triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21(Waf1/Cip1)-transduced NIH3T3 cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p2l(Waf1/Cip1) can also induce senescence-like changes in murine cells.     

Protein phosphatase 2A inhibitors, phoslactomycins. Effects on the cytoskeleton in NIH/3T3 cells

Protein phosphorylation is a key regulatory mechanism of the organization and dynamics of the actin cytoskeleton during cell motility, differentiation, and cytokinesis. The level of protein phosphorylation is dependent on the relative activities of both protein kinases and protein phosphatases. In this paper, we examined the effect of phoslactomycins (PLMs) on the regulation of the cytoskeleton of NIH/3T3 fibroblasts. Treatment of cells with PLM-F (10 microM) induced actin filament depolymerization after 4 h. This effect was reversible and actin filaments were reformed 1 h after removal of the inhibitors. As PLM-F had no effect at all on polymerization of purified actin in vitro, it is thought that PLMs induce actin depolymerization through an indirect mechanism. An in vitro assay showed PLMs inhibited protein phosphatase 2A at lower concentrations (IC50 4.7 microM) than protein phosphatase 1. An in situ phosphorylation assay also revealed that PLM-F treatment stimulated the phosphorylation of intracellular vimentin. These results suggest that phoslactomycins are protein phosphatase 2A-specific inhibitors and that protein phosphatase 2A is involved in regulation of the organization of the actin cytoskeleton.     

Preparation and characteristics of a new transformed cell line G11 by transfection of NIH/3T3 mouse fibroblasts with DNA from the SA7 oncogene of simian adenovirus

Murine fibroblasts NIH 3T3 were transfected with the plasmid pASP containing simian adenovirus oncogene insertion. Focus forming transformants were cloned with a final dilution technique and a new cell line G11 was created as a result. Transformed status of this cell line is evidenced by changes in morphology, specific cytochemical and adhesion properties, ability to grow in semisolid agar and FCS concentration growth independence. Presence of intact integrated E1a-region of adenovirus SA7 oncogene was shown by blot-hybridization technique. Transformed status of G11 cells can be explained by integration of SA7 oncogene, that is evidenced indirectly by the increased resistance to heat shock.     

Antioxidants-induced rearrangements of actin cytoskeleton in 3T3 and 3T3-SV40 fibroblasts

Effect of antioxidants on actin cytoskeleton in 3T3 fibroblasts and 3T3 fibroblasts transformed with SV40 virus (3T3-SV40 cells) was studied. Antioxidants used were as follows: N-acetyl-L-cysteine (NAC), (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and glutathione in the reduced form (GSH). Both NAC and OTZ are precursors of GSH in the cell, but, in contrast to NAC, OTZ reduces inside the cell forming L-cysteine. The presence of NAC (5-20 mM) in the culture medium of both cell types resulted in loosening of monolayer, fragmentation of stress fibers, and the appearance of amorphous actin structures. As 3T3-SV40 cells contain less actin stress fibers than 3T3 cells, the NAC-induced rearrangements of actin cytoskeleton were stronger in these cells than in 3T3 cells. In contrast to NAC, OTZ (10-20 mM) did not destroy monolayer and did not induce any visible disappearance of stress fibers either in 3T3 or 3T3-SV40 cells. However, in the presence of OTZ, amorphous actin-containing structures were observed in 3T3-SV40 cells. The effect of glutathione on both cell types was similar to that of NAC. The time required for GSH-induced alterations of actin cytoskeleton (about 5 h) was consistent with the increase in the intracellular level of reactive oxygen species (4 h after addition of GSH to the culture medium). Upon removal of the antioxidants from the medium, actin filament structures were reconstructed. However, within 24 h after withdrawal of NAC or GSH, only a partial reconstruction of stress fibers was observed in 3T3 cells. On the contrary, 3T3-SV40 cells demonstrated formation of well-structured actin fibers similar to normal fibroblasts. These results suggest that GSH can act as a pro-oxidant in the absence of oxidative stress.     

Senescence-like changes induced by expression of p21~(Waf1/Cip1) in NIH3T3 cell line

P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/Cip1 involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21Waf1/Cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1 is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/Cip1 expression. Induction of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21Waf1/Cip1-transduced NIH3T3 cells expressed β-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p21Waf1/Cip1 can also induce senescence-like changes in murine cells.     

Decreased actin and tubulin synthesis in 3T3 cells after transformation by SV40 virus.

Actin and tubulin are major protein constituents of 3T3 and SV40 virus-transformed 3T3 cells. We have fractionated growing, confluent and SV403T3 cells into particulate and soluble fractions using conditions designed to sediment microtubules, actin filaments or membrane associated actin or tubulin. The ratio of particulate to soluble actin synthesized in growing or confluent 3T3 cells is 2 to 1, while the ratio is reversed in transformed cells. There is also a 60% decrease in particulate tubulin synthesis in SV403T3 cells when compared with that in normal cells. Similar results are obtained when total actin and tubulin amounts are determined. The half-lives of actin, tubulin and total protein are over 3 days in growing 3T3 and SV40 cells and decrease over two-fold in confluent 3T3 cells. The significance of these results with respect to loss of contact inhibition and development of malignancy by these cells after transformation is discussed.     

Identification and expression of human CD81 gene on murine NIH/3T3 cell membrane

The human CD81 (hCD81) molecule has been identified as a putative receptor for hepatitis C virus (HCV). In this study, eukaryotic expression vector pCDM8-hCD81 containing hCD81 cDNA and pSV2neo helper plasmid was used to cotransfect with lipofectamine into murine fibroblast cell line NIH/3T3 to establish an hCD81-expressing cell line. Resistant cell clones were obtained 20 days after the selection with neomycin (600 micro/ml) and then cultured as monoclones. The expression of the transfected hCD81 gene in the cells was verified by RT-PCR and flow cytometry analyses. One of the selected cell clones showed obvious expression of hCD81 and was named NIH/3T3-hCD81. Competitive inhibition tests indicated that the binding of monoclonal anti-hCD81 (JS-81) to NIH/3T3-hCD81 cells was inhibited by recombinant HCV E2 protein, suggesting that the expressed hCD81 molecules on NIH/3T3-hCD81 cells maintain natural conformation of binding to HCV E2. The transfected NIH/3T3-hCD81 cells should be of great potential value in studies on HCV attachment and onset of infection.     

Altered expression and distribution of the cytoskeletal-associated p35 protein in NIH 3T3 cells transformed with the Harvey sarcoma virus v-ras oncogene

1. Cytoskeletal events associated with retroviral oncogene (v-ras)-mediated transformation were studied in NIH 3T3 fibroblasts and their v-ras-transfected counterparts (3T3/H-1 cells). 2. Abnormal microfilament networks seen in 3T3/H-1 cells reflected significant decreases (approximately 90%) in two cytoskeletal-associated proteins (tropomyosin-1, p35). Neither actin content nor actin mRNA levels were altered, however, v-ras transfectants. 3. p35 mRNA activity in both NIH 3T3 and 3T3/H-1 cells was similar although differential compartmentalization of p35 to the detergent-resistant cytoskeletal fraction was evident only in normal fibroblasts. 4. Proper cytoskeletal organization may be a factor in the regulation of p35 mRNA translation in situ or influence the stability of p35 independent of translational rate.     

N-terminally truncated Vav induces the formation of depolymerization-resistant actin filaments in NIH 3T3 cells.

The Dbl family proto-oncogene vav is a guanine nucleotide exchange factor (GEF) for Rho family GTPases. Deletion of the N-terminus of Vav, harboring the single calponin homology (CH) domain, activates Vav's transforming potential, suggesting an important role of the CH domain in influencing Vav function. Since calponin binds actin, it has been suggested that the CH domain may mediate association with the actin cytoskeleton. In this study we have analyzed the subcellular localization and investigated the putative actin association of the Vav protein using enhanced green fluorescent protein (EGFP) fusion constructs. Our data show that both EGFP-tagged full length Vav and the CH domain-depleted EGFPvav 143-845 construct localize throughout the cytoplasm but fail to colocalize with F-actin. However, the latter construct of Vav was more strongly retained in the Triton-insoluble cytoskeleton fraction than full length Vav. Whereas removal of the CH domain had no apparent influence on the subcellular localization of Vav, deletion of the SH domains caused nuclear localization, indicating that Vav contains a functional nuclear localization signal. Expression of N-terminally truncated Vav constructs caused depolarization of fibroblasts and triggered the bundling of actin stress fibers into parallel arrays in NIH 3T3 cells. Notably, the parallel actin bundles showed prolonged resistance to the actin polymerization antagonists cytochalasin B and latrunculin B. These data point towards a regulatory role for the CH domain in Vav and suggest an actin cross-linking or bundling protein as a downstream effector molecule of vav-mediated signalling pathways.     

The v-fms oncogene induces factor-independent growth and transformation of the interleukin-3-dependent myeloid cell line FDC-P1.

The normal cellular counterpart of the v-fms oncogene product is a receptor for the mononuclear phagocyte colony-stimulating factor, CSF-1. An interleukin-3 (IL-3)-dependent mouse myeloid cell line, FDC-P1, was infected with a murine retrovirus vector containing v-fms linked to a gene encoding resistance to neomycin (neo). Infected cells selected for resistance to the aminoglycoside G418 contained few proviral DNA copies per haploid genome, expressed low levels of the v-fms-coded glycoprotein, remained IL-3 dependent for growth, and were nontumorigenic in nude mice. In contrast, infected cells selected for their ability to grow in the absence of IL-3 contained an increased number of proviral insertions, expressed high levels of the v-fms-coded glycoprotein, and were tumorigenic in nude mice. The IL-3-independent cells expressed IL-3 receptors of comparable number and affinity to those detected in uninfected FDC-P1 cells and did not produce a growth factor able to support replication of the parental cells. Thus, the synthesis of high levels of the v-fms gene product in FDC-P1 cells abrogated their requirement for IL-3 and rendered the cells tumorigenic by a nonautocrine mechanism. The data suggest that v-fms encodes a promiscuous tyrosine kinase able to transform cells of the myeloid lineage that do not normally express CSF-1 receptors.