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At least 19 recordsLinked to original sources
PubMed1975

Difference in transport of leucine in attached and suspended 3T3 cells.

3T3 cells in subconfluent culture take up leucine through a transport system which has a relatively high affinity for leucine (M system). When the culture becomes confluent, the M system is turned off and leucine is transported by another system which has a low affinity for leucine (S system). The M system is reactivated by transferring the cells into subconfluent cultures. In suspension cultures 3T3 cells, initiated from confluent cultures, the M system is not activated and leucine is transported by the S system. In cells suspended from subconfluent culture, the M system continues to operate at a high level for four hours and then is gradually turned off. Tumor virus transformed 3T3 cells (SV3T3 and Py3T3) grow quite well in suspension culture and transport leucine both in monolayer and suspension through a high affinity system, with a high Vmax value. A derivative of 3T3, 3T3/41, which grows in suspension much more slowly than tumor virus transformed 3T3 cells, also takes up leucine through a high affinity transport system both in monolayer and suspension but its Vmax value is lower than that of the transformed cells.

Animals↗
PubMed1992

Coculture of newborn rat skin epidermal cells with Swiss 3T3 cells: effect of the phases of 3T3 cells on attachment, growth and keratin synthesis of epidermal cells.

Swiss albino mouse 3T3 cells in various states were inoculated onto one side of Millipore filters. The other side of the filter was then coated with type I collagen and inoculated with newborn rat skin epidermal cells. On coculture of these cells, the attachment, growth and keratin synthesis of epidermal cells were found to depend on the state of the 3T3 cells: 3T3 cells in the stationary phase of growth were the most effective, followed by those in the logarithmic growth phase, those in the lag phase and plasmolyzed fibroblasts being only slightly effective. The effects of 3T3 cells in different states correlated well with their abilities to synthesize type IV collagen, but not type I collagen: with an increase in type IV collagen synthesis by the 3T3 cells, attachment of epidermal cells to the cell support, and their growth and synthesis of keratins increased. This culture system is concluded to mimic conditions in skin in vivo, and therefore to be suitable for studies on the effects of fibroblasts on the growth of epidermal cells.

3T3 Cells↗
PubMed1983

Phosphate and the regulation of DNA replication in normal and virus-transformed 3T3 cells.

3T3 cells were cultured in media with different phosphate concentrations and the effects on DNA synthesis were examined. Even a modest phosphate depletion markedly inhibited DNA synthesis and cell multiplication in proliferating cultures. Furthermore, the decrease in the proportion of DNA-synthesizing cells observed after phosphate starvation followed the same time-course as the decrease seen after serum starvation. Cells starved to quiescence in a medium with a 100-fold decrease in phosphate concentration remained viable but non-proliferating for up to 3 weeks, i.e. they had entered a state of quiescence comparable with that seen after serum starvation. Addition of phosphate to phosphate-depleted cultures restored DNA synthesis within 24h. Furthermore, the kinetics of [3H]thymidine labelling after phosphate addition were nearly identical with the labelling kinetics following addition of serum to serum-depleted cultures. In contrast, phosphate deprivation had no inhibitory effects on DNA synthesis in simian-virus-40-transformed 3T3 cells. Furthermore, the inhibitory effects on DNA synthesis in such cells caused by a complete removal of serum could not be further enhanced by decreasing the phosphate concentration in the culture medium.

Animals↗
PubMed1981

The effects of endothelial cell-conditioned media on the proliferation of aortic smooth muscle cells and 3T3 cells in culture.

Normal aortic endothelial cells cultured in serum-free medium elaborate a factor(s) which cause the proliferation of smooth muscle cells or 3T3 cells grown in medium containing plasma-derived serum. For smooth muscle cell growth plasma factors are required in addition to the endothelial cell-conditioned medium whereas 3T3 cells will grow in the presence of endothelial cell-conditioned medium alone. Injured, growing cultures of endothelial cells do not appear to elaborate any more of the mitogen than normal, quiescent endothelial cells. The mitogenic factor is as yet poorly characterized but is heat stable at 56 degree C for 30 minutes. These results suggest that endothelial cell-smooth muscle cell interactions may contribute to the intimal smooth muscle cell proliferation which is a characteristic of the atherosclerotic lesion.

Animals↗
PubMed1984

Endogenous hyaluronate-cell surface interactions in 3T3 and simian virus-transformed 3T3 cells.

3T3 cells have a large, pericellular coat which contains 30 times more hyaluronate than the amount of cell surface hyaluronate associated with simian virus 40-transformed 3T3 (SV-3T3) cells. On the other hand, SV-3T3 cells have high affinity binding sites for exogenously added hyaluronate, whereas 3T3 cells have much lower affinity sites. Removal of cell surface hyaluronate from SV-3T3 cells by treatment with hyaluronidase caused a reproducible increase in their maximum binding capacity for exogenous hyaluronate but no significant change in binding affinity or specificity. For 3T3 cells, however, the maximum amount of binding decreased and the affinity of binding increased after hyaluronidase treatment. When endogenous cell surface hyaluronate was labeled metabolically and then the cells incubated in the presence of exogenous unlabeled hyaluronate, the labeled cell surface hyaluronate was quantitatively displaced from the SV-3T3 cells but was not displaced from the 3T3 cells. Chondroitin sulfate and heparin did not displace cell surface hyaluronate from either cell type. Membranes isolated from SV-3T3 cells bound hyaluronate specifically and with high affinity, whereas membranes from 3T3 cells did not consistently bind a significant amount of hyaluronate. We conclude from these studies that the retention of endogenous hyaluronate on the surface of SV-3T3 cells is mediated by binding sites similar to those detected by the addition of exogenous hyaluronate, and the mechanism of retention of endogenous hyaluronate on the surface of 3T3 cells differs from SV-3T3 cells.

Animals↗
PubMed2002

Human adrenocortical cell xenotransplantation: model of cotransplantation of human adrenocortical cells and 3T3 cells in scid mice to form vascularized functional tissue and prevent adrenal insufficiency.

To establish an experimental model for replacement of endocrine organ function by xenotransplantation, human adrenocortical cells from postnatal donors were transplanted beneath the kidney capsule of adrenalectomized scidmice together with mitomycin C-treated 3T3 cells that secrete FGF. Adrenocortical cells from seven donors, male and female, ranging from 6-50 years of age, were used. 12 of 13 animals survived > 16 days following surgery. After 50 days they were sacrified to allow assessment of the histology and ultrastructure of tissue formed from the transplanted cells. Only 1 of 23 adrenalectomized sham-operated animals survived > 16 days. In all surviving animals, vascularized adrenocortical tissue formed at the site of transplantation. Cortisol, the normal human glucocorticoid, was present in the plasma of these animals, replacing corticosterone, the mouse glucocorticoid. Some animals, but not most, had measurable aldosterone. The tissue formed from the transplanted cells showed histological and ultrastructural features of normal adrenal cortex. Mitochondria had tubulo-vesicular cristae and there were prominent microvilli between cells. Tissues had a well-developed vasculature, sometimes with large sinusoidal vessels. Proliferation in the transplant tissues was very low. These results show that tissue formed from transplanted human adrenocortical cells is able to replace the essential functions of the adrenal gland in scid mice. This demonstrates that transplanted human endocrine cells can functionally replace a surgically removed endocrine organ in a host animal.

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↗
PubMed1984

Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells.

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short- and long-lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA-3T3) or Rous sarcoma virus (RS-3T3), degraded short- and long-lived proteins at similar rates. Simian virus 40 (SV-3T3)- and benzo(a)pyrene (BP-3T3)-transformed cells had slightly lower rates of degradation of both short- and long-lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long-lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long-lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.

Animals↗
PubMed1977

FITC-labelled antibody staining of tropomyosin-containing fibrils in smooth, cardiac and skeletal muscle cells, prefusion myoblasts, fibroblasts, endothelial cells and 3T3 cells in culture.

FITC-labelled antibodies to purified chicken gizzard smooth muscle tropomyosin were prepared and used to stain muscle and non-muscle cells in culture. Skeletal muscle myoblasts stained both diffusely throughout the cytoplasm and in fine filamentous structures. Once myotubes developed the staining was localized exclusively in the I-band region of the myofibrils. Similarly, cardiac muscle cells stained in the I-band alone. Primary and subcultured smooth muscle cells, irrespective of their state of differentiation, stained exclusively in long, straight fibrils. The staining of the fibrils was interrupted with stained regions 1-2 micrometer long and unstained spacings 0.5 micrometer. Interrupted fibrils were also observed in fibroblasts and endothelial cells, however their staining reaction was very weak (almost indistinguishable from that with pre-immune serum) and they were few in number. 3T3 cells demonstrated moderate staining in interrupted fibrils. Sheaths of very fine fibrils staining with a similar intensity were also found throught the cytoplasm. Interruptions in these fine fibrils were often aligned to give the whole cell a striated appearance. Sheaths of fibrils were not found in the other cell types studied.

Animals↗
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↗
PubMed1981

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines.

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.

Affinity Labels↗
PubMed1984

Transformation-associated changes in nuclear-coded mitochondrial proteins in 3T3 cells and SV40-transformed 3T3 cells.

Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased amounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-coded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33 degrees C) compared to the restrictive temperature (39.5 degrees C).

Animals↗
PubMed1976

Population density and regulation of cell division in 3T3 cells. I. Inorganic phosphate levels, uptake and release.

Triggering mechanisms for initiating density dependent inhibition of cell division in 3T3 cell monolayers are activated approximately two to three population doublings prior to cessation of cell division at monolayer confluency. This activation occurs at a critical contact cell density of approximately 8 X 10(3) cells/cm2. During this period there are selective controls on transport and storage of required low molecular weight nutrients. A possible correlation between orthophosphate and rates of cell division has been investigated. We have demonstrated a relationship between cellular concentrations of orthophosphate and initiation of density dependent inhibition of cell division. Prior to critical intercellular contact, the [Pi] in 3T3 is 10 mM. During critical contact, this concentration is quickly reduced to approximately 2 mM and remains at this concentration to confluency. Similar alterations do not occur in Py 3T3 cells, which maintain a concentration of approximately 2 mM Pi regardless of cell density. After confluent 3T3 cells are released from inhibition of cell division the [Pi] must increase several-fold before DNA synthesis commences. These are physiological changes in 3T3 cellular [Pi] as a function of cell density, and cannot be attributed to nutrient depletion, altered transport of Pi into the cell, increased [ATP], or increased [PPi] levels. The controlled modulation of [Pi] may regulate glycolysis and coordinate counter-ion changes (Ca++) may regulate mitochondrial activity.

Adenosine Triphosphate↗
PubMed1979

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction.

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: 1) In both cases the cells are arrested in the G1 portion of the cell cycle; 2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.

Animals↗
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↗
PubMed2001

The effect of heat shock on amino acid transport and cell volume in 3T3 cells.

In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44 degrees C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response.

3T3 Cells↗
PubMed1999

bcl-X(S)-induced cell death in 3T3 cells does not require or induce caspase activation.

Using a tetracycline-regulated expression system, we have shown that expression of bcl-X(s) is sufficient to induce acute cell death in 3T3 cells, and that the manner in which these cells die is both morphologically and biochemically different from Fas/CD95-induced apoptosis. bcl-X(s) expression causes loss of the inner mitochondrial membrane potential (deltapsim) but does not induce caspase activation. Loss of viability, as determined by mitochondrial function and ethidium bromide exclusion, was not inhibited by the broad-spectrum caspase inhibitor zVAD-fmk or by expression of a dominant negative caspase 9 (9DN). However, zVAD-fmk was efficacious in inhibiting cell death triggered by an activating anti-Fas/CD95 antibody. In addition, bcl-X(s) does not possess the 5th and 6th alpha-helices (thought to be the membrane-spanning domains in bcl-2, bcl-X(L), and bax) and, therefore, should not be able to form membrane channels, thus eliminating this possible mechanism of action. The finding that bcl-X(s) kills 3T3 cells without caspase activation, along with the absence of membrane spanning domains in bcl-X(s), may, therefore, represent a novel cell death pathway for the pro-death bcl-2 family members.

3T3 Cells↗
PubMed1979

Cell cycle control by Ca++-ions in mouse 3T3 cells and in transformed 3T3 cells.

Total cellular calcium levels do not change when 3T3-4a cells stop proliferating due to serum depletion, or when serum-arrested quiescent cells are incubated for up to 44 hours in calcium-deficient medium (approximately 10 micrometer Ca++). Upon stimulation with dialyzed serum cells enter S and progress through at least one cycle even at extremely low calcium levels in the culture medium (greater than or equal to 10 micrometer). Cells divide until a final cell density is attained which is proportional to the calcium concentration in the medium and cells reversibly arrest in G1. Cells which arrested in G1 in medium containing less than or equal to 26 micrometer Ca++ in the presence of excess serum can be stimulated to enter S in response to added calcium after a prereplicative phase of 14 to 16 hours. Serum does not affect 45Ca-uptake in these cells. Benzo[a]pyrene transformed 3T3 (BP3T3) cells have a 100--200 times lower Ca++-requirement than 3T3 cells but arrest in G1 at low Ca++ levels. In contrast, SV40-virus transformed 3T3 (SV3T3) cells that grow without restriction in monolayer cultures have even lower Ca++-requirements for growth than BP3T3 cells and have no Ca++-sensitive restriction point. Therefore, 3T3 and BP3T3 cells have retained the capacity to sense intracellular Ca++-pool sizes and to arrest in G1 at subthreshold cellular Ca++-levels.

Calcium↗