Lysosomal inhibitors stimulate resting NIH 3T3 cells to proliferate

Lysosomal inhibitors (amino acid methyl esters) and platelet-derived growth factor stimulate resting NIH 3T3 cells to enter the S period. Incubation of cells in medium containing lysosomal inhibitors causes an increase in protein accumulation and does not disrupt lysosomes. The results indicate that proliferative homeostasis depends partially on the metabolic status of the cell and that catabolic processes activated in resting cells negatively influence prereplicative reactions.     

The stimulation of the transition of resting NIH 3T3 cells to proliferation under exposure to lysosomal inhibitors

Lysosomal inhibitors (amino acid methyl esters) and the platelet-derived growth factor stimulate the resting NIH 3T3 cells to enter the S period. Incubation of cells in the medium containing lysosomal inhibitors enhances protein accumulation and seemingly does not disrupt lysosomes. These results indicate that proliferative homeostasis depends on the metabolic status of cells and that catabolic processes activated in resting cells negatively influence the prereplicative reactions.     

Protein synthesis in 3T3 cells stimulated to proliferate at various rates

Reproducible conditions were defined for using rates of leucine incorporation as a valid measure of rates of de novo protein synthesis in mouse 3T3 cells. Upon stimulation of quiescent cultures, rates of de novo synthesis of proteins increased and pool levels of amino acids decreased in proportion to the concentration of serum in the stimulating medium. Rates of de novo protein synthesis (per cell) exhibited a biphasic pattern of increase. These rates approached a plateau value at the end of the lag phase and increased again as cells entered S phase. This pattern of behaviour helps to explain the observed relationships between cell growth (increase in mass) and cell proliferation (increase in cell number).     

Inability of human alveolar macrophages to stimulate resting T cells correlates with decreased antigen-specific T cell-macrophage binding

Alveolar macrophages (AM) from the majority of human volunteers are defective antigen presenting cells (APC) in T cell proliferation assays despite the display by the cells of HLA-D region antigens. We have confirmed that AM secrete relatively little interleukin 1 (IL 1), but addition of exogenous IL 1 did not improve the capacity of AM to initiate antigen-induced T cell proliferation. Thus, the presence of HLA-D region antigens and IL 1 is not sufficient to enable an accessory cell to act as an APC. We developed a T cell-accessory cell binding assay to investigate early events in T cell activation. AM demonstrated a diminished capacity as compared with monocytes to bind antigen-specific T cell clones. Nevertheless, AM often induced proliferation of T cell clones as effectively as monocytes, indicating that antigen display was intact. The inefficiency of AM in bind T cell clones correlated with their reduced capacity to induce resting T cells to express IL 2 receptors, secrete IL 2, and proliferate in response to antigen. Indirect immunofluorescence established that similar percentages of AM and monocytes expressed LFA molecules, but the density of the molecules was greater on monocytes than AM. A role for LFA antigens in the physical binding of T cells to monocytes was demonstrated by blocking antigen-specific binding with a monoclonal antibody to LFA-1 antigen. LFA-1 antibody also blocked the low levels of specific binding between AM and T cell clones, indicating that LFA-1-ligand interactions were operative between these two cell types. These studies indicate that there are critical cell membrane characteristics that promote binding of T cells to APC in addition to T cell receptor-antigen interactions. This combination of nonspecific and specific interactions leads to avid T cell-APC binding that may be essential for activation of resting T cells. Furthermore, we postulate that the failure to AM to act as effective APC results from an inability to bind T cells efficiently.     

Intestinal epithelial cells from inflammatory bowel disease patients preferentially stimulate CD4+ T cells to proliferate and secrete interferon-gamma

Previous studies have suggested that intestinal epithelial cells (IECs) have the capacity to function as nonprofessional antigen presenting cells that in the normal state preferentially activate CD8+ T cells. However, under pathological conditions, such as those found in inflammatory bowel disease (IBD), persistent activation of CD4+ T cells is seen. The aim of this study was to determine whether the IBD IECs contribute to CD4+ T cell activation. Freshly isolated human IECs were obtained from surgical specimens of patients with or without IBD and cocultured with autologous or allogeneic peripheral blood T lymphocytes. Cocultures of normal T cells and IECs derived from IBD patients resulted in the preferential activation of CD4+ T cell proliferation that was associated with significant IFN-gamma, but not IL-2, secretion. Cytokine secretion and CD4+ T cell proliferation was inhibited by pretreatment of the IBD IECs with the anti-DR MAb L243. In contrast, normal IECs stimulated the proliferation and cytokine secretion by CD4+ T cells to a significantly lesser degree than IBD IECs. Furthermore, blockade of human leukocyte antigen-DR had a lesser effect in the normal IEC-CD4+ T cell cocultures. We conclude that IECs can contribute to the ongoing CD4+ T cell activation seen in IBD. We suggest that the apparent differences between the secreted levels of IFN-gamma indicate that it may play a dual role in intestinal homeostasis, in which low levels contribute to physiological inflammation whereas higher levels are associated with an uncontrolled inflammatory state.     

The surface topography of the NIH 3T3 line in proliferating and resting states

The surface topography of resting (serum-deprived) and proliferating (serum-stimulated) NIH 3T3 monolayer cell cultures has been examined by scanning electron microscopy. During G1 and S periods of the cell cycle the cells exhibited well pronounced surface microvilli localized mainly in the perinuclear zone, whereas serum deprivation led to a relatively smooth surface with few microvilli. The observed differences are not likely to be associated with the degree of cell spreading over the substrate, rather reflecting metabolic peculiarities of proliferating and resting cells.     

DNA synthesis in heterokaryons obtained by the fusion of proliferating cells of the NIH 3T3 line with cells in transition to the resting state

NIH 3T3 mouse fibroblasts cultured in the medium containing 0.5% serum for 2, 4, 8, 24, 48 and 72 hours were fused to cells stimulated for proliferation by replacing the medium with a fresh one containing 10% serum; DNA synthesis was studied in monokaryons, homo- and heterodikaryons using radioautography and double-labelling technique. Cells that were cultured in the medium with a low serum content for 72 hours exerted their inhibitory effect on the entry of stimulated nuclei into the S period in heterodikaryons, whereas cells deprived of serum for shorter periods failed to exert this effect. It thus appears that in cell fusion studies with NIH 3T3 cells, the effects of endogenous growth inhibitor(s) produced by resting cells may be revealed not earlier than by the 3rd day of serum depletion.     

Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate

Whole blood serum is widely recognized as essential for the growth of diploid cells in culture. Dermal fibroblasts and arterial smooth muscle cells fail to proliferate in culture in the presence of serum derived from platelet-poor plasma. Platelet-poor plasma serum is capable of maintaining monkey arterial smooth muscle cells quiescent in culture at either low (1.5 x 10(3)) or high (2.0 x 10(4)) population densities. The proportion of cell traversing the cell cycle under these conditions was approximately 3%. Equal numbers of quiescent smooth muscle cells initiated DNA synthesis and cell division when treated with whole blood serum or with an equivalent quantity of platelet-poor plasma serum supplemented with a factor(s) derived from a supernate obtained after exposure of human platelets to purified thrombin in vitro.     

Inhibition of epidermal growth factor receptor functions by tyrosine kinase inhibitors in NIH3T3 cells.

Epidermal growth factor (EGF) induces transformed phenotypes in EGF receptor-overexpressing NIH3T3 (ER12) cells. Tyrosine kinase inhibitors such as erbstatin and its stable analogue methyl 2,5-dihydroxycinnamate inhibited the EGF-induced phenotypic changes in these cells; while 5'-O-methylerbstatin, an inactive analogue, did not. Methyl 2,5-dihydroxycinnamate inhibited intracellular tyrosine kinase activity in EGF-treated ER12 cells. Methyl 2,5-dihydroxycinnamate also delayed the EGF-induced DNA synthesis from the quiescent phase ER12 cells without showing irreversible cytotoxicity. It inhibited the DNA synthesis most efficiently at the early G1 phase. Thus, tyrosine kinase inhibitors may modify malignant phenotypes in EGF receptor-overexpressing neoplasms.     

Cell cycle-related differences in susceptibility of NIH/3T3 cells to ribonucleases

Microinjection of Onconase or RNase A into NIH/3T3 cells was used to study the intracellular actions of these two proteins. Onconase preferentially killed actively growing cells in both microinjection and cell culture experiments. Moreover, agents that increased the number of cells in S phase such as serum or microinjected signal transduction mediators (Ras, protein kinase C, and mitogen-activated protein kinase) enhanced Onconase cytotoxicity. Conversely, agents that decreased these proliferative pathways (dibutyryl cAMP and protein kinase A) correspondingly diminished Onconase cytotoxicity in microinjection experiments. These results were also mimicked in cell culture experiments since log-phase v-ras-transformed NIH/3T3 cells were more sensitive to Onconase (IC50 of 7 microg/ml) than parental NIH/3T3 fibroblasts (IC50 of 40 microg/ml). Based on those data we postulated that Onconase-mediated cell death in NIH/3T3 cells was related to events occurring at two or more points in the cell cycle preferentially associated with late G1/S and S phases. In contrast, quiescent NIH/3T3 cells were more sensitive to microinjected RNase A than log phase cells and positive mediators of proliferative signal transduction did not enhance RNase A-mediated cytotoxicity. Taken together, these results demonstrate that these two RNases use different pathways and/or mechanisms to elicit cytotoxic responses in NIH/3T3 cells. Predictions formulated from these studies can be tested for relevance to RNase actions in different target tumor 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.     

Selective cytotoxicity of azatyrosinamides against ras-transformed NIH 3T3 cells

This study aims to develop novel azatyrosinamide compounds structurally modified from ras-specific antioncogenic azatyrosine. Analogues 4-15 were prepared and their inhibition on the growth of wild-type and ras-transformed NIH 3T3 cell lines was compared. Compound 12 was found to be the most active with IC50 16.5+/-2.2 microM which is 458-fold more potent than that of azatyrosine. The selective toxicity, defined as IC(50 wild-type)/IC(50 ras-transformed) for this compound was 138.5.     

Simulated microgravity influences circadian rhythm of NIH3T3 cells

Gravity heavily influences living organisms on earth including their circadian rhythm, which is fundamentally important for coordinately physiology in organisms as diverse as cyanobacteria, fungus and humans. Numerous researches have revealed that microgravity in outer space can affect circadian rhythm of astronauts and rodent animals, but the mechanism remains unknown. Using rotary cell culture system to simulate microgravity environment, we investigated the role of simulated microgravity in regulating the circadian rhythm of NIH3T3 cells. Our experiments found that simulated microgravity can not only influence the mRNA level of some core circadian genes, but also modify the circadian rhythm of Per1 and Per2 synchronized after phorbol myristate acetate treatment. Remarkably, MEK/ERK pathway was transiently activated after a 2-h simulated microgravity treatment, with a significant upregulation of Kras, Raf1 and p-ERK1/ERK2. Moreover, U0126, a selective inhibitor of MEK/ERK pathway, could disrupt the circadian rhythm of Per1 and Per2 synchronized after simulated microgravity treatment. Together, our results unveil that simulated microgravity could act like a zeitgeber to influence the circadian rhythm of NIH3T3 by acting on MEK/ERK pathway, indicating that MEK/ERK pathway may act as a bridge which connects cells mechanotransduction pathway and circadian rhythm regulation.     

Cytotoxicity of the isoflavone genistein in NIH 3T3 cells

Genistein is one of the naturally occurring isoflavones present in plants such as soybeans and is commonly found in a variety of human foods. A number of studies indicated that this class of compounds exerts anticancerogenic and antimutagenic effects in various in vitro systems and in vivo animal models. We studied the effects of genistein on NIH 3T3 cells in in vitro models. The isoflavone genistein has been identified as having antiproliferative and apoptotic effects on various malignant cell types derived from solid tumors. Therefore, the cytotoxic and apoptotic properties of this compound were studied by MTT assay and Hoechst 33258/propidium iodide staining technique. The morphological changes of cells were examined in inverted fluorescent microscope. The oxidation of protein thiol groups and thiobarbituric-acid-reactive species (TBARS) was also determined. The cells were exposed to different concentrations of genistein (0-90 microM) after 24 h of incubation. The results revealed that genistein in concentrations higher than 20 microM significantly reduced cell viability, caused cell morphological changes and induced apoptotic and necrotic cell death. Oxidative modification of protein increased in the cells exposed to genistein in a dose- and time-dependent manner. In conclusion, our preliminary in vitro studies demonstrate the damaging effects of genistein on the mouse embryonic fibroblast cell line.     

Image analysis of Feulgen-stained c-H-ras-transformed NIH/3T3 cells

Feulgen-DNA content, nuclear phenotypes, and levels of chromatin condensation were evaluated by image analysis in NIH/3T3 cells transformed with the c-H-ras oncogene of T24 cells. Three nuclear phenotypes, differing from those of untransformed control cells and defined in terms of patterns of chromatin condensation, were demonstrated microspectrophotometrically for the tumor cells. Polyploidy could only be observed in nuclei with extensive and deeply stained areas covered with condensed chromatin, i.e., only in a small fraction of the tumor cell nuclear population. The increased chromatin condensation that appeared with cell transformation affected the euchromatin zones. The image analysis provided data that, compared with those obtained in other situations involving cell transformation, could be relevant to the understanding of changes in chromatin supraorganization related to tumorigenesis and to tumor cell diagnosis.     

Transformation of NIH 3T3 cells by enhanced PAR expression

Prostate androgen regulated (PAR) is a 1038bp novel gene located on chromosome 1 in epidermal differentiation complex. The gene is ubiquitously expressed in normal tissues and is overexpressed in most of their malignant counterparts. PAR cellular function is unknown. Here we report the effect of increased PAR expression induced by transfection of PAR cDNA on NIH3T3 cell phenotype. PAR-NIH3T3 transfectants expressing 3- to 4-fold higher PAR levels compared to controls grew faster in tissue cultures, formed colonies in soft agar, and exhibited a shortening of G1 and S phases of cell cycle and formed tumors in SCID mice. Transfection of NIH3T3 cells with increased ectopic PAR expression with a 22 mer oligonucleotide in antisense orientation with PAR mRNA abrogated their ability to form colonies in soft agar. The data presented here along with our previously reported results on DU145 cells transfected with antisense PAR cDNA suggest that PAR gene behaves like a proto-oncogene.     

Both dendritic cells and macrophages can stimulate naive CD8 T cells in vivo to proliferate, develop effector function, and differentiate into memory cells

The generation of T cell immunity requires the acquisition and presentation of Ag on bone marrow-derived APCs. Dendritic cells (DC) are believed to be the most potent bone marrow-derived APCs, and the only ones that can stimulate naive T cells to productively respond to Ags. Because macrophages (Mphi) are bone marrow-derived APCs that are also found in tissues and lymphoid organs, can acquire and present Ag, and can express costimulatory molecules, we have investigated their potential to stimulate primary T cell responses in vivo. We find that both injected Mphi and DCs can migrate from peripheral tissues or blood into lymphoid organs. Moreover, injection of peptide-pulsed Mphi or DCs into mice stimulates CD8 T cells to proliferate, express effector functions including cytokine production and cytolysis, and differentiate into long-lived memory cells. Mphi and DCs stimulate T cells directly without requiring cross-presentation of Ag on host APCs. Therefore, more than one type of bone marrow-derived APC has the potential to prime T cell immunity. In contrast, another bone marrow-derived cell, the T lymphocyte, although capable of presenting Ag and homing to the T cell areas of lymphoid organs, is unable to stimulate primary responses. Because Mphi can be very abundant cells, especially at sites of infection and inflammation, they have the potential to play an important role in immune surveillance and the initiation of T cell immunity.     

Onset of DNA replication in nuclei of proliferating and resting NIH 3T3 fibroblasts following fusion

NIH 3T3 mouse fibroblasts arrested in medium containing 0.5% serum were fused with stimulated cells taken at 2-h intervals after replacing the medium with one containing 10% serum, and DNA synthesis was studied in mono-, homo- and heterokaryons using radioautography with double-labelling technique. The presence of a resting nucleus in a common cytoplasm with a stimulated nucleus from the prereplicative period has an inhibitory effect on the entry of the stimulated nucleus into the S period in medium containing either 0.5 or 10% serum, but ongoing DNA synthesis continues. After a 24-h stay in a common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterokaryons still persists, at least for 2 h following stimulation. Preincubation of resting cells with cycloheximide for 4 h abolishes their ability to suppress DNA synthesis in stimulated nuclei.The data suggest that resting cells produce an endogenous inhibitor of cell proliferation, whose formation depends upon the synthesis of protein. When stimulated, the cells can proliferate only after decreasing the level of this inhibitor. The results obtained are consistent with the idea of a negative control of cell proliferation.     

DNA synthesis in the nuclei of stimulated NIH 3T3 cells in heterodikaryons obtained by the fusion of these cells with resting cells treated with cycloheximide

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts preincubated with cycloheximide (7.5 micrograms/ml) were fused with stimulated cells taken 10 hours after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in nuclei of heterodikaryons, homodikaryons, and monokaryons, using radioautography with double-labeling technique. Preincubation of resting cells with the inhibitor of protein synthesis cycloheximide for 4, 3, 2, but not for 1 or 0.5 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei in heterodikaryons. Three hours after the removal of cycloheximide from the media, the resting cells acquire once again the inhibitory effect towards the entry of stimulated nuclei into the S-period. The data suggest that the resting cells may produce a labile endogenous inhibitor of cell proliferation, and support the idea on the active metabolic processes occurring in the resting cells.