Cellular content of ribosomal RNA in relation to the progression and competence signals governing proliferation of 3T3 and SV40-3T3 cells

The method for differential fluorescence staining of cellular RNA and DNA by acridine orange (AO) was optimized for 3T3 and SV40-3T3 cells. Cellular contents of DNA and of ribosomal RNA (rRNA) were determined by dual-channel flow cytometry during cell-density-dependent proliferation and after stimulation of quiescent cells. With increasing density of 3T3 cells, cellular content of rRNA decreases by about 60%, whereas SV40-3T3 cells do not exhibit a significant dependence of rRNA content on cell density. 3T3 cells stimulated early after becoming quiescent resume reaccumulation of rRNA after a delay of only 4 h, whereas cells maintained at quiescence for several days exhibit a delay of about 12 h before a significant rise of rRNA is observed. The extent of rise of cellular rRNA content after different regimens of stimulation of quiescent 3T3 cells does not correlate well with the fraction of cells entering the cell cycle. These and other reported instances of discordance between rRNA content and stimulation into the cell cycle are resolved by showing that of the two signals governing entry into the cell cycle only the progression signal, but not the competence signal is associated with reaccumulation of cellular rRNA. The present results are consistent with the progression function being in essence the achievement of a threshold number of ribosomes per cell, which in conjunction with the competence signal is sufficient for initiation of the cell cycle.     

Properties and Functions of a New Nucleolar Protein,Surf-6, in 3T3 Mouse Cells

The localization of the specific protein Surf-6 from nucleoli of eukaryotic cells in mitosis and its sensitivity to the treatment of cells with RNase A and DNase I in situ were studied. It was shown that, in interphase nucleoli of 3T3 mouse cells, Surf-6 is probably associated with RNA and practically is not associated with DNA. In mitosis, Surf-6 appears in forming nucleoli after the known RNA-binding proteins fibrillarin and B23/nucleofozmin, which are involved in the early and late stages of the assembly of ribosomal particles, respectively. These observations and the regularities of migration of early and late proteins of ribosome assembly to nucleoli in the telophase of mitosis led us to the presumption that Surf-6 is involved in the terminal stages of the assembly of ribosomal particles in murine cells. An immunoblot analysis of the Surf-6 content in synchronized 3T3 cells showed for the first time that Surf-6 is present at all stages of the cell cycle but its content markedly decreases when cells enter the G0 period. Conversely, the activation of cells for proliferation is accompanied by an increase in the Surf-6 content. These observations allow one to regard Surf-6 as a marker of the cell proliferative state and suggest its implication in the regulation of the cell cycle.     

Properties and functions of a new nucleolar protein, Surf-6, in 3T3 mouse cells

The localization of the specific protein Surf-6 from nucleoli of eukaryotic cells in mitosis and its sensitivity to the treatment of cells with RNase A and DNase I in situ were studied. It was shown that, in interphase nucleoli of 3T3 mouse cells, Surf-6 is probably associated with RNA and practically is not associated with DNA. In mitosis, Surf-6 appears in forming nucleoli after the known RNA-binding proteins fibrillarin and B23/nucleofozmin, which are involved in the early and late stages of the assembly of ribosomal particles, respectively. These observations and the regularities of migration of early and late proteins of ribosome assembly to nucleoli in the telophase of mitosis led us to the presumption that Surf-6 is involved in the terminal stages of the assembly of ribosomal particles in murine cells. An immunoblot analysis of the Surf-6 content in synchronized 3T3 cells showed for the first time that Surf-6 is present at all stages of the cell cycle but its content markedly decreases when cells enter the G0 period. Conversely, the activation of cells for proliferation is accompanied by an increase in the Surf-6 content. These observations allow one to regard Surf-6 as a marker of the cell proliferative state and suggest its implication in the regulation of the cell cycle. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.     

2-amino-isobutyric acid and 3-O-methyl-D-glucose transport in 3T3, SV 40-transformed 3T3 and revertant cell lines.

In order to further investigate the connection between transport and growth control, 3T3 cells, SV40 transformed 3T3 cells (SV101), and three revertant cell lines derived from SV101 which have regained certain manifestations of growth control were used. Transport rates of 2-amino-isobutyric acid and 3-O-methyl-D-glucose were measured in sparse, confluent, serum-starved, and serum-stimulated cultures. As shown before, cessation of 3T3 cell growth in G0 under conditions of confluence or serum deprivation was associated with reduced rates of transport for both compounds, whereas the density and serum dependence of growth and transport was largely eliminated in SV101. The density revertant F1SV101, which has regained density regulation of growth similar to 3T3 cells, has also regained density regulation of transport. Neither growth nor transport were serum dependent. The serum revertants AgammaSV7 and LsSV6 have regained both density and serum regulation of growth, but not according to the original mechanism of 3T3 cells of entry into a Go state. Transport was high under conditions of confluence or serum deprivation. Thus for these cells rates of transport were not reduced simply as a consequences of slower cell growth nor were low transport rates responsible for growth arrest. The data are consistent with the possibility that growth arrest specifically in the G0 state could shut off a number of cellular activities, including transport.     

Only one of the two signals required for initiation of the cell cycle is associated with cellular accumulation of ribosomal RNA

Two intracellular functions are elicited by the serum component in culture media in order to initiate the cell cycle: "competence" and "progression". Although both functions have to be present simultaneously for start of cell division, it is shown here for Swiss 3T3 cells that only one of them, the progression signal, is associated with reaccumulation of ribosomal RNA. This result points to the cellular complement of ribosomes as one of the limiting parameters for cell division.     

Uncoupling of RNA and DNA synthesis after plasma stimulation of G0-arrested BALB/C-3T3 cells.

The addition of whole serum to G0-arrested, confluent Balb/c-3T3 cells induces them to progress through G1 and synthesize DNA after a 12-h lag period. Prior to the onset of DNA synthesis, RNA is synthesized and RNA content increases. Serum has been fractionated into two sets of growth factors: a platelet-derived growth factor present in heat-treated (100 degrees C) platelet extracts and platelet-poor plasma. Addition of whole serum, platelet-derived growth factor or platelet-poor plasma induces quiescent cells to increase their cytoplasmic RNA content, but the cells treated with platelet-poor plasma do not synthesize DNA. Messenger RNA content increases within 2 h after stimulation with whole serum or platelet-poor plasma, and after 18 h, mRNA has accumulated to a greater degree than rRNA.     

Induction of replicative senescence by 5-azacytidine: fundamental cell kinetic differences between human diploid fibroblasts and NIH-3T3 cells

Abstract. To analyse the putative role of methylation of cytosine residues in the nuclear DNA as a regulatory step during cellular ageing, we incubated ageing human amniotic fluid derived fibroblast-like cells and non-ageing NIH-3T3 cells with 5-azacytidine. BrdUrd/Hoechst and acridine orange (AO) flow cytometry was used to compare the effects of the base analogue on cell proliferation and cell differentiation. In NIH-3T3 cultures, 96 h exposures to 4 μM 5-azacytidine caused diminished cell proliferation due to cell arrest in the G₁ compartments of the second and third cell cycles of serum stimulated cells. The exit from the G₀/G₁ compartment was not affected. The 5-azacytidine induced cell kinetic disturbances were unstable in NIH-3T3 cultures, such that pre-treated cells reverted to normal cell cycle transit within 2–3 days after termination of treatment. In contrast, 5-azacytidine pre-treated amniotic fluid derived fibroblast-like cell cultures showed persistently elevated G₂ phase arrests and delayed G₀/G₁ phase exit kinetics, which explain the premature cessation of proliferation observed in these primary cultures. In both cell systems, 5-azacytidine exposed cultures showed elevated numbers of G₁ phase cells with increased RNA content as revealed by AO flow cytometry. Again, this effect was reversible in NIH-3T3 cells but not in amniotic fluid derived fibroblast-like cells. These contrasting responses to 5-azacytidine are likely to reflect intrinsic differences in methylation patterns or de novo methylase activity between ageing cell strains and non-ageing cell lines.     

Study of the mode of action of ribosomal vaccines from Klebsiella and Streptococcus pneumoniae and their ribonucleic and protein fractions using passive immunization

The vaccinating potency of ribosomal fractions of Klebsiella pneumoniae and Streptococcus pneumoniae as well as their ribosomal RNA and protein fractions has been studied with respect to their ability to induce cellular or humoral immunity. Experiments with transfer of serum or spleen cells from vaccinated animals have shown that anti-Klebsiella immunity is essentially cellular, while streptococcal immunity is exclusively humoral. Results have been discussed as a function of differential results for the various fractions under study.     

Effect of T4 infection on initiation of protein synthesis and messenger specificity of initiation factor 3

No alteration in the messenger specificity of initiation factor 3 (IF-3) is observed upon T4 phage infection of several strains of Escherichia coli. IF-3 present in the 1.0 m NH₄Cl washes of ribosomes from T4-infected cells supports the translation of f2 RNA and T4 late mRNA with the same degree of efficiency as the IF-3 in the ribosomal washes obtained from uninfected cells. At high concentrations the ribosomal washes obtained from T4-infected cells are more inhibitory for both f2 RNA- and T4 late mRNA-directed protein synthesis than the ribosomal washes from uninfected cells. Furthermore, this increased inhibition is also observed in the poly(U)-directed synthesis of polyphenylalanine. These data suggest that translational controls exerted at the level of IF-3 probably do not account for the alterations in protein synthesis observed upon T4 infection.     

Uridine transport and phosphorylation in 3T3 and CHO cells with induced cell proliferation

In the serum-deficient medium, the cultured Swiss 3T3 and CHO-K1 cells transit to the resting state. The rates of uridine phosphorylation and RNA synthesis in these cells are lowered. After the addition of fresh medium containing 10% serum, cell proliferation is induced. At the early stage of cell entrance into the cell cycle uridine transport through the cell plasma membrane remains unchanged in both cultures. During the 1st hour after serum addition the rate of uridine phosphorylation increases in 3T3 cells to remain practically unchanged in CHO-K1 cells. At this time, RNA synthesis in cells increases almost twofold in both cultures. A correlation has been revealed between the initial level of uridine phosphorylation in 3T3 cells and the percentage of its maximal elevation after serum addition. No such a correlation was observed for CHO-K1 cells. The rate of uridine phosphorylation in arrested CHO-K1 cells is higher than that in 3T3 cells. It has been included that the initial increase of uridine phosphorylation during serum stimulation may be not obligatory for all cell types, but depends on the level of uridine kinase activity before serum addition to the cells.     

Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

We have used an antibody against RNA polymerase I to investigate the role of rRNA synthesis and/or accumulation in the control of cell proliferation. The antibody was microinjected directly into the nuclei of quiescent Swiss 3T3 cells that were subsequently stimulated with serum. Under the experimental conditions used, the microinjection of the antibody against RNA polymerase I (RNA pol I) caused a 50–70% decrease in nucleolar RNA synthesis that lasted for at least 17 h, a >90% inhibition in the accumulation of nucleolar RNA, and a 70% inhibition in the accumulation of total cellular RNA. A control IgG, similarly microinjected into Swiss 3T3 cells had no inhibitory effect on either the synthesis or accumulation of nucleolar and cellular RNA. Despite the dramatic effect on the synthesis and accumulation of ribosomal RNA (rRNA) the antibody against RNA pol I was totally ineffective in inhibiting the entry into S phase of serum-stimulated Swiss 3T3 cells. Cells depleted of cellular RNA by metaphase arrest also entered S phase with subnormal amounts of cellular RNA. The results of these experiments clearly indicate that a normal rate of nucleolar RNA synthesis, and a normal rate of accumulation of total cellular RNA are not a prerequisite for the entry of cells into S phase.     

Aluminum ions stimulate DNA synthesis in quiescent cultures of Swiss 3T3 and 3T6 cells

Micromolar concentrations of AI3+ are shown to be strongly mitogenic for quiescent cultures of Swiss 3T3 and 3T6 cells. AI3+ caused a striking shift in the dose-response curve for the effect of fetal bovine serum on 3H-thymidine incorporation. In the absence of serum the mitogenic effect of aluminum was greatly potentiated by insulin or cholera toxin, but not epidermal growth factor or 12-0-tetradecanoyl-phorbol-13-acetate. The stimulation of DNA synthesis was maximal by 15-20 microM AI3+ X AI3+ at 100 microM had no inhibitory effect on DNA synthesis. AI3+ had no significant effect on cellular cyclic adenosine monophosphate in the presence or absence of insulin or an inhibitor of cyclic nucleotide phosphodiesterases.     

Apparent heterogeneity in the response of quiescent swiss 3T3 cells to serum growth factors: implications for the transition probability model and parallels with "cellular senescence" and "competence"

When subconfluent, Swiss 3T3 cells made quiescent by serum deprivation are stimulated with low concentrations of serum (ca. 1%), only a proportion of them (roughly 50%) enter S phase despite daily replacement with fresh, low-serum medium. The cells that fail to enter S phase are not incapable of doing so, since most of them initiate DNA synthesis after transfer to 10% serum. It would appear that individual cells vary in their growth factor requirements. Using time-lapse cinemicroscopy a few of the cells that respond to low serum were seen to give rise to several generations of progeny, while the majority of cells failed to divide at all, or divided once at most. Despite this, differences between cells in growth factor requirements do not seem to be heritable in the long term, since attempts to enrich for responding cells by prolonged culture in 1% serum have been unsuccessful. Rather, it would appear that the capacity to respond to low serum is an unstable property lost after a few generations in low serum. The loss of responsiveness shows parallels with "cellular senescence" and could conceivably result from decay of the platelet-derived growth factor-induced state of "competence." But regardless of why some cells respond to low serum while others do not, it is clear that the kinetics of entry into S phase after serum stimulation of quiescent 3T3 cells are not strictly first-order, since the labelling index plateaus after roughly 3 days at values substantially below 100%. As such, the kinetics, though not contradicting the transition probability model, cannot be taken to support it as was previously thought.     

TNF triggers mitogenic signals in NIH 3T3 cells but induces apoptosis when the cell cycle is blocked

Tumor necrosis factor (TNF) is known to be a mediator of a variety of cellular responses including apoptotic death or proliferation depending on the target cell and the environmental conditions. We show here that TNF triggers both growth and death signals in NIH 3T3 murine fibroblasts. In cells arrested in G(0) by serum deprivation, TNF drives approximately 50% of them to enter the cell cycle, but kills the cells that remain quiescent. The presence of serum prevents toxic effects of TNF, suggesting that TNF can cooperate to drive cells through the cell cycle, but is unable to do so by itself and alternatively it triggers death signals in cells unable to proliferate. Interestingly, TNF induces a similar toxic effect in cells forced to stay at the G(1)/S border, S or M phases. We have explored the TNF apoptotic pathway in arrested cells. This mechanism is not due to the loss of the anti-apoptotic capacity of NFkappaB and is mediated by mitochondria since Bcl-2 overexpression partially inhibits cell death. There are, however, interesting differences in the kinetics of mitochondrial events which indicate that this form of sensitization to TNF leads to an apoptotic mechanism different from that observed after sensitization by RNA synthesis inhibition.     

Calcium transport and exchange in mouse 3T3 and SV40-3T3 cells

The kinetics of Ca++ uptake have been evaluated in 3T3 and SV40-3T3 mouse cells. The data reveal at least two exchangeable cellular compartments in the 3T3 and SV40-3T3 cell over a 50-min exposure to 45Ca++. A rapidly exchanging compartment may represent surface-membrane-localized Ca++ whereas a more slowly exchanging compartment is presumably intracellular. The transition of the 3T3 cell from exponential growth (at 3 day's incubation) to quiescence (at 7 days) is characterized by a 7.5-fold increase in the size of the fast component. Quiescence of the 3T3 cell is also characterized by a 3.2-fold increase in the unidirectional Ca++ influx into the slowly exchanging compartment and a 3.6-fold increase in its size. The increase in size of the slow compartment at quiescence may result from a redistribution of intracellular Ca++ to a more readily exchangeable compartment, possibly reflecting a release of previously bound Ca++. In contrast, no significant change in any of these parameters is observed in the proliferatively active SV40-3T3 cells after corresponding period of incubation, even though these cells attained higher growth densities and underwent postconfluence.