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.     

Interrelation between cellular rRNA content and regulation of the cell cycle of normal and transformed mouse cell lines

The relation between cellular rRNA content, as a measure of cell size, and the regulation of the cell cycle has been investigated for swiss 3T3 and the spontaneously transformed swiss 3T6 cell line. It is shown that the characteristic of percent of quiescent cells stimulated into the cell cycle versus cellular rRNA content is basically different for 3T3 and 3T6 cells: 3T3 cells do not enter the cell cycle below a certain threshold of cellular rRNA content, whereas 3T6 cells start proliferation without a substantial increase of rRNA. These data are interpreted as consistent with transformation of 3T6 cells being in essence their uncoupling from the requirement of normal cells of passing over a threshold of cellular rRNA content (cell size) before initiating DNA-replication.     

Level of ribosomal RNA required for stimulation from quiescence increases during cellular aging in vitro of mammalian fibroblasts

We have investigated the relation between cell size in terms of cellular ribosomal RNA (rRNA) content and proliferation of diploid human and rat embryo fibroblasts during their aging in vitro. During phase III of the proliferative lifespan in vitro, cellular rRNA content increases by a factor of nearly 3. For very different regimes of stimulation of quiescent cells, a strict correlation was observed, between the proportion of cells stimulated and cellular rRNA content, resembling a steep threshold curve. During aging in vitro, these characteristic curves exhibit an essentially parallel shift to higher values of cellular rRNA content (to higher 'thresholds'). Upon establishment as a permanent cell line, the relation between proliferation stimulation and cellular rRNA ceases to change with further subculturing. It is suggested that the essence of transformation of fibroblasts with a myc-type of oncogenes is a reduction and stabilizing of the critical rRNA content required for proliferation.     

Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density.

Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.     

Cycloheximide or puromycin can substitute for PDGF in inducing cellular DNA synthesis in quiescent 3T3 cells

A brief exposure of quiescent (Go) Swiss 3T3 mouse fibroblasts to inhibitors of protein synthesis can replace platelet-derived growth factor in the stimulation of cellular DNA synthesis. When 3T3 cells, after a 6 hr exposure to either cycloheximide or puromycin, are incubated with platelet-poor plasma, a significant percentage of cells enters DNA synthesis. Either inhibition of protein synthesis, or platelet poor plasma by themselves are totally ineffective. A possible mechanism by which inhibitors of protein synthesis may initiate cell cycle progression is through the activation of the c-myc gene.     

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.     

Expression of c-fos in NIH3T3 cells is very low but inducible throughout the cell cycle.

It has previously been shown that the c-fos proto-oncogene is rapidly and transiently induced following growth factor stimulation of quiescent NIH3T3 mouse fibroblasts. To investigate a possible role of c-fos in growth control mechanisms we have studied its expression and inducibility during the NIH3T3 cell cycle. Two major conclusions can be drawn from this analysis. First, expression of c-fos is not cell cycle-regulated, and is barely detectable in all phases of the cycle. Second, cells at different stages of the cell cycle (except for mitosis) are as sensitive to c-fos induction by growth factors as quiescent cells. These observations suggest that induction of the c-fos gene does not play a role during the continuous cycling of NIH3T3 cells, but they are fully compatible with the hypothesis that a function of c-fos may be associated with the induction of competence in fibroblasts. Through such a function c-fos may contribute to moving cells out of the quiescent state.     

Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen.

The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis.     

G1 phase heterogeneity in exponentially growing Swiss 3T3 mouse fibroblasts

The growth rate of normal cultured Swiss 3T3 fibroblasts is function of serum concentration and the fraction of G1 cells, and hence the average residence time in G1, increases with the generation time. Serum contains two sets of factors: competence factors, essentially platelet-derived growth factor (PDGF), which induces competence in quiescent fibroblasts and prevents replicating cells from entering G0, and plasma, which allows progression. The increase in the duplication time and the duration of Gl at low serum concentration could hence be due to the fact that competence factors become limiting. The fraction of non-competent cells, operationally defined as those G1 cells unable to leave G1 in the presence of plasma alone, was determined in populations exponentially growing at serum concentrations between 5 and 20%. To do so exponentially growing cultures were shifted to plasma plus colcemid: one part of the cell population progressed through the cycle and accumulated with a G2 DNA content, whereas non-competent cells remained in G1. Analysis of the DNA distributions performed 24 h after the shift showed that as serum concentration was lowered more cells were found in the non-competent state: they were less than 5% in 20% serum and almost 50% in 5% serum. The non-competent cells constitute a dynamic fraction of the population, since in the presence of serum they can leave Gl and progress in the cycle. These data indicate that one of the steps limiting exponential growth is the acquisition of competence and that this event gives rise to heterogeneity within the G1 population.     

Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.     

Transport of 6-deoxy-D-glucose and D-xylose by untransformed and SV40-transformed 3T3 cells

Transport rates of the nonphosphorylated D-glucose analogs 6-deoxy-D-glucose and D-xylose were measured in quiescent and serum-stimulated cultures of mouse 3T3 cells, in SV40-transformed 3T3 cells (SV101), and in a density revertant cell line derived from SV101 (Fl-SV101). Initial rates of both entry and exit of 6-deoxy-D-glucose and D-xylose were more than threefold higher in serum-stimulated 3T3 and in SV101 cells than they were in quiescent 3T3 cells, but transport rates were not higher in the transformed cells (SV101) than they were in serum-stimulated 3T3. Confluent cultures of Fl-SV101 showed lower rates of transport than serum-stimulated Fl-SV101, but not as low as quiescent 3T3 cells. These data confirm previous findings of others with other analogs that glucose transport is one of the cell functions that is depressed when 3T3 cells enter the quiescent G₀ state, but emphasize that SV40-transformed 3T3 cells do not show higher activity of the D-glucose carrier than do actively growing 3T3 cells. Thus, enhanced glucose transport appears not to be a specific consequence of transformation, but a reflection of the active growth state of the cell.     

Platelet-derived growth factor stimulation of [3H]-glucosamine incorporation in density-arrested BALB/c-3T3 cells

G0/G1 traverse in density-arrested BALB/c-3T3 cells is controlled by multiple serum-derived growth factors. Platelet-derived growth factor (PDGF) initiates a proliferative response, whereas factors present in plasma facilitate progression through G0/G1. In the absence of competence formation, progression factors are unable to stimulate cell cycle traverse. We have identified the stimulation of a biochemical process specific to competence formation in BALB/c-3T3 cells. PDGF treated BALB/c-3T3 cells incorporated 5-10-fold more [3H]-glucosamine (GlcN) into acid-insoluble material as compared to platelet-poor plasma (PPP) treated cultures. Increased GlcN incorporation occurred in density-arrested BALB/c-3T3 cells in response to treatment with other competence factors, fibroblast growth factor, and Ca3 (PO4)2 and was not due to cell-cycle traverse. Stimulation of [3H]-GlcN incorporation by PDGF was time dependent, and increased incorporation of [3H]-GlcN into protein required de novo protein synthesis. Several mechanisms through which PDGF could increase GlcN incorporation into cellular material were examined. Results of these studies suggest an increase in the cellular capacity to glycosylate proteins is a response to or a part of competence formation.     

Increases in cyclic AMP potentiate competence formation in BALB/c-3T3 cells

The ability of platelet-derived growth factor and fibroblast growth factor to stimulate the initiation of DNA synthesis in quiescent BALB/c-3T3 cells was enhanced by cholera toxin. However, the addition of cholera toxin to unsupplemented medium was not mitogenic, nor did cholera toxin increase the mitogenic potential of mediuum supplemented with platelet-poor plasma. The enhancement of serum-induced DNA synthesis by cholera toxin was due to a specific effect on competence formation and not plasma-controlled progression. Cholera toxin increased the rate of competence formation during a transient exposure of quiescent cells to platelet-derived growth factor; this rate was further increased by the addition of isobutylmethylxanthine, a cyclic nucleotide phosphodiesterase inhibitor. Intracellular cyclic AMP concentrations in quiescent BALB/c-3T3 cells were increased 2- to 3-fold after the addition of cholera toxin. The addition of cholera toxin plus 30 m?M isobutylmethylxanthine caused an even greater (7- to 8-fold) increase in the cellular levels of cyclic AMP. That these increases in cyclic AMP concentrations mediated at least part of the increased sensitivity of quiescent cells to competence factors was substantiated by the observation that 0.01 to 1 mM monobutrylcyclic AMP or 8-bromocyclic AMP also caused a concentration-dependent potentiation of competence formation in quiescent cells during a transient exposure to platelet-derived growth factor.     

Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells.

Components of the renin-angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus-transformed SV3T3 cells. With time after infection, renin decreased in Simian virus 40 transformed cells, while it increased steadily in mock-infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells. Angiotensin II stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose-related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2-8) heptapeptide and angiotensin (4-8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment. Our results indicate (1) that angiotensin II may be involved in cell growth regulation, (2) that a negative feedback exist between angiotensin II added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non-specific angiotensinase activity is increased under this condition.     

Control of the Balb/c-3T3 cell cycle by nutrients and serum factors: analysis using platelet-derived growth factor and platelet-poor plasma.

Much controversy regarding the relationship between nutrients and serum in regulation of cell growth can be reconciled by recognizing that serum contains multiple factors which regulate different events in the cell cycle. Serum was fractionated into a platelet-derived growth factor (PDGF), which induces cells to become competent to synthesize DNA, and plasma which allows competent cells to traverse G0/G1 and enter the S phase. Nutrients are not required for the cellular response to PDGF; however amino acids are required for plasma to promote the entry of PDGF-treated, competent cells into S phase. The nutrient independent, PDGF-modulated, growth regulatory event (competence) is located 12 hours prior to the G1/S phase boundary in quiescent, density-arrested Balb/c-3T3 cells. The nutrient dependent, plasma-modulated event is located six hours prior to the G1/S phase boundary and corresponds in concentration of amino acids required for DNA synthesis. Infection of density-arrested Balb/c3T3 cells with SV40 overrides both the nutrient independent and the nutrient dependent growth regulatory events.     

Effect of agents changing the intracellular level of reactive oxygen species on the cell cycle phase distribution in 3T3 and 3T3SV40 cell lines

A comparison of cell cycle phase distribution of 3T3 cells and their transformants 3T3SV40 treated with different substances changing the intracellular level of reactive oxygen species (ROS) has been made. In this study the following glutathione synthesis modulating agents were tested: two precursors of intracellular glutathione, antioxidant N-acetyl-L-cysteine (NAC) (-)-2-oxo-4-thiazolidine-carboxylic acid (OTZ), and inhibitor of glutathione synthesis, DL-buthionine-S, R-sulfoximine (BSO). It has been shown that both NAC (10-20 mM) and OTZ (20 mM) decreased the intracellular level of ROS in both cell lines. OTZ was more potent than NAC. However, only NAC caused changes in cell cycle progression of both cell types in dose-dependent manner. These changes differed in 3T3 and 3T3SV40 cells. Flow cytometric analysis of cell cycle phase distribution indicated that NAC (20 mM) blocked cell cycle in the G1 phase. The G1--arrest was completely reversible after removal of NAC from the medium. NAC (10-20 mM) caused a decrease in S and G2/M phases of transformants 3T3SV40. Moreover, a part of the population died apoptoticaly. Different mechanisms of NAC effect on normal and transformed cells are discussed. It is suggested that there is no strong correlation between cell cycle progression and intracellular level of ROS.     

Colchicine acts as a progression factor to initiate DNA synthesis in quiescent Balb/c 3T3 cells

In quiescent Balb/c 3T3 cells, competence factors such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and platelet-derived growth factor (PDGF) synergize with progression factors such as insulin to initiate DNA synthesis. In this study, we found that colchicine, a microtubule-disrupting agent, acted synergistically with TPA, but not with insulin, to induce the maximal stimulation of DNA synthesis. Colchicine also synergized with PDGF in the presence of epidermal growth factor to elicit nearly the optimal induction of DNA synthesis. Moreover, it acted synergistically with fibroblast growth factor, another competence factor. These results suggest that colchicine acts as a progression factor like insulin in quiescent Balb/c 3T3 cells.     

Non-histone chromosomal proteins from virus-transformed and untransformed 3T3 mouse fibroblasts.

A peak in the non-histone chromosomal protein polyacrylamide gel electrophoresis profiles has been detected which is higher in log phase 3T3 and 3T3/SV40 cells than in density-inhibited 3T3 cells. Radioactive incorporation is substantially higher into this peak in log phase 3T3 than in 3T3/SV40 and density-inhibited 3T3 cells. Reversion of 3T3/SV40 cells with dibutyryl cyclic AMP and theophylline produces increased radioactive incorporation into the peak. Electrophoresis of non-histone chromosomal proteins extracted at different stages of the cell cycle in density inhibited 3T3 cells following serum stimulation shows a cyclic variation in the amount of this peak with maximum accumulation in late G1. In contrast the height of an equivalent peak in synchronously growing 3T3/SV40 cells remains constant throughout the cell cycle. It is postulated that the protein(s) of this peak may have a regulatory role in cell growth.