Effects of polyamine depletion on serum stimulation of quiescent 3T3 murine fibroblast cells

Numerous reports have shown that polyamines are required for cell proliferation. A current model for regulating commitment to DNA replication in cultured fibroblasts stimulated from quiescence by serum addition postulates sequential action by specific growth factors. To temporally localize polyamine-dependent steps within this defined sequence, mouse Balb/c-3T3 fibroblasts were partially depleted of polyamines by treatment with DL-alpha-difluoromethylornithine (DFMO), next rendered quiescent by serum deprivation, then stimulated by 10% serum with or without exogenous putrescine (Pu). Depletion of polyamines was verified by HPLC, and entry of cells into S phase was monitored by autoradiography. After 24 h of incubation with [3H]-thymidine, polyamine-depleted cells had labeling indices similar to quiescent cells if they were serum-stimulated without Pu, but progressed to S phase to the same degree as control cultures if polyamines were restored by adding Pu at the time of serum stimulation. These observations suggested that commitment of quiescent cells to DNA replication may require polyamines. To determine if polyamine-dependent steps occur during the pre-commitment period (up to 12 h after serum stimulation) or only in traverse of G1 (12 h to 24 h, post-commitment), polyamine-depleted quiescent cells were serum-stimulated for 12 h without Pu, then returned to low serum with Pu. Labeling indices of these cultures remained nearly as low as those of unstimulated cells. Reducing serum concentration from 10% to 0.5% at 12 h after stimulation did not effect labeling indices of control cells not depleted of polyamines by DFMO. These results supported the postulated requirement for polyamines during pre-commitment events. However, polyamine-deficient quiescent cells serum-stimulated without Pu for periods longer than 24 h had labeling indices at 36 and 48 h significantly greater than at 24 h. This suggested that polyamine depletion may decrease the rate at which quiescent cells commit to DNA replication, rather than producing an absolute blockade during the pre-commitment period.     

Effect of cycloheximide and growth factors on gene expression in quiescent mouse embryo fibroblasts

Gene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum-arrested and serum-stimulated cells. Addition of platelet-derived growth factor to serum-arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000-dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5-6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein was observed.     

Endogenous membrane phosphorylation increases in serum-stimulated 3T3 cells.

Autophosphorylation of 3T3 cells, utilizing endogenous membrane protein kinase, can be detected by incubating the cells with μgM³²P-ATP. The phosphorylation activity of growing cells is two to four-fold greater than quiescent ones. In this study, the increased phosphorylation activity of serum-stimulated cells was examined. Phosphorylation, measured at times after serum stimulation of quiescent cultures, was found to increase in early G₁ and to reach a maximum prior to DNA synthesis. This increase in stimulated cells was dependent on RNA and protein synthesis but not on DNA synthesis. The increased activity decayed quickly (half-life approximately 2–3 hours) in the presence of cycloheximide, while the basal activity in quiescent cells was relatively unchanged. Insulin, prostaglandin E₁ or prostaglandin F2α were also found to bring about the same increase in phosphorylation as serum, although in contrast with serum they caused only a small percentage of the culture to synthesize DNA. The results suggest that enhanced phosphorylation activity is a G₁ event. It does not depend on subsequent DNA synthesis. Phosphorylation may be one of the biochemical steps in G₁, necessary but not sufficient for cells to move into S phase.     

Intracellular acidification inhibits the proliferative response in BALB/c-3T3 cells

One of the earliest events to occur upon the addition of serum to quiescent cells is an increase in the intracellular pH (pHin). The relationship between this pH change and proliferation is not known. In the present study, we investigate the consequences of acidifying the cytosol using the weak acid, 5', 5"-dimethyl oxazolidine 2,4-dione (DMO). At a concentration of 50 mM, DMO inhibits the serum-induced increases in pHin, DNA synthesis, and cell number. This concentration of DMO is shown not to inhibit the steady-state rate of mitochondrial respiration and not to inhibit DNA synthesis in a pH-independent fashion. The effects of DMO treatments are also shown to be reversible, indicating that this compound is not cytotoxic. These observations indicate that DMO inhibits cell proliferation by lowering intracellular pH. One important event that must occur prior to the initiation of DNA synthesis is an elevated rate of protein synthesis. The rate of protein synthesis in situ is extremely pH sensitive. Addition of 50 mM DMO to serum-stimulated cultures reduces the rate of leucine incorporation to unstimulated levels. These observations suggest that cytoplasmic acidification may inhibit proliferation through its effects on protein synthesis.     

Simultaneous activation of heat shock protein (hsp 70) and nucleolin genes during in vivo and in vitro prereplicative stages of rat hepatocytes.

Rapidly growing cells usually have high levels of ribosome biogenesis. The sequential expression of protooncogenes during the transition of quiescent hepatocytes to the replicative stage was assumed to be followed by activation of cellular genes related to cell growth such as ribosome biosynthesis. First, the expression of major nucleolar protein (nucleolin or C23) and major heat-shock protein (hsp 70) genes was examined during rat liver regeneration. hsp 70 may function in cell growth and has a characteristic nucleolar location after heat shock. Both nucleolin and hsp 70 mRNA began to increase simultaneously after peaks of c-fos and c-myc, showed a peak 6 h after partial hepatectomy, and declined to the control levels around 20 h. That is, the peaks of nucleolin and hsp 70 mRNA precede the peak of ribosome formation (12-20 h) and DNA replication (24 h). Second, the behavior of nucleolin and hsp 70 mRNA was examined in primary cultured hepatocytes during their G0-G1 transition. Although the amounts of c-myc mRNA reached a plateau around 20 h after the initiation of culture and remained at these levels, DNA synthesis has never been found to start without the addition of EGF and insulin to this system. Both nucleolin and hsp 70 mRNA began to increase at around 20 h (prereplicative stage) and simultaneously decreased in inverse proportion to DNA synthesis induced by these growth factors. Thus, it is possible that the simultaneous enhancement of nucleolin and hsp 70 genes as described above is not merely coincidental, but is important biologically during the transition of quiescent hepatocytes to proliferative cells.     

Serum stimulation of potassium fluxes,ouabain binding,and sodium fluxes in quiescent chicken embryo fibroblasts

Growth-contingent alterations in potassium and sodium fluxes, ouabain binding, and potassium ion content were examined following serum stimulation of quiescent, density-inhibited chicken embryo fibroblasts. Serum stimulation resulted in very rapid 1.5- to 1.8-fold increases in ouabain-sensitive potassium influx and lesser 1.4- to 1.5-fold increases in potassium efflux and sodium influx. Potassium influx stimulation was maximal after addition of 5–20% calf serum and was unaffected by cycloheximide inhibition of protein synthesis. Reflecting the slightly greater stimulation of potassium influx versus potassium efflux, potassium ion levels were 10–15% higher in serum-stimulated compared to unstimulated cells. Specific ouabain binding levels in stimulated and unstimulated control cells were initially similar, however, by four hours after stimulation a 40–50% increase in specific ouabain binding was observed. Incubation with ouabain was found also to inhibit later serum-stimulated hexose uptake and thymidine incorporation; this blockage may be a consequence of subnormal potassium levels rather than ouabain inhibition of the serum-stimulated potassium influx.     

The relation between polyoma T-antigen and increased 5S RNA synthesis in cell-free extracts from polyoma-infected mouse kidney cell cultures.

In polyoma-infected mouse kidney cell cultures 5S RNA synthesis began to increase around 16 h, i.e. 7-9 h after the onset of polyoma T-antigen synthesis. The rate of polyoma-induced 5S RNA synthesis reached a maximum plateau around 25 h when it was 1.8-2.0 times higher than in mock-infected parallel cultures. Stimulation of 5S RNA synthesis in vivo thus coincided in time with the increase in total cellular RNA and protein. Cell-free extracts (S100) prepared at 15 h from mock-(S100-M) or polyoma-infected (S100-Py) mouse kidney cell cultures were indistinguishable with respect to protein concentration and 5S RNA synthesis, using a cloned somatic Xenopus borealis 5S gene as template. S100-Py extracted 25 h after infection contained 30% more protein and synthesized 1.5-2.0 times more 5S RNA than S100-M. Complete removal of the polyoma T-antigens from S100-Py by 3 cycles of immunoprecipitation with hamster anti-T serum remained without effect on stimulated 5S RNA synthesis. However, a linear relationship between 5S RNA synthesis and protein concentration of S100-M and S100-Py was observed.     

Growth state and cell cycle dependent phosphorylation of DNA topoisomerase II in Swiss 3T3 cells.

We have investigated the amount of DNA topoisomerase II and phosphorylation of the enzyme in Swiss 3T3 cells during the transition from cell quiescence to proliferation. A relatively high level of phosphorylation was observed with proliferating cells while no or a very low level of phosphorylation was observed with quiescent cells. Phosphoamino acid analysis of the phosphorylated topoisomerase II revealed that the phosphorylated aminoacyl residue was serine. When quiescent cells were stimulated to grow by the addition of serum, DNA synthesis began to increase at 9 h after serum addition, reaching a maximum at 15 h and then declining. The amount of topoisomerase II began to increase at 6 h and reached a maximum at 22-27 h, corresponding to the G2 phase. The phosphorylation of topoisomerase II measured by pulse-labeling gradually increased from 6 to 18 h and reached a maximum at 22 h when the amount of the enzyme was maximum. The level of phosphorylation measured by continuous-labeling increased gradually up to 12 h and markedly up to 28 h, and then declined. The increase in the rate of phosphorylation in the G2 phase was affected by inhibiting DNA synthesis, but the increase in the amount of the enzyme was not. Thus, it was suggested that the regulation of phosphorylation of topoisomerase II differs from that of the amount of the enzyme.     

Induction of the nuclear protein ''cyclin'' in quiescent mouse 3T3 cells stimulated by serum and growth factors. Correlation with DNA synthesis

The effect of serum and growth factors [platelet-derived growth factor (PDGF), fibroblast growth factor (FGF)] on the synthesis of the nuclear protein cyclin and its correlation with DNA synthesis has been studied in quiescent mouse 3T3 cells by means of quantitative two-dimensional gel electrophoresis. Serum must be present in the medium for at least 8-12 h to induce maximal synthesis of cyclin (6- to 7-fold increase compared with quiescent cells). The stimulation of cyclin synthesis is dose-dependent and correlates directly with DNA synthesis. In addition, partially purified PDGF and FGF also induce cyclin and DNA synthesis in a coordinate way. Both growth factors, like serum, exhibit a similar lag phase to induce maximal cyclin (6- to 7-fold) and DNA synthesis (90% of the cells). Pure PDGF at a concentration as low as 10 ng/ml has the same effect as 10% serum. The coordinate induction of cyclin and DNA synthesis can only be observed with growth factors that induce DNA synthesis. These results strengthen the notion that cyclin is an essential component of the events leading to DNA replication.     

Time dependency of a critical effect of EGTA on DNA synthesis in cultures of Swiss 3T3 cells.

In a temporal analysis of the mitogenic response to serum, a critical period has been demonstrated just prior to the onset of replicative DNA synthesis during which transient calcium depletion blocks the subsequent entry of the cells into the S phase of the mitotic cycle. Transient washington of monolayer cultures of 3T3 cells with 2.5 mM EGTA between 6 and 8 h after serum-stimulated initiation of DNA synthesis was found to reduce cell-associated calcium levels and to inhibit thymidine incorporation, whereas similar treatment before (1-5 h) and after (8-9 h) had no detectable effect on either of these parameters when estimated after 21 h incubation. The effects during the chelation-sensitive period were reversed by the subsequent addition of fresh serum.     

Induction of the nuclear protein cyclin in serum-stimulated quiescent 3T3 cells is independent of DNA synthesis

Inhibition of DNA synthesis and cell proliferation of mouse 3T3 cells by aphidicolin did not affect the expression of cyclin, a nuclear protein whose synthesis correlates with cell proliferation, as determined by quantitative two-dimensional gel electrophoresis analysis. Serum stimulation of quiescent 3T3 cells revealed that cyclin synthesis increases shortly before DNA synthesis. Inhibition of DNA synthesis by aphidicolin in serum-stimulated quiescent cells did not affect the increase of cyclin following stimulation. These results demonstrate that cyclin synthesis is not coupled to DNA synthesis and that it is one of the latest events before DNA replication.     

Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNAk synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2-5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]-thymidine incorporation into acid-precipitable material in a dose-dependent manner. The effect of these agents is specific since 8Br5'AMP, 5'AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1-methyl-3-isobutyl xanthine and 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth-promoting effects of the cAMP derivatives were demonstrated by [3H]-thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simultaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) act sas a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.     

U-61,431F, a stable prostacyclin analogue, inhibits the proliferation of bovine vascular smooth muscle cells with little antiproliferative effect on endothelial cells.

The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.     

Sequential stimulation of cellular RNA synthesis in polyoma-infected mouse kidney cell cultures.

Lytic infection with polyoma virus leads in Go-arrested primary mouse kidney cell cultures to a mitotic host response. In the present work we focused our attention on cellular RNA synthesis shortly after onset of polyoma T-antigen synthesis. Onset of polyoma-induced stimulation of 45S pre-rRNA synthesis was determined by hybridization of total cellular RNA with a plasmid (pMrSalB) containing the 5'-end of the mouse ribosomal gene and of the other cellular RNA species by standard biochemical analysis of cellular fractions. The results showed that polyoma-induced stimulation of cellular hnRNA (hnRNP) synthesis, the earliest presently known host cell reaction, preceded onset of stimulated 45S pre-rRNA synthesis and that the latter was paralleled by polyoma-induced stimulation of 5S RNA, tRNA and overall protein synthesis. The polyoma-induced mitotic response is similar to that triggered by simian virus 40 and by certain nonviral mitogens.     

Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 cells

Although variability in the duration of the cell cycle is thought to reflect growth-regulatory processes that control cell cycle progression, the precise timing of the variable period within the G1 phase of the cell cycle has not been defined. In particular, the timing of cell cycle variability in relation to the cell's commitment (R point) to the initiation of DNA synthesis remains controversial. In order to investigate cell cycle variability, indirect immunofluorescence was used to measure the formation of the primary cilium as a possible marker of G1 events in both stimulated quiescent and exponentially growing cells. The primary cilium, an internal "9 + 0" nonmotile structure formed by one of the interphase centrioles, was first detected in postmitotic BALB/c 3T3 cells 5 hr before the initiation of DNA synthesis, an interval similar to that for the reassembly of the primary cilium in serum-stimulated quiescent fibroblasts. This similarity in the timing of ciliation suggests that serum-stimulated quiescent cells reenter the cell cycle in early G1 and recapitulate much of G1. Moreover, the rate of cilia formation in both postmitotic and serum-stimulated quiescent cells was identical to the rate of DNA synthesis initiation. Thus, cell cycle variability occurs before ciliation in both stimulated quiescent and exponentially growing cells. Furthermore, since ciliation also precedes the R point, variability in the centriole cycle occurs before the R point and thus may reflect processes controlling the cell's commitment to the initiation of DNA synthesis.     

Effects of dexamethasone,heat shock,and serum responses on the inhibition of hsc70 synthesis by antisense RNA in NIH 3T3 cells

A dexamethasone (Dex)-inducible antisense RNA expression vector was constructed that contains the 5′-untranslated region and one third of the coding sequence for the bovine hsc70 protein. This vector was used to transfect NIH 3T3 cells from which clonal cell lines expressing hsc70 antisense RNA were developed. Quantitative Northern blot analysis with strand-specific probes was used to demonstrate the Dex-inducible accumulation of hsc70 antisense RNA in proliferating cell cultures and the inhibition of hsc70 RNA levels. Surprisingly, antisense RNA was either much less effective in reducing the amounts of hsc70 RNA in Dex-treated cultures than in untreated controls or cells compensated by producing more hsc70 RNA in response to increasing amounts of antisense RNA. Hsc70 protein synthesis did not decrease in either Dex-treated or untreated cultures: it actually increased, again suggesting the activation of a compensatory response. In Dex-treated cultures subjected to heat shock, hsc70 antisense RNA blocked the induction of hsp70, indicating that newly synthesized RNA was targeted effectively before it became translationally active. To test this hypothesis further, Dex-treated cultures were made quiescent by serum deprivation and then restimulated with serum, which causes a burst of RNA and protein synthesis. Consistent with this hypothesis, increased synthesis of hsc70 was blocked in serum-stimulated cultures expressing antisense RNA. © 1995 Wiley-Liss, Inc.     

Mitogenic and antimitogenic effects of cholera toxin-mediated cyclic AMP levels in 3T3 cells

The effect of time-controlled exposures to cholera toxin (CT) on intracellular levels of cyclic AMP (cAMP) and on the proliferative response of serum-stimulated 3T3 cells was investigated. Continuous exposure to CT caused up to 8-fold raises in cAMP content and inhibited DNA replication by delaying G1-S transition and by reducing the fraction of cells committed to DNA replication. In contrast, short exposures to CT during G0-G1 transition increased the fraction of cells responding to serum stimulation and potentiated the serum-induced morphological changes in the cell monolayer. A short exposure during late G1 phase, however, inhibited the onset of DNA synthesis but had little effect on ongoing DNA replication. The results indicate that cAMP has diverse and opposite effects on two defined restriction points in cell cycle control. Cyclic AMP was positively involved in the acquisition of the state of competence by quiescent cells (G0-G1 transition) but antagonistic on the onset of DNA replication (G1-S transition) in committed cells. The observations reconcile a number of controversial conclusions regarding the role of cAMP in cell cycle control.