Human diploid fibroblasts (HDF) can induce DNA synthesis in cycling HDF but not in quiescent HDF or senescent HDF

HeLa cells in S phase induce DNA synthesis in cycling cells, serum-deprived quiescent cells, and non-replicative senescent cells following cell fusion. In contrast normal human diploid fibroblasts (HDF) do not induce DNA synthesis in either quiescent cells or senescent cells. Instead, the replicative HDF nuclei are inhibited from entering S phase in heterokaryons formed with these two types of non-replicative cells. These differences in the inducing capabilities of normal HDF and HeLa cells raise the question whether normal HDF in S phase can induce DNA synthesis in cycling cells. This paper demonstrates that young HDF in S phase can induce DNA synthesis in cycling HDF. Thus, the hypothesis that initiation of DNA synthesis in cycling cells is positively controlled by inducer molecules appears to be valid for normal HDF as well as for transformed cells such as HeLa.     

Synthesis of the nuclear protein cyclin (PCNA) and its relationship with DNA replication

Synthesis of the nuclear protein cyclin (MW 36 000) and DNA in quiescent mouse fibroblasts is coordinately induced by serum and purified growth factors. Inhibition of DNA synthesis by hydroxyurea or aphidicolin in serum-stimulated quiescent cells does not affect the induction of cyclin. The levels of cyclin synthesis decrease rapidly at the end of the S phase. Immunofluorescence studies reveal that there are dramatic changes in the nuclear distribution of cyclin during S phase and that these depend on DNA synthesis or events during S phase. These observations strengthen the notion that cyclin is an important component of the events leading to DNA replication.     

The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress

In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state.     

The effect of polyoma virus,serum factors,and dibutyryl cyclic AMP on dihydrofolate reductase synthesis,and the entry of quiescent cells into S phase

Three procedures were used to induce dihydrofolate reductase synthesis in quiescent cultures of methotrexate resistant mouse fibroblasts: (1) lytic infection with polyoma virus, (2) growth stimulation by replating cells at lower density in fresh cell culture medium, and (3) the addition of fresh medium to confluent cells. Following polyoma infection, an increase in the percentage of S-phase cells began at approximately 20 hours; dihydrofolate reductase synthesis also increased following a lag of 20 hours or more, and continued to increase throughout the late phase of lytic infection, reaching values nearly fivefold greater than that originally present in the quiescent cells. When quiescent cells received fresh medium (with or without replating), the percentage of cells in S phase began to increase by 10 hours and was accompanied by an increase in dihydrofolate reductase synthesis which reached a maximum by approximately 25 hours. These observations show that the initial entry of cells into S phase following mitogenic stimulation is associated with an induction of dihydrofolate reductase synthesis. Dibutyryl cyclic AMP blocked the stimulation of dihydrofolate reductase synthesis and the increase in the percentage of S-phase cells that resulted from the addition of fresh medium to confluent cells. When dibutyryl cyclic AMP was added at various times following the addition of fresh medium, the block in the induction of dihydrofolate reductase synthesis was correlated with a corresponding block in the increase in S-phase cells. These results suggest that dibutyryl cyclic AMP blocks cells at a point in Gl prior to either the induction of dihydrofolate reductase synthesis or the beginning of S phase. The relationship between the control of dihydrofolate reductase synthesis and entry into S phase suggests some form of coordinate control over these two parameters.     

Cell cycle re-entry of quiescent mammalian nuclei following heterokaryon formation

When 3T3 mouse fibroblasts are made quiescent by serum deprivation and are then fused with tsAF8 hamster fibroblasts synchronized by a combination of high temperature block and hydroxyurea, the nuclei of binucleated heterokaryons which are formed enter S phase asynchronously in media containing low levels of serum. The tsAF8 nuclei of these biphasic heterokaryons enter S phase shortly after fusion, as do the tsAFS nuclei of homokaryons in the same culture. In contrast, the nuclei of the biphasic heterokaryons which have been contributed by quiescent 3T3 enter S phase only after a lag following fusion. This suggests that the quiescent nucleus within the heterokaryon is stimulated by factor(s) from the more advanced cell to re-enter the cell cycle in the absence of serum. In contrast to factors which induce the immediate synthesis of DNA, these factors may be those responsible for the transition of a cell from a non-proliferating to a proliferating state.     

Cell-cycle-dependent expression of human ornithine decarboxylase

A human ornithine decarboxylase (ODC) gene probe has been isolated from a Jurkat T-cell cDNA expression library, sequenced, and used to analyze ODC mRNA levels in untransformed human lymphocytes and fibroblasts stimulated to proliferate by various mitogens. The partial cDNA sequence is 86% homologous to the mouse ODC cDNA, and Northern blots indicate that the human and mouse mRNA species are similar in size. ODC mRNA is barely detectable in quiescent human T lymphocytes and undetectable in density-arrested W138 fibroblasts. Following stimulation of T-lymphocyte proliferation with phytohemagglutinin, the ODC mRNA level rises to a peak around mid G1 phase and decreases as the cells enter S phase. Serum stimulation of density-arrested fibroblasts results in an elevation of the ODC mRNA level which persists throughout the cell cycle. Epidermal growth factor (20 ng/ml) but not insulin (10 mg/ml) or dexamethasone (55 ng/ml) stimulates ODC expression in quiescent W138 fibroblasts. Southern blots suggest that human cells have a single copy of the ODC gene.     

Regulation of the quiescence-induced genes: quiescin Q6, decorin, and ribosomal protein S29

The transition from growth to quiescence is deeply deranged in cancer cells. Expression of the quiescence-induced genes, quiescin Q6, decorin, and S29, was examined in important physiological states and in several cell types. Senescent fibroblasts expressed neither Q6 nor decorin mRNAs. The quiescins were induced in serum-deprived cultures. Trypsinized cells, which rapidly reattached to the culture dish, expressed Q6, S29, and decorin mRNAs at reduced levels, compared to those that remained in suspension. Expression of Q6 and S29 mRNAs in endothelial cells was low in growth phase and high in quiescent cells. Q6 and S29 mRNAs were found in a large variety of human tissues. The quiescin Q6 protein was detected in WI38 cell extracts and in conditioned medium from quiescent cells. A complex regulation of the quiescins by growth and attachment status in specific cell types may be of importance in pathological growth regulation and the development of cancer.     

Characterization of two populations of statin and the relationship of their syntheses to the state of cell proliferation

Statin has previously been identified to be a 57-kD protein present in the nuclei of quiescent and senescent human fibroblasts, but not in their replicating counterparts (Wang, E. 1985. J. Cell Biol. 100: 545-551). In the present report we demonstrate by immunoprecipitation analysis of fractionated cellular extracts the existence of two populations of statin. The Triton X-100-soluble statin is found in replicating sparse cultures as well as in quiescent confluent cultures and quiescent serum-starved cultures of young human fibroblasts, but the Triton X-100-insoluble, nuclear envelope-localized statin is present only in the quiescent cultures. Two-dimensional gel analysis of the immunoprecipitated cellular fractions reveals that both populations of statin have an isoelectric point of 5.3. Pulse-chase experiments show that statin is synthesized as a 57-kD polypeptide and is not processed from a precursor of different molecular mass. Experiments on serum stimulation of quiescent cells show that synthesis of the Triton X-100-insoluble statin decreases rapidly during the transition from the G0 to S phase, and that this decrease is accompanied by a slower reduction in synthesis of the Triton X-100-soluble statin. These results suggest that the cellular expression of the two populations of statin may be associated with the mechanisms controlling the transition between the growing state and the quiescent state and confirm the previous finding that the Triton X-100-insoluble, nuclear envelope-localized statin could be used as a marker for cells arrested at the G0 phase of the cell cycle.     

Elevation of intracellular glutathione content associated with mitogenic stimulation of quiescent fibroblasts

The relationship between total glutathione (GSH) content and cell growth was examined in 3T3 fibroblasts. The intracellular GSH level of actively growing cultures gradually decreases as these cells become quiescent by either serum deprivation or high cell density. Upon mitogenic stimulation of sparse, quiescent (G0/G1) cultures with serum, there is a rapid 2.3-fold elevation in intracellular GSH levels which is maximal by 1 h and returns to baseline by 2 h. This is followed by a more gradual increase in GSH content as cells enter the S phase. In addition, the elevation in GSH content is required for maximum induction of DNA synthesis. Treatments that prevent the early increase in intracellular GSH levels do not affect protein synthesis but result in a reversible dose-dependent decrease in the percent of cells capable of entering S phase. These results indicate that GSH may be important in the regulation of cellular proliferation.     

p45SKP2 promotes p27Kip1 degradation and induces S phase in quiescent cells

The F-box protein p45SKP2 is the substrate-targeting subunit of the ubiquitin-protein ligase SCFSKP2 and is frequently overexpressed in transformed cells. Here we report that expression of p45SKP2 in untransformed fibroblasts activates DNA synthesis in cells that would otherwise growth-arrest. Expression of p45SKP2 in quiescent fibroblasts promotes p27Kip1 degradation, allows the generation of cyclin-A-dependent kinase activity and induces S phase. Coexpression of a degradation-resistant p27Kip1 mutant suppresses p45SKP2-induced cyclin-A-kinase activation and S-phase entry. We propose that p45SKP2 is important in the progression from quiescence to S phase and that the ability of p45SKP2 to promote p27Kip1 degradation is a key aspect of its S-phase-inducing function. In transformed cells, p45SKP2 may contribute to deregulated initiation of DNA replication by interfering with p27Kip1 function.     

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.     

Effects of sodium n-butyrate on entry into S phase in resting rat 3Y1 cells infected with simian virus 40.

In quiescent rat 3Y1 fibroblasts infected with simian virus 40 (SV40), sodium butyrate elongated the time lag before entry into S phase in a concentration-dependent fashion. In spite of the elongated time lags, SV40-infected cells entered S phase in a very synchronous mode, irrespective of the butyrate concentrations. The elongated time lag seemed to be at least partially due to a delayed synthesis and a delayed accumulation of large T antigen caused by butyrate. The entry into S phase was also delayed even when butyrate was added to the cultures after expression of T antigen to an extent sufficient for untreated cells to enter S phase. This suggests that butyrate may also inhibit a cellular event(s) that is required for entry into S phase after expression of the T antigen. In contrast, serum-stimulated cells were more sensitive to butyrate with respect to entry into S phase than SV40-infected cells, and the distribution of the time lag among cell populations increased (i.e., asynchrony in entry into S phase increased) with an increase in the butyrate concentration.     

Uridine transport and phosphorylation in mouse cells in culture: effect of growth-promoting factors, cell cycle transit and oncogenic transformation

The rapid increase in uridine uptake produced by the addition of serum to quiescent cultures of fibroblasts is primarily caused by an enhanced rate of nucleoside phosphorylation. While quiescent and serum-stimulated cells display identical initial rates of transport, they show a considerable change in the composition of the acid-soluble pools labelled with [3H] uridine for five seconds. The radioactivity recovered in the phosphorylated pools increases 2-, 3-, 4- and 6-fold after addition of serum to cultures of Swiss 3T3 cells, tertiary mouse embryo fibroblasts, Swiss 3T6 and Balb 3T3, cells respectively. Furthermore, insulin, a growth factor isolated from medium conditioned by SV40 BHK cells (FDGF) and epidermal growth factor (EGF) also stimulate uridine phosphorylation within minutes. The initial rate of uridine uptake is 2- to 3-fold faster in rapidly growing normal and Simian virus 40 or polyoma virus transformed 3T3 cells as compared to untransformed 3T3 cells in the quiescent state. When quiescent cultures of 3T3 or mouse embryo cells are stimulated to leave G1 and enter into DNA synthesis, transport increases several hours after addition of serum and apparently coincides with the S phase of the cell cycle. The results demonstrate that an increase in uridine phosphorylation is a rapid metabolic response elicited by growth-promoting agents in a variety of cell types and that uridine transport and phosphorylation are independently regulated.     

Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase α revealed the intranuclear localization of DNA polymerase α in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the α-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase a appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase a by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.     

Rapid induction of competence formation is PDGF-isoform specific.

Platelet-derived growth factor (PDGF) stimulates the expression of a number of genes associated with entry of quiescent Balb/c-3T3 fibroblasts into the cell cycle. We determined that two of these genes, c-myc and c-fos, are induced equivalently in medium supplemented with platelet-poor plasma (PPP) and either PDGF-BB or PDGF-AA. The rate at which fibroblasts entered S phase was also similar in PDGF-BB- and AA-treated cells as was the expression of the late G1 gene, thymidine kinase (TK). However, PDGF-AA must be present for a period of 16 h to stimulate the proliferation of 90% of the cells, whereas PDGF-BB was required for only 4 h. Exposure of cells to PDGF-AA for 4 h, a time during which maximum expression of c-fos and c-myc occurred, only induced 20% of the cells in a quiescent population to enter the cell cycle. Therefore, PDGF-AA-mediated expression of the immediate early genes c-fos and c-myc may be necessary but is not sufficient to rapidly stimulate density-arrested Balb/c-3T3 fibroblasts into the competent state. Thus, these data suggest that PDGF-AA and PDGF-BB initiate traverse of the cell cycle by distinct mechanisms.     

Cell cycle requirements for transduction by foamy virus vectors compared to those of oncovirus and lentivirus vectors

Retroviral vectors based on foamy viruses (FV) are efficient gene delivery vehicles for therapeutic and research applications. While previous studies have shown that FV vectors transduce quiescent cell cultures more efficiently than oncoviral vectors, their specific cell cycle requirements have not been determined. Here we compare the transduction frequencies of FV vectors with those of onco- and lentiviral vectors in nondividing and dividing normal human fibroblasts by several methods. FV vectors transduced serum-deprived fibroblast cultures more efficiently than oncoretroviral vectors and at rates comparable to those of lentiviral vectors. However, in these cultures FV vectors only transduced a subpopulation of proliferating cells, as determined by bromodeoxyuridine staining for DNA synthesis. In contrast to lentiviral vectors, FV vectors were unable to transduce human fibroblasts arrested by aphidicolin (G(1)/S phase) or gamma-irradiation (G(2) phase), and a partial cell cycle that included mitosis but not DNA synthesis was required. We could not determine if mitosis facilitated nuclear entry of FV vectors, since cell-free vector preparations contained long terminal repeat circles, precluding their use as nuclear markers. In contrast to oncoviral vectors, both FV and lentiviral vectors efficiently transduced G(0) fibroblasts that were later stimulated to divide. In the case of FV vectors, this was due to the persistence of a stable transduction intermediate in quiescent cells. Our findings support the use of FV vectors as a safe and effective alternative to lentiviral vectors for ex vivo transduction of stem cells that are quiescent during culture but divide following transplantation.     

TPA-induction of c-fos mRNA during the cell cycle of WI-38 human fibroblasts

A brief exposure of quiescent (Go) WI-38 human fibroblasts to the tumor promoter TPA results in an increase in the mRNA levels of c-fos protooncogene. The same effect is produced by exposing to TPA human diploid fibroblasts WI38 synchronized in S phase by treatment with 2.5 mM hydroxyurea. Induction of c-fos mRNA in response to TPA occurs also during the progression of synchronized WI38 throughout the second and third cell cycle, but it is not associated with measurable changes in the cell cycle progression of these cells. These findings suggest that TPA induction of c-fos mRNA levels in proliferating cells is a stimulus specific rather than a function specific event.     

Analysis of the growth factor requirements for stimulation of WI-38 cells after extended periods of density-dependent growth arrest

When cultures of WI-38 human diploid fibroblasts reach high cell densities, they cease to proliferate and enter a viable state of quiescence. WI-38 cells can remain in this quiescent state for long periods of time; however, the longer the cells remain growth arrested, the more time they require to leave G0, progress through G1, and enter S after stimulation with fresh serum. The experiments presented here compare the response of long-term quiescent WI-38 cells (stimulated 26 days after plating) and short-term quiescent WI-38 cells (stimulated 12 days after plating) to treatment with a variety of individual purified growth factors instead of whole serum. Our results show that the qualitative and quantitative growth factor requirements necessary to stimulate G1 progression and entry into S were the same for both short- and long-term quiescent WI-38 cells, in that the same defined medium (supplemented with epidermal growth factor [EGF], recombinant human insulin-like growth factor 1 [IGF-1], and dexamethasone [DEX]) stimulated both populations of cells to proliferate with the same kinetics and to the same extent as serum. However, the long-term quiescent WI-38 cells were found to exhibit a difference in the time during which either serum or these individual growth factors were required to be present during the prereplicative period. We believe that this difference may be the cause of the prolongation of the prereplicative phase after stimulation of long-term density-arrested WI-38 cells.