The metabolism of histone fractions. VI. Differences in the phosphorylation of histone fractions during the cell cycle

Phosphorylation of histone fractions in the presence and absence of DNA synthesis was measured using the new “isoleucine-limiting” method for synchronizing Chinese hamster cells in early G₁-phase. Using preparative electrophoresis, histone f1 phosphorylation was found to be dependent upon cell-cycle position, being absent in G₁-arrested and G₁-traversing cells and active in the S-phase. The absence of f1 phosphorylation in G₁-arrested cells, which are known to exhibit f1 turnover, indicates that f1 phosphorylation is not an obligatory part of the f1 turnover process. In contrast to histone f1, it was found that histone f2a2 phosphorylation is independent of cell-cycle position, occurring with equal magnitude in the G₁-traversing state when DNA synthesis is essentially absent and in the S-phase when DNA synthesis is active. When cells were arrested in the G₁-state by isoleucine deprivation, f2a2 phosphorylation continued to be active, occurring at 56% of the rate observed in the G₁-traversing state. These results indicate that phosphorylation of histone f2a2 is independent of f2a2 synthesis, independent of DNA synthesis, and independent of histone f1 phosphorylation. Because f2a2 is actively phosphorylated in G₁-arrested cells known to be active in the synthesis of various types of RNA (including messenger) as well as in G₁-traversing and S-phase cells, we feel that phosphorylation of histone f2a2 should continue to be considered in models concerning activation of DNA template activity.     

Centriole ciliation is related to quiescence and DNA synthesis in 3T3 cells.

Both DNA and the centriole pairs are replicated once in each cell generation. The cyclic changes in both must be coordinated so that the two centriole pairs can participate in mitosis when the genetic material is to be partitioned to the two daughter cells. One of the centriole pairs also forms a primary (“9 + 0”) cilium sometime during the cell cycle. In this study, we asked whether some aspects of the coordination of the DNA and centriole cycles occur in G₁, a part of the cell cycle when non-neoplastic cells become irreversibly committed to DNA synthesis. We used indirect immunofluorescence with antitubulin antibody to reveal the centriole pairs as a microtubule organizing center with or without a cilium. Quiescent Balb/c and Swiss 3T3 cells in low serum or at high cell density stopped in G₁ with ciliated, probably unduplicated centrioles. When these quiescent 3T3 cells were stimulated to enter DNA synthesis, the centriole's ciliation changed in three phases: first, an initial but transient deciliation within 1–2 hr; second, a return of the cilium by 6–8 hr; and third, a subsequent final deciliation of the centriole coincident with the initiation of DNA synthesis at 12–24 hr.The deciliated and duplicated centrioles subsequently separated in preparation for mitosis. Together with other information, these results imply that centrioles in growing mammalian cells are primarily ciliated in a part of G₁ during which the cells can arrest in suboptimal environmental conditions. Arrests in low serum or at high cell density also occur before centriole replication. These results suggest that deciliation and duplication of the centriole may occur near the time that quiescent cells become irreversibly committed to DNA synthesis. Certain centriole events may therefore be necessary before DNA synthesis can be initiated in 3T3 cells.     

Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up-regulation of oncogenes in S-49 cyc− cells

Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c-AMP in prostaglandin mediated cell cycle arrest, we use the-49 lymphoma variant (cyc^?) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A₁ (dmPGA₁) inhibits DNA synthesis and cell growth in cyc^? cells. DNA synthesis is inhibited 42% by dmPGA₁ (50 μM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the α,β unsaturated ketone ring. Dimethyl PGA₁ is most effective in inhibiting DNA synthesis in cyc^? cells, with prostaglandins PGE₁ and PGB₁ being less potent inhibitors of DNA synthesis. DmPGE₂ caused a significant stimulation of DNA synthesis. S-49 cyc^- variant cells exposed to (30–50 μm) dmPGA₁, arrested in the G₁ phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S-49 cyc^? cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G₁, S, G₂, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S-phase DNA synthesis from the G₁ cell cycle block. The S-49 cyc^? cells are known to have a G₁/S boundary through M phase transition time of 14.8 h, making the location of the prostaglandin cell cycle arrest at or very near the G₁/S interface. The oncogenes, c-fos and c-myc which are normally expressed during G₁ in proliferating cells have a 2–3 fold enhanced expression in prostaglandin G₁ arrested cells. These data using the S-49 variants demonstrate that dmPGA₁ inhibits DNA synthesis and arrests the cell cycle independent of cAMP-mediated effects. The prostaglandin arrested cells maintain the gene expression of a G₁ synchronous cell which suggests a unique molecular mechanism for prostaglandin action in arresting cell growth. These properties indicate that this compound may be an effective tool to study molecular mechanisms of regulation of the cell cycle.     

Cell cycle-dependent gene expression in V point-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells stimulated to proliferate in an amino acid-deficient medium arrest in mid-G₁ at a point termed the V point. Cells released from V point arrest require 6 hr to traverse late G₁ and enter S phase. As data presented here show that mRNA synthesis is needed for 2–3 hr after release of cells from the V point, after which inhibition of mRNA synthesis does not prevent entry into S phase, we used this mid-G₁ arrest protocol to analyze gene expression in late G₁. We found that although stimulation of cells in amino acid-deficient medium did not inhibit the induction of genes expressed in early G₁, genes normally expressed in late G₁ were expressed only after release from the V point. The expression of late G₁ genes in cells released from the V point was temporally similar, in respect to G₁ location, as was seen in stimulation of quiescent G_o cells. As this protocol effectively divides gene expression into early (pre-V point) and late (post-V point) categories, it should be useful in studies of growth factor-modulated events that regulate traverse of late G₁ and commitment to DNA synthesis. In addition, we used c-myb antisense oligonucleotides to show that c-myb expression, which occurs in late G₁, is required for BALB/c-3T3 fibroblasts to traverse late G₁ and initiate DNA synthesis. © 1993 Wiley-Liss, Inc.     

Murine coronavirus replication induces cell cycle arrest in G0/G1 phase

Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G₀/G₁ phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G₀ phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G₁ and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21^Cip1, p27^Kip1, and p16^INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G₁ cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G₁ cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G₀/G₁ phase.     

Potassium transport and content during G1 and S phase following serum stimulation of 3T3 cells.

The effect of serum stimulation on unidirectional and net K flux and their relationship to the initiation of DNA synthesis has been investigated in mouse 3T3 fibroblasts. Stimulation of quiescent 3T3 cells with 20% serum results in the initiation of S phase approximately ten hours after serum addition. During transition from G₁ to S phase distinct changes in K transport and cellular K content occur. Total unidirectional K influx undergoes an immediate 2-fold increase upon serum addition, an observation in qualitative agreement with previous results (Rozengurt and Heppel, 1975). This total increase in unidirectional K influx represents a proportional increase in the active, ouabain sensitive component and the K-K exchange component. The initial increase in total flux is followed by a gradual decline over a 16-hour period to levels approaching those of quiescent cells. Following the initial increase in unidirectional K influx is an approximately 75% increase in cell K on a per milligram protein basis or a 40% increase on a per volume basis. This increase peaks at four to five hours and then declines to initial levels at 10 to 14 hours. Populations of quiescent cells given 20% serum plus 0.5 mM ouabain simultaneously are totally blocked from entering S phase, as determined by the appearance of ³H-thymidine labeled nuclei. However, if the ouabain is removed after six hours these cells then undergo the same changes in unidirectional K influx and content as serum stimulated cells with entrance into S phase retarded by five to six hours. If ouabain is added to serum stimulated cells at six hours, after the increase in K transport and K content have occurred, entrance into S phase is not entirely blocked. In cells stimulated with serum and 0.5 mM dBcAMP plus 1 mM theophylline simultaneously, entrance into S phase is greatly reduced as compared to serum stimulation only. However, the early and late changes in K flux and K content are not substantially altered. This indicates that the K transport events associated with G₁ and early S phase are not directly regulated by changes in cAMP levels which follow serum stimulation.     

Stimulation of the initiation of DNA synthesis and cell division in several cultured mouse cell types : Effect of growth-promoting hormones and nutrients

The ability of prostaglandin F_2α (PGF_2α) and other prostaglandins to stimulate the initiation of DNA synthesis in quiescent cultures of various mouse fibroblastic cell types has been investigated. PGF_2α was found to be more effective than the other prostaglandins. Most cell types, with the exception of BALB/c 3T3, responded to PGF_2α. Addition of PGF_2α in combination with insulin resulted in a synergistic increase in the proportion of cells synthesizing DNA. The effect of nutrients on the stimulation of the initiation of DNA synthesis has been examined in detail; it was found that Swiss 3T3 cells showed a requirement for hypoxanthine and vitamin B₁₂ whereas Swiss 3T6 cells demonstrated a stringent requirement for vitamin B₁₂ only. The effect of prostaglandin precursors, synthetic analogues of the prostaglandin endoperoxides and inhibitors of prostaglandin synthesis was also examined in two cell types. The effect of PGF_2α was compared with that of two polypeptide growth factors, epidermal growth factor (EGF) and fibroblast growth factor (FGF) in Swiss 3T6 cells grown in 0.0025% (v/v) serum. In combination with insulin each of these three growth factors stimulated the initiation of DNA synthesis in approximately the same number of cells.     

Evidence for differences in the mechanism of cell cycle arrest between senescent and serum-deprived human fibroblasts: Heterokaryon and metabolic inhibitor studies

It has previously been shown that serum-deprived, early passage quiescent human diploid fibroblastlike (HDFL) cells are able to inhibit cycling cells from entry into DNA synthesis upon cell fusion. We have found that the degree of inhibition of DNA synthesis in the heterokaryon correlates with the duration of serum deprivation, which is consistent with the suggestion that serum-deprived cells may enter progressively deeper stages of G₀ as they increase their time in quiescence. In contrast to fusions with senescent cells, in heterokaryons between serum-deprived early passage and cycling young cells transient inhibition of protein synthesis with cycloheximide or inhibition of RNA synthesis with 5–6-dichloro-1-β-D-ribofuranosyl benzimidazole (DRB) did not stimulate nuclear [₃H]-thymidine incorporation. These results suggest that differences may exist in the mechanisms responsible for inhibiting cell cycle progression in senescent vs early passage quiescent HDFL cells.     

Factors affecting the formation of phytol and its incorporation into chlorophyll by homogenates of the leaves of the french bean Phaseolus vulgaris

The biosynthesis and phosphorylation of histone fractions were measured in synchronized CHO Chinese hamster cells arrested in late G₁ by hydroxyurea treatment. Hydroxyurea was found to inhibit the initiation of both DNA and histone synthesis, thus confirming the conclusion that it arrests cells in G₁ slightly before the $\text{G}{}_1\text{S}$ boundary. However, hydroxyurea did not inhibit the phosphorylation of histone f1 or histone f2a2. The phosphorylation of histone f1, which normally is absent in early G₁, begins 2 hr prior to DNA synthesis. In the presence of hydroxyurea, f1 phosphorylation occurs on schedule at this same time in G₁, resulting in significant G₁-phase f1 phosphorylation. This offers strong evidence that (a) f1 phosphorylation is not restricted to S phase; (b) “old” f1 which was synthesized in previous cell cycles is phosphorylated in G₁ before “new” f1 which is synthesized in S phase; and (c) G₁-phase f1 phosphorylation does not require new histone or new DNA synthesis.Histone f1 phosphorylation was observed to occur at accelerated rates in S phase over phosphorylation rates observed in late G₁-arrest. Data support the proposal that three different levels of f1 phosphorylation occur during the cell cycle: (1) a G₁-related phosphorylation of “old” f1; (2) an S-related phosphorylation of both “old” and “new” f1; and (3) a superphosphorylation of f1 associated with chromosome condensation during the G₂ to M transition. It is also possible that a limited proportion of f1 may be phosphorylated in G₁, perhaps at the initial DNA synthesis sites, and that an increased proportion of f1 is phosphorylated in S as DNA is synthesized. Similarities between the kinetics of histone f1 phosphorylation and the association of DNA with lipoprotein in synchronized control and hydroxyurea-treated cells suggest an involvement of f1 phosphorylation in cell-cycle-dependent chromatin structural changes.     

Histone phosphorylation in late interphase and mitosis

Histone phosphorylation in late interphase has been investigated employing cells synchronized by the isoleucine-deprivation method, followed by resynchronization at the $\text{G}{}_1\text{S}$ boundary using hydroxyurea. Phosphorylation occurred in both f1 and f2a2 as cells synchronously entered S phase following removal of hydroxyurea. The relative rates of phosphorylation of both species of histone increased in G₂-rich and metaphase-rich cultures. A small amount of histone f3 phosphorylation was also observed in M-rich cultures which was not seen in G₁, S, or G₂-rich cultures. It is concluded that f1 phosphorylation is not dependent on continous DNA replication. These experiments suggest consideration of the concept that f1 phosphorylation is initiated as a preparation for impending cell division.     

Stimulation of DNA synthesis and myo-inositol incorporation in mammalian cells

Phosphatidylinositol (PI) synthesis and its role in controlling the cell cycle has been investigated using fibroblasts and liver cells in culture. PI synthesis as measured by incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material during 0–60 min after serum or growth factor stimulation of serum-starved cells is increased in primary fetal rat liver cells, rat embryo fibroblasts, and 3T3 mouse cells. In contrast, growth stimulation of 3T3 cells and hepatocytes rendered quiescent in G₁ by amino acid starvation is not accompanied by increased incorporation of [³H]-myo-inositol into trichloroacetic acid precipitable material. This suggests that those cells might be arrested at a different point in G₁ than cells arrested by serum depletion. Inhibition of PI synthesis by δ-hexachlorocyclohexane (HCH), a steric analog of myo-inositol, during early times (e.g., 0–4 hr) after growth stimulation, reversibly blocks initiation of DNA synthesis in 3T3 cells. The results support the idea that increased PI synthesis in response to growth stimulation in the cell types studied here is a prerequisite for progression through G₁ and subsequent entry into S phase.     

Regulation of NIH-3T3 cell G1 phase transit by serum during exponential growth

The proliferation rate of mammalian cells is regulated normally in the G₁ phase of the cell cycle. During this phase, it is convenient to assign positive and negative roles to the molecular programs that regulate the duration of G₁ and the phase transition from G₁ to S phase. Density-dependent inhibition of cellular proliferation results in an increase in the duration of G₁. This form of regulation is due to both secreted factors and cell—cell contact. Serum is mitogenic to a variety of mammalian cell types. Because quiescent cells enter S phase as a result of serum addition to culture media, serum is usually regarded as a source of positive regulatory growth factors. We have measured the length of the G₁, S and G₂+ M phases of NIH 3T3 cells during exponential growth as a function of cell density and serum concentration. The G₁ length increases during exponential growth as a function of density while S and G₂+ M are relatively constant. Further, this increase in G₁ phase time, or density mediated negative regulation, is inhibited by increasing serum concentration. This phenotype is saturable between 10% to 20% serum. Serum concentrations above 2.5% are able to increase the rate of cell cycling (decrease the G₁ phase time) by inhibiting density dependent negative regulation of NIH 3T3.     

Differential effects of ACTH or 8-Br-cAMP on growth and replication in a functional adrenal tumor cell line

The effects of ACTH and 8-Br-cAMP on growth and replication of a functional mouse adrenal tumor cell line (Y-1) were investigated. ACTH and 8-Br-cAMP both inhibited DNA synthesis and replication when added to randomly growing cell cultures. ACTH addition and serum deprivation each arrested cells in G₁; an additional point of arrest in G₂ occurred with 8-Br-cAMP. Cells whose growth was arrested in G₁ by ACTH had a significantly larger volume and protein and RNA content compared to cells arrested in G₁ by serum deprivation. When ACTH or 8-Br-cAMP was added with serum to cells arrested by serum deprivation, the wave of DNA synthesis and cell division seen with serum was abolished. ACTH and 8-Br-cAMP had no effect on the serum-induced increases in protein and RNA content, rates of leucine incorporation into protein and uridine incorporation into RNA, and RNA polymerase I activity observed in cells during the pre-replicative period. Partial inhibition of the serum-induced increase in uridine transport occurred. ACTH and cAMP do not appear to inhibit replication by generalized negative pleiotypic effects but rather to inhibit the initiation of DNA synthesis more specifically. The ACTH-arrested Y-1 cell resembles an in vivo hypertrophied adrenal cortical cell.     

Inhibition of BALB/c-3T3 cells in late G1: Commitment to DNA synthesis controlled by somatomedin C

Methylglyoxal bis-(guanylhydrazone) (mGBG) blocked the stimulation of DNA synthesis in quiescent, density-inhibited BALB/c-3T3 cells treated with platelet-derived growth factor (PDGF) and platelet-poor plasma (PPP). Competence formation produced by a transient exposure to PDGF was not effected by mGBG. In contrast, mGBG effectively inhibited the PPP-stimulated progression of competent cells through the G₁ phase of the cell cycle, although maximal inhibition was observed when mGBG was present during both the exposure to PDGF- and PPP-supplemented media. When quiescent cells were treated with PDGF and PPP-supplemented media in the presence of mGBG for 12–18 hours and the mGBG was then removed, cells entered the S phase after a 4 hour lag. The rate of entry into the S phase, but not the time necessary for the cells to progress from the mGBG block into the S phase, was dependent on the concentration of PPP present after removal of the mGBG. Either somatomedin C or insulin, but not epidermal growth factor, fibroblast growth factor, or PDGF were able to substitute for PPP in allowing cells to enter the S phase after the cells were released from the mGBG block. A marked inhibition of (³H)-leucine incorporation in serum-stimulated cultures was produced at mGBG concentrations which caused no decrease in the amount of (³H)-uridine incorporated during a short (15 minute) pulse. The ability of hormones to allow cells to progress to the late G₁ phase and become committed to DNA synthesis after a mGBG inhibition was not related to their ability to restore the normal rate of protein synthesis as determined by (³H)-leucine incorporation.     

Ascorbate potentiates serum-induced S phase entry of quiescent 3T3 cells

Summary Arrested BALB/c 3T3 cells were induced to the G₀-G₁ transition by fetal calf serum (FCS) and S phase entry was measured by [³H]thymidine incorporation as an index of DNA synthesis. [³H]Thymidine uptake was proportional to FCS concentration. Ascorbate (ASC) itself was unable to increase DNA synthesis in these cells but potentiated it in the presence of both 1% and 10% FCS. [³H]Thymidine uptake profile was similar with and without ASC, and showing at 24 h an ASC stimulation of 69% in the presence of 1% FCS and 58% with 10% FCS. These data are discussed in reference to the participation of ASC on plasma membrane energization for membrane translocations and transport.Abbreviations ASC ascorbate - FCS fetal calf serum     

Phosphodiesterase 3A (PDE3A) deletion suppresses proliferation of cultured murine vascular smooth muscle cells (VSMCs) via inhibition of mitogen-activated protein kinase (MAPK) signaling and alterations in critical cell cycle regulatory proteins

Cyclic nucleotide phosphodiesterase 3 (PDE3) is an important regulator of cyclic adenosine monophosphate (cAMP) signaling within the cardiovascular system. In this study, we examined the role of PDE3A and PDE3B isoforms in regulation of growth of cultured vascular smooth muscle cells (VSMCs) and the mechanisms by which they may affect signaling pathways that mediate mitogen-induced VSMC proliferation. Serum- and PDGF-induced DNA synthesis in VSMCs grown from aortas of PDE3A-deficient (3A-KO) mice was markedly less than that in VSMCs from PDE3A wild type (3A-WT) and PDE3B-deficient (3B-KO) mice. The reduced growth response was accompanied by significantly less phosphorylation of extracellular signal-regulated kinase (ERK) in 3A-KO VSMCs, most likely due to a combination of greater site-specific inhibitory phosphorylation of Raf-1^Ser-259 by protein kinase A (PKA) and enhanced dephosphorylation of ERKs due to elevated mitogen-activated protein kinase phosphatase 1 (MKP-1). Furthermore, 3A-KO VSMCs, compared with 3A-WT, exhibited higher basal PKA activity and cAMP response element-binding protein (CREB) phosphorylation, higher levels of p53 and p53 phosphorylation, and elevated p21 protein together with lower levels of Cyclin-D1 and retinoblastoma (Rb) protein and Rb phosphorylation. Adenoviral overexpression of inactive CREB partially restored growth effects of serum in 3A-KO VSMCs. In contrast, exposure of 3A-WT VSMCs to VP16 CREB (active CREB) was associated with inhibition of serum-induced DNA synthesis similar to that in untreated 3A-KO VSMCs. Transfection of 3A-KO VSMCs with p53 siRNA reduced p21 and MKP-1 levels and completely restored growth without affecting amounts of Cyclin-D1 and Rb phosphorylation. We conclude that PDE3A regulates VSMC growth via two complementary pathways, i.e. PKA-catalyzed inhibitory phosphorylation of Raf-1 with resulting inhibition of MAPK signaling and PKA/CREB-mediated induction of p21, leading to G₀/G₁ cell cycle arrest, as well as by increased accumulation of p53, which induces MKP-1, p21, and WIP1, leading to inhibition of G₁ to S cell cycle progression.     

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.     

Regulation of growth of human diploid fibrobalasts by factors elaborated by activated lymphoid cells

Fibroblast growth and synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin-P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose-dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80°C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (α-LT). It was not cytotoxic, as shown by the ⁵¹chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G⁰ or G¹ stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA Synthesis but cannot divide, and they accumulate in G². The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.     

Ornithine decarboxylase induction in cells stimulated to proliferate differs from that in continuously dividing cells

Ornithine decarboxylase activity increases at least 4–5-fold before DNA synthesis both in synchronous cycling cells and in quiescent cells stimulated to proliferate. The purpose of our experiments was to test whether the transient peaks of ornithine decarboxylase activity in both growth situations were biochemically regulated in a similar manner. We found that the regulation of this particular enzyme activity is distinct in two ways. Firstly, the addition of 2mm-hydroxyurea will block the induction of ornithine decarboxylase in continuously dividing Chinese-hamster ovary cells, while having no effect on ornithine decarboxylase induction in stimulated quiescent cells. Hydroxyurea added after the induction occurs has no effect on the enzyme activity. The apparent half-life of the enzyme is not altered in cells treated with hydroxyurea. Hydroxyurea does not affect the enzyme directly, since incubation of cell homogenates with this drug results in no loss of measurable ornithine decarboxylase activity and hydroxyurea does not markedly alter general RNA- or protein-synthesis rates. The inactivation of ornithine decarboxylase activity by hydroxyurea does not resemble the loss of activity observed with a 90min treatment with spermidine. Thiourea, a less potent inhibitor of ribonucleoside diphosphate reductase, will also inhibit ornithine decarboxylase activity, but to a lesser extent. Secondly, the expression of ornithine decarboxylase in quiescent cells stimulated to proliferate is biphasic as these cells traverse G₁ and enter S phase, whereas only one peak of activity is apparent in synchronous cycling G₁-phase cells. The time interval between the first peak of ornithine decarboxylase activity and the onset of DNA synthesis is approx. 5h longer in non-dividing cells stimulated to proliferate than in continuously dividing cells. The results suggest that the regulation of ornithine decarboxylase activity is different in the two growth systems in that the induction of ornithine decarboxylase in continuously dividing cells occurs closer in time to DNA synthesis and is dependent on deoxyribonucleoside triphosphates.