Characteristics of 30-, 63-, and 89-kilodalton proteins whose secretion from mouse fibroblasts is altered by beta-interferon

Quiescent mouse BALB/c-3T3 cells were treated with beta-interferon to induce the secretion of proteins of 30 and 89 kDa and with a platelet-derived growth factor preparation to induce the secretion of a 63-kDa protein. To label the secreted proteins the cultures were supplemented with [35S]methionine after addition of the inducer. The proteins in the culture fluid were fractionated resulting in a radioactively pure 63-kDa protein and 30- and 89-kDa protein preparations with residual minor radioactive impurities. The secreted 89-kDa protein shared at least one characteristic with some interferon-induced cell-associated enzymes: it bound double-stranded RNA tightly. The 63-kDa protein was undetectable in the culture fluid from resting BALB/c-3T3 cells and was barely or not at all detectable in the culture fluids from growing BALB/c-3T3 and NIH 3T3 cells, respectively. The protein was, however, among the three major constitutively secreted proteins in the case of growing Kirsten murine sarcoma virus-transformed NIH 3T3 cells. Treatment with 1000 units/ml beta-interferon decreased the accumulation of the 63-kDa protein in the culture fluid of quiescent BALB/c-3T3 cells which had been treated with a platelet-derived growth factor preparation by over 80% and that in the culture fluid of Kirsten murine sarcoma virus-transformed NIH 3T3 cells by about 50%. This decrease was not a consequence of an inhibition of cell growth.     

Murine mRNA for the beta-subunit of integrin is increased in BALB/c-3T3 cells entering the G1 phase from the G0 state

The amount of murine mRNA for the beta-subunit of integrin is enriched 6-fold when BALB/c-3T3 cells traverse from the G0 state to the G1 phase, whereas it remains at the basal level when the cells are growing continuously. The peak of its appearance is at 10 h after serum stimulation. The increase in integrin mRNA at a specific point in cell proliferation may be correlated with growth-signal transduction.     

Plasma and platelet associated factors act in G1 to maintain proliferation and to stabilize arrested cells in a viable quiescent state : A temporal map of control points in the G1 phase

In medium supplemented with defibrinogenated, platelet-poor human plasma and a low molecular weight growth factor derived from human platelets (PDGF), Swiss 3T3 cells proliferate exponentially with the same cell cycle kinetics as cells cultured in medium supplemented with commercial calf serum. Removal of PDGF from the culture medium arrests proliferating cells in a stable, reversible G0/G1 quiescent state. This arrested state is similar to the known quiescent state induced by deprivation of calf serum in cell exit kinetics and cytoplasmic proteins synthesized. Cells are sensitive to PDGF deprivation only at the beginning of G1. Reduction of the plasma concentration in the culture medium also arrests cells in G1. The resulting arrested population is unstable and exhibits progressive cell death. Reduced levels of plasma block cellular transit through the cell cycle at a median time of approx. 2.1 h following mitosis, approx. 3.3 h prior to S phase initiation. In addition to being required by cycling cells, plasma associated factors are required to maintain G1 cells blocked by PDGF deprivation in a stable quiescent state. Establishment of a stable, viable G0/G1 growth-arrested state, therefore, apparently involves two distinct processes: arrest of cellular proliferation in G1 and stabilization of the arrested cells in a viable quiescent state. Together with previously reported findings on serum and isoleucine starvation, these results provide a temporal map of growth control points in the G1 phase.     

Epidermal growth factor (EGF) is required only during the traverse of early G1 in PDGF stimulated density-arrested BALB/c-3T3 cells

Density-arrested BALB/c-3T3 cells that had received a transient exposure to PDGF and were then transferred to medium containing only EGF and somatomedin C (Sm-C) began DNA synthesis after the G0/G1 lag. Supraphysiological concentrations of insulin could be employed to replace the Sm-C requirement. This G0/G1 lag phase was bisected by the requirement for the exogenous presence of EGF. Our data indicated that EGF was required during the traverse of only the first half of G0/G1 phase (6 h) and not during the traverse of late G1. Subphysiological serum concentrations of Sm-C were also necessary to be present with EGF for progression through early G0/G1; however, traverse of the final half of G0/G1 and commitment to DNA synthesis required the presence of Sm-C. It was found that physiological concentrations of Sm-C were required for the traverse late G1. The requirement for Sm-C for G0/G1 traverse of BALB/c-3T3 cells as opposed to human fibroblasts or glial cells may be due to a difference in endogenous synthesis of an insulin-like growth factor. Our data are in close agreement with previous reports that EGF is only required for approximately the first 8 h during traverse of the G0/G1 phase. The requirement for EGF to be present for the first 6 h of G0/G1 could result from a continued or repetitious event or by more than one distinct EGF-requiring event.     

beta-Interferon alters the pattern of proteins secreted from quiescent and platelet-derived growth factor-treated BALB/c-3T3 cells

Mouse beta-interferon (at a concentration of 100 units/ml or higher) inhibited the platelet-derived growth factor (PDGF)-induced replication of quiescent BALB/c-3T3 cells. The interferon treatment did not inhibit, but slightly enhanced, the accumulation of the following three PDGF-induced RNAs: myc RNA, JE RNA, and KC RNA. The treatment with interferon changed the pattern of secreted proteins from quiescent cells and from cells treated with partially purified PDGF; it inhibited the accumulation of the PDGF-induced proteins (including proteins of 63 and 32 kDa) and it induced the accumulation of several other proteins (including proteins of 89, 31.5, 30, and 10.5 kDa) in both quiescent and also in PDGF-treated cells.     

Cellular ras activity is required for passage through multiple points of the G0/G1 phase in BALB/c 3T3 cells.

Microinjection experiments demonstrated a requirement for cellular ras activity late in G1. In this study, we used two separate methods to identify an additional requirement for cellular ras activity early in the G0/G1 phase of the cell cycle. Quiescent BALB/c cells were injected with anti-ras antibody prior to stimulation with serum. The cells would therefore be inhibited in progression through the cell cycle at the earliest point requiring ras function. Alternatively, cells were inhibited in late G1 as in previous studies by injecting anti-ras several hours after serum addition to quiescent cells. The injected cultures were then treated with chemical cell cycle inhibitors known to function in mid-G1. Cells injected with anti-ras prior to serum stimulation were retained at a point of ras requirement prior to the execution point of the chemical inhibitor, while cells injected 3 to 5 h after serum stimulation were retained at a point of ras requirement downstream of the execution point of the chemical inhibitor. To confirm these results, quiescent BALB/c cells were injected with anti-ras antibody prior to or several hours following serum addition. In this case, however, second injections of oncogenic ras or adenoviral E1A protein were performed to overcome the inhibitory effects of the anti-ras antibody. Cells injected prior to serum addition were clearly inhibited at an early point of Ras requirement since they required 5 or 6 h longer to enter S phase than cells injected with anti-ras antibody after serum addition.     

Epidermal growth factor (EGF) and somatomedin C regulate G1 progression in competent BALB/c-3T3 cells

The activity in platelet-poor plasma that allowed density-arrested BALB/c-3T3 cells rendered competent by a transient exposure to platelet-derived growth factor (PDGF) to traverse G1 and enter the S phase has been termed progression activity. Epidermal growth factor (EGF) and somatomedin C-supplemented medium was shown to be capable of replacing the progression activity of 5% platelet-poor plasma (PPP) for competent density-inhibited BALB/c-3T3 cells. Exposure of competent cells to medium supplemented with EGF and somatomedin C reduced the 12 h minimum G1 lag time found in plasma-supplemented medium by 2 h. It is suggested that the reduction in the minimum time required for progression through G1 is due to the availability of free, unbound somatomedin C. Complete G1 traverse required both EGF and somatomedin C; however, the traverse of the last 6 h of G1 and entry into the S phase required only somatomedin C. Though EGF and somatomedin C could replace the G1 phase progression activity of plasma, medium supplemented with EGF and somatomedin C did not support complete cell cycle traverse or growth of sparse cultures of BALB/c-3T3 cells.     

Intracellular univalent cations and the regulation of the BALB/c-3T3 cell cycle

Addition of serum to density-arrested BALB/c-3T3 cells causes a rapid increase in uptake of Na+ and K+, followed 12 h later by the onset of DNA synthesis. We explored the role of intracellular univalent cation concentrations in the regulation of BALB/c-3T3 cell growth by serum growth factors. As cells grew to confluence, intracellular Na+ and K+ concentrations ([Na+]i and [K+]i) fell from 40 and 180 to 15 and 90 mmol/liter, respectively. Stimulation of growth of density-inhibited cells by the addition of serum growth factors increased [Na]i by 30% and [K+]i by 13-25% in early G0/G1, resulting in an increase in total univalent cation concentration. Addition of ouabain to stimulated cells resulted in a concentration-dependent steady decrease in [K+]i and increase in [Na+]i. Ouabain (100 microM) decreased [K+]i to approximately 60 mmol/liter by 12 h, and also prevented the serum- stimulated increase in 86Rb+ uptake. However, 100 microM ouabain did not inhibit DNA synthesis. A time-course experiment was done to determine the effect of 100 microM ouabain on [K+]i throughout G0/G1 and S phase. The addition of serum growth factors to density-inhibited cells stimulated equal rates of entry into the S phase in the presence or absence of 100 microM ouabain. However, in the presence of ouabain, there was a decrease in [K+]i. Therefore, an increase in [K+]i is not required for entry into S phase; serum growth factors do not regulate cell growth by altering [K+]i. The significance of increased total univalent cation concentration is discussed.     

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.     

Cl- channel blockers inhibit transition of quiescent (G0) fibroblasts into the cell cycle

Modulation of ion permeability during the cell cycle is one of the key events in cell cycle progression. We have compared the effects of K+ and Cl- channel blockers on the cell cycle in synchronous and asynchronous NIH3T3 cells. The Cl- channel blocker 5-N-2-(3-phenylpropylamino) benzoic acid (NPPB; 0.2 mM) inhibited entry into S phase in synchronous cells but not in asynchronous cells, while the K+ channel blocker 4-aminopyridine (4-AP) showed similar inhibitory effects in both conditions. In NIH3T3 cells synchronized by serum deprivation/replenishment, G0-to-G1 transition occurred within 8 h after serum addition, and the G1/S checkpoint at 10-14 h. NPPB applied only at 0-8 or 8-14 h after serum addition inhibited entry into S phase. Cl- permeability measured as 125I efflux increased at 4 and 10 h after serum addition. Ki-67-negative cells, which represent quiescent G0 phase cells, progressively decreased in number until 8 h after serum addition. The Cl- channel blockers (NPPB and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid [DIDS]) but not the K+ channel blocker (4-AP) significantly decreased the rate of reduction in number of Ki-67-negative cells. These data indicate that an increase in Cl- permeability plays an important role in reentry of quiescent cells into the proliferating phase, in addition to the known effects on passage through the G1/S checkpoint.     

Cyclic GMP metabolism in relation to the regulation of cell growth in BALB/c3T3 cells.

BALB/c3T3 cell homogenates have guanylate cyclase in both 105000 g paniculate and soluble fraction. The activity in particulate fraction was much higher than that in the soluble fraction. Both enzyme activities were 2- to 3-fold greater in the resting state (G0 phase) than in the logarithmically growing state. In addition, cGMP-phosphodiesterase activity was 2-fold greater in resting than in growing cells. When G0-arrested cells entered into the G1 phase by serum addition, cGMP levels rapidly increased, whereas guanylate cyclase activities did not change within 30 min after serum addition. Four hours after serum addition, these activities had, however, decreased to one third and remained at that low level throughout the G1 phase. The relationship between cell growth and guanylate cyclase activity is discussed.     

Elevated intracellular concentrations of cyclic AMP inhibited serum-stimulated, density-arrested BALB/c-3T3 cells in mid G1

The stimulation of DNA synthesis in quiescent, density-arrested BALB/c-3T3 cells by platelet-derived growth factor in plasma-supplemented medium was inhibited by the presence of isobutylmethylxanthine (IBMX) and cholera toxin, although neither IBMX or cholera toxin when used alone inhibited the stimulation of DNA synthesis. The cells were reversibly inhibited in mid G1 at a point 6 hr prior to the initiation of DNA synthesis. The inhibition of cell cycle traverse was associated with a 10-15 fold increase in cellular cyclic AMP concentration over basal levels. The reversal of this inhibition by removal of IBMX was correlated with a dramatic decrease in cyclic AMP levels. The traverse of G1 and the initiation of DNA synthesis after release from the cholera toxin and IBMX inhibition was dependent on the presence of plasma in the medium. Either somatomedin C (10-20 ng/ml) or insulin (10(-6)-10(-5) M) completely replaced the plasma requirement for late G1 progression and entry into S phase. Once the inhibited cells were released from the IBMX and cholera toxin block a subsequent increase in cyclic AMP did not prevent entry into S phase. The presence of cholera toxin alone inhibited the stimulation of human dermal fibroblasts. The elevation of intracellular cyclic AMP levels in the human dermal fibroblasts by cholera toxin was two to three fold greater than that found in the BALB/c-3T3 cells in the presence of cholera toxin and the IBMX.     

Modulation of urokinase plasminogen activator gene expression during the transition from quiescent to proliferative state in normal mouse cells.

We have investigated the regulation of urokinase (u-PA) mRNA in quiescent mouse fibroblasts and keratinocytes stimulated to divide by the addition of serum or epidermal growth factor (EGF), respectively. Serum stimulation of quiescent fibroblasts (BALB/c 3T3 or Swiss 3T3) results in an early and transient increase of u-PA mRNA level, which precedes by several hours the onset of DNA synthesis. A similar response is elicited by EGF stimulation of quiescent keratinocytes. The increase of u-PA mRNA parallels that of c-myc mRNA, does not require protein synthesis and is at least in part due to increase in template activity of the u-PA gene. Induction of terminal differentiation of mouse keratinocytes results in a decrease of u-PA mRNA which parallels the decrease of thymidine incorporation. In conclusion, variation in the level of u-PA mRNA is seen during G0/G1 transition and correlates with the proliferative state of these normal mouse cells.     

Fibroblast growth regulatory factor inhibits DNA synthesis in BALB/c 3T3 cells by arresting in G1

A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated fibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late G1 or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early G1 showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the G1. Further, the regulatory factor delayed the progression of G0/G1-arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early G1, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.     

Induction of cell division in BALB/c-3T3 cells by phorbol myristate acetate or bovine serum: effects of inhibitors of cyclic AMP phosphodiesterase and Na+-K+-ATPase.

Cell division is induced in stationary cultures of BALB/c-3T3 mouse embryo cells without renewal of medium by addition of the tumor promoter, phorbol myristate acetate (PMA), or bovine serum. The addition of dbcAMP (10(-3) M) or other inhibitors of cAMP phosphodiesterase, papaverine (6.7 X 10(-6) M), Persantin (5 X 10(-5) M) or RO-20-1724 (10(-4) M), prevents cell replication induced by PMA or serum. In contrast, ouabain (10(-4) M) and N,N'-dicyclohexylcarbodiimide (10(-5) M), inhibitors of Na+-K+-ATPase activity, block the PMA-stimulated effect but do not inhibit serum-stimulated cell division. Several stages in the cell cycle are sensitive to dbcAMP addition. One is early in the G1 phase at the time of reinitiation of the cell cycle from a stationary (Go) phase, a second is associated with the G1-S transition, and a third with passage of cells from a post-S phase to mitosis. Based on observations of early morphological changes, responses of plasma membrane enzymes and effects of enzyme inhibitors, the stimulation of cell division in BALB/c-3T3 cells by PMA or serum appears to involve several membrane functions which may act in a cooperative manner.     

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.     

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.     

Autocrine induction of major histocompatibility complex class I antigen expression results from induction of beta interferon in oncogene-transformed BALB/c-3T3 cells.

By varying growth conditions, we identified a novel mechanism of autocrine regulation of major histocompatibility complex (MHC) class I gene expression by induction of beta interferon gene expression in transformed BALB/c-3T3 cells. Low-serum conditions enhanced MHC class I antigen expression in v-rasKi- and v-mos-transformed BALB/c-3T3 cells but not in untransformed BALB/c-3T3 cells. Transformed and untransformed cells grown under standard serum conditions (10% bovine calf serum) expressed similar cell surface levels of MHC class I antigens. However, low-serum conditions (0.5% bovine calf serum) induced four- to ninefold increases in cell surface levels of MHC class I antigens in both v-rasKi- and v-mos-transformed cells but not in untransformed cells. These increases in MHC class I gene expression were seen at both the mRNA and cell surface protein levels and involved not only the heavy-chain component of the class I antigens but also beta 2 microglobulin. Beta 1 interferon mRNA and beta interferon-inducible 2',5'-oligoadenylate synthetase mRNA were induced by growth under low-serum conditions in transformed BALB/c-3T3 cells, and antibodies to beta interferon blocked the induction of MHC class I antigen expression by serum deprivation in these cells. These results demonstrate that growth under low-serum conditions leads to induction of beta interferon expression in oncogene-transformed cells which then directly mediates autocrine enhancement of MHC class I gene expression.     

Inhibition of early platelet-derived growth factor responses in BALB/c-3T3 cells by interferon

Stimulation of total inositol phosphate production, alteration of cytosolic free calcium [( Ca++]i), vinculin disruption from adhesion plaques, and DNA synthesis caused by PDGF were examined in normal and INF pretreated density arrested BALB/c-3T3 fibroblasts. In normal cells, PDGF caused an increase in total inositol phosphates, a rapid, transient increase in [Ca++]i, disappearance of vinculin from adhesion plaques, and stimulation of DNA synthesis. Pretreatment of cells with INF inhibited PDGF-stimulated increases in [Ca++]i, vinculin disruption from adhesion plaques, and DNA synthesis, but had no effect on PDGF-induced increase in total inositol phosphate levels. These findings suggest that INF prevents entry of quiescent BALB/c-3T3 cells into G1 by inhibiting PDGF-induced release of Ca++ from intracellular stores.