The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.     

Activation of the G2 cell cycle checkpoint enhances survival of epithelial cells exposed to hyperoxia

Reactive oxygen species produced during hyperoxia damage DNA, inhibit proliferation in G1- through p53-dependent activation of p21(Cip1/WAF1/Sdi1), and kill cells. Because checkpoint activation protects cells from genotoxic stress, we investigated cell proliferation and survival of the murine type II epithelial cell line MLE15 during hyperoxia. These cells were chosen for study because they express Simian large and small-T antigens, which transform cells in part by disrupting the p53-dependent G1 checkpoint. Cell counts, 5-bromo-2'-deoxyuridine labeling, and flow cytometry revealed that hyperoxia slowed cell cycle progression after one replication, resulting in a pronounced G2 arrest by 72 h. Addition of caffeine, which inactivates the G2 checkpoint, diminished the percentage of hyperoxic cells in G2 and increased the percentage in sub-G1 and G1. Abrogation of the G2 checkpoint was associated with enhanced oxygen-induced DNA strand breaks and cell death. Caffeine did not affect DNA integrity or viability of cells exposed to room air. Similarly, caffeine abrogated the G2 checkpoint in hyperoxic A549 epithelial cells and enhanced oxygen-induced toxicity. These data indicate that hyperoxia rapidly inhibits proliferation after one cell cycle and that the G2 checkpoint is critical for limiting DNA damage and cell death.     

SPARC inhibits epithelial cell proliferation in part through stimulation of the transforming growth factor-beta-signaling system

Secreted protein, acidic and rich in cysteine (SPARC) is a multifunctional secreted protein that regulates cell-cell and cell-matrix interactions, leading to alterations in cell adhesion, motility, and proliferation. Although SPARC is expressed in epithelial cells, its ability to regulate epithelial cell growth remains largely unknown. We show herein that SPARC strongly inhibited DNA synthesis in transforming growth factor (TGF)-beta-sensitive Mv1Lu cells, whereas moderately inhibiting that in TGF-beta-insensitive Mv1Lu cells (i.e., R1B cells). Overexpression of dominant-negative Smad3 in Mv1Lu cells, which abrogated growth arrest by TGF-beta, also attenuated growth arrest stimulated by SPARC. Moreover, the extracellular calcium-binding domain of SPARC (i.e., SPARC-EC) was sufficient to inhibit Mv1Lu cell proliferation but not that of R1B cells. Similar to TGF-beta and thrombospondin-1, treatment of Mv1Lu cells with SPARC or SPARC-EC stimulated Smad2 phosphorylation and Smad2/3 nuclear translocation: the latter response to all agonists was abrogated in R1B cells or by pretreatment of Mv1Lu cells with neutralizing TGF-beta antibodies. SPARC also stimulated Smad2 phosphorylation in MB114 endothelial cells but had no effect on bone morphogenetic protein-regulated Smad1 phosphorylation in either Mv1Lu or MB114 cells. Finally, SPARC and SPARC-EC stimulated TGF-beta-responsive reporter gene expression through a TGF-beta receptor- and Smad2/3-dependent pathway in Mv1Lu cells. Collectively, our findings identify a novel mechanism whereby SPARC inhibits epithelial cell proliferation by selectively commandeering the TGF-beta signaling system, doing so through coupling of SPARC-EC to a TGF-beta receptor- and Smad2/3-dependent pathway.     

Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia

Prolonged exposure to hyperoxia inhibits cell proliferation in G1 via increased expression of p21. While p21 inhibits proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis, it can also directly lower PCNA abundance; however, it is unclear whether loss of PCNA contributes to growth arrest. Here, we investigate how PCNA loss affects ability of p21 to exert G1 growth arrest of lung epithelial cells exposed to hyperoxia. In A549 cells that express p21 and growth arrest in G1 during hyperoxia, small interfering RNA (siRNA) knockdown of p21 led to G1 checkpoint bypass, increased cell death, and restoration of PCNA expression. Conditional overexpression of the PCNA binding domain of p21 in H1299 cells that do not normally express p21, or exposure to hyperoxia, caused a time-dependent loss of PCNA. Titrating PCNA levels using siRNA to approximate the low amount observed in cells expressing p21 resulted in S phase arrest. While lowering PCNA by itself caused S phase arrest, the combination of hyperoxia and siRNA against PCNA dramatically reduced PCNA abundance resulting in G1 arrest. G1 growth arrest was markedly enhanced upon the addition of p21 to these cells. Our findings suggest a model in which reducing expression of the abundant protein PCNA allows the less abundant protein p21 to be more effective at suppressing the processivity functions of remaining PCNA, thereby fully exerting the G1 checkpoint. Given that high p21 expression is often associated with lower PCNA abundance, our findings are suggestive of a global growth inhibitory mechanism involving p21-mediated PCNA suppression.     

The antiproliferative effect of type beta transforming growth factor occurs at a level distal from receptors for growth-activating factors

Transforming growth factor-beta (TGF beta) from human platelets blocks the ability of Mv1Lu mink lung epithelial cells to grow in response to serum mitogens, epidermal growth factor (EGF), or insulin. The phenotypic response of Mv1Lu cells to TGF beta is characterized by a flat, very enlarged cell morphology and a markedly increased production and accumulation of extracellular matrix fibronectin. The ability of TGF beta to alter the ligand binding or signal transducing activity of mitogen receptors in Mv1Lu cells has been examined. In contrast to NRK-49F rat fibroblasts, Mv1Lu cells do not respond to TGF beta with a decrease in the affinity or a change in the number of cell surface receptors for EGF. Soluble extracts from Mv1Lu cells contain a protein kinase activity which selectively phosphorylates ribosomal protein S6; this S6 kinase activity is elevated severalfold minutes after exposure of cells to mitogens. This kinase activity has been used as the parameter to measure the signaling ability of EGF receptors and insulin receptors in cells treated with TGF beta. We find that TGF beta does not alter the basal level of S6 kinase activity or its elevation by EGF or insulin. In TGF beta-treated cells rendered insensitive to the growth-promoting action of EGF, the parameters of elevation of S6 kinase activity by EGF are similar to those of control, growth-competent cells. The results suggest that TGF beta inhibits cell proliferation by acting at a level distal from the receptors for growth-activating factors.     

The Cdk and PCNA domains on p21Cip1 both function to inhibit G1/S progression during hyperoxia

This study investigates molecular mechanisms underlying cell cycle arrest when cells are exposed to high levels of oxygen (hyperoxia). Hyperoxia has previously been shown to increase expression of the cell cycle regulators p53 and p21. In the current study, we found that p53-deficient human lung adenocarcinoma H1299 cells failed to induce p21 or growth arrest in G(1) when exposed to 95% oxygen. Instead, cells arrested in S and G(2). Stable expression of p53 restored induction of p21 and G(1) arrest without affecting mRNA expression of the other Cip or INK4 G(1) kinase inhibitors. To confirm the role of p21 in G(1) arrest, we created H1299 cells with tetracycline-inducible expression of enhanced green fluorescent protein (EGFP), EGFP fused to p21 (EGFp21), or EGFP fused to p27 (EGFp27), a related cell cycle inhibitor. The amino terminus of p21 and p27 bind cyclin-dependent kinases (Cdk), whereas the carboxy terminus of p21 binds the sliding clamp proliferating cell nuclear antigen (PCNA). EGFp21 or EGFp27, but not EGFP by itself, restored G(1) arrest during hyperoxia. When separately overexpressed, the amino-terminal Cdk and carboxy-terminal PCNA binding domains of p21 each prevented cells from exiting G(1) during exposure. These findings demonstrate that exposure in vitro to hyperoxia exerts G(1) arrest through p53-dependent induction of p21 that suppresses Cdk and PCNA activity. Because PCNA also participates in DNA repair, these results raise the possibility that p21 also affects repair of oxidized DNA.     

Secretory phospholipase A(2) induces apoptosis via a mechanism involving ceramide generation

Secretory phospholipase A(2) (sPLA(2)) plays important roles in cellular signaling and various biological events. In this study, we examined the biological effects and the potential signaling mechanism of purified sPLA(2) in MV1Lu cells. Three types of snake venom sPLA(2) were purified and their enzymatic activities were characterized by using various lipid substrates prepared from [3H]-myristate-labeled cells and by determining their effects on the induction of arachidonic acid (AA) release. The purified sPLA(2) induced apoptosis in Mv1Lu cells in a dose- and time-dependent manner, and was associated with a rapid increase in the intracellular ceramide level. Similar apoptotic effects were observed in Mv1Lu cells treated with exogenous ceramide analog, C(2)- and C(8)-ceramide. Moreover, treatment of cells with sphingomyelinase (SMase), which reduced the intracellular SM level, enhanced the apoptotic response to sPLA(2)s. sPLA(2)s also displayed an inhibitory effect on bradykinin-induced phospholipase D (PLD) activity, which can be imitated by exogenous ceramide. Our data indicate that sPLA(2) induces cell apoptosis via a mechanism involving increased ceramide generation.     

Novel Cdk inhibitors restore TGF-beta sensitivity in cdk4 overexpressing epithelial cells

Transforming growth factor-beta (TGF-beta) is a potent mitogen that effects a wide variety of cells by blocking cell growth. TGF-beta acts by interacting with components of cell cycle machinery to cause G1 arrest and in mink lung epithelial cells (Mv1Lu) it does so by inhibiting Cdk4 synthesis. Overexpression of Cdk4 in these cells (B7) renders them resistant to the effects of TGF-beta. Here we report that two novel Cdk inhibitors (pyridopyrimidines) that not only inhibit Cdk4 and Cdk2 in an in vitro kinase assay but also, in the absence of TGF-beta, block growth of Mv1Lu cells in G1 more efficiently than their B7 (overexpressing Cdk4) counterparts. Interestingly, these inhibitors restored sensitivity of B7 cells towards TGF-beta. This may have implications for the treatment of tumors that have lost TGF-beta responsiveness due to deregulated cellular growth in vivo. These Cdk inhibitors could therefore be used in conjunction with TGF-beta to understand the mechanism of growth arrest in normal versus tumour cells.     

The protein phosphatase 1 regulator PNUTS is a new component of the DNA damage response

The function of protein phosphatase 1 nuclear-targeting subunit (PNUTS)--one of the most abundant nuclear-targeting subunits of protein phosphatase 1 (PP1c)--remains largely uncharacterized. We show that PNUTS depletion by small interfering RNA activates a G2 checkpoint in unperturbed cells and prolongs G2 checkpoint and Chk1 activation after ionizing-radiation-induced DNA damage. Overexpression of PNUTS-enhanced green fluorescent protein (EGFP)--which is rapidly and transiently recruited at DNA damage sites--inhibits G2 arrest. Finally, γH2AX, p53-binding protein 1, replication protein A and Rad51 foci are present for a prolonged period and clonogenic survival is decreased in PNUTS-depleted cells after ionizing radiation treatment. We identify the PP1c regulatory subunit PNUTS as a new and integral component of the DNA damage response involved in DNA repair.     

Hepatocyte growth factor releases mink epithelial cells from transforming growth factor beta1-induced growth arrest by restoring Cdk6 expression and cyclin E-associated Cdk2 activity

Transforming growth factor beta (TGF-beta) potently suppresses Mv1Lu mink epithelial cell growth, whereas hepatocyte growth factor (HGF) counteracts TGF-beta-mediated growth inhibition and induces Mv1Lu cell proliferation (J. Taipale and J. Keski-Oja, J. Biol. Chem. 271:4342-4348, 1996). By addressing the cell cycle regulatory mechanisms involved in HGF-mediated release of Mv1Lu cells from TGF-beta inhibition, we show that increased DNA replication is accompanied by phosphorylation of the retinoblastoma protein and alternative regulation of cyclin-Cdk-inhibitor complexes. While TGF-beta treatment decreased the expression of Cdk6, this effect was counteracted by HGF, followed by partial restoration of cyclin D2-associated kinase activity. Notably, HGF failed to prevent TGF-beta induction of p15 and its association with Cdk6. However, HGF reversed the TGF-beta-mediated decrease in Cdk6-associated p27 and cyclin D2-associated Cdk6, suggesting that HGF modifies the TGF-beta response at the level of G1 cyclin complex formation. Counteraction of TGF-beta regulation of Cdk6 by HGF may in turn affect the association of p27 with Cdk2-cyclin E complexes. Though HGF did not differentially regulate the total levels of p27 in TGF-beta-treated cells, p27 immunodepletion experiments suggested that upon treatment with both growth factors, less p27 is associated with Cdk2-cyclin E complexes, in parallel with restoration of the active form of Cdk2 and the associated kinase activity. The results demonstrate that HGF intercepts TGF-beta cell cycle regulation at multiple points, affecting both G1 and G1-S cyclin kinase activities.     

Trifluoperazine stimulates ionizing radiation induced cell killing through inhibition of DNA repair

The effect of trifluoperazine (TFP), a phenothiazine derivative antipsychotic drug, on ionizing radiation (IR) induced cell killing through inhibition of DNA repair was investigated in human cell lines. In clonogenic survival assay, TFP augmented IR induced cell killing. Also, TFP enhanced micronucleus formation in irradiated human lymphocytes. The effect of TFP and other known DNA repair inhibitors like wortmannin and caffeine, on irradiated cells, was compared by MTT assay. On the other hand, TFP failed to increase the toxicity induced by H2O2. Repair of DNA double strand breaks induced by IR was markedly inhibited by TFP, as determined by field inversion gel electrophoresis (FIGE). Further, TFP increased radiation induced apoptosis, which was accompanied by enhanced G2/M arrest. Thus, our results strongly suggest that TFP inhibits repair of DNA damage induced by IR, which significantly implicates the possibility of using TFP as an adjuvant to radiotherapy.     

Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A

The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.     

Enhancement of radiation cytotoxicity by UCN-01 in non-small cell lung carcinoma cells

Thoracic ionizing radiation is a standard component of combined-modality therapy for locally advanced non-small cell lung cancer. To improve low 5-year survival rates (5- 15%), new strategies for enhancing the effectiveness of ionizing radiation are needed. The kinase inhibitor UCN-01 has multiple cell cycle effects, including abrogation of DNA damage-induced S- and G(2)-phase arrest, which may limit DNA repair prior to mitosis. To test the hypothesis that therapy-induced cell cycle effects would have an impact on the efficacy of a combination of UCN-01 plus ionizing radiation, the cell cycle responses of the non-small cell lung cancer cell lines Calu1 (TP53-null) and A549 (wild-type TP53) to 2 Gy ionizing radiation were correlated with clonogenic survival after irradiation plus UCN-01. Irradiated cells were exposed to UCN-01 simultaneously and at 3-h increments after irradiation. In Calu1 cells but not A549 cells, sequence-dependent potentiation of radiation by UCN-01 was observed, with maximal interaction occurring when UCN-01 was administered 6 h after irradiation. This coincided with the postirradiation time with the greatest depletion of cells from G(1). Abrogation of G(2) arrest was observed regardless of TP53 status. The role of TP53 was investigated using siRNA to achieve gene silencing. These studies demonstrated that radiation plus UCN-01 was more effective in cells with diminished TP53 activity, associated with a reduced G(1) checkpoint arrest. These studies indicate that simultaneous elimination of multiple DNA damage-induced checkpoints in G(1), S and G(2) may enhance the effects of radiation and that drug scheduling may have an impact on clinical efficacy.     

DNA damage during reoxygenation elicits a Chk2-dependent checkpoint response

Due to the abnormal vasculature of solid tumors, tumor cell oxygenation can change rapidly with the opening and closing of blood vessels, leading to the activation of both hypoxic response pathways and oxidative stress pathways upon reoxygenation. Here, we report that ataxia telangiectasia mutated-dependent phosphorylation and activation of Chk2 occur in the absence of DNA damage during hypoxia and are maintained during reoxygenation in response to DNA damage. Our studies involving oxidative damage show that Chk2 is required for G2 arrest. Following exposure to both hypoxia and reoxygenation, Chk2-/- cells exhibit an attenuated G2 arrest, increased apoptosis, reduced clonogenic survival, and deficient phosphorylation of downstream targets. These studies indicate that the combination of hypoxia and reoxygenation results in a G2 checkpoint response that is dependent on the tumor suppressor Chk2 and that this checkpoint response is essential for tumor cell adaptation to changes that result from the cycling nature of hypoxia and reoxygenation found in solid tumors.     

The modulation of apoptosis by cyclic AMP involves Akt and epidermal growth factor receptor

Adenosine 3',5'-cyclic monophosphate (cAMP) and transforming growth factor-beta are important regulators of many biological processes. In this study we investigated the effect and its potential mechanism of cAMP on transforming growth factor-beta1- and serum deprivation-induced apoptosis in Mv1Lu cells. Transforming growth factor-beta1 treatment or serum deprivation induces apoptotic response in Mv1Lu cells. Forskolin, a cAMP-elevating agent, or 8-Bromo-cAMP (8-B-cAMP), a cell permeable cAMP analogue, inhibited the cell proliferation and markedly enhanced apoptosis induced by transforming growth factor-beta1, but completely suppressed serum deprivation-induced apoptosis. Furthermore, forskolin decreased the Akt phosphorylation, and the inhibition of phosphatidylinositol-3 kinase by LY294002 sensitized Mv1Lu cells to transforming growth factor-beta1-induced apoptosis. In addition, forskolin treatment induced tyrosine phosphorylation of epidermal growth factor receptor. Inhibition of epidermal growth factor receptor by specific inhibitor PD153035 blocked the cAMP-mediated suppression of serum deprivation-induced apoptosis. The results indicate that cAMP exerts its opposite effects in transforming growth factor-beta1- and serum deprivation-induced apoptosis via a mechanism involving the modulation of signaling components of phosphatidylinositol-3-kinase/Akt and epidermal growth factor receptor in Mv1Lu cells.     

Distinct roles for Galpha(i)2 and Gbetagamma in signaling to DNA synthesis and Galpha(i)3 in cellular transformation by dopamine D2S receptor activation in BALB/c 3T3 cells

Control of cell proliferation depends on intracellular mediators that determine the cellular response to external cues. In neuroendocrine cells, the dopamine D2 receptor short form (D2S receptor) inhibits cell proliferation, whereas in mesenchymal cells the same receptor enhances cell proliferation. Nontransformed BALB/c 3T3 fibroblast cells were stably transfected with the D2S receptor cDNA to study the G proteins that direct D2S signaling to stimulate cell proliferation. Pertussis toxin inactivates G(i) and G(o) proteins and blocks signaling of the D2S receptor in these cells. D2S receptor signaling was reconstituted by individually transfecting pertussis toxin-resistant Galpha(i/o) subunit mutants and measuring D2-induced responses in pertussis toxin-treated cells. This approach identified Galpha(i)2 and Galpha(i)3 as mediators of the D2S receptor-mediated inhibition of forskolin-stimulated adenylyl cyclase activity; Galpha(i)2-mediated D2S-induced stimulation of p42 and p44 mitogen-activated kinase (MAPK) and DNA synthesis, whereas Galpha(i)3 was required for formation of transformed foci. Transfection of toxin-resistant Galpha(i)1 cDNA induced abnormal cell growth independent of D2S receptor activation, while Galpha(o) inhibited dopamine-induced transformation. The role of Gbetagamma subunits was assessed by ectopic expression of the carboxyl-terminal domain of G protein receptor kinase to selectively antagonize Gbetagamma activity. Mobilization of Gbetagamma subunits was required for D2S-induced calcium mobilization, MAPK activation, and DNA synthesis. These findings reveal a remarkable and distinct G protein specificity for D2S receptor-mediated signaling to initiate DNA synthesis (Galpha(i)2 and Gbetagamma) and oncogenic transformation (Galpha(i)3), and they indicate that acute activation of MAPK correlates with enhanced DNA synthesis but not with transformation.     

Apoptosis of mink lung epithelial cells by co-treatment of low-dose staurosporine and transforming growth factor-beta1 depends on the enhanced TGF-beta signaling and requires the decreased phosphorylation of PKB/Akt

We demonstrate how co-treatment of low-dose staurosporine (STS) and TGF-beta1, which alone have little effect on cell death, markedly induces apoptosis in Mv1Lu mink lung epithelial cells, but not in its clonal variant R1B cells lacking functional TGF-beta signaling. This process was associated with mitochondria-dependent apoptosis and the enhanced TGF-beta/Smad signaling in Mv1Lu cells. When R1B cells were infected with adenovirus carrying wild-type ALK5, a functional TGF-beta type I receptor gene, the apoptotic cell death was significantly restored in these cells following co-treatment of low-dose STS and TGF-beta1. Treatment of Mv1Lu cells with both low-dose STS and TGF-beta1 decreased the activity of phospho-Akt, which is involved in cell survival signal. In addition, pre-treatments of PI3 kinase inhibitors, LY294002 and wortmannin, further increased the apoptosis of MvlLu cells induced by co-treatment of low-dose STS and TGF-beta1. And overexpression of constitutively active Akt (myr-Akt) using adenoviral expression system inhibited the apoptotic cell death of Mv1Lu cells by about 50% upon co-treatment of low-dose STS and TGF-beta1. These results suggest that co-treatment of low-dose STS and TGF-beta1 induces apoptosis of mink lung epithelial cells by enhancing TGF-beta signaling and in part suppressing cytoprotective signaling.     

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.