Glucocorticoid-induced G1 arrest and the release effect of epidermal growth factor on the human salivary gland adenocarcinoma cell

Dexamethasone (1 microM) decreased the distribution of cells in S phase (about 75%) and increased that of G1 cells (1.1-fold) in the DNA histogram of human submandibular salivary gland adenocarcinoma cells (HSG) reversibly. In synchronized cells at G1 phase, glucocorticoid delayed the initiation of DNA synthesis by about 3-4 h. The conditioned medium (50%) or exogenous human epidermal growth factor (EGF, 10 ng/ml) significantly nullified these effects by glucocorticoids. These results suggested that glucocorticoids arrested the cells at G1 phase, which implied the inhibition of production of some progressive factor, probably EGF, in the cell cycle of HSG.     

S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus.

We examined replication of the autonomous parvovirus Aleutian mink disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.     

G1shortening following unbalanced growth a specific v. nonspecific effect

The synthesis and abundance of proteins were examined in synchronous populations of HeLa cells under conditions in which the lengthening of S phase, by inhibiting DNA synthesis, resulted in shortening of G1 in the subsequent generation. Mitotically collected cells were resynchronized by incubation with 3 microM aphidicolin from 3 to 12 h after mitotic selection; they were blocked again at various times thereafter to induce unbalanced growth. Cells were labelled with [35S]-methionine before and after release from the block to study the changes in protein synthesis. Triton X-100 soluble and insoluble proteins were analysed by 7-15% gradient SDS-PAGE, and radioactivity incorporation was quantified by liquid-scintillation counting. The degree of G1 shortening correlated with S phase position, increasing gradually from early S and reaching maximum when cells were blocked half-way through S phase. Synthesis of proteins of 120, 66, and 51 kDa was stimulated, and synthesis of a new protein of 57kDa was observed, in cells in which DNA synthesis had been blocked in mid-S. These proteins also showed increased accumulation. These results suggest that the shortening of G1, induced by the prior arrest of cell-cycle progression, is associated with synthesis of specific proteins rather than the non-specific accumulation of cellular proteins through unbalanced growth.     

Abrogation of growth arrest signals by human papillomavirus type 16 E7 is mediated by sequences required for transformation.

Cells arrest in the G1 or G0 phase of the cell cycle in response to a variety of negative growth signals that induce arrest by different molecular pathways. The ability of human papillomavirus (HPV) oncogenes to bypass these signals and allow cells to progress into the S phase probably contributes to the neoplastic potential of the virus. The E7 protein of HPV-16 was able to disrupt the response of epithelial cells to three different negative growth arrest signals: quiescence imposed upon suprabasal epithelial cells, G1 arrest induced by DNA damage, and inhibition of DNA synthesis caused by treatment with transforming growth factor beta. The same set of mutated E7 proteins was able to abrogate all three growth arrest signals. Mutant proteins that failed to abrogate growth arrest signals were transformation deficient and included E7 proteins that bound retinoblastoma protein in vitro. In contrast, HPV-16 E6 was able to bypass only DNA damage-induced G1 arrest, not suprabasal quiescence or transforming growth factor beta-induced arrest. The E6 and E7 proteins from the low-risk virus HPV-6 were not able to bypass any of the growth arrest signals.     

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.     

Regulation of p27Kip1 by intracellular iron levels

Enhanced intracellular iron levels are essential for proliferation of mammalian cells. If cells have entered S phase when iron is limiting, an adequate supply of deoxynucleotides cannot be maintained and the cells arrest with incompletely replicated DNA. In contrast, proliferating cells that are not in S phase, but have low iron pools, arrest in late G1. In this report the mechanism of iron-dependent G1 arrest in normal fibroblasts was investigated. Cells were synchronized in G0 by contact inhibition and serum deprivation. Addition of serum caused the cells to re-enter the cell cycle and enter S phase. However, if the cells were also treated with the iron chelator deferoxamine, S phase entry was blocked. This corresponded to elevated levels of the cyclin dependent kinase inhibitor p27^Kip1 and inhibition of CDK2 activity. Expression of other cell cycle regulatory proteins was not affected, including the induction of cyclins D1 and E. When the quiescent serum starved cells were supplemented with a readily usable form of iron in the absence of serum or any other growth factors, a significant population of the cells entered S phase. This was associated with downregulation of p27^Kip1 and increased CDK2 activity. Using an IPTG-responsive construct to artificially raise p27^Kip1 levels blocked the ability of iron supplementation to promote S phase entry. Thus it appears that p27^Kip1 is a mediator of G1 arrest in iron depleted Swiss 3T3 fibroblasts. We propose that this is part of an iron-sensitive checkpoint that functions to ensure that cells have sufficient iron pools to support DNA synthesis prior to entry into S phase.     

Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells

A cDNA library was screened for plant proliferating-cell nuclear antigen (PCNA) from Catharanthus roseus (periwinkle). A lambda gt11 cDNA library was constructed using poly(A)-rich RNA isolated from the cells in the S phase. A cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ-1, which contains PCNA-related gene sequences. The cDNA contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29,765 Da. When conservative substitutions were included, a high degree of similarity (about 85%) was observed between the predicted amino acid sequence of periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA was undetectable or very weak in quiescent cells, such as phosphate-starved cells, auxin-starved cells and cells in the stationary phase. In the synchronous progression of the cell cycle induced by the addition of phosphate or auxin, the active accumulation of periwinkle PCNA mRNA was observed preferentially in the S phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells at the G1 phase, an increase in the level of PCNA mRNA was observed. The partial inhibition of protein synthesis at the G1 phase by a protein inhibitor, anisomycin, caused the arrest of cells in the G1 phase. No increase of the level of periwinkle PCNA mRNA was observed in cells arrested at the G1 phase by the inhibition of protein synthesis. These results indicate that the induction of mRNA for periwinkle PCNA occurred independently of the initiation of DNA replication, but that synthesis of certain proteins at the G1 phase was required for the induction of periwinkle PCNA mRNA at the S phase.     

Serum-dependent regulation of proliferation of cultured rat fibroblasts in G1 and G2 phases

We reported that: (i) 3Y1tsF121 cells, a temperature-sensitive (ts) mutant of rat 3Y1 fibroblasts, are reversibly arrested either in the G1 or in the G2 phase, at the nonpermissive temperature. (ii) Cells retain the ability to resume proliferation at the permissive temperature after prolonged arrest in the G1 phase (for 5 days), whereas they lose it after prolonged arrest in the G2 phase (over 24 h). (iii) The G1 arrest is overcome at the nonpermissive temperature by the addition of fresh serum (H. Zaitsu and G. Kimura (1984) J. Cell. Physiol. 119, 82; (1985) J. Cell. Physiol. 124, 177). In the present study, the G2 arrest was overcome by exposing the cells to fresh serum, at the nonpermissive temperature. The G2 arrest occurred only at a higher cell density than that of the G1 arrest. The efficiency of the overcome was higher in the case of the G2 arrest than in case of the G1 arrest. When cells synchronized at the G1/S border by aphidicolin at the permissive temperature were released from the block, they divided in the absence of serum, at the permissive temperature. Even if they had passed through the previous G2 phase in a very high concentration of fresh serum at the permissive temperature, mitotic cells did not enter the S phase in the absence of serum, even at the permissive temperature. When the cells arrested in the G1 phase (not in G0) due to the ts defect were incubated in the absence of serum at the permissive temperature, only 34% entered the S phase and only 15% divided. These results suggest that (i) the ts defect in 3Y1tsF121 limiting cellular proliferation in both the G1 and the G2 phases is probably due to a single mutational event, and is a serum-requiring event. (ii) Preparation of the serum-requiring event which is required for the G2 traverse is completed in the G1 phase, under ordinary conditions. (iii) However, cells are able to fulfill the serum-requiring event in the G2 phase as well as in the G1 phase when the preparation is below the required level. (iv) The commitment to DNA synthesis is not necessarily a commitment to cell division. (v) Cells are arrested in the G1 phase more safely and more effectively than in the G2 phase, by the serum-related mechanism.     

Deoxyribonucleotide metabolism and cyclic AMP resistance in hydroxyurea-resistant S49 T-lymphoma cells

We investigated the cell cycle regulation of deoxyribonucleoside triphosphate (dNTP) metabolism in hydroxyurea-resistant (HYUR) murine S49 T-lymphoma cell lines. Cell lines 10- to 40-fold more hydroxyurea-resistant were selected in a stepwise manner. These HYUR cells exhibited increased CDP reductase activity (5- to 8-fold) and increased dNTP pools (up to 5-fold) that appeared to result from increased activity of the M2 subunit (binding site of hydroxyurea) of ribonucleotide reductase. These characteristics remained stable when the cells were grown in the absence of hydroxyurea for up to 2 years. In both wild type and hydroxyurea-resistant cell populations synchronized by elutriation, dCTP and dTTP pools increased in S phase, whereas dATP and dGTP pools generally remained the same or decreased, suggesting that allosteric effector mechanisms were operating to regulate pool sizes. Additionally, CDP reductase activity measured in permeabilized cells increased in S phase in both wild type and hydroxyurea-resistant cells, suggesting a nonallosteric mechanism of increased ribonucleotide reductase activity during periods of active DNA synthesis. While wild type S49 cells could be arrested in the G1 phase of the cell cycle by dibutyryl cyclic AMP, hydroxyurea-resistant cell lines could not be arrested in the G1 phase by exogenous cyclic AMP or agents that elevate the concentration of endogenous cyclic AMP. These data suggest that cyclic AMP-generated G1 arrest in S49 cells might be mediated by the M2 subunit of ribonucleotide reductase.     

Yph1p,an ORC-interacting protein: potential links between cell proliferation control,DNA replication,and ribosome biogenesis

Immunoprecipitation of the origin recognition complex (ORC) from yeast extracts identified Yph1p, an essential protein containing a BRCT domain. Two Yph1p complexes were characterized. Besides ORC, MCM proteins, cell-cycle regulatory proteins, checkpoint proteins, 60S ribosomal proteins, and preribosome particle proteins were found to be associated with Yph1p. Yph1p is predominantly nucleolar and is required for 60S ribosomal subunit biogenesis and possibly for translation on polysomes. Proliferating cells depleted of Yph1p arrest in G(1) or G(2), with no cells in S phase, or significantly delay S phase progression after release from a hydroxyurea arrest. Yph1p levels decline as cells commit to exit the cell cycle, and levels vary depending on energy source. Yph1p may link cell proliferation control to DNA replication, ribosome biogenesis, and translation on polysomes.     

Antiproliferative effect of brief exposure to cholera toxin in vascular smooth muscle cells: role of cAMP and protein kinase A.

The effect of cholera toxin (CTX), an activator of the adenylate cyclase-coupled G protein alpha(s) subunit, was studied on cultured vascular smooth muscle cell (VSMC) proliferation. Continuous exposure (48 h) to CTX as well as 2-min pretreatment of VSMC with CTX led to the same level of cAMP production, inhibition of DNA synthesis, and arrest in the G1 phase without induction of necrosis or apoptosis in VSMC. Protein kinase A (PKA) activity in CTX-pretreated cells was transiently elevated by 3-fold after 3 h of incubation, whereas after 48 h it was reduced by 2-fold compared with baseline values without modulation of the expression of its catalytic alpha subunit. The PKA inhibitors H89 and KT 5720 did not protect VSMC from the antiproliferative effect of CTX. Two-dimensional electrophoresis was used to analyze the influence of CTX on protein phosphorylation. After 3 h of incubation of CTX-pretreated cells, we observed both newly-phosphorylated and dephosphorylated proteins (77 and 50 protein species, respectively). After 24 h of incubation, the number of phosphorylated proteins in CTX-treated cells was decreased to 39, whereas the number of dephosphorylated proteins was increased to 106. In conclusion, brief exposure to CTX leads to full-scale activation of cAMP signaling and evokes VSMC arrest in the G1 phase.     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Analysis of BHK cell growth kinetics after microinjection of catalytic subunit of cyclic AMP-dependent protein kinase.

The effect of catalytic subunit (C) of cyclic AMP-dependent protein kinase on cell growth kinetics of BHK cells was assessed by microinjection with chicken erythrocyte ghosts as vehicles for introduction of the protein into the cytosol of large populations of cells. The advantage in using chicken erythrocytes for microinjection is that the inactive erythrocyte nuclei serve as a probe for identifying and analyzing microinjection events. By utilizing this procedure, BHK cells were microinjected with an amount of C that was 5- to 10-fold greater than their endogenous levels. Growth kinetics were analyzed by [3H]thymidine incorporation and autoradiography. Cells were stained after autoradiography to more clearly reveal the chicken nuclei, and at each time point, cells were categorized into four groups: (i) not microinjected, not in S phase, (ii) not microinjected, in S phase, (iii) microinjected, not in S phase, (iv) microinjected, in S phase. Those cells not microinjected served as internal controls. Two experimental protocols were used to test the notion that C is involved in blocking cell progression through G1 phase of the cell cycle. First, cells were arrested in G0 phase by serum deprivation, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum or purified growth factors. Second, cells were collected in mitosis, microinjected with C or control proteins, and stimulated to proceed to S phase by the addition of serum. The results of these studies indicate that a 5- to 10-fold increase in the intracellular concentration of C is not a sufficient signal to arrest cell growth in G1 phase. Thus, growth-inhibitory effects of cyclic AMP on BHK cells are unlikely to be the result of activation of cyclic AMP-dependent protein kinase.     

Both low and high concentrations of staurosporine induce G1 arrest through down-regulation of cyclin E and cdk2 expression

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.     

Non-histone protein synthesis during G1 phase and its relation to DNA replication.

The kinetics of non-histone chromosomal protein (NHCP) synthesis were studied in Chinese hamster ovary (CHO) plateau phase cells stimulated to proliferate and were compared to NHCP synthesis kinetics in two populations of synchronous G1 traversing cells. In all cases, NHCP synthesis rates increase 3- to 5-fold as cells traversed G1 and attained maximum values one hour before semi-conservative DNA replication began. Similar to results in synchronous G1 cells, the molecular weight distributions of the NHCP fraction from stimulated plateau phase cells underwent only minor changes, measured by sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis, as these cells moved toward S phase. Yet, during this progression after plateau phase and in the transition from early G1 to late G1 in synchronous cells, the total NHCP fraction increased significantly (1.5-2-fold) in amount per cell. These data indicate that plateau phase cells are similar to early G1 cells both in terms of their amounts of non-histone per cell and in their subsequent NHCP synthesis kinetics as they move toward S phase. These results extend previous findings which suggested that NHCP synthesis was coupled to DNA replication and demonstrate that the increased NHCP synthesis and accumulation in chromatin may be a biochemical marker for G1 progression.     

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.     

G1- and S-phase syntheses of histones H1 and H1o in mitotically selected CHO cells: utilization of high-performance liquid chromatography

We have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [3H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O4; the total HC1O4-soluble, Cl3CCO2H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns [Gurley, L. R., D'Anna, J. A., Blumenfeld, M., Valdez, J. G., Sebring, R. J., Donahue, D. K., Prentice, D. A., & Spall, W. D. (1984) J. Chromatogr. 297, 147-165]. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases (3.6 +/- 0.5)-fold as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases (3.1 +/- 0.2)-fold so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure.     

Regulation of purine biosynthesis in G1 phase-arrested mammalian cells

The effects of G1 phase growth arrest on purine biosynthesis were studied in cultured S49 T lymphoma cells. Incubations of wildtype S49 cells for 18 hr with dibutyryl cyclic AMP or forskolin, two agents which induced G1 arrest, reduced the rates of purine biosynthesis by 95%. Time course and concentration dependence studies indicated that the decrease in rates of purine biosynthesis correlated with the extent of G1 phase arrest. Similar studies with somatic cell mutants deficient in some component of cyclic AMP action or metabolism indicated that the depression in purine synthetic rates required G1 arrest and did not result from cell death. Rates of RNA and DNA synthesis were also markedly diminished in the growth arrested cells. Measurements of purine rates in the presence of azaserine indicated that the block in purine biosynthesis was prior to the formation of phosphoribosylformylglycinamide. Additionally, the activities of adenylosuccinate synthetase and IMP dehydrogenase were diminished in G1 arrested cells. The levels of all controlling enzymes, substrates, and cofactors, however, were not diminished in G1 arrested cells. Despite diminished rates of purine biosynthesis, the amounts of intracellular nucleotides in G1 cells were equivalent to those in exponentially growing cells. However, the concentrations of intracellular nucleotides were 30-50% higher in the growth arrested cells. These results suggested that perturbations in the consumption of nucleotides via inhibition of nucleic acid synthesis have profound effects on the purine pathway and indicated the importance of feedback inhibition by nucleotides in the regulation of purine synthesis in situ.