IGF-I stimulates human intestinal smooth muscle cell growth by regulation of G1 phase cell cycle proteins

Autocrine production of insulin-like growth factor-I (IGF-I) regulates growth of human intestinal muscle cells by activation of distinct phosphatidylinositol 3-kinase (PI3-kinase)-dependent and ERK1/2-dependent pathways. The aim of the present study was to determine the mechanisms by which IGF-I regulates the G(1) phase of the cell cycle and muscle cell proliferation. Incubation of quiescent cells with IGF-I stimulated time-dependent cell cycle progression measured by using fluorescence-activated cell sorting analysis and by incorporation of [(3)H]thymidine. Studies using a microarray-based approach were used initially to identify genes expressed in human intestinal muscle encoding proteins known to participate in the G(1) phase of the cell cycle that were regulated by IGF-I. Incubation of muscle cells for 24 h with IGF-I elicited greater than fivefold increase in the expression of cyclin D1 and greater than twofold increase in retinoblastoma protein (Rb1). IGF-I elicited a time-dependent increase in cyclin D1 protein levels mediated jointly by ERK1/2-dependent and PI3-kinase-dependent mechanisms. Increase in cyclin D1 levels was accompanied by a time-dependent increase in cyclin D1-dependent cyclin-dependent kinase-4 (CDK4) activity. IGF-I also elicited a rapid time-dependent increase in Rb-(Ser807/811) phosphorylation, the specific target of the cyclin D(1)-dependent CDK4 kinase, and a slower increase in total Rb protein levels. We conclude that IGF-I stimulates G(1) phase progression, DNA synthesis, and cell proliferation of human intestinal smooth muscle cells. Effects of IGF-I on proliferation are mediated jointly by ERK1/2-dependent and PI3-kinase-dependent pathways that regulate cyclin D1 levels, CDK4 activity, and Rb activity.     

Regulation of the G1 phase of the mammalian cell cycle

In any multi-cellular organism,the balance between cell division and cell death maintains a constant cell number.Both cell division cycle and cell death are highly regulated events.Whether the cell will proceed through the cycle or not,depends upon whether the conditions required at the checkpoints during the cycle and fulfilled.In higher eucaryotic cells,such as mammalian cells,signals that arrest the cycle usually act at a G1 checkpoint.Cells that pass this restriction point are committed to complete the cycle.Regulation of the G1 phase of the cell cycle is extremely complex and involves many different families of proteins such as retinoblastoma family,cyclin dependent kinases,cyclins,and cyclin kinase inhibitors.     

Prespore cell fate bias in G1 phase of the cell cycle in Dictyostelium discoideum

By generating a population of Dictyostelium cells that are in the G1 phase of the cell cycle we have examined the influence of cell cycle status on cell fate specification, cell type proportioning and its regulation, and terminal differentiation. The lack of observable mitosis during the development of these cells and the quantification of their cellular DNA content suggests that they remain in G1 throughout development. Furthermore, chromosomal DNA synthesis was not detectable these cells, indicating that no synthesis phase had occurred, although substantial mitochondrial DNA synthesis did occur in prespore cells. The G1-phase cells underwent normal morphological development and sporulation but displayed an elevated prespore/prestalk ratio of 5.7 compared to the 3.0 (or 3:1) ratio normally observed in populations dominated by G2-phase cells. When migrating slugs produced by G1-phase cells were bisected, each half could reestablish the 5.7 (or 5.7:1) prespore/prestalk ratio. These results demonstrate that Dictyostelium cells can carry out the entire developmental cycle in the G1 phase of the cell cycle and that passage from G2 into G1 phase is not required for sporulation. Our results also suggest that the population asymmetry provided by the distribution of cells around the cell cycle at the time of starvation is not strictly required for cell type proportioning. Finally, when developed together with G2-phase cells, G1-phase cells preferentially become prespore cells and exclude G2-phase cells from the prespore-spore cell population, suggesting that G1-phase cells have an advantage over G2-phase cells in executing the spore cell differentiation pathway.     

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.     

Indomethacin-induced G1/S phase arrest of the plant cell cycle

In animal systems, indomethacin inhibits cAMP production via a prostaglandin-adenylyl cyclase pathway. To examine the possibility that a similar mechanism occurs in plants, the effect of indomethacin on the cell cycle of a tobacco bright yellow 2 (TBY-2) cell suspension was studied. Application of indomethacin during mitosis did not interfere with the M/G1 progression in synchronized BY-2 cells but it inhibited cAMP production at the beginning of the G1 phase and arrested the cell cycle progression at G1/S. These observations are discussed in relation to the putative involvement of cAMP biosynthesis in the cell cycle progression in TBY-2 cells.     

Polycystin-1 induced apoptosis and cell cycle arrest in G0/G1 phase in cancer cells

Studies have shown that polycystin-1, encoded by PKD1, the major ADPKD, may have a central role in regulating both apoptosis and proliferation, which could prevent the malignant transformation of affected cells. However, as a putative tumor suppressor, direct studies on the possibility that polycystin-1 may play a role in cancer cells' biological properties have not yet been reported. We have demonstrated that the apoptosis of cancer cells was induced by overexpression of polycystin-1. After transfection with polycystin-1, three cancer cell lines, HepG2, A549, and SW480, showed significantly increased apoptosis compared with the respective control groups. This was accompanied by cell cycle arrest at G(0)/G(1) phase, whereas cell proliferation was not significantly affected. Overexpression of polycystin-1 induces apoptosis in cancer cells, at least partially, through Wnt and a caspase-dependent pathway.     

Novel antitumor artemisinin derivatives targeting G1 phase of the cell cycle

Modification of artemisinin structure led us to the discovery of a novel class of antitumor compounds. These artemisinin derivatives containing cyano and aryl groups showed potent antiproliferative effect in vitro against P388 and A549 cells. This activity was reflected in P388 murine leukemia by an accumulation of cells in G1 phase, and induction of apoptosis.     

Ecdysone-induced accumulation of mosquito cells in the G1 phase of the cell cycle

We have established baseline conditions for investigating the interaction of the insect steroid hormone 20-hydroxyecdysone (20E) with the cell cycle in the C7-10 cell line from the mosquito, Aedes albopictus. As is the case with Drosophila melanogaster cells, treatment of C7-10 cells with 20E inhibits proliferation. In the presence of 10⁻⁶ M 20E, a gradual decline in cell number is typically apparent at 24 h. Media components such as phenol red and the potential presence of endogenous steroids in serum have no effect on the response to 20E. Pre-treating the cells with 10⁻⁸ M 20E, with or without an intervening hormone-free period, did not alter the response to 10⁻⁶ M 20E. However, replenishment of the medium appeared to synchronize the response to 10⁻⁶ M 20E, causing an abrupt and complete cessation of cell division by 48 h. Flow cytometry over a 20 h period showed a decrease in the proportion of cells in S within 4-6 h after exposure to 20E. By 6-10 h, a transient increase in G2 was followed by the accumulation of more than 70% of the cells in G1. These data suggest that after treatment with 20E, cells complete the ongoing cycle before arresting in G1. Consistent with the decrease in the proportion of cells in S and G2, western blots show that levels of cyclin A, which is required during the S phase of the cycle, decreased in 20E-treated cells.     

Coordination of mitochondrial bioenergetics with G1 phase cell cycle progression

Relatively little is known regarding how energetic demand during cell proliferation is sensed or coordinated with mitochondrial metabolism. Here we demonstrate that cell cycle progression through G1 is associated with a significant increase in mitochondrial membrane potential (?Ψm) and respiration. We used this change in metabolic rate to isolate cells in G1 with low and high levels of mitochondrial membrane potential (?ΨmL and ?ΨmH). Biochemical and functional studies demonstrate that ?ΨmL and ?ΨmH cells display the distinct characteristics of early and late G1 phase, respectively. We further demonstrate that the metabolic rate in G1 reflect levels of the mTOR-raptor complex as well as susceptibility to rapamycin-induced cell cycle delay. In conclusion, our data suggests a coupling of mitochondrial bioenergetics and G1 progression and points to the mTOR signaling pathway as a potential molecular coordinator of these two processes.     

Complementation between two temperature-sensitive mammalian cell mutants, each defective in the G1 phase of the cell cycle

Two mammalian temperature-sensitive (ts) G1 cell cycle mutants of different species origin (Syrian hamster and mouse) have been tested for complementation using somatic cell hybrid analysis. All hamster-mouse hybrid clones tested were found to exhibit normal growth properties at the restrictive temperature, while neither mutant alone was capable of normal growth at this temperature. The two mutant lines therefore complement for growth in a somatic cell hybrid and most likely represent ts lesions in different cellular functions specific to the G1 phase of the cell cycle.     

Integrating the MAP kinase signal into the G1 phase cell cycle machinery

Growth factors and the extracellular matrix provide the environmental cues that control the proliferation of most cell types. The binding of growth factors and matrix proteins to receptor tyrosine kinases and integrins, respectively, regulates several cytoplasmic signal transduction cascades, among which activation of the mitogen-activated protein kinase cascade, ras --> Raf --> MEK --> ERK, is perhaps the best characterized. Curiously, ERK activation has been associated with both stimulation and inhibition of cell proliferation. In this review, we summarize recent studies that connect ERK signaling to G1 phase cell cycle control and suggest that the cellular response to an ERK signal depends on both ERK signal intensity and duration. We also discuss studies showing that receptor tyrosine kinases and integrins differentially regulate the ERK signal in G1 phase.     

Glucose and glutamine metabolism control by APC and SCF during the G1-to-S phase transition of the cell cycle

Recent studies have given us a clue as to how modulations of both metabolic pathways and cyclins by the ubiquitin system influence cell cycle progression. Among these metabolic modulations, an aerobic glycolysis and glutaminolysis represent an initial step for metabolic machinery adaptation. The enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and glutaminase-1 (GLS1) maintain a high abundance in glycolytic intermediates (for synthesis of non-essential amino acids, the use of ribose for the synthesis of nucleotides and hexosamine biosynthesis), as well as tricarboxylic acid cycle intermediates (replenishing the loss of mitochondrial citrate), respectively. On the one hand, regulation of these key metabolic enzymes by ubiquitin ligases anaphase-promoting complex/cyclosome (APC/C) and Skp1/cullin/F-box (SCF) has revealed the importance of anaplerosis by both glycolysis and glutaminolysis to overcome the restriction point of the G1 phase by maintaining high levels of glycolytic and glutaminolytic intermediates. On the other hand, only glutaminolytic intermediates are necessary to drive cell growth through the S and G2 phases of the cell cycle. It is interesting to appreciate how this reorganization of the metabolic machinery, which has been observed beyond cellular proliferation, is a crucial determinant of a cell’s decision to proliferate. Here, we explore a unifying view of interactions between the ubiquitin system, metabolic activity, and cyclin-dependent kinase complexes activity during the cell cycle.     

Altered cell cycle kinetics, gene expression, and G1 restriction point regulation in Rb-deficient fibroblasts.

Fibroblasts prepared from retinoblastoma (Rb) gene-negative mouse embryos exhibit a shorter G1 phase of the growth cycle and smaller size than wild-type cells. In addition, the mutant cells are no longer inhibited by low levels of cycloheximide at any point in G1 but do remain sensitive to serum withdrawal until late in G1. Certain cell cycle-regulated genes showed no temporal or quantitative differences in expression. In contrast, cyclin E expression in Rb-deficient cells is deregulated in two ways. Cyclin E mRNA is generally derepressed in mutant cells and reaches peak levels about 6 h earlier in G1 than in wild-type cells. Moreover, cyclin E protein levels are higher in the Rb-/- cells than would be predicted from the levels of its mRNA. Thus, the selective growth advantage conferred by Rb gene deletion during tumorigenesis may be explained in part by changes in the regulation of cyclin E. In addition, the mechanisms defining the restriction point of late G1 may consist of at least two molecular events, one cycloheximide sensitive and pRb dependent and the other serum sensitive and pRb independent.     

Mimosine reversibly arrests cell cycle progression at the G1-S phase border

It has previously been demonstrated that the compound mimosine inhibits cell cycle traverse in late G1 phase prior to the onset of DNA synthesis (Hoffman BD, Hanauske-Abel HM, Flint A, Lalande M: Cytometry 12:26-32, 1991; Lalande M: Exp Cell Res 186:332-339, 1990). These results were obtained by using flow cytometric analysis of DNA content to compare the effects of mimosine on cell cycle traverse with those of aphidicolin, an inhibitor of DNA polymerase alpha activity. We have now measured the incorporation of bromodeoxyuridine into lymphoblastoid cells by flow cytometry to determine precisely where the two inhibitors act relative to the initiation of DNA synthesis. It is demonstrated here that mimosine arrests cell cycle progression at the G1-S phase border. The onset of DNA replication occurs within 15 min of releasing the cells from the mimosine block. In contrast, treatment with aphidicolin results in the accumulation of cells in early S phase. These results indicate that mimosine is a suitable compound for affecting the synchronous release of cells from G1 into S phase and for analyzing the biochemical events associated with this cell cycle phase transition.     

G1/S control of anchorage-independent growth in the fibroblast cell cycle

We have developed methodology to identify the block to anchorage-independent growth and position it within the fibroblast cell cycle. Results with NRK fibroblasts show that mitogen stimulation of the G0/G1 transition and G1-associated increases in cell size are minimally affected by loss of cell anchorage. In contrast, the induction of G1/S cell cycle genes and DNA synthesis is markedly inhibited when anchorage is blocked. Moreover, we demonstrate that the anchorage-dependent transition maps to late G1 and shortly before activation of the G1/S p34cdc2-like kinase. The G1/S block was also detectable in NIH-3T3 cells. Our results: (a) distinguish control of cell cycle progression by growth factors and anchorage; (b) indicate that anchorage mediates G1/S control in fibroblasts; and (c) identify a physiologic circumstance in which the phenotype of mammalian cell cycle arrest would closely resemble Saccharomyces cerevisiae START. The close correlation between anchorage independence in vitro and tumorigenicity in vivo emphasizes the key regulatory role for G1/S control in mammalian cells.     

Cytophotometric evaluation of the transition of cells from the G0 phase to the G1 phase of the cell cycle

Feulgen stained nuclei of PHA-stimulated human blood lymphocytes were used for cytophotometric chromatin pattern analysis. Similar distributions of low optical density values indicating the predominance of diffuse chromatin were obtained for G1, S and G2 cells. Condensed chromatin was predominant in G0 and M nuclei. Integral versus average optical densities scatter plots analyses permitted one to distinguish cells undergoing different phases of cell cycle including G0 and G1.     

T-kininogen inhibits fibroblast proliferation in the G(1) phase of the cell cycle

By using synthetic protease inhibitors, several investigators have demonstrated that cysteine proteinases are required for cell proliferation. Kininogens are potent and specific physiological inhibitors of cysteine proteinases. We have used several mouse fibroblast-derived cell lines that express biologically active T-kininogen under the control of the mouse metallothionein promoter to test its effect on cell proliferation. Our results indicate that expression of T-kininogen results in diminished proliferative capacity, as measured by reduced cell numbers, both in logarithmically growing cultures and in G(0) cells induced to proliferate in response to serum. Furthermore, both fluorescence-activated cell sorting (FACS) analysis and incorporation of radioactive precursors into DNA suggest that the cells are unable to progress from G(0) through the S phase of the cell cycle in response to serum stimulation. However, we find that T-kininogen-expressing cell lines are still capable of responding to growth factors present in the serum, both by activating the ERK pathway and by expressing early genes, such as c-Fos and c-Jun. Thus, our results suggest that inhibition of cysteine proteinases by T-kininogen leads to inhibition of cell proliferation between the G(1) and S phases of the cell cycle.