Identification of a restriction point at the M/G1 transition in CHO cells

The regulation of cell cycle progression in normal mammalian cells is dependent on the presence of growth factors. In their absence, non-transformed cells will stop dividing and enter the quiescent state (G0). We show here that in Chinese hamster ovary cells, at least two serum-dependent points exist during G1 that lead to different cellular responses. The first point is located immediately after mitosis and is suggested to link with apoptosis. The second point is located late in G1, and probably corresponds with the classic restriction point R. Cells depleted of serum after the first restriction point will not stop randomly in G1 but continue G1 progression until they reach the late restriction point, as marked by translocation of p42^MAPkinase (ERK2) to the nucleus.Received 18 September 2003; received after revision 11 December 2003; accepted 19 December 2003     

Cell fate determination during G1 phase progression

During the cell cycle, a cell may encounter one of five different fates: it can proliferate, differentiate, become quiescent or senescent, or go into apoptosis. The initiation of such fates is often seen in the G1 phase. The aim of this review is to describe an integrative model of G1 phase progression and cell fate determination. Along the G1 phase, the cell will encounter an early checkpoint after which apoptosis can result. For a quiescent state and for differentiation, the cell will exit G1 before the restriction point and a subsequent differentiation checkpoint will decide the fate of the cell, quiescence or differentiation. After the restriction point, the cell can be arrested in response to stress stimuli, such as telomere depletion, and a decision between senescence and apoptosis occurs. Received 19 June 2007; received after revision 23 July 2007; accepted 17 August 2007     

Inhibitors of phosphatidylinositol 3-kinase activity prevent cell cycle progression and induce apoptosis at the M/G1 transition in CHO cells

Phosphatidylinositol 3-kinase (PI3-kinase) activity has been implicated in regulating cell cycle progression at distinct points in the cell cycle by preventing cell cycle arrest or apoptosis. In this study, the role of PI3-kinase activity during the entire G1 phase of the ongoing cell cycle was studied in Chinese hamster ovary (CHO) cells synchronized by mitotic shake-off. We show that inhibition of PI3-kinase activity during and 2 h after mitosis inhibited cell cycle progression into S phase. In the presence of the PI3-kinase inhibitor wortmannin or LY294002, cells were arrested during early G1 phase, leading to the expression of the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PI3-kinase activity is required for progression through the M/G1 phase. In the absence of PI3-kinase activity, cells are induced for apoptosis in this particular phase of the cell cycle. Received 7 September 2005; received after revision 26 October 2005; accepted 11 November 2005     

Revisiting retinoblastoma protein phosphorylation during the mammalian cell cycle

It is widely accepted that phosphorylation of the retinoblastoma (Rb) protein during the G1 phase of the mammalian division cycle is a major control element regulating passage of cells into S phase and through the division cycle. The experiments supporting G1-phase-specific Rb phosphorylation and the historical development of this idea are reviewed. By making a rigorous distinction between 'growth cessation' and the phenomena of 'cell cycle exit' or 'G1-phase arrest', the evidence for the G1-phase-specific phosphorylation of Rb protein is reinterpreted. We show that the evidence for G1-phase phosphorylation of Rb rests on few experiments and a chain of reasoning with some weak links. Evidence is reviewed that growth conditions regulate the phosphorylation of Rb. A growth-regulated control system that is independent of the cell cycle explains much of the evidence adduced to support cycle-specific phosphorylation of Rb. We propose that additional experimental evidence is needed to decide whether there is a G1-phase-specific phosphorylation of Rb protein. Received 16 October 2000; received after revision 13 November 2000; accepted 15 November 2000     

Regulation of phagocyte migration and recruitment by Src-family kinases

Src-family kinases (SFKs) regulate different granulocyte and monocyte/macrophage responses. Accumulating evidence suggests that members of this family are implicated in signal transduction pathways regulating phagocytic cell migration and recruitment into inflammatory sites. Macrophages with a genetic deficiency of SFKs display marked alterations in cytoskeleton dynamics, polarization and migration. This same phenotype is found in cells with either a lack of SFK substrates and/or interacting proteins such as Pyk2/FAK, c-Cbl and p190RhoGAP. Notably, SFKs and their downstream targets also regulate monocyte recruitment into inflammatory sites. Depending on the type of assay used, neutrophil migration in vitro may be either dependent on or independent of SFKs. Also neutrophil recruitment in in vivo models of inflammation may be regulated differently by SFKs depending on the tissue involved. In this review we will discuss possible mechanisms by which SFKs may regulate phagocytic cell migratory abilities.     

Prolactin and growth hormone signal transduction in lymphohaemopoietic cells

The peptide hormones, prolactin (PRL) and growth hormone (GH), are known to regulate numerous target tissues. Among such targets are cells of the immune system, including T cells, B cells, macrophages and natural killer cells. We have cloned a panel of PRL- and GH-inducible T cell genes for use in studies to understand how these hormones through the expression of these genes modulate the biology of immune function cells. This article focuses on the signalling pathways emanating from the PRL receptor (PRL-R) and GH receptor (GH-R), and the expression of PRL-inducible target genes.     

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

T-cell signal transduction and the role of protein kinase C

The T lymphocyte has a vital part to play in maintaining the host response to bacterial and viral infection and also appears to play a key pathological role in autoimmune diseases such as rheumatoid arthritis. In this review, we summarize the signalling pathways which trigger antigen-driven T-cell proliferation and examine the evidence which suggests that protein kinase C (PKC) is fundamental to this process. Finally, we discuss the therapeutic potential that PKC inhibitors may have in the treatment of autoimmune disease. Received 31 March 1998; received after revision 19 May 1998; accepted 19 May 1998     

K252a: A new blocker of the cell-cycle at G1 phase in a human hepatoma cell line

The administration of 200 nM K252a to HuH7 suppressed the proliferation of the cells almost completely. The uptake of [³H]thymidine was inhibited, and flow cytometry revealed only one peak at 2C on day 3 after treatment with 100 nM K252a. The expression of proto-oncogene c-myc was not reduced. Despite the blockage at G1, both the size of the cells and the amount of cell protein had increased by 4 times by day 3 after treatment with K252a, while the cells secreted albumin and -fetoprotein into the medium as usual. These results show that K252a can increase the cell size of HuH7 without losing its function by blocking the cell cycle at G1 phase.     

Regulation of mitochondrial oxidative phosphorylation by second messenger-mediated signal transduction mechanisms

The mitochondrial oxidative phosphorylation system is responsible for providing the bulk of cellular ATP molecules. There is a growing body of information regarding the regulation of this process by a number of second messenger-mediated signal transduction mechanisms, although direct studies aimed at elucidating this regulation are limited. The main second messengers affecting mitochondrial signal transduction are cAMP and calcium. Other second messengers include ceramide and reactive oxygen species as well as nitric oxide and reactive nitrogen species. This review focuses on available data on the regulation of the mitochondrial oxidative phosphorylation system by signal transduction mechanisms and is organised according to the second messengers involved, because of their pivotal role in mitochondrial function. Future perspectives for further investigations regarding these mechanisms in the regulation of the oxidative phosphorylation system are formulated. Received 11 December 2005; received after revision 14 January 2006; accepted 6 February 2006     

Species-specific differences in the mitogenic activity of heparin-binding growth factors in the sera of various mammals

Sera from different mammalian species displayed great differences in mitogenic activity, as measured by stimulation of DNA synthesis in BALB/c 3T3 cells (3T3 cells). Among the sera examined, fetal bovine serum was least active, and increasing activity was detected in calf serum, human serum, rat serum and mouse serum, in that order. Rat and mouse sera exhibited extremely high mitogenic activity with 3T3 cells, but when TIG-1 human fetal lung fibroblasts were used for the DNA assay instead, the activity levels of all of the sera were lower, and the differences between them were smaller. To determine the reasons for these differences, the heparin-binding growth factors in each serum were separated on a heparin affinity column. Five peaks of DNA-stimulating activity were obtained. Three of these were found in all sera examined, with both 3T3 cells and TIG-1 cells. Two other peaks were found only with 3T3 cells; one was peculiar to rat and mouse sera, with extremely high activity in the rat, and the other was specific to fetal serum. The dependence of the activity of these peaks on the cells used for the test was confirmed using normal rat lung fibroblasts and immortalized rat kidney cells. These findings adequately explain the species-specific differences in mitogenic activity of whole sera, and the variation in activity depending on the cells used for assay of DNA synthesis.     

Post-translational regulation of the tumor suppressor p27KIP1

Mitogenic signals stimulate cell division by activating cyclin/cyclin-dependent kinase (CDK) complexes. Their timely regulation ensures proper cell cycle progression. It is therefore not surprising that cyclin/CDK complexes are integrators of multiple signals from both the extracellular environment and intracellular cues. Important regulators of cyclin/CDKs are the CDK inhibitors that have attracted attention due to their association with disease. p27^KIP1 is a CDK inhibitor that controls CDK activity throughout the cell cycle. As a CDK inhibitor, p27^KIP1 has tumor suppressor activity. Besides CDKs, p27^KIP1 regulates additional cellular processes, including cell motility, some of which seem to mediate oncogenic activities of p27^KIP1. These activities of p27^KIP1 are regulated through multiple phosphorylation sites, targeted by several signal transduction pathways. Understanding functions and regulation of p27^KIP1 will be important to determine which isoform of p27^KIP1 has anti- or pro-tumorigenic activities. Such knowledge might be of prognostic value and may offer novel therapeutic windows. Received 26 May 2008; accepted 17 June 2008     

Unscheduled CDK1 activity in G1 phase of the cell cycle triggers apoptosis in X-irradiated lymphocytic leukemia cells

Cyclin-dependent kinase 1 (CDK1) is a major component of the cell cycle progression engine. Recently, several investigations provided evidence demonstrating that unscheduled CDK1 activation may also be involved in apoptosis in cancerous cells. In this article, we demonstrate that X-ray irradiation induced G1 arrest in MOLT-4 lymphocytic leukemia cells, the arrest being accompanied by reduction in the activity of CDK2, but increased CDK1 activity and cell apoptosis in the G1 phase. Interestingly, this increase in CDK1 and apoptosis by ionizing radiation was prevented by pretreatment with the CDK1 inhibitor, roscovitine, suggesting that CDK1 kinase activity is required for radiation-induced apoptotic cell death in this model system. Furthermore, cyclin B1 and CDK1 were detected co-localizing and associating in G1 phase MOLT-4 cells, with the cellular lysates from these cells revealing a genotoxic stress-induced increase in CDK1 phosphorylation (Thr-161) and dephosphorylation (Tyr-15), as analyzed by postsorting immunoprecipitation and immunoblotting. Finally, X-irradiation was found to increase Bcl-2 phosphorylation in G1 phase cells. Taken together, these novel findings suggest that CDK1 is activated by unscheduled accumulation of cyclin B1 in G1 phase cells exposed to X-ray, and that CDK1 activation, at the wrong time and in the wrong phase, may directly or indirectly trigger a Bcl-2-dependent signaling pathway leading to apoptotic cell death in MOLT-4 cells. Received 30 March 2006; received after revision 23 June 2006; accepted 24 August 2006 J. Wu and Y. Feng contributed equally to this work.     

Regulation of the cell cycle following DNA damage in normal and Ataxia telangiectasia cells

A proportion of the population is exposed to acute doses of ionizing radiation through medical treatment or occupational accidents, with little knowledge of the immedate effects. At the cellular level, ionizing radiation leads to the activation of a genetic program which enables the cell to increase its chances of survival and to minimize detrimental manifestations of radiation damage. Cytotoxic stress due to ionizing radiation causes genetic instability, alterations in the cell cycle, apoptosis, or necrosis. Alterations in the G1, S and G2 phases of the cell cycle coincide with improved survival and genome stability. The main cellular factors which are activated by DNA damage and interfere with the cell cycle controls are: p53, delaying the transition through the G1-S boundary; p21^WAF1/CIPI, preventing the entrance into S-phase; proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), blocking DNA replication; and the p53 variant protein p53as together with the retinoblastoma protein (Rb), with less defined functions during the G2 phase of the cell cycle. By comparing a variety of radioresistant cell lines derived from radiosensitive ataxia talangiectasia cells with the parental cells, some essential mechanisms that allow cells to gain radioresistance have been identified. The results so far emphasise the importance of an adequate delay in the transition from G2 to M and the inhibition of DNA replication in the regulation of the cell cycle after exposure to ionizing radiation.     

Survival of BSC-1 cells through the maintenance of cell volume brought about by epidermal growth factor depends on attachment to the substratum

Summary Addition of epidermal growth factor to culture medium without calf serum suppressed the increase in cell volume and then enhanced the survival of BSC-1 cells attached to culture dishes. However, these effects of epidermal growth factor were not observed in the case of cells on dishes coated with heat-denatured bovine serum albumin.     

Myosin regulation in the migration of tumor cells and leukocytes within a three-dimensional collagen matrix

The migration of cells is a complex regulatory process which results in the generation of motor forces through the reorganization of the cytoskeleton. Here we present a comparative study of the expression and involvement of myosin in the regulation of the physiological migration of leukocytes and the pathological migration of tumor cells. We show that the involvement of myosin in the migration is distinct in these two cell types. In leukocytes, the activity of non-muscle myosin II is essential for both the spontaneous (matrix-induced) migration and the migration induced by ligands to G protein-coupled receptors, i.e. chemokines and neurotransmitters. In contrast, spontaneous tumor cell migration is largely independent of non-muscle myosin II activity, whereas the norepinephrine-induced migration is completely inhibited by either direct inhibition of non-muscle myosin II or of the kinases phosphorylating the myosin light chain, namely ROCK or the calcium/calmodulin-dependent myosin light-chain kinase.Received 31 August 2004; accepted 26 October 2004     

The role of growth hormone and insulin-like growth factors in the immune system

Growth hormone (GH) and insulin-like growth factor I (IGF-I) can modulate the development and function of the immune system. In this chapter, we present data on the expression of receptors for GH and IGFs and the in vitro and in vivo effects of these proteins. We show that expression of GH and IGFs in the immune system opens up the possibility that these proteins are not only involved in endocrine control of the immune system but can also play a role as local growth and differentiation factors (cytokines). Endocrine control of GH could be direct or mediated via endocrine or autocrine/paracrine IGF-I. In addition, GH can act as an autocrine or paracrine factor itself. Furthermore, IGF-I in the immune system has been shown to be regulated by cytokines, such as interleukin-1 and interferon-γ, alluding to a cytokine-like function of IGF-I. In addition to data on the function of GH and IGF-I in the immune system, we present new findings which imply a possible function of IGF-II and IGF-binding proteins.     

Soluble factors and the development of rod photoreceptors

Photoreceptors are the most abundant cell type in the vertebrate neural retina. Like the other retinal neurons and the Müller glia, they arise from a population of precursor cells that are multipotent and intrinsic to the retina. Approximately 10 years ago, several studies demonstrated that retinal precursor cells (RPCs) are competent to respond to environmental factors that promote cell type determination and differentiation. Since those studies, significant effort has been directed at identifying the molecular nature of these environmental signals and understanding the precise mechanisms they employ to drive RPCs towards the different retinal fates. In this review, we describe the recent progress toward understanding how environmental factors influence the development of vertebrate rod photoreceptors.     

Activation of the human FP prostanoid receptor disrupts mitosis progression and generates aneuploidy and polyploidy

Studies have shown prostaglandin F_2α to be an endogenous tumor promoter in mouse models of skin carcinogenesis; however, the mechanisms by which PGF_2α affects cell cycle events remain unknown. Here we performed cell cycle analyses on HEK cells stably expressing the human FP receptor and found that treatment with PGF_2α delays mitosis and is associated with an increased expression of cyclin B1 and Cdc2 kinase activity. In addition, multipolar spindles and misaligned chromosomes were observed in a significant proportion of cells treated with PGF_2α. Defective cytokinesis was also observed which resulted in gross aneuploidy and polyploidy. Expression of dominant negative Rho attenuated the cell cycle delay and prevented the generation of micronuclei following treatment with PGF_2α. This suggests that FP receptor activation of Rho signaling by PGF_2α can interfere with nuclear division. Aneuploidy is associated with genomic instability and may underlie the tumor-promoting properties of PGF_2α. Received 7 July 2005; received after revision 22 October 2005; accepted 11 November 2005