Interferon-alpha inhibits interleukin-3-induced proliferation of Ba/F3 cells in a protein kinase R-dependent manner

We have previously shown that interferon-alpha (IFN-alpha) inhibits proliferation of Ba/F3 cells by interfering with the action of the mitogen interleukin-3 (IL-3) [Cell Signal 11 (1999) 769]. Here, we have characterised the role of protein kinase R (PKR), an IFN-alpha-inducible enzyme, in the mediation of IL-3-antagonistic IFN-alpha effects. Downregulation of PKR expression by antisense oligonucleotide treatment blocked IFN-alpha-induced growth inhibition. Reduction of PKR levels and overexpression of a dominant-negative PKR mutant correlated with diminished inhibitory IFN-alpha effects on the IL-3-dependent expression of a luciferase reporter construct, GAS-luc. Furthermore, increased nuclear levels of STAT1 (bound in ISGF3 complexes) were observed in PKR-depleted cells cultured with or without IFN-alpha. Together, our data indicate an essential role of PKR in the mediation of IL-3-antagonistic IFN-alpha effects on Ba/F3 cells. They also suggests that activation of STAT1, an essential mediator of IFN effects, is insufficient for growth inhibition if PKR is not expressed.     

Redox control of G(1)/S cell cycle regulators during nitric oxide-mediated cell cycle arrest

Redox regulation of cell cycle progression during nitric oxide (NO) mediated cytostasis is not well-understood. In this study, we investigated the role of the intracellular antioxidant glutathione (GSH) in regulating specific signaling events that are associated with NO-mediated cell cycle arrest. Manipulation of intracellular GSH content through pharmacological inhibition of glutamate-cysteine ligase (GCL) indicated that GSH depletion potentiated nitrosative stress, DNA damage, phosphorylation of the tumor suppressor p53 (Ser-18) and upregulation of p21(cip1/waf1) upon NO stimulation. However, we found that neither overexpression of a dominant negative p53 nor pharmacological inhibition of p53 with cyclic pifithrin-alpha (cPFT-alpha) was sufficient to reverse NO-mediated cell cycle arrest or hypophosphorylation of retinoblastoma protein (Rb). We found that the decrease in cyclin D1 levels induced by NO was GSH-sensitive implying that the redox regulation of NO-mediated cytostasis was a multifaceted process and that both p53/p21(cip1/waf1) and p53 independent cyclin D1 pathways were involved. Together, our results demonstrate that GSH serves as an important component of cellular protective mechanisms against NO-derived nitrosative stress to regulate DNA damage checkpoint.     

Landomycin A inhibits DNA synthesis and G1/S cell cycle progression.

Landomycin A was found to inhibit the uptake of [3H]thymidine into DNA in murine smooth muscle cells indicating decreased DNA synthesis. Subsequent studies showed that landomycin A inhibits cell cycle progression.     

Troglitazone inhibits growth of MCF-7 breast carcinoma cells by targeting G1 cell cycle regulators

Peroxisome proliferator activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily. Ligand activation of PPARgamma has been shown to cause growth arrest in several human tumor cell types, but the underlying molecular mechanism has not been elucidated. We report here that the PPARgamma ligand troglitazone (TRO) inhibited MCF-7 cell proliferation by blocking events critical for G1 --> S progression. Flow cytometry demonstrated that TRO at 20 microM increased the percentage of cells in G1 from 51 to 69% after 24 h. Accumulation of cells in G1 was accompanied by an attenuation of Rb protein phosphorylation associated with decreased CDK4 and CDK2 activities. Inhibition of CDK activity by TRO correlates with decreased protein levels for several G1 regulators of Rb phosphorylation (cyclin D1, and CDKs 2, 4, and 6). Overexpression of cyclin D1 partially rescued MCF-7 cells from TRO-mediated G1 arrest. Targeting of G1 regulatory proteins, particularly cyclin D1, and the resulting induction of G1 arrest by TRO may provide a novel antiproliferative therapy for human breast cancer.     

Human NDR kinases control G(1)/S cell cycle transition by directly regulating p21 stability

The G(1) phase of the cell cycle is an important integrator of internal and external cues, allowing a cell to decide whether to proliferate, differentiate, or die. Multiple protein kinases, among them the cyclin-dependent kinases (Cdks), control G(1)-phase progression and S-phase entry. With the regulation of apoptosis, centrosome duplication, and mitotic chromosome alignment downstream of the HIPPO pathway components MST1 and MST2, mammalian NDR kinases have been implicated to function in cell cycle-dependent processes. Although they are well characterized in terms of biochemical regulation and upstream signaling pathways, signaling mechanisms downstream of mammalian NDR kinases remain largely unknown. We identify here a role for human NDR in regulating the G(1)/S transition. In G(1) phase, NDR kinases are activated by a third MST kinase (MST3). Significantly, interfering with NDR and MST3 kinase expression results in G(1) arrest and subsequent proliferation defects. Furthermore, we describe the first downstream signaling mechanisms by which NDR kinases regulate cell cycle progression. Our findings suggest that NDR kinases control protein stability of the cyclin-Cdk inhibitor protein p21 by direct phosphorylation. These findings establish a novel MST3-NDR-p21 axis as an important regulator of G(1)/S progression of mammalian cells.     

p27Kip1 inhibits the cell cycle through non-canonical G1/S phase-specific gatekeeper mechanism

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 has been shown to regulate cellular proliferation via inhibition of CDK activities. It is now recognized that p27Kip1 can regulate cellular processes through non-canonical, CDK-independent mechanisms. We have developed an inducible p27Kip1 model in cultured cells to explore CDK-independent p27Kip1 regulation of biological processes. We present evidence that p27Kip1 can function in a CDK-independent manner to inhibit entry and/or progression of S phase. Even though this p27Kip1 mechanism is non-canonical it does requires the intact cyclin-binding motif in p27Kip1. We suggest a mechanism similar to that proposed in post-mitotic neural cells whereby p27Kip1 functions to coordinate growth arrest and apoptosis. Our hypothesis supports the concept that p27Kip1 is a gatekeeper for the entry and progression of S phase through interaction with specific protein(s) or via binding to specific DNA sequences in a CDK-independent manner.     

A unified model for the G1/S cell cycle transition

Efficient S phase entry is essential for development, tissue repair, and immune defences. However, hyperactive or expedited S phase entry causes replication stress, DNA damage and oncogenesis, highlighting the need for strict regulation. Recent paradigm shifts and conflicting reports demonstrate the requirement for a discussion of the G1/S transition literature. Here, we review the recent studies, and propose a unified model for the S phase entry decision. In this model, competition between mitogen and DNA damage signalling over the course of the mother cell cycle constitutes the predominant control mechanism for S phase entry of daughter cells. Mitogens and DNA damage have distinct sensing periods, giving rise to three Commitment Points for S phase entry (CP1-3). S phase entry is mitogen-independent in the daughter G1 phase, but remains sensitive to DNA damage, such as single strand breaks, the most frequently-occurring lesions that uniquely threaten DNA replication. To control CP1-3, dedicated hubs integrate the antagonistic mitogenic and DNA damage signals, regulating the stoichiometric cyclin: CDK inhibitor ratio for ultrasensitive control of CDK4/6 and CDK2. This unified model for the G1/S cell cycle transition combines the findings of decades of study, and provides an updated foundation for cell cycle research.     

PCLAF promotes neuroblastoma G1/S cell cycle progression via the E2F1/PTTG1 axis

PCLAF (PCNA clamp-associated factor), also known as PAF15/ KIAA0101, is overexpressed in most human cancers and is a predominant regulator of tumor progression. However, its biological function in neuroblastoma remains unclear. PCLAF is extremely overexpressed in neuroblastoma and is associated with poor prognosis. Through the analysis of various data sets, we found that the high expression of PCLAF is positively correlated with increased stage and high risk of neuroblastoma. Most importantly, knocking down PCLAF could restrict the proliferation of neuroblastoma cells in vitro and in vitro. By analyzing RNA-seq data, we found that the enrichment of cell cycle-related pathway genes was most significant among the differentially expressed downregulated genes after reducing the expression of PCLAF. In addition, PCLAF accelerated the G1/S transition of the neuroblastoma cell cycle by activating the E2F1/PTTG1 signaling pathway. In this study, we reveal the mechanism by which PCLAF facilitates cell cycle progression and recommend that the PCLAF/E2F1/PTTG1 axis is a therapeutic target in neuroblastoma.Subject terms: Cancer epigenetics, Cell growth     

Human cytomegalovirus infection inhibits cell cycle progression at multiple points, including the transition from G1 to S.

Human cytomegalovirus inhibits the growth of human foreskin fibroblast cells by 12 h after infection. Analysis of the cellular DNA content of infected cells by flow cytometry demonstrated that cytomegalovirus does not arrest cell cycle progression at a single point. At least two blockages occur, one of which is in the G1 phase of the cell cycle. The G1 arrest introduced by cytomegalovirus infection blocks S-phase entry after serum stimulation.     

The positive circadian regulators CLOCK and BMAL1 control G2/M cell cycle transition through Cyclin B1

We previously identified a tight bidirectional phase coupling between the circadian clock and the cell cycle. To understand the role of the CLOCK/BMAL1 complex, representing the main positive regulator of the circadian oscillator, we knocked down Bmal1 or Clock in NIH3T3^3C mouse fibroblasts (carrying fluorescent reporters for clock and cell cycle phase) and analyzed timing of cell division in individual cells and cell populations. Inactivation of Bmal1 resulted in a loss of circadian rhythmicity and a lengthening of the cell cycle, originating from delayed G2/M transition. Subsequent molecular analysis revealed reduced levels of Cyclin B1, an important G2/M regulator, upon suppression of Bmal1 gene expression. In complete agreement with these experimental observations, simulation of Bmal1 knockdown in a computational model for coupled mammalian circadian clock and cell cycle oscillators (now incorporating Cyclin B1 induction by BMAL1) revealed a lengthening of the cell cycle. Similar data were obtained upon knockdown of Clock gene expression. In conclusion, the CLOCK/BMAL1 complex controls cell cycle progression at the level of G2/M transition through regulation of Cyclin B1 expression.     

Inhibition of G1 to S cell cycle progression by BCCIP beta

The BCCIP alpha protein was identified as a BRCA2 and CDKN1A (p21, or p21(Waf1/Cip1)) Interacting Protein. It binds to a highly conserved domain proximate to the C-terminus of BRCA2 protein and the C-terminal domain of the CDK-inhibitor p21. Previous reports showed that BCCIP alpha enhances the inhibitory activity of p21 toward CDK2 and that BCCIP alpha inhibits the growth of certain tumor cells. Here we show that a second isoform, BCCIP beta, also binds to p21 and inhibits cell growth. The growth inhibition by BCCIP beta can be partially abrogated in p21 deficient cells. Overexpression of BCCIP beta delays the G1 to S progression and results in an elevated p21 expression. These data suggest BCCIP beta as a new regulator for the G1-S cell cycle progression and cell growth control.     

Wobble inosine tRNA modification is essential to cell cycle progression in G(1)/S and G(2)/M transitions in fission yeast

Inosine (I) at position 34 (wobble position) of tRNA is formed by the hydrolytic deamination of a genomically encoded adenosine (A). The enzyme catalyzing this reaction, termed tRNA A:34 deaminase, is the heterodimeric Tad2p/ADAT2.Tad3p/ADAT3 complex in eukaryotes. In budding yeast, deletion of each subunit is lethal, indicating that the wobble inosine tRNA modification is essential for viability; however, most of its physiological roles remain unknown. To identify novel cell cycle mutants in fission yeast, we isolated the tad3-1 mutant that is allelic to the tad3(+) gene encoding a homolog of budding yeast Tad3p. Interestingly, the tad3-1 mutant cells principally exhibited cell cycle-specific phenotype, namely temperature-sensitive and irreversible cell cycle arrest both in G(1) and G(2). Further analyses revealed that in the tad3-1 mutant cells, the S257N mutation that occurred in the catalytically inactive Tad3 subunit affected its association with catalytically active Tad2 subunit, leading to an impairment in the A to I conversion at position 34 of tRNA. In tad3-1 mutant cells, the overexpression of the tad3(+) gene completely suppressed the decreased tRNA inosine content. Notably, the overexpression of the tad2(+) gene partially suppressed the temperature-sensitive phenotype and the decreased tRNA inosine content, indicating that the tad3-1 mutant phenotype is because of the insufficient I(34) formation of tRNA. These results suggest that the wobble inosine tRNA modification is essential for cell cycle progression in the G(1)/S and G(2)/M transitions in fission yeast.     

Association between the cell cycle and neural crest delamination through specific regulation of G1/S transition

Delamination of premigratory neural crest cells from the dorsal neural tube depends both upon environmental signals and cell-intrinsic mechanisms and is a prerequisite for cells to engage in migration. Here we show that avian neural crest cells synchronously emigrate from the neural tube in the S phase of the cell cycle. Furthermore, specific inhibition of the transition from G1 to S both in ovo and in explants blocks delamination, whereas arrest at the S or G2 phases has no immediate effect. Thus, the events taking place during G1 that control the transition from G1 to S are necessary for the epithelial to mesenchymal conversion of crest precursors.     

Porcine testicular extract inhibits T cell proliferation by blocking cell cycle transition from G1 phase to S phase

Since T cells express diverse sex steroid hormone receptors, they might be a good model to evaluate the effects of sex steroid hormones on immune modulation. Porcine testicular extract contains several sex steroid hormones and may be useful to study the effects of sex steroid hormones during T cell activation. We have examined the effects of the porcine testicular extract on T cell activation: proliferation and secretion of cytokines (IL-2 and IFN-γ) by activated T cells were severely decreased after treatment with porcine testicular extract. The extract produced an immunosuppressive effect and inhibited the proliferation of activated T cells by blocking the cell cycle transition from the G(1) phase to S phase. These effects were mediated by a decrease in the expression of cyclin D1 and cyclin E and constitutive expression of p27(KIP1) after T cell activation.     

Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition

Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly. We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z. Strezoska, D. G. Pestov, and L. F. Lau, Mol. Cell. Biol. 20:5516-5528, 2000). Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts. In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase. Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased. The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point. Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing. These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle. We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner.     

Maitotoxin,a calcium channel activator,inhibits cell cycle progression through the G1/S and G2/M transitions and prevents CDC2 kinase activation in GH4C1 cells

Calcium regulates progression through several checkpoints in the cell cycle, including the G1/S-phase transition, G2/M-phase transition, and exit from mitosis. In the GH₄C₁ rat pituitary cell line, calcium mobilizing polypeptides and calcium channel activation inhibit cell proliferation. This report examines the effects of maitotoxin (MTX), an activator of type L voltage-dependent calcium channels (L-VDCC), on calcium influx and cell cycle progression in GH₄C₁ cells. MTX causes both a block from G1 to S-phase and a concentration-dependent accumulation of cells in G2+M. MTX does not increase the mitotic index; thus, sustained calcium channel activation by MTX results in an accumulation of cells in G2. In order to temporally localize the MTX-induced G2 block relative to cell cycle regulatory events at the G2/M transition, we assessed the relative activity of two cell cycle regulatory protein kinases, CDC2 and CDK2, in MTX-treated cells. CDC2-specific histone kinase activity in MTX-treated cells is lower than either in cells blocked in mitosis with the microtubule destabilizing agent demecolcine or in randomly cycling cells. In contrast, the activity of CDK2 is highest in MTX-treated cells, consistent with a G2 block prior to CDC2 activation. Together, these results implicate calcium as an intracellular signal required for progression through G2 phase of the cell cycle prior to CDC2 kinase activation. © 1996 Wiley-Liss, Inc.     

Simian immunodeficiency virus Nef protein delays the progression of CD4+ T cells through G1/S phase of the cell cycle

Human and simian immunodeficiency virus (HIV and SIV, respectively) infections are characterized by gradual depletion of CD4+ T cells. The underlying mechanisms of CD4+ T-cell depletion and HIV and SIV persistence are not fully determined. The Nef protein is expressed early in infection and is necessary for pathogenesis. Nef can cause T-cell activation and downmodulates cell surface signaling molecules. However, the effect of Nef on the cell cycle has not been well characterized. To determine the role of Nef in the cell cycle, we investigated whether the SIV Nef protein can modulate cell proliferation and apoptosis in CD4+ Jurkat T cells. We developed a CD4+ Jurkat T-cell line that stably expresses SIV Nef under the control of an inducible promoter. Alterations in cell proliferation were determined by flow cytometry using stable intracytoplasmic fluorescent dye 5- and 6-carboxyfluorescein diacetate succinimidyl ester and bromodeoxyuridine incorporation. Apoptotic cell death was measured by annexin V and propidium iodide staining. Our results demonstrated that SIV Nef inhibited Fas-induced apoptosis in these cells and that the mechanism involved upregulation of the Bcl-2 protein. SIV Nef suppressed CD4+ T-cell proliferation by inhibiting the progression of cells into S phase of the cell cycle. Suppression involved an upregulation of cyclin-dependent kinase inhibitors p21 and p27 and the downregulation of cyclin D1 and cyclin A. In summary, inhibition of apoptosis by Nef can lead to persistence of infected cells and can support viral replication. In addition, a Nef-mediated delay in cell cycle progression may contribute to CD4+ T-cell anergy/depletion seen in HIV and SIV disease.