The tumour suppressor gene product APC blocks cell cycle progression from G0/G1 to S phase.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum-induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell-cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down-regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin-CDK complexes.     

The human cytomegalovirus UL82 gene product (pp71) accelerates progression through the G1 phase of the cell cycle

As viruses are reliant upon their host cell to serve as proper environments for their replication, many have evolved mechanisms to alter intracellular conditions to suit their own needs. For example, human cytomegalovirus induces quiescent cells to enter the cell cycle and then arrests them in late G(1), before they enter the S phase, a cell cycle compartment that is presumably favorable for viral replication. Here we show that the protein product of the human cytomegalovirus UL82 gene, pp71, can accelerate the movement of cells through the G(1) phase of the cell cycle. This activity would help infected cells reach the late G(1) arrest point sooner and thus may stimulate the infectious cycle. pp71 also induces DNA synthesis in quiescent cells, but a pp71 mutant protein that is unable to induce quiescent cells to enter the cell cycle still retains the ability to accelerate the G(1) phase. Thus, the mechanism through which pp71 accelerates G(1) cell cycle progression appears to be distinct from the one that it employs to induce quiescent cells to exit G(0) and subsequently enter the S phase.     

Heparin prevents vascular smooth muscle cell progression through the G1 phase of the cell cycle

To gain insight into the mechanism of the antiproliferative effects of heparin on vascular smooth muscle cells (SMC), the influence of this glycosaminoglycan on cell cycle progression and the expression of the c-fos, c-myc, and c-myb proto-oncogenes and two other growth-regulated genes was examined. SMC, synchronized by a serum-deprivation protocol, enter S phase 12-16 h after serum stimulation. Pretreatment with heparin for 48 h blocked the induction of histone H3 RNA, an S phase-expressed product, and prevented cell replication. Thus, heparin prevents entry of cells into S phase. Conversely, heparin had essentially no effect on changes in expression of the c-fos and c-myc proto-oncogenes during the G0 to G1 transition. Normal increases in c-fos and c-myc RNA were observed 30 min and 2 h following serum addition, respectively. However, the increase in expression of the mRNA of the c-myb proto-oncogene and the mitochondrial ATP/ADP carrier protein, 2F1, which begins to occur 8 h following serum addition to SMC, was completely inhibited by heparin. Two-dimensional polyacrylamide gel electrophoresis of the products of a rabbit reticulocyte cell-free translation of RNA isolated at various times confirmed this temporal assessment of the effects of heparin. These results suggest that heparin does not inhibit cell proliferation by blocking the G0 to G1 transition. Rather, heparin may affect a critical event in the mid-G1 phase of the cell cycle which is necessary for subsequent DNA synthesis.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

WWOX suppresses prostate cancer cell progression through cyclin D1-mediated cell cycle arrest in the G1 phase

WW domain-containing oxidoreductase (WWOX) has been reported to be a tumor suppressor in multiple cancers, including prostate cancer. WWOX can induce apoptotic responses to inhibit tumor progression, and the other mechanisms of WWOX in tumor suppression have also been reported recently. In this study, we found significant down-regulation of WWOX in prostate cancer specimens and prostate cancer cell lines compared with the normal controls. In addition, an ectopically increased WWOX expression repressed tumor progression both in vitro and in vivo. Interestingly, overexpression of WWOX in 22Rv1 cells led to cell cycle arrest in the G1 phase but did not affect sub-G1 in flow cytometry. GFP-WWOX overexpressed 22Rv1 cells were shown to inhibit cell cycle progression into mitosis under nocodazole treatment in flow cytometry, immunoblotting and GFP fluorescence. Further, cyclin D1 but not apoptosis correlated genes were down-regulated by WWOX both in vitro and in vivo. Restoration of cyclin D1 in the WWOX-overexpressed 22Rv1 cells could abolish the WWOX-mediated tumor repression. In addition, WWOX impair c-Jun-mediated cyclin D1 promoter activity. These results suggest that WWOX inhibits prostate cancer progression through negatively regulating cyclin D1 in cell cycle lead to G1 arrest. In summary, our data reveal a novel mechanism of WWOX in tumor suppression.     

Integrin-specific activation of Rac controls progression through the G(1) phase of the cell cycle

Adhesion to fibronectin through the alpha5beta1 integrin enables endothelial cells to proliferate in response to growth factors, whereas adhesion to laminin through alpha2beta1 results in growth arrest under the same conditions. On laminin, endothelial cells fail to translate Cyclin D1 mRNA and activate CDK4 and CDK6. Activated Rac, but not MEK1, PI-3K, or Akt, rescues biosynthesis of cyclin D1 and progression through the G(1) phase. Conversely, dominant negative Rac prevents these events on fibronectin. Mitogens promote activation of Rac on fibronectin but not laminin. This process is mediated by SOS and PI-3K and requires coordinate upstream signals through Shc and FAK. These results indicate that Rac is a crucial mediator of the integrin-specific control of cell cycle in endothelial cells.     

Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle.

Three mouse cyclin-like (CYL) genes were isolated, two of which are regulated by colony-stimulating factor 1 (CSF-1) during the G1 phase of the macrophage cell cycle. CSF-1 deprivation during G1 leads to rapid degradation of CYL proteins (p36CYL) and correlates with failure to initiate DNA synthesis. However, after entering S phase, macrophages no longer require CSF-1 and can complete cell division without expressing CYL genes. During G1, p36CYL is phosphorylated and associates with a polypeptide antigenically related to p34cdc2. The timing of p36CYL expression, its rapid turnover in the absence of CSF-1, and its phosphorylation and transient binding to a cdc2-related polypeptide suggest that CYL genes may function during S phase commitment.     

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.     

The latency-related gene of bovine herpesvirus 1 encodes a product which inhibits cell cycle progression.

Bovine herpesvirus 1 (BHV-1) establishes a latent infection in the sensory ganglionic neurons of cattle. The exclusive viral RNA expressed in a latent infection is the latency-related (LR) RNA, suggesting that it regulates some aspect of a latent infection. During the course of a productive infection, alphaherpesviruses induce certain events which occur during cell cycle progression. Consequently, we hypothesized that a BHV-1 infection might induce events in neurons which occur during cell cycle progression. In agreement with this hypothesis, cyclin A was detected in neurons of trigeminal ganglia when rabbits were infected. Neuronal cell cycle progression or inappropriate expression of cyclin A leads to apoptosis, suggesting that a viral factor inhibits the deleterious effects of cyclin A expression. The BHV-1 LR gene inhibited cell cycle progression and proliferation of human osteosarcoma cells. Antibodies directed against cyclin A or the LR protein coprecipitated the LR protein or cyclin A, respectively, suggesting that the two proteins interact with each other. We conclude that LR gene products inhibit cell cycle progression and hypothesize that this activity enhances the survival of infected neurons.     

The phosphorylation of retinoblastoma gene product in human myeloid leukemia cells during the cell cycle.

Counterflow centrifugal elutriation and immunoblotting techniques were used to study the expression of the retinoblastoma (RB) gene during the cell cycle of BV173 chronic myeloid leukemia (CML) cells. Our data showed that Rb protein started to be phosphorylated at early G1 phase, became hyperphosphorylated when cells progressed to late G1 and S phases during cell cycle, and remained hyperphosphorylated throughout S and G2/M phases. Our data suggest that Rb phosphorylation starts at a more distal point to the G1/S phase boundary in human myeloid leukemia BV173 cells rather than at a point more proximal to the G1/S boundary, as seen in HeLa cells.     

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.     

The puc1 cyclin regulates the G1 phase of the fission yeast cell cycle in response to cell size

Eukaryotic cells coordinate cell size with cell division by regulating the length of the G1 and G2 phases of the cell cycle. In fission yeast, the length of the G1 phase depends on a precise balance between levels of positive (cig1, cig2, puc1, and cdc13 cyclins) and negative (rum1 and ste9-APC) regulators of cdc2. Early in G1, cyclin proteolysis and rum1 inhibition keep the cdc2/cyclin complexes inactive. At the end of G1, the balance is reversed and cdc2/cyclin activity down-regulates both rum1 and the cyclin-degrading activity of the APC. Here we present data showing that the puc1 cyclin, a close relative of the Cln cyclins in budding yeast, plays an important role in regulating the length of G1. Fission yeast cells lacking cig1 and cig2 have a cell cycle distribution similar to that of wild-type cells, with a short G1 and a long G2. However, when the puc1(+) gene is deleted in this genetic background, the length of G1 is extended and these cells undergo S phase with a greater cell size than wild-type cells. This G1 delay is completely abolished in cells lacking rum1. Cdc2/puc1 function may be important to down-regulate the rum1 Cdk inhibitor at the end of G1.     

The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element

The retinoblastoma susceptibility gene product, Rb, is suspected to suppress cell growth. Rb is a 110-114 kd nuclear phosphoprotein. We have previously demonstrated that SV40 T antigen binds only to unphosphorylated Rb, and not pp112-114Rb, the family of phosphorylated Rb. Here we demonstrate the cell cycle-dependent phosphorylation of Rb. In G0/G1 cells, virtually all the Rb is unphosphorylated. In contrast, during S and G2, it is largely, if not exclusively, phosphorylated. Rb phosphorylation occurs at the G1/S boundary in several cell types tested. A 14 residue peptide, corresponding to the SV40 T domain required for transformation, is able to compete effectively with SV40 T for binding to p110Rb. We propose a model to explain how Rb may suppress cell growth by acting as a cell cycle regulatory element.     

DEPDC1 is a novel cell cycle related gene that regulates mitotic progression

DEPDC1 is a recently identified novel tumor-related gene that is upregulated in several types of cancer and contributes to tumorigenesis. In this study, we have investigated the expression pattern and functional implications of DEPDC1 during cell cycle progression. Expression studies using synchronized cells demonstrated that DEPDC1 is highly expressed in the mitotic phase of the cell cycle. Immunofluorescence assays showed that DEPDC1 is predominantly localized in the nucleus during interphase and is redistributed into the whole cell upon nuclear membrane breakdown in metaphase. Subsequently, siRNA-mediated knockdown of DEPDC1 caused a significant mitotic arrest. Moreover, knockdown of DEPDC1 resulted in remarkable mitotic defects such as abnormal multiple nuclei and multipolar spindle structures accompanied by the upregulation of the A20 gene as well as several cell cycle-related genes such as CCNB1 and CCNB2. Taken together, our current observations strongly suggest that this novel cancerous gene, DEPDC1, plays a pivotal role in the regulation of proper mitotic progression. [BMB Reports 2015; 48(7): 413-418]     

Isolation of a human X chromosome-linked gene essential for progression from G1 to S phase of the cell cycle

The tsBN462 cell line, a temperature-sensitive (ts) mutant isolated from the hamster cell line, BHK21/13, cannot progress into S phase at 39.5 degrees C, following the release from isoleucine deprivation. The mutant cells were transfected with high molecular weight (HMW) DNA from human KB cells, and several human DNA bands were found to be conserved through three cycles of ts+ transformation. Conserved human DNA was isolated from the cosmid library of the secondary ts+ transformant (K-1-1), using 32P-labelled total human DNA as a probe. The isolated human DNA covers about 70 kb of human DNA flanked with hamster DNA, and originates from the human X chromosome. The middle part (56 kb) of the isolated human DNA was conserved through the primary, secondary and tertiary ts+ transformation, without gross rearrangement.     

Extracellular matrix-dependent myosin dynamics during G1-S phase cell cycle progression in hepatocytes

Cell spreading and proliferation are tightly coupled in anchorage-dependent cells. While adhesion-dependent proliferation signals require an intact actin cytoskeleton, and some of these signals such as ERK activation have been characterized, the role of myosin in spreading and cell cycle progression under different extracellular matrix (ECM) conditions is not known. Studies presented here examine changes in myosin activity in freshly isolated hepatocytes under ECM conditions that promote either proliferation (high fibronectin density) or growth arrest (low fibronectin density). Three different measures were obtained and related to both spreading and cell cycle progression: myosin protein levels and association with cytoskeleton, myosin light chain phosphorylation, and its ATPase activity. During the first 48 h in culture, corresponding with transit through G1 phase, there was a six-fold increase in both myosin protein levels and myosin association with actin cytoskeleton. There was also a steady increase in myosin light chain phosphorylation and ATPase activity with spreading, which did not occur in non-spread, growth-arrested cells on low density of fibronectin. Myosin-inhibiting drugs blocked ERK activation, cyclin D1 expression, and S phase entry. Overexpression of the cell cycle protein cyclin D1 overcame both ECM-dependent and actomyosin-dependent inhibition of DNA synthesis, suggesting that cyclin D1 is a key event downstream of myosin-dependent cell cycle regulation.