ALG-2 knockdown in HeLa cells results in G2/M cell cycle phase accumulation and cell death

ALG-2 (apoptosis-linked gene-2 encoded protein) has been shown to be upregulated in a variety of human tumors questioning its previously assumed pro-apoptotic function. The aim of the present study was to obtain insights into the role of ALG-2 in human cancer cells. We show that ALG-2 downregulation induces accumulation of HeLa cells in the G2/M cell cycle phase and increases the amount of early apoptotic and dead cells. Caspase inhibition by the pan-caspase inhibitor zVAD-fmk attenuated the increase in the amount of dead cells following ALG-2 downregulation. Thus, our results indicate that ALG-2 has an anti-apoptotic function in HeLa cells by facilitating the passage through checkpoints in the G2/M cell cycle phase.     

Anthraquinone derivatives induce G2/M cell cycle arrest and apoptosis in NTUB1 cells

Thirteen anthraquinone derivatives 5-17 including two 3-(3-alkylaminopropoxy)-9,10-anthraquinone (NHA) derivatives 5 and 6, and 11 1-hydroxy-3-(3-alkylaminopropoxy)-9,10-anthraquinone (MHA) derivatives 7-17 were synthesized, evaluated for cytotoxicities against two cancer cell lines, and assayed the generation of reactive oxygen species (ROS) in NTUB1 cells (a human bladder carcinoma cell line). Compound 9 bearing a pyrrolidinyl group induced the stronger cytotoxic effect than those of other synthesized NHA and MHA derivatives. Exposure of NTUB1 cells to 9, 13, and 17 for 24h significantly increased the production of ROS, respectively. Flow cytometric analysis exhibited that the exposure of NTUB1 cells to the selective 9 led to the G2/M phase arrest accompanied by an increase of apoptotic cell death after the incubation for 24h. Compound 9 induced up-regulation of cyclinB1 and p21 expressions. Biological results suggested that the induction of G2/M arrest, apoptosis, and cell death by 9 may associate with increased expression of p21 and cyclin B1, elevation of Bax and p53 levels, and generation of ROS in the cell. In conclusion, these series of compounds may be used as anticancer agents.     

The role of Beclin 1 in IR-induced crosstalk between autophagy and G2/M cell cycle arrest

ObjectivesBeclin 1 is a well-established core mammalian autophagy protein. Autophagy has been demonstrated to play roles in cellular responses to DNA damage, such as cell cycle regulation and apoptosis. In the present study, we investigated the exact mechanism by which Beclin 1 acts as a bridge between autophagy and cell cycle, when cells are exposed to ionizing radiation (IR).Materials and methodsWestern blotting and coimmunoprecipitation were performed to investigate protein expression levels and interactions. Immunofluorescence was used to monitor the localization and distribution of the indicated proteins. The levels of apoptosis and cell cycle changes were evaluated by flow cytometry. Double thymidine deoxyribonucleoside (TdR) blocking was conducted to differentiate G2 from mitotic delay. In vitro kinase assays using ATM kinase were performed to elucidate the specific phosphorylation site in Beclin 1.ResultsIn this study, we show that Beclin 1 knockdown reduces IR-induced autophagy. IR enhanced Beclin 1/PIK3CIII complex activity as demonstrated by the results of coimmunoprecipitation and immunofluorescence assays. An investigation to assess the possible relationship between autophagy and G2/M arrest showed that, similar to the autophagy inhibitor 3MA, Beclin 1 knockdown delayed IR-induced G2/M arrest. Furthermore, the interactions between Beclin 1 and several G2/M checkpoint-related proteins, namely, PLK1 and CDC25C, were observed to increase. In addition, we observed that both 3MA and Beclin 1 inhibition decreased IR-induced apoptosis. Regarding the potential mechanism associated with this phenomenon, we showed that IR induced the interaction between Beclin 1 and Tip60 as well as their redistribution. Furthermore, we demonstrated that Beclin 1 T57 may be a targeted phosphorylation site for ATM.ConclusionsIn the present study, we demonstrate the crucial and intricate roles of Beclin 1 in IR-induced autophagy, G2/M cell cycle arrest, and apoptosis. Additionally, Tip60 and ATM were identified as important molecular regulators of Beclin 1. Our findings show the precise mechanism of crosstalk between IR-induced autophagy and G2/M cell cycle arrest.     

Adenosine induces G2/M cell‐cycle arrest by inhibiting cell mitosis progression

Cellular adenosine accumulates under stress conditions. Few papers on adenosine are concerned with its function in the cell cycle. The cell cycle is the essential mechanism by which all living things reproduce and the target machinery when cells encounter stresses, so it is necessary to examine the relationship between adenosine and the cell cycle. In the present study, adenosine was found to induce G₂/M cell‐cycle arrest. Furthermore, adenosine was found to modulate the expression of some important proteins in the cell cycle, such as cyclin B and p21, and to inhibit the transition of metaphase to anaphase in mitosis.     

Phthalocyanine-mediated photodynamic therapy induces cell death and a G0/G1 cell cycle arrest in cervical cancer cells

We have developed a series of novel photosensitizers which have potential for anticancer photodynamic therapy (PDT). Photosensitizers include zinc phthalocyanine tetra-sulphonic acid and a family of derivatives with amino acid substituents of varying alkyl chain length and degree of branching. Subcellular localization of these photosensitizers at the phototoxic IC(50) concentration in human cervical carcinoma cells (SiHa Cells) was similar to that of the lysosomal dye Lucifer Yellow. Subsequent nuclear relocalization was observed following irradiation with 665nm laser light. The PDT response was characterized using the Sulforhodamine B cytotoxicity assay. Flow cytometry was used for both DNA cell cycle and dual Annexin V-FITC/propidium iodide analysis. Phototoxicity of the derivatives was of the same order of magnitude as for tetrasulphonated phthalocyanine but with an overall trend of increased phototoxicity with increasing amino acid chain length. Our results demonstrate cell death, inhibition of cell growth, and G(0)/G(1) cell cycle arrest during the phthalocyanine PDT-mediated response.     

Targeted deletion of the Tsg101 gene results in cell cycle arrest at G1/S and p53-independent cell death

The tumor susceptibility gene 101 (Tsg101) was originally discovered in a screen for potential tumor suppressors using insertional mutagenesis in immortalized fibroblasts. To investigate essential functions of this gene in cell growth and neoplastic transformation, we derived primary mouse embryonic fibroblasts from Tsg101 conditional knockout mice. Expression of Cre recombinase from a retroviral vector efficiently down-regulated Tsg101. The deletion of Tsg101 caused growth arrest and cell death but did not result in increased proliferation and cellular transformation. Inactivation of p53 had no influence on the deleterious phenotype, but Tsg101(-/-) cells were rescued through expression of exogenous Tsg101. Fluorescence-activated cell sorting, proliferation assays, and Western blot analysis of crucial regulators of the cell cycle revealed that Tsg101 deficiency resulted in growth arrest at the G(1)/S transition through inactivation of cyclin-dependent kinase 2. As a consequence, DNA replication was not initiated in Tsg101-deficient cells. Our results clearly demonstrate that Tsg101 is not a primary tumor suppressor in mouse embryonic fibroblasts. However, the protein is crucial for cell proliferation and cell survival.     

G2/M cell cycle arrest by an N-acetyl-D-glucosamine specific lectin from Psathyrella asperospora

A new N-acetyl-D-glucosamine (GlcNAc) specific lectin was identified and purified from the fruiting body of the Australian indigenous mushroom Psathyrella asperospora. The functional lectin, named PAL, showed hemagglutination activity against neuraminidase treated rabbit and human blood types A, B and O, and exhibited high binding specificity towards GlcNAc, as well as mucin and fetuin, but not against asialofetuin. PAL purified to homogeneity by a combination of ammonium sulfate precipitation, chitin affinity chromatography and size exclusion chromatography, was monomeric with a molecular mass of 41.8 kDa, was stable at temperatures up to 55 °C and between pH 6–10, and did not require divalent cations for optimal activity. De novo sequencing of PAL using LC-MS/MS, identified 10 tryptic peptides that revealed substantial sequence similarity to the GlcNAc recognizing lectins from Psathyrella velutina (PVL) and Agrocybe aegerita (AAL-II) in both the carbohydrate binding and calcium binding sites. Significantly, PAL was also found to exert a potent anti-proliferative effect on HT29 cells (IC₅₀ 0.48 μM) that was approximately 3-fold greater than that observed on VERO cells; a difference found to be due to the differential expression of cell surface GlcNAc on HT29 and VERO cells. Further characterization of this activity using propidium iodine staining revealed that PAL induced cell cycle arrest at G₂/M phase in a manner dependent on its ability to bind GlcNAc.     

Exposure of single-stranded telomeric DNA causes G2/M cell cycle arrest in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Cdc13p is a single-stranded TG(1-3) DNA binding protein that protects telomeres and maintains telomere length. A mutant allele of CDC13, cdc13-1, causes accumulation of single-stranded TG(1-3) DNA near telomeres along with a G(2)/M cell cycle arrest at non-permissive temperatures. We report here that when the single-stranded TG(1-3) DNA is masked by its binding proteins, such as S. cerevisiae Gbp2p or Schizosaccharomyces pombe Tcg1, the growth arrest phenotype of cdc13-1 is rescued. Mutations on Gbp2p that disrupt its binding to the single-stranded TG(1-3) DNA render the protein unable to complement the defects of cdc13-1. These results indicate that the presence of a single-stranded TG(1-3) tail in cdc13-1 cells serves as the signal for the cell cycle checkpoint. Moreover, the binding activity of Gbp2p to single-stranded TG(1-3) DNA appears to be associated with its ability to restore the telomere-lengthening phenotype in cdc13-1 cells. These results indicate that Gbp2p is involved in modulating telomere length.     

Sulforaphane-induced G2/M phase cell cycle arrest involves checkpoint kinase 2-mediated phosphorylation of cell division cycle 25C

Previously, we showed that sulforaphane (SFN), a naturally occurring cancer chemopreventive agent, effectively inhibits proliferation of PC-3 human prostate cancer cells by causing caspase-9- and caspase-8-mediated apoptosis. Here, we demonstrate that SFN treatment causes an irreversible arrest in the G(2)/M phase of the cell cycle. Cell cycle arrest induced by SFN was associated with a significant decrease in protein levels of cyclin B1, cell division cycle (Cdc) 25B, and Cdc25C, leading to accumulation of Tyr-15-phosphorylated (inactive) cyclin-dependent kinase 1. The SFN-induced decline in Cdc25C protein level was blocked in the presence of proteasome inhibitor lactacystin, but lactacystin did not confer protection against cell cycle arrest. Interestingly, SFN treatment also resulted in a rapid and sustained phosphorylation of Cdc25C at Ser-216, leading to its translocation from the nucleus to the cytoplasm because of increased binding with 14-3-3beta. Increased Ser-216 phosphorylation of Cdc25C upon treatment with SFN was the result of activation of checkpoint kinase 2 (Chk2), which was associated with Ser-1981 phosphorylation of ataxia telangiectasia-mutated, generation of reactive oxygen species, and Ser-139 phosphorylation of histone H2A.X, a sensitive marker for the presence of DNA double-strand breaks. Transient transfection of PC-3 cells with Chk2-specific small interfering RNA duplexes significantly attenuated SFN-induced G(2)/M arrest. HCT116 human colon cancer-derived Chk2(-/-) cells were significantly more resistant to G(2)/M arrest by SFN compared with the wild type HCT116 cells. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in irreversible G(2)/M arrest by SFN. Activation of Chk2 in response to DNA damage is well documented, but the present study is the first published report to link Chk2 activation to cell cycle arrest by an isothiocyanate.     

Delta 12-prostaglandin J2 mimics heat shock in inducing cell cycle arrest at G1 phase

Using a human neuroblastoma cell line GOTO, the effects of delta 12-prostaglandin (PG) J2 on the modulation of cell cycle progression and protein synthesis were examined in comparison with those caused by heat shock (HS). delta 12-PGJ2 induced G1 arrest, the peak of which was obtained at 24 h and continued for 72 h. HS was found to induce G1 arrest earlier than delta 12-PGJ2. Furthermore, sequential HS could maintain G1 arrest. delta 12-PGJ2 induced the synthesis of several heat shock proteins (HSPs) in a manner similar to HS. Using immunoblot analysis, HSP72 was detected prior to inducing G1 arrest and accumulated during the subsequent 72h. The content of HSP72 induced by HS also correlated well with the induction, release, and maintenance of G1 arrest. In addition, both delta 12-PGJ2 and HS induced HSP72 mRNA and simultaneously suppressed N-myc mRNA expression. These results suggest that delta 12-PGJ2 and HS regulate cell cycle progression of GOTO cells via similar mechanisms.     

Rotenone-induced G2/M cell cycle arrest and apoptosis in a human B lymphoma cell line PW.

Concentrations of rotenone (ROT) that block electron flow through mitochondrial complex I (100 nM) did not significantly alter either cell viability or the growth of PW cells. However, 10- to 50-fold higher concentrations (1-5 microM) were found to induce a dose-dependent cell cycle arrest predominantly at the G2/M stage of the cycle and apoptosis. Apoptosis was dependent on the cell cycle arrest, since apoptosis but not the G2/M arrest was prevented with the broad spectrum caspase inhibitor zVADfmk. Biochemical features of apoptosis included mitochondrial cytochrome c release, reactive oxygen species generation, and the activation of procaspase 3. Thus, ROT inhibition of mitochondrial electron transport may be insufficient to induce apoptosis in PW cells. Instead, apoptosis in these cells occurs as a consequence of disruption of the cell cycle and is only indirectly dependent upon mitochondrial electron transport.     

Hepatic stellate cell activation in vitro: cell cycle arrest at G2/M and modification of cell motility

Hepatic fibrosis is a common response to chronic liver injury and is characterized by increased production of extracellular matrix components, whose major part is produced by hepatic stellate cells activated by inflammatory mediators to proliferate and migrate into the injured regions. GRX cells are a model of hepatic stellate cells characterized as myofibroblasts by morphological and biochemical criteria. We have recently shown that they respond to inflammatory mediators and cytokines present in the concanavalin A-activated spleen cell supernatant (SCS) by quantitative changes in the expression of intermediate filaments. The present study investigated the effects of SCS and TNF-alpha on the GRX cell proliferation and on the organization of the actin cytoskeleton. SCS and TNF-alpha diminished the culture cell density, with an increase of cell [(3)H]thymidine incorporation and of cellular protein content, indicating an arrest in the G2/M phase of the cell cycle, which was reversible 48 h after removal of SCS. This effect was abrogated by dibutiryl-cAMP. Actin cytoskeleton reorganization was observed after 24 h treatment, indicating increased cell motility. Our results suggest that inflammation-dependent activation of stellate cells occurs in ordered interaction and coordination of proinflammatory agents. The increase of cAMP levels activates the conversion of lipocytes into myofibroblasts and increases the number of cells that can participate in repair. Since cAMP retains cells in the G1 phase, cytokines of the TNF-alpha group are required for cell proliferation inducing the entry into the S phase. The progression through the G2/M checkpoint is mediated again by increased cAMP levels.     

Silencing of ZFP36L2 increases sensitivity to temozolomide through G2/M cell cycle arrest and BAX mediated apoptosis in GBM cells

Molecular Biology Reports - Despite the advancements in primary brain tumour diagnoses and treatments, the mortality rate remains high, particularly in glioblastoma (GBM). Chemoresistance,...     

2‐methoxyestradiol‐induced cell death in osteosarcoma cells is preceded by cell cycle arrest

The article to which this erratum refers was published in J. Cell. Biochem. 104: 1937–1945, 2008. © 2008 Wiley‐Liss, Inc.     

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.     

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells

BackgroundPrevious studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown.MethodsWe generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics.ResultsPC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP.ConclusionsSuppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells.General significanceOur results highlight the possibility of the use of PC as an anti-cancer drug target.     

Downregulation of PLK4 expression induces apoptosis and G0/G1‐phase cell cycle arrest in keloid fibroblasts

ObjectivesKeloids are benign fibroproliferative tumors that display many cancer‐like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo‐like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids.Materials and MethodsWe evaluated the expression of PLK4 in keloids and adjacent normal skin tissue samples. Then, we established PLK4 knockdown and overexpression cell lines in keloid fibroblasts (KFs) and normal skin fibroblasts (NFs), respectively, to investigate the roles of PLK4 in the regulation of proliferation, migration, invasion, apoptosis, and cell cycle in KFs. Centrinone B (Cen‐B), a highly selective PLK4 inhibitor, was used to inhibit PLK4 activity in KFs to evaluate the therapeutic effect on KFs.ResultsWe discovered that PLK4 was overexpressed in keloid dermal samples and KFs compared with adjacent normal skin samples and NFs derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by Cen‐B suppressed KF growth, induced KF apoptosis via the caspase‐9/3 pathway, and induced cell cycle arrest at the G0/G1 phase in vitro.ConclusionsThese findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen‐B is a promising candidate drug for keloid treatment.

Keloids are benign fibroproliferative tumors that display many cancer‐like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo‐like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids. Here, we discovered that PLK4 is a potential target for the treatment of keloids. PLK4 was overexpressed in keloid dermal samples and keloid fibroblasts (KFs) compared with adjacent normal skin samples and normal skin fibroblasts derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by a highly selective inhibitor, centrinone B (Cen‐B), suppressed KF growth, induced KF apoptosis via the caspase‐9/3 pathway, and induced cell cycle arrest at the G0/G1 phase via the p53/p21/Cyclin D1 pathway in vitro. These findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen‐B is a promising candidate drug for keloid treatment.     

Centipedegrass extract enhances radiosensitivity in melanoma cells by inducing G2/M cell cycle phase arrest

Molecular Biology Reports - Melanoma is aggressive, highly metastatic, and potentially fatal. In the case of patients with advanced melanoma, it is difficult to expect a good prognosis, since this...