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Abstract: Numerous anti-cancer agents inhibit cell cycle progression via a p53-dependent mechanism; however, many of these carcinostatic substances are toxic. Here, we show that ascochlorin, an isoprenoid antibiotic, is a non-toxic anti-cancer agent that induces G1 arrest via the induction of p21WAF1/CIP1 in a c-Myc, but not a p53, dependent manner. Ascochlorin has a broad spectrum of anti-tumor and anti-metastatic activities, but the molecular mechanism by which it inhibits cell cycle progression of cancer cells remains to be elucidated. We demonstrated that cytostatic G1 arrest by ascochlorin is mainly associated with the upregulation of p21WAF1/CIP1, and the downregulation of c-Myc. Furthermore, we used a chromatin immunoprecipitation assay, RNA interference, and p53-deficient cells to verify that p21WAF1/CIP1 induction by ascochlorin is related to transcriptional repression of c-Myc. Ascochlorin abolished pRB hyperphosphorylation, which resulted in the inactivation of E2F transcriptional activity. These results suggest that ascochlorin induces G1 arrest via the p53-independent suppression of c-Myc. Thus, we reveal a role for ascochlorin in inhibiting tumor growth via G1 arrest, and identify a novel regulatory mechanism for c-Myc. [Copyright &y& Elsevier]

Abstract: Fertilization relieves the oocyte from a cell cycle arrest, inducing progression towards mitotic cycles. While the signalling pathways involved in oocyte to embryo transition have been widely investigated, how they specifically trigger DNA replication is still unclear. We used sea urchin eggs whose oocytes are arrested in G1 to investigate in vivo the molecular mechanisms regulating initiation of replication after fertilization. Unexpectedly, we found that CDC6, Cdt1 and MCM3, components of the pre-replication complexes (pre-RC) which license origins for replication, were already loaded on female chromatin before fertilization. This is the first demonstration of a cell cycle arrest in metazoan in which chromatin is already licensed for replication. In contrast pre-RC assemble on chromatin post-fertilization as in other organisms. These differences in the timing of pre-RC assembly are accompanied by differences in Cdk2 requirement for DNA replication initiation between female and male chromatin post-fertilization. Finally, we demonstrated that a concomitant inhibition of MAP kinase and ATM/ATR pathways releases the block to DNA synthesis. Our findings provide new insight into the mechanisms contributing to the release of G1 arrest and the control of S-phase entry at fertilization. [Copyright &y& Elsevier]

Abstract: Tylophorine, a representative phenanthroindolizidine alkaloid from Tylophora indica plants, exhibits anti-inflammatory and anti-cancerous growth activities. However, the underlying mechanisms of its anti-cancer activity have not been elucidated and its effects on cell cycle remain ambiguous. Here, we reveal by asynchronizing and synchronizing approaches that tylophorine not only retards the S-phase progression but also dominantly arrests the cells at G1 phase in HepG2, HONE-1, and NUGC-3 carcinoma cells. Moreover, tylophorine treatment results in down regulated cyclin A2 expression and overexpressed cyclin A2 rescues the G1 arrest by tylophorine. Thus, we are the first to report that the downregulated cyclin A2 plays a vital role in G1 arrest by tylophorine in carcinoma cells. [Copyright &y& Elsevier]

Abstract: hSNF5/Ini1 is a core component of the SWI/SNF complex and the gene is frequently mutated in aggressive pediatric rhabdoid tumors. Mechanisms of the malignant transformation, however, remain poorly understood. We analyzed HeLa cells treated with siRNA to the hSNF5/Ini1 mRNA. The resulting efficient and long-term suppression caused characteristic cell enlargement, cell cycle arrest in G1 phase, and subsequent modest apoptosis. Gene expression profiling of the hSNF5-down-regulated cells by cDNA microarray analysis revealed that a limited number of p53-responsive genes, especially p21, were up-regulated. The p53 protein level was also greatly enhanced, suggesting that loss of hSNF5/Ini1 induces a p53 signaling pathway irrelevant to the chk1/2 phosphorylation pathway. Some rhabdoid tumors with very low or no ARF expression were induced to undergo cell enlargement, growth arrest, and, in one case, apoptosis by ectopic expression of the p14ARF protein. These results may in part account for molecular mechanisms of rhabdoid tumor formation. [Copyright &y& Elsevier]

Abstract: The Hepatitis B virus X (HBx) protein has been strongly implicated in the carcinogenesis of hepatocellular carcinoma (HCC). However, effects of the HBx protein on cell proliferation and cell death are controversial. This study investigates the effects of the HBx protein on liver regeneration in two independent lines of HBx transgenic mice, which developed HCC at around 14 to 16 months of age. High mortality, lower liver mass restoration, and impaired liver regeneration were found in the HBx transgenic mice post-hepatectomy. The levels of alanine aminotransferase and α-fetoprotein detected post-hepatectomy increased significantly in the HBx transgenic livers, indicating that they were more susceptible to damage during the regenerative process. Prolonged activation of the immediate-early genes in the HBx transgenic livers suggested that the HBx protein creates a strong effect by promoting the transition of the quiescent hepatocytes from G0 to G1 phase. However, impaired DNA synthesis and mitosis, as well as inhibited activation of G1, S, and G2/M markers, were detected. These results indicated that HBx protein exerted strong growth arrest on hepatocytes and imbalanced cell-cycle progression resulting in the abnormal cell death; this was accompanied by severe fat accumulation and impaired glycogen storage in the HBx transgenic livers. In conclusion, this study provides the first physiological evidence that HBx protein blocks G1/S transition of the hepatocyte cell-cycle progression and causes both a failure of liver functionality and cell death in the regenerating liver of the HBx transgenic mice. [Copyright &y& Elsevier]

Abstract: The effects of activating endogenous protein kinase C (PKC) on cell proliferation and the cell cycle were investigated by treating the breast cancer cell line SKBR-3 with phorbol 12-myristate 13 acetate (PMA). This inhibited cell growth in a concentration-dependent manner, causing a marked arrest of cells in G1. Pre-treatment with GF109203X completely blocked the antiproliferative effect of PMA, and pre-treatment with the PKCδ inhibitor rottlerin partially blocked it. Infecting SKBR-3 cells with an adenovirus vector containing wild-type PKCδ, WTPKCδAdV, had similar effects on PMA. Infecting the cells with a dominant-negative PKCδAdV construct blocked the growth inhibition induced by PMA. Downstream of PKC, PMA treatment inhibited extracellular signal-regulated kinase mitogen-activated protein kinase phosphorylation, up-regulated c-jun NH2-terminal kinase phosphorylation, and inhibited retinoblastoma (Rb) phosphorylation. These results strongly implicated PKC (mainly PKCδ) in the G1 arrest induced by PMA and suggested PKC as a target for breast cancer treatment. [Copyright &y& Elsevier]

Abstract: The F9 murine embryonal carcinoma cell line provides an attractive system for studying epithelial differentiation and antiproliferative processes. We have recently established F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4α and shown that HNF-4α triggers the gene expression of tight-junction molecules, occludin, claudin-6, and claudin-7, as well as formation of functional tight junctions and polarized epithelial morphology (Exp. Cell Res. 286, [2003] 288). Since these events were very similar to those induced by retinoids, we investigated whether HNF-4α, like retinoid receptors, was involved in the control of cell proliferation. We herein show that HNF-4α up-regulates expression of the p21 gene, but not the p15, p16, p18, p19, or p27 gene, in a p53-independent manner, and inhibits cell growth in F9 cells. Similar results were observed in rat lung endothelial cells, in which expression of HNF-4α is conditionally induced by doxycycline. Furthermore, we demonstrate, by reporter assay, that HNF-4α significantly elevates the transcriptional activity of the p21 promoter. Since, HNF-4α is expressed not only in the liver but also in organs containing epithelial cells, such as kidney, intestine, pancreas, and stomach, it might also play critical roles in the regulation of epithelial morphogenesis and proliferation in these organs. [Copyright &y& Elsevier]

K. Orihara, S. Biro, S. Hamasaki, H. Eto, M. Miyata, Y. Ikeda and C. Tei. Hyperthermia at 43°C for 2 h Inhibits the Proliferation of Vascular Smooth Muscle Cells, but not Endothelial Cells. Journal of Molecular and Cellular Cardiology (2002) 34, 1205–1215. Restenosis after angioplasty is one of the most critical problems of the various interventional therapies for myocardial ischemia. It has been difficult to prevent the vascular smooth muscle cells (VSMCs) proliferation resulting in restenosis. The goal of this study was to prove the treatment by hyperthermia to be effective in suppressing VSMC''s proliferation in vitro. When just-stimulated VSMCs, which were incubated for 2 h after 5% FBS stimulation to quiescent VSMCs, were exposed to hyperthermia (43°C, 2 h), the cell cycle progression to S and G2/M phase was significantly delayed 24 h after 5% FBS stimulation. And another 24 h later, cell death was observed partly (19%) of heat-treated VSMCs. Nonetheless, hyperthermia under the same conditions did not result in the death of quiescent VSMCs, and did not inhibit the proliferation of cultured bovine aortic endothelial cells (BAECs). In addition, we found that hyperthermia (43°C, 2 h) elevated p27Kip1 over the amount induced in confluent VSMCs. Much elevation of p27Kip1, which is a negative regulator of G1/S progression, may play a role in heat-induced G1 arrest of VSMCs. In conclusion, we have found that hyperthermia (43°C, 2 h) inhibited the proliferation of the dividing VSMCs mainly due to G1 arrest with neither inhibiting the generation of BAECs nor damaging quiescent VSMCs. Hence, our data suggest that hyperthermia may be clinically applicable for the prevention of restenosis. [Copyright &y& Elsevier]

Miconazole (MIC), a promising oral antifungal agent, has been used worldwide in the treatment of superficial mycosis. In this study, we demonstrated that MIC dose dependently arrested various human cancer cells at the G0/G1 phase of the cell cycle. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated by MIC treatment in COLO 205 cells. Electrophoretic mobility gel shift assays showed that the nuclear extracts of the MIC-treated COLO 205 cells exerted a significant binding between wild-type p53 and its consensus-binding site present in the p21/Cip1 promoter. These results suggested that the p53-associated signaling pathway is involved in the regulation of MIC-induced cancer cell growth arrest. By immunoblot analysis, we demonstrated that cyclin D3 and cyclin-dependent kinase-4 (CDK4) protein levels were inhibited by MIC treatment in the cancer cells. Significant therapeutic effect was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with MIC (50 mg/kg ip). The protein expression of p53 was significantly increased in MIC-treated tumor tissues by immunohistochemical staining and Western blotting analysis. DNA fragmentation and TUNEL assay were performed and demonstrated that apoptosis occurred in tumor tissues treated with MIC. Our study provides the novel mechanisms of antitumor effects of MIC and such results may have significant applications for cancer chemotherapy. [Copyright &y& Elsevier]

In an attempt to determine whether exposure to extremely low frequency (ELF) electromagnetic fields can affect cells, Ku80-deficient cells (xrs5) and Ku80-proficient cells (CHO-K1) were exposed to ELF electromagnetic fields. Cell survival, and the levels of the apoptosis-related genes p21, p53, phospho-p53 (Ser15), caspase-3 and the anti-apoptosis gene bcl-2 were determined in xrs5 and CHO-K1 cells following exposure to ELF electromagnetic fields and X-rays. It was found that exposure of xrs5 and CHO-K1 cells to 60 Hz ELF electromagnetic fields had no effect on cell survival, cell cycle distribution and protein expression. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields for 5 h after irradiation significantly inhibited G1 cell cycle arrest induced by X-rays (1 Gy) and resulted in elevated bcl-2 expression. A significant decrease in the induction of p53, phospho-p53, caspase-3 and p21 proteins was observed in xrs5 cells when irradiation by X-rays (8 Gy) was followed by exposure to 5 mT ELF magnetic fields. Exposure of xrs5 cells to the ELF electromagnetic fields for 10 h following irradiation significantly decreased X-ray-induced apoptosis from about 1.7% to 0.7%. However, this effect was not found in CHO-K1 cells within 24 h of irradiation by X-rays alone and by X-rays combined with ELF electromagnetic fields. Exposure of xrs5 cells to 60 Hz ELF electromagnetic fields following irradiation can affect cell cycle distribution and transiently suppress apoptosis by decreasing the levels of caspase-3, p21, p53 and phospho-p53 and by increasing bcl-2 expression. [Copyright &y& Elsevier]

Aging is characterized by the progressive decline of physiology at the cell, tissue and organism level, leading to an increased risk of mortality. Proteotoxic stress, mitochondrial dysfunction and genomic instability are considered major universal drivers of cell aging, and accumulating evidence establishes clear biunivocal relationships among these key hallmarks. In this regard, the finite lifespan of the budding yeast, together with the extensive armamentarium of available analytical tools, has made this single cell eukaryote a key model to study aging at molecular and cellular levels. Here we review the current data that link proteostasis to cell cycle progression in the budding yeast, focusing on senescence as an inherent phenotype displayed by aged cells. Recent advances in high-throughput systems to study yeast mother cells while they replicate are providing crucial information on aging-related processes and their temporal interdependencies at a systems level. In our view, the available data point to the existence of multiple feedback mechanisms among the major causal factors of aging, which would converge into the loss of proteostasis as a nodal driver of cell senescence and death. [ABSTRACT FROM AUTHOR]

In recent years, anaplastic lymphoma kinase (ALK) rearrangement-positive anaplastic large cell lymphoma (ALCL) has rising morbidity and mortality. Unfortunately, no ALK inhibitor has been approved by the FDA for single treatment of ALK rearrangement-positive ALCL. In this study, we investigated the antitumor effect of ZYY, a novel ALK inhibitor, showing a strong growth inhibitory effect on Karpas299 cells in vitro and in vivo. Specifically, ZYY significantly reduced the mRNA and protein expression of ALK and its downstream signaling proteins in Karpas299 cells. Furthermore, ZYY induced G1 phase arrest and promoted apoptosis in Karpas299 cells. Furthermore, we demonstrated that ZYY-induced apoptosis was mainly related to the mitochondria-dependent endogenous pathway. In vitro studies further showed that ZYY induced autophagy in Karpas299 cells, along with increased levels of the autophagy-related proteins, including LC3II and Beclin-1. Moreover, knockdown Beclin-1 and application of autophagy inhibitor chloroquine potentiated ZYY-induced cytotoxicity and apoptosis in vitro , indicating that cytoprotective autophagy might be triggered by ZYY in Karpas299 cells. Taken together, the novel ALK inhibitor ZYY has tremendous potential for treating human ALCL, and a combination of autophagy and ALK inhibition could effectively elicit potent antitumor effects. • ZYY is a novel synthetic ALK inhibitor derived from ceritinib. • ZYY inhibits Karpas299 cell growth in vitro and in vivo without evident toxicity. • ZYY induces apoptosis and autophagy in Karpas299 cells. • Autophagy inhibition enhances ZYY-induced Karpas299 cell apoptosis and cytotoxicity. [ABSTRACT FROM AUTHOR]