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Parkin (PK) is an E3-ligase harboring tumor suppressor properties that has been associated to various cancer types including glioblastoma (GBM). However, PK is also a transcription factor (TF), the contribution of which to GBM etiology remains to be established. Methods: The impact of PK on GBM cells proliferation was analyzed by real-time impedance measurement and flow cytometry. Cyclins A and B proteins, promoter activities and mRNA levels were measured by western blot, luciferase assay and quantitative real-time PCR. Protein-protein and protein-promoter interactions were performed by co-immunoprecipitation and by ChIP approaches. The contribution of endogenous PK to tumor progression in vivo was performed by allografts of GL261 GBM cells in wild-type and PK knockout mice. Results: We show that overexpressed and endogenous PK control GBM cells proliferation by modulating the S and G2/M phases of the cell cycle via the trans-repression of cyclin A and cyclin B genes. We establish that cyclin B is regulated by both E3-ligase and TF PK functions while cyclin A is exclusively regulated by PK TF function. PK invalidation leads to enhanced tumor progression in immunocompetent mice suggesting an impact of PK-dependent tumor environment to tumor development. We show that PK is secreted by neuronal cells and recaptured by tumor cells. Recaptured PK lowered cyclins levels and decreased GBM cells proliferation. Further, PK expression is decreased in human GBM biopsies and its expression is inversely correlated to both cyclins A and B expressions. Conclusion: Our work demonstrates that PK tumor suppressor function contributes to the control of tumor by its cellular environment. It also shows a key role of PK TF function in GBM development via the control of cyclins in vitro and in vivo . It suggests that therapeutic strategies aimed at controlling PK shuttling to the nucleus may prove useful to treat GBM., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Aim: To investigate the relationship between paracetamol and expression levels of cyclooxygenase-2, cyclin B, cell viability and apoptosis in glioblastoma cell line., Material and Methods: The A172 glioblastoma cells were treated with different concentrations of paracetamol and phosphate buffer saline as a vehicle for 24, 48, and 72 hours. Cell viability was detected by MTT. Bax, procaspase 3, COX-2 and Cyclin B expressions were detected using Western blotting., Results: A paracetamol treatment of 0.5 mg/mL for 24, 48, and 72 hours led to a 14%, 31%, and 37% decrease in cell viability. The expression of COX-2 and cyclin B levels decreased by 36% and 52% respectively, after treatment with 0.5 mg/mL paracetamol. Treatment with 0.5 mg/mL and 1 mg/mL paracetamol significantly induced the expression of cleaved caspase 3, procaspase 3 and Bax proteins compared to the control group (60%, 40%, 21%, %100, 18%, 17%, respectively)., Conclusion: The results of our study showed that paracetamol has antitumoral effects on glioblastoma cells and this activity was induced by different signaling pathways.

Neural stem/progenitor cells (NSPCs) express higher levels of poly(ADP-ribose) polymerase 1 (PARP1) than mouse embryonic fibroblasts (MEFs). Inhibition of PARP induces the expression of several genes in the p53 signaling pathway, including p21, which is critical for cell cycle control at the G1/S phase, triggers apoptosis, and suppresses cell cycle progression in NSPCs. However, upon the up-regulation of p21, the cell cycle does not arrest at any specific phase. In the present study, the expression of genes specific to the G1/S and G2/M phases of the cell cycle were analyzed following treatment with PJ34 (N-[6-oxo-5,6-dihydro-phenanthridin-2-yl]-N,N-dimethylacetamide), an inhibitor of PARP. PJ34 treatment dramatically down-regulated cyclin B1 expression in NSPCs, but not in MEFs, which was confirmed by a promoter assay. Down-regulation of FoxM1 and B-MYB revealed that the down-regulation of cyclin B occurs at the transcriptional level. GADD45 was also specifically up-regulated in NSPCs. Taken together, the activation of p53 by PJ34 treatment in NSPCs induced changes in the expression of genes involved in the cell cycle. Fluorescence-activated cell sorting analysis revealed that PJ34 treatment suppressed G2/M to G1 progression in NSPCs, but not in MEFs. These data indicate that PJ34 treatment inhibits cyclin expression at the mRNA level and suppresses cell cycle progression in NSPCs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Few researches on increase of chemotherapy sensitivity by microRNA (miRNA) were reported. We aim to investigate exact role of miR-381 in chemotherapy sensitivity of 5-fluorouracil (5-FU) in renal cancer cells., Methods: We investigated the cell survival, cell-cycle and apoptosis of 786-O and HK-2 cells treated with miR-381 and 5-FU. IC50 of 5-FU was calculated. To study apoptosis and G2/M arrest, we determined pHH3, mitotic index and caspase-3/7 activity., Results: We showed that miR-381 combined with 5-FU inhibited proliferation and potentiated the anti-tumour efficacies of 5-FU at tolerated concentration in vitro. miR-381 combined with 5-FU led to Cdc2 activation, mitotic catastrophe, and cell apoptosis through inhibitory WEE1. WEE1 was also validated as the direct target of miR-381. IC50 of 5-FU decreased significantly in the presence of miR-381., Conclusion: miR-381 increases sensitivity of 786-O cells to 5-FU by inhibitory WEE1 and increase of Cdc2 activity.

Background and Objective: Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated., Material and Methods: Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively., Results: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS., Conclusion: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases., (© 2012 John Wiley & Sons A/S.)

Aim: To evaluate the effect of TEGDMA on cell cycle progression as well as alterations of cell cycle-related gene and protein expression., Methodology: Human dental pulp cells were exposed to 0-5 mmol L(-1) TEGDMA for 24 h. Cytotoxicity was evaluated by 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell cycle progression was analysed by propidium iodide (PI) flow cytometry. Cell death pathway was surveyed by annexin V/PI dual-staining flow cytometry. The mRNA expression of cell cycle-related genes (cdc2, cyclinB1 and p21) and COX-2 was evaluated by reverse transcriptase-polymerase chain reaction, and their protein expression was evaluated by Western blotting. The production of PGE(2) and PGF(2α) in the culture medium was determined by enzyme-linked immunosorbent assay., Results: Triethylene glycol dimethacrylate inhibited cellular growth and induced cell cycle deregulation in dental pulp cells. High-dose exposure provoked both necrotic and apoptotic cell death. The gene and protein expression of cdc2, cyclin B1 and cdc25C declined obviously whilst cells treated with 2.5 mmol L(-1) TEGDMA concurrent with the elevated expression of p21. The mRNA and protein expression of COX-2, along with production of PGE(2) and PGF(2α), are drastically raised by 2.5-5 mmol L(-1) TEGDMA., Conclusions: Triethylene glycol dimethacrylate induced cytotoxicity, cell cycle arrest and apoptosis in dental pulp cells, which was associated with the decline of cdc2, cyclin B1, cdc25C expression and elevation of p21 expression. Concomitantly, COX-2 expression, PGE(2) and PGF(2α) production increased. These effects may contribute to explain the pulpal damage and inflammation induced by TEGDMA after operative procedures., (© 2012 International Endodontic Journal.)

In this study, we incubated human A549 lung cancer cells with quercetin-metabolite-enriched plasma (QMP) obtained from Mongolian gerbils 2 h after quercetin feeding (100 mg/kg body wt/week). We investigated the effects of QMP on the growth of A549 cells and the possible mechanisms for these effects. We found that QMP but not control plasma (CP) reduced the cell growth in A549 cells. QMP led to cell cycle arrest at the G (2)/M phase by downregulating the expression of cdk1 and cyclin B. QMP but not CP or quercetin itself significantly increased PPAR- γ expression (p < 0.05), which was accompanied by an increase of phosphatase and tensin homologue deleted on the chromosome ten level and a decrease of phosphorylation of Akt. Furthermore, quercetin-3-glucuronide and quercetin-3'-sulfate also significantly increased PPAR- γ expression in A549 cells. GW9662, a PPAR- γ antagonist, significantly suppressed the effects of 10 % QMP on cell proliferation and on the expression of cyclin B and cdk1. Taken together, these data suggest that the activation of PPAR- γ plays an important role, at least in part, in the antiproliferative effects of quercetin metabolites., (© Georg Thieme Verlag KG Stuttgart · New York.)

Scope: We investigated the inhibition of pulmonary tumor formation through treatment with curcumin in transgenic mice., Methods and Results: In this study, a strain of transgenic mice carrying human vascular endothelial growth factor A₁₆₅ (hVEGF-A₁₆₅) gene to induce pulmonary tumor was used as an in vivo cancer therapy model. We found that curcumin significantly reduced hVEGF-A₁₆₅ overexpression to normal, specifically in Clara cells of the lungs of transgenic mice, and suppressed the formation of tumors. In addition, we demonstrated a relationship between curcumin treatment and the expression of VEGF, EGFR, ERK2, and Cyclin A at the transcriptional and translational levels. We also noticed a reduction of Cyclin A and Cyclin B after curcumin treatment that had an effect on the cell cycle. Curcumin-induced inhibition of Cyclin A and Cyclin B likely results in decreased progression through S and G2/M phases. These results demonstrated that the expression of proteins involved in the S to M phase transition in transgenic mice is suppressed by curcumin., Conclusion: A Data suggest that a blockade of the cell cycle may be a critical mechanism for the observed effects on vasculogenesis and angiogenesis following treatment with curcumin., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: 17-(demethoxy), 17-allylamino geldanamycin (17-AAG) suppresses growth in some cancers by inhibiting Heat shock protein 90 (Hsp90). We examined the effects of 17-AAG-mediated Hsp90 inhibition on human hepatocellular carcinoma (HCC) growth in vitro and in vivo., Methods: Human HCC cell lines, Hep3B and HuH7, were exposed to 17-AAG and cell viabilities and apoptosis were determined. Cell cycle profiles were analyzed and the G(2)/M cell cycle checkpoint proteins cdc2 and cyclin B1 were examined. Studies were performed to determine whether 17-AAG-mediated cdc2 decrease was due to altered gene expression, transcription, or protein degradation. The effects of 17-AAG on Hep3B and HuH7 xenograft growth in athymic nude mice were also examined., Results: Hep3B and HuH7 treated with 17-AAG versus untreated controls showed decreased cell viability and increased apoptosis. Cells treated with 17-AAG also showed an increased fraction in G(2)/M phase and an associated decrease in cdc2 through protein degradation rather than through other mechanisms. Hsp90 inhibition by 17-AAG also decreased HCC xenograft growth in association with decreased cdc2 expression., Conclusions: 17-AAG-mediated inhibition of Hsp90 abrogates human HCC cell growth in vitro and in vivo through cdc2 decrease, which in turn induces G(2)/M cell cycle arrest and apoptosis. Hsp90 is a mediator of HCC growth and survival and its inhibition may serve as a potential treatment.

We recently demonstrated that treatment of three leukemic cell lines with an aqueous extract of cinnamon (CE) for 24 h produced dose-dependent arrests in the G2/M phase of the cell cycle. To accomplish the goal of understanding underlying mechanisms, we selected the cell line most responsive to the CE treatment to study the effects of the extract on signaling molecules regulating cell cycle progression. Cell cycle analyses were conducted on treated versus nontreated cells from 0-6 h. The percentages of cells in G2/M in CE-treated cells increased significantly from 11.0+/-1.0 to 23.6+/-1.4 after 6 h, while the percentage for nontreated cells remained unchanged (12.3+/-0.8). Multiparametric flow cytometric analyses were used to associate activation of p38 mitogen-activated protein kinase (MAPK) with cells arrested in G2/M, the size of these cells, and the presence or absence of cyclin B1. After 4 h, there was a 26% increase in the activated phosphorylated form of p38 MAPK in CE-treated cells compared with the nontreated control cells, with larger cells showing the greater increases. Although the proportion of CE-treated cells in G2/M was higher than controls, this population was shown to be less positive for cyclin B1 than the control G2/M population. Our results demonstrate that CE significantly modulated two signaling proteins, p38 MAPK and cyclin B, that regulate progression through G2/M. Overall, the data provide evidence that CE affects proliferation in a leukemic cell line by disrupting critical phosphorylating/dephosphorylating signaling events that propel cells through the G2/M phase.

Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) for thymidine substitution instead of BrdU (5-bromo-2'-deoxyuridine) in cell proliferation assays has recently been proposed. However, the effect of EdU on cell viability, DNA synthesis, and cell cycle progression and consequently its usability for dynamic cell proliferation analysis in vitro has not been explored. We compared the effect of EdU and BrdU incorporation into SK-BR-3 and BT474 breast cancer cells and the impact on cell cycle kinetics, cell viability, and DNA damage. We found that EdU can be used not only for pulse but also for continuous cell labeling and henceforth in high resolution EdU/Hoechst quenching assays. BrdU and EdU proliferation assays based on click chemistry revealed comparable results. However, cell viability of SK-BR-3 breast cancer cells was highly affected by long term exposure to EdU. Both SK-BR-3 as well as BT474 cells show cell cycle arrests upon long term EdU treatment whereas only SK-BR-3 cells were driven into necrotic cell death by long term exposure to EdU. In contrast BT474 cells appeared essentially unharmed by EdU treatment in terms of viability. Consequently using EdU enables highly sensitive and quantitative detection of proliferating cells and facilitates even continuous cell cycle assessment. Nevertheless, potential cellular susceptibility needs to be individually evaluated.

Arsenic compounds have been used as anti-cancer agents in traditional Chinese medicine. Ionizing radiation (IR) is one of the most effective tools in the clinical treatment of cancer. The induction of apoptotic cell death is a significant mechanism of tumor cells under the influence of radio-/chemotherapy, and resistance to these treatments has been linked to some cancer cell lines with a low propensity for apoptosis. A combination of different anti-tumoral treatment modalities is advantageous in limiting non-specific toxicity often observed by an exceedingly high dose of single regimen. The present study aimed at investigating the enhanced effects and mechanisms in cell cycle distribution and apoptosis of U937 cells, a human pre-monocytic leukemia cell line lacking functional p53 protein, after combination treatment with irradiation and As(2)O(3). Our results indicated that combined treatment led to activation of cdc-2, which is related to the expression of cyclin B. In addition, combined treatment increased apoptotic cell death in U937 cells, which is correlated with the induction of mitotic arrest, the increase in intracellular reactive oxygen species (ROS) generation, the decrease in B-cell leukemia/lymphoma 2 (Bcl-2) and B-cell leukemia/lymphoma XL (Bcl-XL) levels, the loss of mitochondria membrane potential, and the activation of caspase-3. We found that combining radiation and As(2)O(3) may be an effective strategy against p53-deficient leukemia cells.

A number of polyamine analogs are currently used in various clinical trials as cancer treatment and it is important to investigate their effects not only on cancer cells but also on normal cells. Treatment with polyamine analogs depletes cells of polyamines and inhibits cell proliferation, but the analogs cannot take over the normal function of the natural polyamines in the cell. In this study, the normal-like breast epithelial cell line MCF-10A was treated with the polyamine analog N',N"-diethylnorspermine (DENSPM). The cells were then studied using a bromodeoxyuridine- DNA flow cytometry method as well as western blot. The ability of both normal-like and breast cancer cells to recover from DENSPM treatment was also studied. DENSPM treatment of MCF-10A cells resulted in a prolongation of the S and G2 +M phases, followed by a G1/S block. The p53/p21/RB1 pathway was involved in the G1/S block as shown by increased levels of p53 and p21 detected by western blot. Decreased levels of cyclin E1, cyclin A2, and cyclin B1 in DENSPM-treated cells can explain the prolongation of cell cycle phases that occurred before the G1/S block. We also show that MCF-10A cells rapidly recover from DENSPM-induced growth inhibition in contrast to four human breast cancer cell lines. The goal of cancer treatment is to cause minimal and reversible damage to normal cells, while cancer cells should be eliminated. Altogether, the data show that treatment with polyamine analogs spares normal cells, while negatively affecting the cancer cells.

We have studied in vivo, the effects of physiological androgen (11-ketotestosterone: 11-KT and testosterone: T) concentrations on the growth of cod previtellogenic oocytes and steroidogenic gene expression patterns. Immature female Atlantic cod were injected three times (days 0, 7 and 14) with 0.05, 0.5 and 5 mg/kg of 11-KT and T. The control group was injected with the carrier solvent (ethanol diluted 1:10 in sunflower oil). Quantitative histological analyses demonstrated growth and development of previtellogenic oocytes after exposure to androgens. The oocyte developmental effect of androgens was more pronounced in fish receiving 11-KT. Quantitative PCR analysis demonstrated dose- and androgen-specific modulation of mRNA expression for genes involved in steroidogenesis (StAR (steroidogenic acute regulatory) protein, P450scc (P450-mediated cholesterol side-chain cleavage), 20beta-HSD (20beta-hydroxysteroid dehydrogenase)) and cell growth control, namely--opioid growth factor receptor (OGF-R), progesterone receptor protein p23 (PR23P) and apoptosis-inducing TAF9-like domain 1 (TAF9). Messenger RNA species associated with the zona pelucida, namely--the zona pellucida protein A domain (ZPA) and egg envelope glycoprotein (EeG) were modulated based on dose and androgen type. Cyclin-B mRNA expression was not affected by androgen exposure. Interestingly, we showed recently that these transcripts were responsive to in vitro androgen exposure in previtellogenic cod ovary. In conclusion, the present study adds further information regarding the effects of androgens on the development of previtellogenic oocytes, suggesting androgen control of early oocyte growth in cod. The enhanced effects of 11-KT on oocyte growth support our hypothesis that non-aromatizable androgens play significant roles in the regulation of early previtellogenic oocyte growth and development.

Background: In this study, we investigated the effects of 2-(2-fluorophenyl)-6,7-methylenedioxyquinolin-4-one (CHM-1) on cell viability, cell cycle arrest and apoptosis in CT-26 murine colorectal adenocarcinoma cells., Methods: For determining cell viability, the MTT assay was used. CHM-1 promoted G2/M arrest by PI staining and flow cytometric analysis. Apoptotic cells were evaluated by DAPI staining. We used CDK1 kinase assay, Western blot analysis and caspase activity assays for examining the CDK1 activity and proteins correlated with apoptosis and cell cycle arrest. The in vivo anti-tumor effects of CHM-1-P were evaluated in BALB/c mice inoculated with CT-26 cells orthotopic model., Results: CHM-1 induced CT-26 cell viability inhibition and morphologic changes in a dose-dependent and time-dependent manner and the approximate IC50 was 742.36 nM. CHM-1 induced significant G2/M arrest and apoptosis in CT-26 cells. CHM-1 inhibited the CDK1 activity and decreased CDK1, Cyclin A, Cyclin B protein levels. CHM-1 induced apoptosis in CT-26 cells and promoted increasing of cytosolic cytochrome c, AIF, Bax, BAD, cleavage of pro-caspase-9, and -3. The significant reduction of caspase-9 and -3 activity and increasing the viable CT-26 cells after pretreated with caspase-9 and -3 inhibitor indicated that CHM-1-induced apoptosis was mainly mediated a mitochondria-dependent pathway. CHM-1-P improved mice survival rate, and enlargement of the spleen and liver metastasis were significantly reduced in groups treated with either 10 mg/kg and 30 mg/kg of CHM-1-P and 5-FU in comparison to these of CT-26/BALB/c mice., Conclusions: Taken together, CHM-1 acted against colorectal adenocarcinoma cells in vitro via G2/M arrest and apoptosis, and CHM-1-P inhibited tumor growth in vivo.

We reported previously that oroxylin A, a natural product isolated from Scutellariae Radix, was a potent apoptosis inducer of human hepatoma HepG2 cells. In this study, cell-cycle arrest of BGC-823 human gastric carcinoma cells caused by oroxylin A has been investigated. Based on our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of oroxylin A caused an irreversible arrest in the G2/M phase of the cell cycle. Western blot analysis demonstrated that oroxylin A-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in cdc2/p34, cyclin B1 and cyclin A expression. In addition, oroxylin A-treated cells decreased the expression of Cdk7, which was responsible for the low expression of M phase promoting factor (cyclin B1/Cdc2). The results suggested that oroxylin A induced G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells.

In mammalian cells, the H2AX histone is rapidly phosphorylated upon the induction of DNA double strand breaks and promotes their repair, which is required for preserving genomic integrity. Etoposide is an inhibitor of DNA topoisomerase II, which causes DNA breaks and induces H2AX phosphorylation. To elucidate whether H2AX may affect cellular sensitivity to etoposide, we studied the response to this agent in immortalized embryonic fibroblasts derived from H2AX knockout mice. Clonogenic assays in cells treated with the drug revealed a greater sensitivity of H2AX null cells compared to wild-type cells, possibly due to the persistence of a higher number of DNA breaks, as detected with the comet assay. In both cell lines, etoposide induced micronuclei formation and nuclear fragmentation; however, in H2AX deficient cells nuclear fragmentation was observed at a lower drug concentration. Flow cytometric analysis showed that etoposide induced a G2/M cell cycle arrest in both cell lines, which occurred at lower drug concentrations in H2AX deficient cells. G2/M arrest was paralleled by an accumulation of cyclin A and cyclin B1, suggesting that treated cells are not able to complete cell cycle correctly and undergo cell death. Taken together, our observations suggest that H2AX takes part to the cellular response to etoposide and confirm its role in the maintenance of genome stability.

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimer's disease. In this study, we report that the expressions of Swedish double mutation of amyloid precursor protein (Swe-APP) or of the APP intracellular domain (AICD) into nerve growth factor (NGF)-differentiated PC12 cells or rat primary cortical neurons increased mRNA and protein levels of cyclin D1 and cyclin B1. Treatment with lithium chloride (a glycogen synthase kinase-3beta inhibitor) down-regulated cyclin B1 induced by Swe-APP expression but up-regulated cyclin D1 expression induced by Swe-APP, suggesting that glycogen synthase kinase-3beta activity is involved in these expression changes of cyclins D1 and B1. Swe-APP, which is a prevailing cause of familial Alzheimer's disease, is well known to increase amyloid beta peptide production both in vitro and in vivo, but the underlying molecular means whereby it leads to the pathogenesis of AD remains unknown. The finding that cyclin D1 and B1 expressions were up-regulated by Swe-APP in in vitro cultured cells was substantiated in the brain tissues of Tg2576 mice, which harbor the Swe-APP mutation. These results suggest that some disturbances in cell cycle regulation may be involved in Swe-APP or AICD-induced neurodegeneration and that these contribute to the pathogenesis of AD.

Lithium, a therapeutic agent for bipolar disorder, can induce G2/M arrest in various cells, but the mechanism is unclear. In this article, we demonstrated that lithium arrested hepatocellular carcinoma cell SMMC-7721 at G2/M checkpoint by inducing the phosphorylation of cdc2 (Tyr-15). This effect was p53 independent and not concerned with the inhibition of glycogen synthase kinase-3 and inositol monophosphatase, two well-documented targets of lithium. Checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2/M arrest, was at least partially responsible for the lithium action. The lithium-induced phosphorylation of cdc2 and G2/M arrest was abrogated largely by SB218078, a potent Chk1 inhibitor, as well as by Chk1 siRNA or the over-expression of kinase dead Chk1. Furthermore, lithium-induced cdc25C phosphorylation in 7721 cells and in vitro kinase assay showed that the activity of Chk1 was enhanced after lithium treatment. Interestingly, the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) kinase. This is because no elevated phosphorylation on Chk1 (Ser-317 and Ser-345) was observed after lithium treatment. Moreover, caffeine, a known ATM/ATR kinase inhibitor, relieved the phosphorylation of cdc2 (Tyr-15) by hydroxyurea, but not that by lithium. Our study's results revealed the role of Chk1 in lithium-induced G2/M arrest. Given that Chk1 has been proposed to be a novel tumor suppressor, we suggest that the effect of lithium on Chk1 and cell cycle is useful in tumor prevention and therapy., (2008 Wiley-Liss, Inc.)

Diallyl disulfide (DADS), a major organosulfur compound of garlic oil, is known to have an anticancer effect on human cancer cells. However, the exact mechanisms of this anticancer activity remain unclear. Here, we investigate the effects of DADS on cell cycle progression in human colon cancer HCT-116 cells by exploring the role played by regulatory molecules such as p53 and cyclin B1. Treatment of HCT-116 cells by DADS induced a marked growth inhibition with a slight reduction in viability and induced transient cell cycle arrest in the G2/M phase. Cyclin B1 is thought to play an important role in this process, as the DADS-induced G2/M phase arrest occurs with the increase of cyclin B1 expression. DADS also significantly induced the expression of p53, which contributes to cell cycle arrest in cancer cells, at a late time-point of 24 h. In addition, knockdown of p53 by siRNA did not affect cell cycle arrest, its reversibility, or the expression of cyclin B1 in the G2/M phase induced by DADS. Based on these results we conclude that, with the dynamic expression of cyclin B1, DADS induces reversible cell cycle arrest in the G2/M phase of HCT-116 cells through a p53-independent mechanism.

The steroidogenic acute regulatory (StAR) protein and cytochrome P450-mediated cholesterol side-chain cleavage (P450scc) have been localized in most steroidogenic organs and are rapidly synthesized in response to acute tropic hormone stimulation. In this study, we present the development of cod previtellogenic oocyte in vitro culture system, histological and molecular methods for evaluating the effects of endocrine disruptors such as nonylphenol (NP) on steroid hormone levels, the StAR protein and P450scc. In addition, expression pattern of cyclin-B was studied, because of cyclin B's role as an indicator of oocyte growth in fish. The in vitro previtellogenic oocyte culture technique was based on an agarose floating method. Tissue was cultured in a humidified incubator at 10 degrees C for 4, 7, 14 and 21 d with different concentrations of nonylphenol (0 (control), 1, 10, 50 and 100 microM) dissolved in ethanol (0.3%). Gene expressions were detected using validated real-time polymerase chain reaction (PCR) with specific primers. Immunohistochemistry of the StAR protein and P450scc were performed using antisera prepared against synthetic peptide for both proteins. Estradiol-17beta (E2) and 11-ketotestosterone (11-KT) tissue levels were estimated using enzyme immunoassay. Our data show that nonylphenol produced a unique and consistent concentration-specific pattern of modulation for the StAR protein, P450scc and cyclin-B gene expression at day 14 after exposure. This pattern is generally described as increasing from 0 (control) to 1 and 10 microM, and decreased at 50 and 100 microM. The observed changes in the StAR protein, P450scc and cyclin-B levels showed a direct relationship with changes in 11-KT levels at day 14 after exposure. Cellular localization of StAR and P450scc were specific to the follicular cells of previtellogenic oocytes, but with no differences in staining intensities. No significant change in oocyte diameter was observed between the exposure groups. Our data reveal some novel aspects of nonylphenol effects on maturation and oocyte growth in teleosts, suggesting impaired steroidogenesis and hormonal imbalance with potential consequences for the vitellogenic process and overt fecundity.

Agaricus blazei is well known as a traditional medicinal mushroom and it has been shown to exhibit immunostimulatory and anti-cancer activity. However, the cellular and molecular mechanism of cell cycle arrest and apoptosis of cancer cells is poorly understood. In this study, we have investigated whether A. blazei extract (ABE) exerts anti-proliferative and apoptotic effects on human gastric epithelial AGS cells. It was found that ABE could inhibit cell growth in a dose-dependent manner, which was associated with the arrest of G2/M phase and the induction of apoptotic cell death. Flow cytometric analysis showed that ABE could cause an arrest at the G2/M phase of the cell cycle, which is closely correlated to decreased cyclin B1 and cdc2 levels. Furthermore, this compound induced apoptosis through up-regulation of Bax and the activation of caspases with down-regulation of XIAP and cIAP-1, but not cIAP-2, and a capase-3 inhibitor could block cell death and apoptotic body formation. These data clearly indicate that ABE-induced apoptosis is associated with caspase-3 activation. In summary, the growth inhibition of ABE is highly related to cell cycle arrest at the G2/M phase and the induction of caspase-3-dependent apoptosis in human gastric epithelial AGS cells.

Microtubules are crucial targets for cancer chemotherapeutic drugs, and new microtubule-directed agents are of continued interest in drug development. A novel microtubule-directed agent, ethyl-2-[N-rho-chlorobenzyl-(2'-methoxy)]-anilino-4-oxo -4, 5-dihydro-furan-3-carboxylate, was identified. The compound, designated K2154, inhibited cell proliferation, with IC(50) values of 10.3, 15.3, 9.6, 11.2, 12.8 and 12.1 muM in prostate cancer PC-3, hepatocellular carcinoma Hep3B, non-small cell lung cancer A549, colorectal cancer HT29 and HCT116, and P-glycoprotein-rich breast cancer NCI/ADR-RES cells, respectively. Because NCI/ADR-RES cells were susceptible to inhibition by K2154, it indicated that this compound is a poor substrate for P-glycoprotein. In this study, PC-3 cells were used to identify the anticancer mechanisms of K2154. K2154 induced an arrest of the cell cycle at G2/M phase and a subsequent increase of hypodiploid phase in PC-3 cells, whereas it only induced a moderate level of G2/M arrest with little increase of hypodiploid phase in normal prostate cells. K2154 inhibited microtubule assembly in both in vitro turbidity assay and in vivo microtubule spin-down experiment. Immunochemical examination showed that K2154 caused formation of abnormal mitotic characteristics with bipolar spindles, particularly, in beta(II)- and beta(III)-tubulin staining. It also induced several pathways, including cyclin B1 up-regulation, dephosphorylation on Tyr(15) and phosphorylation on Thr(161) of Cdk1 and Cdc25C phosphorylation, and roscovitine (a Cdk1 inhibitor) significantly inhibited K2154-induced apoptosis, suggesting a pro-apoptotic role of Cdk1. Phosphorylation of Bcl-2 and Bcl-xL and cleavage of Mcl-1, together with activation of caspase-9 and -3, indicated that mitochondrial pathway played a central role in K2154-mediated apoptotic cell death. Additionally, AIF contributed to a late phase of K2154-induced apoptotic pathway. In conclusion, it is suggested that K2154 displays an anticancer activity through a target on microtubules and a subsequent signaling cascade on cell cycle regulation and apoptotic machinery.

A unique in vitro system has been developed in our lab that consists of normal enterocytes derived from the rat ileum (IEC-18 cells) and their transformed derivatives with c-K-ras (R1 cells), anti-sense bak (B3 cells) and cyclin D1 (D1 cells). R1 and B3 cells express high level of COX-2 protein and PGE2. IEC 18 and D1 cells express negligible amount of COX-2, and produce very low level of PGE2. A relatively low dose of celecoxib (5-10 microM) induced G2/M arrest, followed by induction of apoptosis in the transformed but not in the normal cells. Down-regulation of cyclin B1 and up-regulation of p21 expressions independent of p53 might have cause this cell cycle block. Growth inhibition was related to COX-2 function with 90-95% reduction in PGE2 production. These findings may be of clinical importance, since low concentration of celecoxib can be achieved in human serum following standard anti-inflammatory (100-200 mg bid) regime.

Bmyb is a ubiquitously expressed transcription factor involved in cellular proliferation and cancer. Loss of bmyb function in the zebrafish mutant crash&burn (crb) results in decreased cyclin B1 expression, mitotic arrest and genome instability. These phenotypic observations in crb mutants could be attributed to the decreased expression of cyclin B1, a cell-cycle regulatory protein that is responsible for driving cell progression from G2 through mitosis. To identify small molecules that interact with the bmyb pathway, we developed an embryo-based suppressor screening strategy. In 16 weeks we screened a diverse approximately 16,000 compound library, and discovered one previously unknown compound, persynthamide (psy, 1), that suppressed bmyb-dependent mitotic defects. Psy-treated embryos showed an S-phase delay, and knockdown of the cell-cycle checkpoint regulator ataxia telangiectasia--and Rad-related kinase (ATR) abrogated the suppression of crb. The DNA synthesis inhibitors aphidicolin (2) and hydroxyurea (3) also suppressed crb. S-phase inhibition upregulated cyclin B1 mRNA, promoting the progression of cells through mitosis. Our study demonstrates that chemical suppressor screening in zebrafish can identify compounds with cell-cycle activity and can be used to identify pathways that interact with specific cell-cycle phenotypes.

Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass G1 but completely arrest in G2. p21CIP1 and p27KIP1 inhibit Cyclin A-Cdk2 activity and sequester Cyclin B1-Cdk1 in inactive complexes in the nucleus. This response is alleviated by mitogen restimulation or inactivation of p53. Thus, our results disclose a cell cycle arrest mechanism in G2 that restricts the proliferative capacity of mitogen-deprived cells that have lost the G1 restriction point. The involvement of p53 provides a rationale for the synergism between loss of Rb and p53 in tumorigenesis.

Selenomethionine (SeMet) is being tested alone and in combination with other agents in cancer chemoprevention trials. However, the molecular targets and the signaling mechanism underlying the anticancer effect of this compound are not completely clear. Here, we provide evidence that SeMet can induce cell-growth arrest and that the growth inhibition is associated with S-G2/M cell-cycle arrest. Coincidentally with the cell-cycle arrest, we observed a striking increase in cyclin B as well as phosphorylation of the cyclin-dependent kinase Cdc2. Since activation of the mitogen-activated protein kinase (MAPK) cascade has been associated with cell-cycle arrest and growth inhibition, we evaluated the activation of extracellular signal-regulated kinase (ERK). We found that SeMet induced phosphorylation of the MAPK ERK in a dose-dependent manner. We also demonstrate phosphorylation of ribosomal S6 kinase (p90RSK) by SeMet. Additionally, we show phosphorylation of histone H3 in a concentration-dependent manner. Furthermore, the phosphorylation of p90RSK and histone H3 were both antagonized by the MEK inhibitor U0126, implying that SeMet-induced phosphorylation of p90RSK and histone H3 are at least in part ERK pathway dependent. Based on these results, we propose that SeMet induced growth arrest and phosphorylation of histone H3 are mediated by persistent ERK and p90RSK activation. These new data provide valuable insights into the biological effects of SeMet at clinically relevant concentrations.

8-Chloro-cyclic AMP (8-Cl-cAMP) is known to be most effective in inducing growth inhibition and differentiation of a number of cancer cells. Also, its cellular metabolite, 8-Cl-adenosine was shown to induce growth inhibition in a variety of cell lines. However, the signaling mechanism that governs the effects of 8-Cl-cAMP and/or 8-Cl-adenosine is still uncertain and it is not even sure which of the two is the key molecule that induces growth inhibition. In this study using mouse fibroblast DT cells, it was found that adenosine kinase inhibitor and adenosine deaminase could reverse cellular growth inhibition induced by 8-Cl-cAMP and 8-Cl-adenosine. And 8-Cl-cAMP could not induce growth inhibition in the presence of phosphodiesterase (PDE) inhibitor, but 8-Cl-adenosine could. We also found that protein kinase C (PKC) inhibitor could restore this growth inhibition, and both the 8-Cl-cAMP and 8-Cl-adenosine could activate the enzymatic activity of PKC. Besides, after 8-Cl-cAMP and 8-Cl-adenosine treatment, cyclin B was down-regulated and a CDK inhibitor, p27 was up-regulated in a time-dependent manner. These results suggest that it is not 8-Cl-cAMP but 8-Cl-adenosine which induces growth inhibition, and 8-Cl-cAMP must be metabolized to exert this effect. Furthermore, there might exist signaling cascade such as PKC activation and cyclin B down-regulation after 8-Cl-cAMP and 8-Cl-adenosine treatment., (Copyright 2004 Wiley-Liss, Inc.)

Jasmonic acid (JA) plays a crucial role in plant fertility and defense responses. It exerts an inhibitory effect on plant growth when applied exogenously. This effect seems to be somehow related to a negative regulation of cell cycle progression in the meristematic tissues. In this report, we focus on the molecular events that occur during JA-induced G2 arrest. We demonstrate that JA prevents the accumulation of B-type cyclin-dependent kinases and the expression of cyclin B1;1, which are both essential for the initiation of mitosis. This feature suggests the existence of an early G2 checkpoint that is affected by JA.

Vertebrate oocytes proceed through meiosis I before undergoing a cytostatic factor (CSF)-mediated arrest at metaphase of meiosis II. Exit from MII arrest is stimulated by a sperm-induced increase in intracellular Ca2+. This increase in Ca2+ results in the destruction of cyclin B1, the regulatory subunit of cdk1 that leads to inactivation of maturation promoting factor (MPF) and egg activation. Progression through meiosis I also involves cyclin B1 destruction, but it is not known whether Ca2+ can activate the destruction machinery during MI. We have investigated Ca2+ -induced cyclin destruction in MI and MII by using a cyclin B1-GFP fusion protein and measurement of intracellular Ca2+. We find no evidence for a role for Ca2+ in MI since oocytes progress through MI in the absence of detectable Ca2+ transients. Furthermore, Ca2+ increases induced by photorelease of InsP3 stimulate a persistent destruction of cyclin B1-GFP in MII but not MI stage oocytes. In addition to a steady decrease in cyclin B1-GFP fluorescence, the increase in Ca2+ stimulated a transient decrease in fluorescence in both MI and MII stage oocytes. Similar transient decreases in fluorescence imposed on a more persistent fluorescence decrease were detected in cyclin-GFP-injected eggs undergoing fertilization-induced Ca2+ oscillations. The transient decreases in fluorescence were not a result of cyclin B1 destruction since transients persisted in the presence of a proteasome inhibitor and were detected in controls injected with eGFP and in untreated oocytes. We conclude that increases in cytosolic Ca2+ induce transient changes in autofluorescence and that the pattern of cyclin B1 degradation at fertilization is not stepwise but exponential. Furthermore, this Ca2+ -induced increase in degradation of cyclin B1 requires factors specific to mature oocytes, and that to overcome arrest at MII, Ca2+ acts to release the CSF-mediated brake on cyclin B1 destruction.

Cyclin B1 is the regulatory subunit of M-phase promoting factor, and proper regulation of cyclin B1 is essential for the initiation of mitosis. Increasing evidence indicates that the deregulation of cyclin B1 is involved in neoplastic transformation, suggesting the suppression of cyclin B1 could be an attractive strategy for antiproliferative therapy. In the present work, we analysed the impact of small interfering RNAs (siRNAs) targeted to cyclin B1 on different human tumor cell lines. Cyclin B1 siRNAs reduced the protein level of cyclin B1 in HeLa, MCF-7, BT-474 and MDA-MB-435 tumor cells and efficiently reduced the kinase activity of Cdc2/cyclin B1 in HeLa cells. siRNA-treated cells were arrested in G2/M phase in all tumor cell lines tested. Proliferation of tumor cells from different origins was suppressed by 50-80% 48 h after transfection and apoptosis was increased from 5 to 40-50%. Furthermore, tumor cells showed less colony-forming ability after siRNA treatment. In contrast, primary human umbilical vein endothelial cells exhibited only a slight change in cell cycle, and neither apoptosis nor clear inhibition of proliferation was observed after cyclin B1 siRNA treatment for 48 h. These results indicate that siRNAs against cyclin B1 could become a powerful antiproliferative tool in future antitumor therapy.

It has previously been demonstrated that gingival fibroblasts derived from nifedipine-reactive patients (nifedipine responders) show a greater cell proliferation rate than those from nifedipine non-reactive patients (nifedipine non-responders) in the presence of 1 microM nifedipine. The aim of the present study was to characterize cell cycle differences between nifedipine responder and non-responder fibroblast cells and determine the effect of basic fibroblast growth factor (bFGF) on cell cycle progression. Further, the effect of bFGF on cyclins A, B1, D1, E, and CDKs 1, 2, 4, 6 mRNA expression in responder and non-responder cells was investigated. A population of nifedipine responder cells underwent progression to S and G2/M phases from G0/G1 phase in the presence of 10% fetal calf serum or 10 ng/ml bFGF was greater than nifedipine non-responder cells. mRNA expression of cyclins A, B1, D1, E and CDKs 1, 2, 4, 6 in the presence of 10 ng/ml bFGF was generally greater in nifedipine responder cells than non-responder cells. These results indicate that nifedipine responder cells may be more susceptible to growth factors such as bFGF with a resultant increase in expression of cyclins and CDKs in responder compared with non-responder cells.

The cell-transforming potential of 1,2-dibromoethane and folpet, two widely used agricultural pesticides that are potential sources of environmental pollution, has been previously ascribed to their promoting activity. In this study, we investigated whether BALB/c 3T3 transformation by these chemicals was associated with the deregulation of signals involved in cell-cycle progression and in cell-cycle checkpoint induction. We found that two BALB/c 3T3 cell clones transformed by in vitro medium-term (8-week) exposure to the carcinogens had a constitutive acceleration of cell transition from G(1) to S phase and an abrogation of the radiation-induced G(1)/S checkpoint. These events involved multiple signals; in particular, the inhibitors of cyclin/cyclin-dependent kinase complexes p21 and p27 were significantly down-modulated and the positive regulators of cell-cycle progression cyclin D(3) and E were up-modulated. As anticipated for cells where the G(1)/S checkpoint was abrogated, the transformed cells exhibited a significant reinforcement of the radiation-induced G(2)/M checkpoint, the only checkpoint remaining to protect genomic integrity. However, cyclin A(1) and B(1) coexpression and cyclin A(1) overexpression were found despite the G2 arrest in irradiated cells and these signals likely attenuate the G(2)/M checkpoint. These alterations to normal cell cycling may promote the emergence of both numerical and structural chromosomal abnormalities and their tolerance. Such a condition could play a key role in neoplastic transformation and be crucial in tumor progression. Furthermore, cyclin A(1) overexpression may play an autonomous role in the neoplastic transformation of BALB/c 3T3 cells, as it does in other cell types of mesenchymal origin., (Copyright 2003 Wiley-Liss, Inc.)

Formation of an active nuclear cyclin B1-CDK1 complex is a highly intricate procedure requiring many different levels of regulation. Each of these regulatory steps represents a potential target for controlling cell proliferation. Accumulation of threshold levels of cyclin B1 protein at the G2/M transition requires the cooperation of various promoter elements, possibly the activation of several transcription factors, enhanced cyclin B1 mRNA stability and, in some cases, translational activation of dormant mRNA. Binding of cyclin B1 to its inactive partner, CDK1, initiates conformational changes allowing CDK1 to alter its phosphorylation status and to become an active kinase. Lastly, the active cyclin B1-CDK1 complex must translocate to the nucleus to begin phosphorylating nuclear substrates. These phosphorylation events are necessary for mitotic onset. While cyclin B1 is capable of shuttling from the nucleus to the cytoplasm throughout interphase, mitotic onset requires phosphorylation of cyclin B1 within the CRS region, thereby enhancing import and inhibiting export of the cyclin B1-CDK1 complex. Elucidating the role of mediators controlling cyclin B1-CDK1 translocation at the onset of mitosis is essential in developing drug targets for cell cycle control.

The short-chain fatty acid (SCFA) butyrate is produced via anaerobic bacterial fermentation within the colon and is thought to be protective in regard to colon carcinogenesis. Although butyrate (C4) is considered the most potent of the SCFA, a variety of other SCFA also exist in the colonic lumen. Butyrate is thought to exert its cellular effects through the induction of histone hyperacetylation. We sought to determine the effects of a variety of the SCFA on colon carcinoma cell growth, differentiation and apoptosis. HT-29 or HCT-116 (wild-type and p21-deleted) cells were treated with physiologically relevant concentrations of various SCFA, and histone acetylation state was assayed by acid-urea-triton-X gel electrophoresis and immunoblotting. Growth and apoptotic effects were studied by flow cytometry, and differentiation effects were assessed using transient transfections and Northern blotting. Propionate (C3) and valerate (C5) caused growth arrest and differentiation in human colon carcinoma cells. The magnitude of their effects was associated with a lesser degree of histone hyperacetylation compared with butyrate. Acetate (C2) and caproate (C6), in contrast, did not cause histone hyperacetylation and also had no appreciable effects on cell growth or differentiation. SCFA-induced transactivation of the differentiation marker gene, intestinal alkaline phosphatase (IAP), was blocked by histone deacetylase (HDAC), further supporting the critical link between SCFA and histones. Butyrate also significantly increased apoptosis, whereas the other SCFA studied did not. The growth arrest induced by the SCFA was characterized by an increase in the expression of the p21 cell-cycle inhibitor and down-regulation of cyclin B1 (CB1). In p21-deleted HCT-116 colon cancer cells, the SCFA did not alter the rate of proliferation. These data suggest that the antiproliferative, apoptotic and differentiating properties of the various SCFA are linked to the degree of induced histone hyperacetylation. Furthermore, SCFA-mediated growth arrest in colon carcinoma cells requires the p21 gene.

Extravillous cytotrophoblasts are specialised epithelial cells of the placenta that proliferate or invade the maternal decidua. Little is known about the mechanisms that regulate these processes. Here the effects of several insulin and insulin-like growth factor-I (IGF-I) doses, either singly or in synergy with serum, on human chorionic gonadotropin-beta (hCG-beta) secretion (RIA), proliferation (cell counting, cyclin B(1) levels) and invasion [Matrigel invasion assay, secretion of matrix metalloproteinases (MMP) 2 and 9] were investigated. The choriocarcinoma cell lines BeWo, JAR and JEG-3 served as models for first trimester human trophoblasts. Both growth factors altered hCG-beta secretion and proliferation dependent on the cell line. Insulin stimulated proliferation in JAR cells and, to a lesser extent, in JEG-3 cells, and when cultured in serum-free medium, BeWo was not affected. Invasion was not affected although proMMP-2 levels in culture medium were altered under some conditions. A strong synergistic effect with serum was noted. In the presence of serum both growth factors reduced proliferation and invasion in a similar fashion. Since the cell models differ by their degree of differentiation, the data demonstrate that the effects of insulin and IGF-I strongly depend on serum and the degree of differentiation. It can be speculated that IGF-I can take on tasks of insulin in the regulation of trophoblast functions under conditions of insulinopenia.

PRL is essential for normal lobulo-alveolar growth of the mammary gland and may contribute to mammary cancer development or progression. However, analysis of the mechanism of action of PRL in these processes is complicated by the production of PRL within mammary epithelia. To examine PRL actions in a mammary cell-specific context, we selected MCF-7 cells that lacked endogenous PRL synthesis, using PRL stimulation of interferon-gamma-activated sequence-related PRL response elements. Derived clones exhibited a greater proliferative response to PRL than control cells. To understand the mechanism, we examined, by Western analysis, levels of proteins essential for cell cycle progression as well as phosphorylation of retinoblastoma protein. The expression of cyclin D1, a critical regulator of the G1/S transition, was significantly increased by PRL and was associated with hyperphosphorylation of retinoblastoma protein at Ser(780). Cyclin B1 was also increased by PRL. In contrast, PRL decreased the Cip/Kip family inhibitor, p21, but not p16 or p27. These studies demonstrate that PRL can stimulate the cell cycle in mammary epithelia and identify specific targets in this process. This model system will enable further molecular dissection of the pathways involved in PRL-induced proliferation, increasing our understanding of this hormone and its interactions with other factors in normal and pathogenic processes.

KARs, new semisynthetic antitumor bis-indole derivatives, were found to be inhibitors of tubulin polymerization with lower toxicity than vinblastine or vincristine, used in chemotherapy. Here, we compare the effect of KARs with those of vinblastine and vincristine on cell viability, cell proliferation, and cell cycle in neuroblastoma cell line (SH-SY5Y). At concentrations of the different compounds equivalent in causing 50% of inhibition of cell growth, KARs induced a complete arrest in the G2/M phase, whereas vinblastine and vincristine induced a partial arrest in both G0/G1 and G2/M. Moreover, a combination of KAR-2 and W13 (an anticalmodulin drug) qualitatively caused a similar arrest in both G0/G1 and G2/M than vinblastine. Levels of cyclin A and B1 were higher in KARs-treated cells than in vinblastine- or vincristine-treated cells. Cdc2 activity was much higher in KAR-2 than in vinblastine-treated cells, indicating a stronger mitotic arrest. The effect of KAR2 and vinblastine on microtubules network was analyzed by immunostaining with anti-tubulin antibody. Results indicated that KAR-2-induces the formation of aberrant mitotic spindles, with not apparent effect on interphase microtubules, whereas vinblastine partially destroyed interphase microtubules coexisting with normal and aberrant mitotic spindles.

The cytotoxic activity of the imidazoacridinone C1311 was assessed on two ovarian cancer cell lines (A2780, OAW42) and one osteogenic sarcoma cell line (U2-OS) and their sublines (A2780Cp8, OAW42-MER and U2-OS-R) with experimentally induced resistance to cisplatin. A 1-h exposure to C1311 significantly inhibited the growth of all cell lines, with IC50 values ranging from 0.50 +/-0.11 to 4.10+/-0.36 microM. No or only partial cross-resistance was found between C1311 and cisplatin in the different cell lines. Treatment with equitoxic (IC50) C1311 concentrations consistently induced accumulation of cells in the G2M phase. The cyclin B1-associated p34(cdc2) kinase activity in cells arrested in G2M was superimposable to that of control cells in the OAW42-MER and U2-OS cell lines, whereas a reduction of cdc2 catalytic activity was observed in OAW42 and U2-OS-R cells. Exposure to C1311 (IC50) induced apoptosis in the U2-OS and U2-OS-R cell lines, whereas in the OAW42 and OAW42-MER cell lines there was a negligible percentage of apoptotic cells. In U2-OS, U2-OS-R and OAW42 cells, C1311 induced an increase in p53 expression and an increase in p21waf1 protein, whereas p53 failed to transactivate p21waf1 in OAW42-MER cells. An almost complete abrogation of bcl-2 was observed in U2-OS-R cells in correspondence with the peak of apoptosis induction. Our results indicate that C1311 is active against human ovarian cancer and osteogenic sarcoma cells and is not cross-resistant with CDDP. Moreover, C1311 blocks cells in the G2M phase and induces apoptosis in a small percentage of osteogenic sarcoma cells.

Background: Proliferation of synovial cells is considered to play a key role in rheumatoid arthritis (RA). Using paclitaxel, a unique antineoplastic agent known to suppress collagen-induced arthritis, we conducted an in vitro study of cell kinetics on cultured synovial cells from patients with RA., Methods: Alterations of the cell cycle of cultured fibroblast-like synovial cells (FLSs) from patients with RA were studied using flow cytometry and laser scanning cytometry. Apoptosis and accumulation of cyclin concerning effects of paclitaxel were detected., Results: Paclitaxel induced arrest of the cell cycle at G2/M phase and apoptosis in FLSs. The late stage of apoptosis was determined by the positivity of terminal deoxynucleotidyl transferase assay. Morphological observation by combined usage of both annexin V and propidium iodide on FLSs on a slide glass showed early apoptotic changes in detail. FLSs arrested at G2/M phase showed marked accumulation of cyclin B1. The effects of paclitaxel decreased on FLSs, which diminished proliferative activity., Conclusions: These data indicate that paclitaxel induces cell arrest at G2/M phase followed by apoptosis in human FLSs, which have high proliferative activity, and possible therapeutic effects of paclitaxel on RA., (Copyright 2001 Wiley-Liss, Inc.)

Human immunodeficiency virus (HIV)-infection is characterized by loss of CD4+ T cells associated with high levels of immune activation, T-cell proliferation, and lymphocyte apoptosis. To investigate the role of intrinsic perturbations of cell-cycle control in the immunopathogenesis of acquired immunodeficiency syndrome (AIDS), we studied the expression of cell-cycle-dependent proteins in lymphocytes from HIV-infected patients. Cyclin B1 expression, Nucleolar Organizer Regions (NORs) number, and NORs area of distribution were all consistently increased in HIV-infected patients, but returned to normal after effective antiretroviral therapy, suggesting that viral replication is directly implicated in the genesis of the observed changes. Analysis of cyclin B1 intracellular turnover showed that the increased cyclin B1 expression is (1) caused by defective degradation in the presence of normal rates of synthesis, and (2) is temporally associated with decreased levels of ubiquitination. After in vitro activation of lymphocytes from healthy individuals, cyclin B1 and cdc25 expression and ubiquitination, p34 cdc2 activity, NORs morphology, and C23/nucleolin localization showed a 72- to 96-hour cyclic pattern that led to a biologic state similar to baseline. On the contrary, complex but consistent changes of the same indices followed activation of T lymphocytes from HIV-infected patients, resulting in a 5-fold increase in apoptosis. Overall, our data indicate that a profound dysregulation of cell-cycle control is present in lymphocytes from HIV-infected patients. This finding may provide a novel biologic link between immune activation, accelerated lymphocyte turnover, and increased apoptosis during HIV infection.

Combination of selecting agents that act on different cellular mechanisms is a common strategy in cancer chemotherapy. GL331 is a new potent topoisomerase II (Topo II) poison; distinctly, paclitaxel is a microtubule-interfering cancer chemotherapeutic agent. In this study, we intended to evaluate the efficacy of combining GL331 with paclitaxel in cell killing and apoptotic induction in nasopharyngeal carcinoma NPC-TW01 cells. By MTT and internucleosomal DNA cleavage assays, we found that pretreatment or simultaneous treatment of NPC-TW01 cells with GL331 could significantly interfere with paclitaxel's cell killing and apoptosis-inducing activity. When the administration schedule was reversed, the cytotoxicity of GL331 was attenuated by paclitaxel pretreatment. The anti-cancer activity produced by combining GL331 with paclitaxel was obviously lower than the addition of the activities of two individual agents. NPC-TW01 cells were treated with GL331 and 3H-labeled paclitaxel simultaneously or with GL331 before 3H-labeled paclitaxel. In both conditions, GL331 did not reduce the [3H]paclitaxel level in the cells, suggesting that GL331's interference with paclitaxel's cell-killing and apoptosis-inducing efficacy did not result from any inhibition of cellular uptake or retention of paclitaxel. In addition, we found that GL331-induced perturbation of cell cycle progression dramatically over-rode the patterns of mitotic arrest induced by paclitaxel, and the mechanism could be the inhibition of cyclin B1/CDC2 kinase and MAD2 checkprotein activities.

Diversity-oriented organic synthesis offers the promise of advancing chemical genetics, where small molecules are used to explore biology. While the split--pool synthetic method is theoretically the most effective approach for the production of large collections of small molecules, it has not been widely adopted due to numerous technical and analytical hurdles. We have developed a split--pool synthesis leading to an array of stock solutions of single 1,3-dioxanes. The quantities of compounds are sufficient for hundreds of phenotypic and protein-binding assays. The average concentration of these stock solutions derived from a single synthesis bead was determined to be 5.4 mM in 5 microL of DMSO. A mass spectrometric strategy to identify the structure of molecules from a split--pool synthesis was shown to be highly accurate. Individual members of the 1,3-dioxane library have activity in a variety of phenotypic and protein-binding assays. The procedure developed in this study allows many assays to be performed with compounds derived from individual synthesis beads. The synthetic compounds identified in these assays should serve as useful probes of cellular and organismal processes.

The bisindole indirubin has been described, more than 30 years ago, as being clinically active in the treatment of human chronic myelocytic leukaemia. However, the underlying mechanism of action has remained unclear. We have reported previously that indirubin and its analogues are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, we investigated the influence of indirubin and derivatives on CDK1/cyclin B kinase in human tumour cells at concentrations known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G(1)/G(0)phase of the cell cycle in the presence of 2 microM indirubin-3'-monoxime. At higher drug concentrations (> or = 5 microM) an increase of the cell population in the G(2)/M phase is additionally observed. Cells synchronized in G(2)/M phase by nocodazole remain arrested in the G(2)/M phase after release, in the presence of indirubin-3'-monoxime (> or =5 microM). After 24 h treatment with 10 microM indirubin-3'-monoxime a sub-G(2)peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concentrations of indirubin-3'-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a reduction of cyclin B in complex with CDK1 is observed. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumour cells is a major mechanism by which indirubin derivatives exert their potent antitumour efficacy., (Copyright 2001 Cancer Research Campaign.)

The deoxyadenosine-resistant mouse leukemia L1210 cell line (Y8) has previously been shown to be more sensitive to apoptosis induced by DNA damaging agents and by protein synthesis inhibitors than the parental wild-type L1210 (WT) cells. These responses occur independently of p53 as both cell lines lack wild-type p53 function. Recent evidence suggests that a serine/threonine kinase is involved in the divergent cellular responses of the WT and Y8 cells. In the present study, the effects of 7-hydroxystaurosporine (UCN-01), a relatively specific serine/threonine kinase inhibitor, were examined in the WT and Y8 cells. Both cell lines were equally sensitive to the growth inhibitory effects of UCN-01. However, the Y8 cells accumulated in G0/G1 and became apoptotic. Apoptosis induced by UCN-01 in the Y8 cells was mediated by a caspase-3-like activity which could be partially blocked by Ac-DEVD-CHO, a caspase-3 inhibitor. UCN-01 did not alter the phosphorylation status of cdc2 nor cyclin B1 and cdc2 protein levels in either cell line.

To demonstrate secretion of cumulus expansion-enabling factor (CEEF) by porcine oocytes, we used an interspecies testing system. Porcine oocytes were used to condition culture medium, and the presence of CEEF was tested using mouse oocytectomized complexes (OOX), which require CEEF for expansion. Follicle-stimulating hormone-stimulated expansion and synthesis of hyaluronic acid (HA) by mouse OOX were assessed after 18 h of culture in media conditioned by porcine oocytes: 1) at different stages of maturation and 2) in which maturation was inhibited with a specific inhibitor of cdk-kinases, butyrolactone I. Fully grown (GV-germinal vesicle), late-diakinesis (LD), metaphase I (MI), and metaphase II (MII) oocytes were prepared by culture of oocyte-cumulus complexes (OCC) for 0, 22, 27, and 42 h, respectively. To block GV breakdown, porcine oocytes were cultured for 27 h in medium supplemented with butyrolactone I (50 microM). Medium conditioned by oocytes in GV, LD, and after butyrolactone I block allowed full expansion of >90% of mouse OOX, whereas oocytes in MI and MII caused disintegration of mouse OOX without cumulus mucification. To measure synthesis of HA by cumulus cells, 25 mouse OOX were cultured in the conditioned media in the presence of 2.5 microCi of D-[6-(3)H]glucosamine hydrochloride. After 18 h, incorporation of the [(3)H]glucosamine into HA was determined either in complexes (retained HA) or in medium plus complexes (total HA). Total HA accumulation by mouse OOX was not different from that of intact OCC. However, oocytes in GV, LD, and after butyrolactone I treatment enabled mouse OOX to retain significantly more HA within the complex than oocytes in MI and MII. The results indicate that secretion of factors that promote the retention of HA within the complex is developmentally regulated during oocyte maturation.

The second most prevalent urological malignancy in middle aged and elderly men is bladder cancer, with 90% of the cases being transitional cell carcinomas. The success of current systemic and intravesical therapeutic agents, such as cisplatin, thiotepa, Adriamycin, mitomycin C, and bacillus Calmette-Guerin, is limited with recurrence rates reduced to 17-44%. In addition, most of these agents require instrumentation of the urinary tract and are delivered at a significant cost and potential morbidity to the patient. Fluroquinolone antibiotics such as ciprofloxacin, which can be administered p.o., may have a profound effect in bladder cancer management. This is primarily based on limited in vitro studies on tumor cells derived from transitional cell carcinoma of the bladder that revealed a dose- and time-dependent inhibition of cell growth by ciprofloxacin at concentrations that are easily attainable in the urine of patients. However, the mechanism(s) by which ciprofloxacin elicits its biological effects on bladder cancer cells is not well documented. Our experimental data confirm previous studies showing the in vitro cell growth inhibition of the transitional cell carcinoma of the bladder cell line HTB9 and further showed the induction of cell cycle arrest at the S/G2-M checkpoints. In addition, we found down-regulation of cyclin B, cyclin E, and dephosphorylation of cdk2 in ciprofloxacin-treated bladder tumor cells. There was also an up-regulation of Bax, which altered the Bax:Bcl-2 ratio, which may be responsible for mitochondrial depolarization reported to be involved prior to the induction of apoptosis. The cyclin-dependent kinase inhibitor p21WAF1 level was found to be decreased within 12 h of ciprofloxacin treatment and disappeared completely when HTB9 cells were treated with 200 microg/ml ciprofloxacin for 24 h. The down-regulation of p21WAF1 closely correlated with poly(ADP-ribose) polymerase cleavage and CPP32 activation. Recent studies revealed that p21WAF1 protects cells from apoptosis by arresting them in G1 and further binds to pro-caspase-3, preventing its activation and thus, inhibiting the apoptotic cascade. Hence, the down-regulation of p21WAF1, together with the alterations in Bax and cdk2 as observed in our studies, may define a novel mechanism by which ciprofloxacin inhibits tumor cell growth and induces apoptotic cell death. The results of our current studies provide strong experimental evidence for the use of ciprofloxacin as a potential preventive and/or therapeutic agent for the management of transitional cell carcinoma of the bladder.

Epidemiological studies have shown lower incidence of breast and prostate cancers in Asian populations consuming a traditional diet rich in soy. Protection from these cancers was attributed to the isoflavones, particularly genistein and daidzein found in vivo as the major metabolites of soy isoflavones. However, the role of isoflavones in head and neck cancer is less clear. In our previous studies we reported that genistein can induce cell growth inhibition by arresting the cells at S/G2-M phases, and also induces apoptosis in HN4 squamous cell carcinoma of the head and neck cell line (HNSCC). In this report we show that these changes are accompanied by the down-regulation of Cdk1, and CyclinB1, and up-regulation of the cyclin dependent kinase (Cdk) inhibitor p21WAF1, which may be responsible for the induction of cell cycle arrest and apoptosis. The evidence for the induction of apoptosis was supported by the appearance of DNA ladder as reported previously, and further supported by our current results on the cleavage of poly-ADP-ribose polymerase (PARP), hallmark of apoptosis. This was also accompanied by the up-regulation of Bax, with modest down-regulation of Bcl-2 protein expression, which changes the balance between pro- and anti-apoptosis molecules in favor of pro-apoptosis. Furthermore, we also observed down-regulation and degradation of Cdc25C, which is a marker of cell proliferation, and plays important role in CyclinB-Cdk1 complex activation. The down-regulation followed by the degradation of Cdc25C is an indicator of G2/M arrest and anti-proliferation effects of genistein. Collectively, these data provide strong molecular evidence for the anti-tumor activity of genistein in HNSCC cells.

Staurosporine has been reported to cause arrest of cells in G1 phase at low concentration and in G2 phase at high concentration. This raises the question of why the effects of staurosporine on the cell cycle depend on the applied concentration. In order to verify these multiple functions of staurosporine in Meth-A cells, we used cyclin E as a landmark of G1/S transition, cyclin B as a landmark of G2/M transition and MPM2 as a hallmark of M phase. We found that staurosporine arrested cells in G1 phase at a low concentration (20 nM) and in G2/M phase at a high concentration (200 nM). However, 200 nM staurosporine increased the expression of cyclin B and cdc2 proteins, suggesting that the cells progressed through the G2/M transition, and increased the expression of MPM2 protein, indicating that the cells entered M phase. Moreover, 200 nM staurosporine increased the expression of p53 and p21 proteins and inhibited the expression of cyclin E and cdk2 proteins, suggesting that the cells were arrested in the G1 phase of the next cycle. Morphological observation showed similar results as well. These data suggest that the G2/M accumulation induced by 200 nM staurosporine does not reflect G2 arrest, but rather results from M phase arrest, followed by progression from M phase to the G1 phase of the next cycle without cytokinesis, and finally arrest of the cells in G1 phase.