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Tocotrienols, a subgroup within the vitamin E family of compounds, have shown antiproliferative and anticancer properties, however, the molecular basis of these effects remains to be elucidated. In this study, the effect of 3-tocotrienol on cell cycle arrest was assessed by studying the retinoblastoma protein (Rb) levels and phosphorylation status, levels of E2F (a transcription factor critically involved in the G1/S-phase transition of the mammalian cell cycle; originally identified as a DNA-binding protein essential for early region 1A-dependent activation of the adenovirus promoter designated E2), and other cell cycle controlling proteins in estrogen receptor-negative MDA-MB-231 breast cancer cells. The cell growth assay demonstrated that exposure of the MDA-MB-231 cells to 6-tocotrienol (1-20 microM) resulted in a dose- and time-dependent inhibition of cell growth as compared with vehicle treated cells and the magnitude of growth inhibition was higher at 10 and 20 microM treatment for 48 and 72 h. The phosphorylation status of Rb plays a central role in the control of the cell cycle at the G0/G1-phase. delta-Tocotrienol treatment reduced the total Rb and its phosphorylation at the Ser780, Ser795, Ser 807/811 and Thr826 positions in a dose- and time-dependent fashion. The site-specific inhibition of the phosphorylation of Rb by delta-tocotrienol was tightly associated with a marked reduction in the expression of cyclin D1 and its regulatory partner cyclin-dependant kinase 4 (CDK4), which is responsible for the phosphorylation of Rb at Ser780, Ser795, Ser 807/811 and Thr826. In addition, delta-tocotrienol also reduced the expression of E2F that occurred simultaneously with the loss of Rb phosphorylation and inhibition of cell cycle progression. Interestingly, delta-tocotrienol also caused a marked reduction in the expression of G2/M regulatory proteins including cyclin B1 and CDK1. To the best of our knowledge, this study was the first to reveal that the target of cell proliferative inhibitory action of delta-tocotrienol in a model estrogen receptor-negative human breast cancer cell line MDA-MB-231 is mediated by the loss of cyclin D1 and associated suppression of site-specific Rb phosphorylation, suggesting its future development and use as an anticancer agent.

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.

A simple negative feedback loop of interacting genes or proteins has the potential to generate sustained oscillations. However, many biological oscillators also have a positive feedback loop, raising the question of what advantages the extra loop imparts. Through computational studies, we show that it is generally difficult to adjust a negative feedback oscillator's frequency without compromising its amplitude, whereas with positive-plus-negative feedback, one can achieve a widely tunable frequency and near-constant amplitude. This tunability makes the latter design suitable for biological rhythms like heartbeats and cell cycles that need to provide a constant output over a range of frequencies. Positive-plus-negative oscillators also appear to be more robust and easier to evolve, rationalizing why they are found in contexts where an adjustable frequency is unimportant.

Purpose: Medulloblastoma is the most common malignant embryonal brain tumor in children. The current clinical risk stratification to select treatment modalities is not optimal because it does not identify the standard-risk patients with resistant disease or the unknown number of high-risk patients who might be overtreated with current protocols. The aim of this study is to improve the risk stratification of medulloblastoma patients by using the expression of multiple prognostic markers in combination with current clinical parameters., Experimental Design: Candidate prognostic markers were selected from literature or from medulloblastoma expression data. Selected genes were immunohistochemically analyzed for their prognostic value using medulloblastoma tissue arrays containing 124 well-characterized patient samples., Results: Protein expression analyses showed that the combined expression of three genes was able to predict survival in medulloblastoma patients. Low MYC expression identified medulloblastoma patients with a very good outcome. In contrast, concomitant expression of LDHB and CCNB1 characterized patients with a very poor outcome. Multivariate analyses showed that both expression of MYC and the LDHB/CCNB1 gene signature were strong prognostic markers independent of the clinical parameters metastasis and residual disease. Combined analysis of clinical and molecular markers enabled greater resolution of disease risk than clinical factors alone., Conclusions: A molecular risk stratification model for medulloblastoma patients is proposed based on the signature of MYC, LDHB, and CCNB1 expression. Combined with clinical variables, the model may provide a more accurate basis for targeting therapy in children with this disease.

The effects of 17 beta-estradiol and progesterone were evaluated separately and in combination, on the growth, survival, and cell cycle dynamics of SW-13 human adrenal carcinoma cells in culture. Both hormones significantly decreased cell survival, with dose response curves at four days demonstrating EC (50)s estimated at 1.2 x 10 (-5) M for 17 beta-estradiol and 4.8 x 10 (-6) M for progesterone. Flow cytometry studies of these cultures indicated a strong G2/M blocking effect of both steroids, either individually or in combination; the effects of progesterone and of both agents together were substantially greater than the effect with 17 beta-estradiol alone. The sub-G1 region of the flow cytometry profile was significantly enhanced by exposure to 17 beta-estradiol and even more by progesterone. Sub-G1 "apoptosis" was confirmed by fragmented and condensed nuclear chromatin staining using a standard DAPI fluorescence assay. The expression of the critical cell cycle regulatory proteins cyclin B1 and D1 were significantly decreased by each hormone, with the influence of progesterone again predominating. These data demonstrate that high doses of 17 beta-estradiol and progesterone have inhibitory and apoptotic effects on SW-13 human adrenal carcinoma cells IN VITRO. The observed effects are associated with declines in cyclin B1 and D1 expression as well as a block in G2/M.

Suppression of Cdc2 activity is essential for DNA damage-induced G2 arrest. In the present study, we elucidated regulatory mechanism of Cdc2 activity during radiation-induced transient G2 arrest. Exposure of the cells to gamma-radiation (4 Gy) led to a transient increase of cells in G2 at 12 h rather than M phase and then the cells resumed cell cycle progression from the G2 arrest. However, the levels of cyclin B1 and Cdc2 activity were increased in the whole cell extracts at 12 h. Despite cyclin B1 induction and increased level of Cdc2 activity after irradiation the activities in the nuclear fractions were transiently decreased at 12 h and returned to control levels by 24-48 h, demonstrating transient inhibition of nuclear translocation of cyclin B1 in response to radiation. Moreover, inhibitory phosphorylation of the Cdc2 on Tyr15 and the Cdc25C on Ser216 were increased concomitant with transient G2 arrest. The level of phosphorylated Wee1 and its activity were also markedly increased at 12 h after irradiation. In addition, radiation caused nuclear accumulation of p21(CIP1/WAF1) at 12 h, resulting in increased-binding of p21(CIP1/WAF1) to Cdc2. Nuclear p21(CIP1/WAF1) protein level and its binding to Cdc2 gradually returned to control level when the cells resumed cell cycle progression. However, total protein level of p21(CIP1/WAF1) continued to increase until 48 h after irradiation. Collectively, these results indicate that the suppression of nuclear import of cyclin B1, the induction of Wee1 kinase activity, and the transient nuclear accumulation of p21(CIP1/WAF1) may play important roles in the transient cell cycle delay in response to ionizing radiation.

The meiotic maturation of oocyte and spermatocyte in animals is controlled by the maturation promotion factor (MPF), a complex of Cdc2 and cyclin B proteins. To better understand the mechanism of oocyte and spermatocyte maturation in fish, the expression of cyclin B1 (CB1), B2 (CB2) and Cdc2 kinase during oogenesis and spermatogenesis in rainbow trout were examined at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that the amount of CB1 and CB2 mRNA was greater at previtellogenesis and late vitellogenesis stages, but less at early vitellogenesis stage and during early embryogenesis. Cdc2 mRNA was continuously present throughout the processes of oogenesis and early embryogenesis except for a decline at early vitellogenesis. In situ hybridization analysis indicated that CB1, CB2 and Cdc2 transcripts were present in oocytes of different developmental stages as well as in all spermatogenic cells except for spermatogonia. Immunohistochemical analysis revealed that CB1 protein was absent in vitellogenic oocytes, but present in young previtellogenic and mature oocytes. In contrast, CB2 and Cdc2 proteins were present at all stages oocyte development. Similarly, CB2 and Cdc2 proteins were present throughout spermatogenesis, whereas CB1 protein was only detected in spermatogonia and spermatocytes, but not in spermatids. Thus, it appears that CB1, CB2 and Cdc2 transcripts have similar expression patterns during oogenesis and spermatogenesis, but CB1 protein varies in amount during these processes. These data suggest that CB1 may have a leading role in the regulation of meiotic maturation of oocytes and spermotocytes.

Cyclin B1, a key component in the control of cell cycle progression from G(2) to M phase, has been implicated in tumorigenesis and the development of malignancy. However, the underlying mechanism by which cyclin B1 acts as an important oncogenic molecule remains largely unknown. Here we show that ectopic expression of cyclin B1 promotes cell proliferation, enhances cell motility and migration and results in increased ability of cells extravasating through the capillary endothelium. Interestingly, isogenic esophageal squamous cell carcinoma (ESCC) cells overexpressing cyclin B1 reveal strong invasive growth and high potential of metastasis to lung in xenograft mice. Suppression of cyclin B1 expression via small interfering RNA approach in high-metastatic esophagus carcinoma cells specifically inhibits their ability to metastasize from the primary ESCC to lung. Notably, altered expression of epithelial markers and mesenchymal markers were observed in the cells overexpressing cyclin B1, suggesting that cyclin B1 contributes to metastasis probably by promoting an epithelial-mesenchymal transition. These results establish a mechanistic link between cyclin B1 and ESCC metastasis and provide novel insight into understanding of cyclin B1 in the development of ESCC malignancy.

The change of the expression of Cyclins in neurons of rats after focal cerebral ischemia was investigated. Ischemia was induced by temporary middle cerebral artery occlusion (MCAO). The experimental rats induced by MCAO were sacrificed on 7th and 14th day after reperfusion. The brain was taken out at 7th and 14th day after injury, and the expression of Cyclin D1, E, A and B1 in neurons of cerebral cortex or hippocampal CA1 region was detected by immunofluorescence and confocal microscope. The results showed that after MCAO, in the ipsilateral CA1 subfield of hippocampus the expression of Cyclin D1, E, A and B1 in neurons was significantly gradually up-regulated at 7th and 14th day after reperfusion (P<0.05) as compared with that in control group. In the ipsilateral cerebral cortex the expression of Cyclin D1 and B1 in neurons was notably gradually down-regulated at 7th and 14th day, and that of Cyclin E and A was significantly up-regulated at 14th day after reperfusion as compared with that in control group (all P<0.05). It was concluded that there was a differential sensitivity among neurons from different brain regions to ischemic injury. But all of them re-enter into cell cycle after MCAO.

Norcantharidin (NCTD), a demethylated form of cantharidin, is currently used as an anticancer drug in China, but five newly synthesized derivatives have not been tested for antitumor efficacy. In this study, we investigated the in-vitro and in-vivo activity of five derivatives on Bel-7402, HeLa and PC-3M1E8 cell lines on a sulfarhodamine B assay. All of the derivatives showed significant antiproliferative activity, hence we elected to study further one of them, NCTD-Nd3II, in an in-vivo mouse model, and to examine its effects on cell cycle and protein expression. NCTD-Nd3II inhibited H22 tumors in mice in a dose-dependent manner with low toxicity. Flow cytometry results showed that apoptosis and G2/M cell cycle arrest contributed to the cytotoxic and cytostatic effects of NCTD-Nd3II. Further studies showed that Bax and p21 protein expression was upregulated, whereas cyclin B1, Cdc-2 and Bcl-2 protein expression was downregulated. Our findings show that NCTD-Nd3II might be a promising chemotherapeutic agent for hepatomas.

Cancer of the prostate gland (PCA) is the most common invasive malignancy and is the second leading cause of cancer-related death in males. The polyphenolic constituents of black tea have gained considerable attention as chemopreventive agents. Many studies have shown that black tea reduces the risk of several cancer types. In the present study, we studied the effect of a black tea polyphenol, theaflavin (TF), on cellular proliferation and cell death in the human prostate cancer cell line, PC-3. We showed that TF inhibits cell proliferation in a dose- and time-dependent manner. Studies on cell cycle progression have shown that the anti-proliferative effect of TF is associated with an increase in the G2/M phase of PC-3 cells. Western blot results showed that TF-induced G2/M phase arrest was mediated through the inhibition of cyclin-regulated signaling pathways. TF induces cyclin kinase inhibitor p21(waf1/cip1) expression and inhibits cdc25C and cyclin B expression. Increased exposure time to TF caused apoptosis of PC-3 cells, which was associated with up-regulation of the pro-apoptotic proteins Bax, caspase-3 and caspase-9 and down-regulation of anti-apoptotic protein Bcl-2. The role of caspase-induced apoptosis was further confirmed by a reduction in mitochondria membrane potential and the appearance of a DNA laddering pattern. Thus, it can be concluded that TF acts as an effective anti-proliferative agent by modulating cell growth regulators in prostate cancer cells.

Propolis is a resinous substance collected by bees (Apis mellifera) from various tree buds which they then use to coat hive parts and to seal cracks and crevices in the hive. Propolis, a known ancient folk medicine, has been extensively used in diet to improve health and to prevent disease. In the present study, we have evaluated the effects of ethanolic extracts of Brazilian propolis group l2 and bud resins of botanical origin (B. dracunculifolia), and propolis group 3 on proliferation of metastasis (DU145 and PC-3) and primary malignant tumor (RC58T/h/SA#4)-derived human prostate cancer cells. The strongest inhibition was observed in propolis group 3 (sample #3) extracts whereas moderate growth inhibition was observed in human prostate epithelial cells. In the RC58T/h/SA#4 cells, resins of botanical origin of propolis group 12 (sample #1) and propolis group 12 (sample #2) induced growth inhibition that was associated with S phase arrest whereas propolis group 3 (sample #3) induced growth inhibition that was associated with G2 arrest. The mechanisms of cell cycle effects of propolis were investigated. The resins of botanical origin of propolis group 12 and propolis group 12 showed similar inhibition of cyclin D1, CDK4 and cyclin B1 expression. Propolis group 3 showed higher induction of p21 expression but no inhibition of cyclin D1, CDK4 and cyclin B1 expression. The results obtained here demonstrate that the Brazilian propolis extracts have significant inhibitory effect on proliferation of human prostate cancer cells. Inhibition was achieved through regulation of protein expression of cyclin D1, B1 and cyclin dependent kinase (CDK) as well as p21. Our results indicate that the Brazilian propolis extracts show promise as chemotherapeutic agents as well as preventive agents against prostate cancer.

After reports on regression of cancer in humans and animals infected with microbial pathogens date back more than 100 years, much effort has been spent over the years in developing wild type or attenuated bacterial and purified bacterial proteins for the treatment of cancer. Pseudomonas aeruginosa exotoxin A (ETA) is known to inhibit cell growth and trigger significant cell death in various cancer cells. Although ETA induces apoptosis of cancer cells, its exact mechanism of action is not yet known. Four different assays were performed in this study: morphological assessment of apoptotic cells, cell cytotoxicity, cell cycle analysis and Western blot analysis. The proliferation and survival in the cells treated with ETA was decreased. In addition, percentages of apoptotic HeLa S(3) cells treated with ETA were increased. ETA-induced apoptosis rates were confirmed to have increased in a dose-dependent manner through annexin V binding assay. Flow cytometric analysis was examined to ascertain whether ETA could regulate cell cycle in HeLa S(3) cells. ETA treatment demonstrated that the expression of 14-3-3delta proteins was increased, while expression of cdc and cyclin B proteins was decreased, suggesting that ETA induces cell cycle arrest and then progresses to apoptosis. Therefore, these results suggest that P. aeruginosa ETA induced apoptosis in HeLa S(3) cells.

Background: The epidermal growth factor receptor (EGFR) is frequently overexpressed in uterine cervix carcinoma. The role of the pre-treatment EGFR expression levels and the changes of expression induced by ionizing radiation (IR) have not been conclusively defined., Patients and Methods: The staining intensity (SI) and labeling index (LI) of EGFR were determined in 38 patients by immunohistochemistry (IHC). Biopsies were taken before after 1 week of RT. EGFR expression was correlated with cell cycle, apoptosis and angiogenesis., Results: Before RT, 87% and after 1 week of RT, 95% of samples were positive for EGFR (p=0.2). Two patterns were observed, either increasing or decreasing expression after initiating RT. An increase of the EGFR SI was seen in 63% of patients from a mean of 57 SI (SD+/-60) before RT to 142 SI (SD+/-80.8) (p=0.001) during RT. In 32% of cases, EGFR decreased from 165 SI before (SD+/-83.0) to 75 SI (SD+/-73.0) (p< or =0.001) during RT. Two of five (5%) patients negative for EGFR before RT remained negative. An increase of the RT-induced EGFR LI was associated with reduced microvessel density (MVD) (p=0.02). Changes of the EGFR LI did neither correlate with cell cycle arrest nor apoptosis., Conclusions: EGFR expression changes unpredictably during RT. The implications of changing EGFR during RT remain to defined. Repeated biopsies and EGFR reassessment during RT may help to better define EGFR-targeted treatment.

In the present study, we examined the expression and cytolocalization of protein phosphatase type 1 (PP1) isoforms and nucleolin in human osteoblastic cell line MG63 cells at two boundaries in the cell cycle. We treated MG63 cells with hydroxyurea and nocodazole to arrest the cells at the G(1)/S and G(2)/M boundaries, respectively. As judged from the results of Western blot analysis, PP1 isoforms were expressed differently at each boundary of the cell cycle. Nucleolin was also shown to have a different expression pattern at each boundary. In the hydroxyurea-treated cells, nucleolus-like bodies were bigger in size and decreased in number compared with those in asynchronized cells. However, the subcellular localization of PP1s and nucleolin was not changed. Anti-nucleolin antibody interacted with 110-kDa and 95-kDa proteins present in asynchronized cells and in the cells treated with hydroxyurea. Treatment of the cells with nocodazole decreased the level of the 95-kDa form of nucleolin. In the nocodazole-treated cells, it was impossible to distinguish the distribution of each protein. The phosphorylation status of nucleolin in the cell cycle arrested samples was examined by 2D-IEF-PAGE followed by Western blot analysis. In the case of asynchronized cells or hydroxyurea-treated ones, nucleolin was located at a basic isoelectric point (dephosphorylated status); whereas in the G(2)/M arrest cells, the isoelectric point of nucleolin shifted to an acidic status, indicating that nucleolin was phosphorylated. The present results indicate that PP1 and nucleolin were differently expressed at G(1)/S and G(2)/M boundaries of the cell cycle and acted in a different fashion during cell-cycle progression., ((c) 2005 John Wiley & Sons, Ltd.)

Chemotherapeutic drugs ideally should take advantage of the differences between transformed and normal cells and induce apoptosis only in cancer cells. One such difference may be the overexpression of cyclin B1 protein in cancer cells, which is required for the proper progression through mitosis. Previously, we showed that treatment of human prostate cancer cells with 2-methoxyestradiol (2-ME) or docetaxel results in an accumulation of cyclin B1 protein and an increase in cyclin B1 kinase activity, followed by induction of apoptotic cell death. Inhibition of cyclin B1 kinase lowers apoptosis induced by 2-ME and docetaxel. In this study, we established a positive correlation between cyclin B1 protein and apoptosis induced by chemotherapy in prostate cancer cells. There is minimal cyclin B1 and induction of apoptosis by chemotherapy in nontransformed cells. LNCaP and PC-3 prostate cancer cells stably overexpressing cyclin B1 are more sensitive to apoptosis induced by chemotherapy. LNCaP cells expressing cyclin B1 small interfering RNA to lower cyclin B1 protein or dominant negative cyclin-dependent kinase 1 to inhibit cyclin B1 kinase show a decrease in apoptosis. Increased sensitivity to apoptosis by overexpression of cyclin B1 may be due to lower Bcl-2, higher p53, and decreased neuroendocrine differentiation. We suggest that a cancer-specific mechanism whereby 2-ME and docetaxel may exert anti-prostate cancer activity is the deregulated activation of cyclin B1 kinase, leading to the induction of apoptotic cell death. Our results also suggest that higher levels of cyclin B1 in prostate cancer cells may be a good prognostic marker for chemotherapy.

The cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) exerts a growth inhibitory effect on cancer cells, and this effect is linked to the induction of apoptosis or cell cycle arrest. Induction of apoptosis by 15d-PGJ(2) is associated with the down-regulation of anti-apoptotic proteins. G(0)-G(1)-->S phase progression is inhibited by 15d-PGJ(2) via the degradation of cyclin D1. In this study, we further investigated the mechanism by which 15d-PGJ(2) inhibits cancer cell growth by using the breast cancer cell lines MCF-7 and T-47D. Treatment with 20 microM 15d-PGJ(2) for 72 h completely blocked the growth in both cell lines. However, the proportions of apoptotic MCF-7 and T-47D cells were 21.1% and 40.9%, respectively, indicating that the induction of apoptosis did not appear to fully account for growth inhibition by 15d-PGJ(2). Cell cycle analysis using cells synchronized at the G(0)-G(1) or S phase revealed that 15d-PGJ(2) blocked not only G(0)-G(1)-->S phase progression but also G(2)-M phase progression. The expression of both cyclins D1 and B1 was decreased by 15d-PGJ(2). Furthermore, 15d-PGJ(2) inhibited aurora-B kinase activity, which coincided with the down-regulation of survivin. Thus, 15d-PGJ(2) induced cell cycle arrest at the G(2)-M phase via inhibition of cyclin B1 expression and aurora-B kinase activity. We conclude that survivin may be an important target for 15d-PGJ(2), and its down-regulation may lead to a decrease in aurora-B kinase activity.

In the Drosophila embryo, Nanos and Pumilio collaborate to repress the translation of hunchback mRNA in the somatic cytoplasm. Both proteins are also required for repression of maternal Cyclin B mRNA in the germline; it has not been clear whether they act directly on Cyclin B mRNA, and if so, whether regulation in the presumptive somatic and germline cytoplasm proceeds by similar or fundamentally different mechanisms. In this report, we show that Pumilio and Nanos bind to an element in the 3' UTR to repress Cyclin B mRNA. Regulation of Cyclin B and hunchback differ in two significant respects. First, Pumilio is dispensable for repression of Cyclin B (but not hunchback) if Nanos is tethered via an exogenous RNA-binding domain. Nanos probably acts, at least in part, by recruiting the CCR4-Pop2-NOT deadenylase complex, interacting directly with the NOT4 subunit. Second, although Nanos is the sole spatially limiting factor for regulation of hunchback, regulation of Cyclin B requires another Oskar-dependent factor in addition to Nanos. Ectopic repression of Cyclin B in the presumptive somatic cytoplasm causes lethal nuclear division defects. We suggest that a requirement for two spatially restricted factors is a mechanism for ensuring that Cyclin B regulation is strictly limited to the germline.

Purpose: Cyclin B2, a G(2)-M cyclin, is overexpressed in colorectal adenocarcinomas compared with the normal mucosa. This study examined the level of cyclin B2 overexpression according to the histologic findings and investigated the mechanism(s) and clinical implications of cyclin B2 overexpression in colorectal adenocarcinomas., Experimental Design: The immunoreactivity of the polyclonal antibodies to cyclin B2 was determined in colorectal cancer cells. The transcriptional regulation of cyclin B2 by NF-Y was analyzed using an in vitro transfection assay and an in vivo chromatin immunoprecipitation assay. The proliferative activity of the colorectal cancer cells in relation to cyclin B2 overexpression was further examined., Results: The cytoplasmic distribution of cyclin B2 immunoreactivity was positive in 42 of 65 (64.6%) cases of colorectal adenocarcinoma, and the level was similar regardless of the histologic type. A dominant-negative form of NF-YA effectively inhibited the cyclin B2 promoter activity, and NF-Y was found to bind three conserved CCAAT boxes in the cyclin B2 promoter in colorectal adenocarcinoma cells. Tumor cells with a higher functional cyclin B2 activity grew faster than those with a lower activity. Furthermore, there was a correlation between the cells showing immunoreactivity to cyclin B2 and those containing the proliferating cell nuclear antigen, a G1-S cyclin, which is also downstream of NF-Y in colorectal adenocarcinoma cells., Conclusions: Cyclin B2 seems to be a molecular marker of a colorectal adenocarcinoma and that its up-regulation and coordinate expression of the other cell cycle-related genes by NF-Y might contribute to tumor cell proliferation by accelerating cell cycle progression.

Low-grade central osteosarcoma is a rare type of osteosarcoma with peculiar clinical radiographic and microscopic features. The aim of this article is to report and discuss a case of low-grade central osteosarcoma in the mandible of a 42-year-old woman. The patient reported sensing mild pain and tooth mobility for a period of 4 years, despite continuous dental treatment. Radiographic evaluation showed a mixed radiopaque/radiolucenct lesion in the body, ramus, coronoid process, and condyle of the left side of the mandible. Destruction of the mandibular cortex in the area was also observed. After incisional biopsy, the patient underwent hemimandibulectomy. Microscopic findings showed a tumor exhibiting spindle cells with nuclear hyperchromasia and no mitotic activity, irregular osteoid formation, and soft tissue infiltration. The immunohistochemical analysis of the expression of Ki-67, Cyclin B1, and PCNA proteins (cellular proliferation markers) revealed a very low Ki-67+ and Cyclin B1+ cell index (mean 7% and 3%, respectively), but a moderate number of PCNA+ cells (mean 49%). The 2 years of clinical and imaging postoperative follow-up showed no evidence of recurrence. Clinicians should be aware of these lesions, because histopathologicially low-grade central osteosarcoma may be misinterpreted as fibrous dysplasia.

The effects of morin (3,5,7,20,40-pentahydroxyflavone) on human leukemia HL-60 cells in vitro were investigated and the molecular mechanisms of morin-induced G2/M arrest and apoptosis in HL-60 cells were examined. Morin induced morphological changes and decreased the percentage of viable cells via induction of G2/M-phase arrest and apoptosis. Morin-induced G2/M-phase arrest was accompanied by the promotion of p21 and Wee1, and decreased levels of Cdc25c and cyclins A and B1 complex. Morin-induced apoptosis in HL-60 cells was also confirmed by flow cytometric assay, DNA gel electrophoresis for DNA fragmentation and DAPI staining. Morin induced apoptosis in time- and dose-dependent manners. Morin-induced apoptosis was associated with elevated intracellular reactive oxygen species (ROS) increased and Ca2+ production; decreased the levels of mitochondria membrane potential (deltapsi(m)) and increased caspase-3 activation. Collectively, these results suggest that the morin-induced apoptosis in HL-60 cells may result from the activation of caspase-3 and intracellular Ca2+ release, as well as the mitochondria membrane potential pathway.

Background: Cell division or cytokinesis, which results from a series of events starting in metaphase, is the mechanism by which the mother cell cytoplasm is divided between the two daughter cells. Hence it is the final step of the cell division cycle. The aim of the present study was to demonstrate that mammalian cells undergoing cytokinesis can be sorted selectively by flow cytometry., Materials and Methods: Cultures of HeLa cells were arrested in prometaphase by nocodazole, collected by mitotic shake-off and released for 90 min into fresh medium to enrich for cells undergoing cytokinesis. After ethanol fixation and DNA staining, cells were sorted based on DNA content and DNA fluorescence signal height., Results: We define a cell population that transiently accumulates when synchronized cells exit mitosis before their entry into G1. We show that this population is highly enriched in cells undergoing cytokinesis. In addition, this population of cells can be sorted and analyzed by immunofluorescence and western blotting., Conclusions: This method of cell synchronization and sorting provides a simple means to isolate and biochemically analyze cells in cytokinesis, a period of the cell cycle that has been difficult to study by cell fractionation.

The Drosophila PAN GU (PNG) kinase complex regulates the developmental translation of cyclin B. cyclin B mRNA becomes unmasked during oogenesis independent of PNG activity, but PNG is required for translation from egg activation. We find that although polyadenylation of cyclin B augments translation, it is not essential, and a fully elongated poly(A) is not required for translation to proceed. In fact, changes in poly(A) tail length are not sufficient to account for PNG-mediated control of cyclin B translation and of the early embryonic cell cycles. We present evidence that PNG functions instead as an antagonist of PUMILIO-dependent translational repression. Our data argue that changes in poly(A) tail length are not a universal mechanism governing embryonic cell cycles, and that PNG-mediated derepression of translation is an important alternative mechanism in Drosophila.

Although cyclin G1 has been implicated in certain p53-related biological phenomena, other aspects of its function remain unclear. Here we report hitherto unknown mechanism by which cyclin G1 increases radiation sensitivity by regulating the level of cyclin B1. Overexpression of cyclin G1 was observable in lung carcinoma tissues. Irradiation of human lung cells with cyclin G1 overexpression resulted in increased cell death and gamma-H2AX foci suggesting that cyclin G1 rendered the cells more susceptible to DNA damage. Enhanced radiosensitivity by cyclin G1 was correlated with increased cyclin B1, CDC2/cyclin B1 complex, and MPM2. Cell cycle synchronization clearly showed coexpression of cyclin G1 and cyclin B1 in G2/M phase. Depletion of cyclin G1 by interference RNA revealed that cyclin G1 regulated transcription of cyclin B1 in a p53-independent manner, and confirmed that the increased mitotic cells and cell death by cyclin G1 were dependent upon cyclin B1. Therefore, our data suggest that cyclin G1 enhanced radiation sensitivity by overriding radiation-induced G2 arrest through transcriptional upregulation of cyclin B1.

We have previously shown that MBP-1 acts as a general transcriptional repressor, and forced expression of MBP-1 exerts an anti-proliferative effect on a number of human cancer cells. In this report, we have investigated the role of endogenous MBP-1 in cell growth regulation. For this, we generated human prostate cancer cells (PC3) stably transfected with short hairpin RNA targeting MBP-1. We have observed retarded growth and longer doubling time of MBP-1 knockdown PC3 cells as compared with control mock-transfected PC3 cells. Fluorescence-activated cell sorter analysis suggested that PC3 cells expressing MBP-1-specific small interfering RNA accumulated during G2/M phase of the cell cycle. Further analysis suggested that depletion of MBP-1 was associated with reduction of cyclin A and cyclin B1 expression when compared with that of the control cells. A delayed induction of cyclin A and B1 expression was observed in MBP-1-depleted PC3 cells (PC3-4.2) upon serum stimulation, although the level of expression was much lower than that of control PC3 cells. Supplementation of MBP-1 in PC3-4.2 cells restored cyclin A and cyclin B1 expression. Together, these results suggest that knockdown of MBP-1 in prostate cancer cells perturbs cell proliferation by inhibiting cyclin A and cyclin B1 expression.

The E2F transcription factors play a critical role in coordinating transcription of specific genes essential for G1-S transition. In early G1, the retinoblastoma protein (pRB) becomes phosphorylated by cyclin-dependent kinases, disrupting pRB binding to E2F-1-3, allowing "free" E2F to regulate genes involved in proliferation. In the present study, we used a tetracycline E2F-1 inducible U2OS osteosarcoma cell line to investigate the effect of increasing levels of E2F-1 on the cytotoxicity of various chemotherapeutic drugs. Upon overexpression of E2F-1, there was no detectable change in cytotoxicity to doxorubicin, cisplatin, 5-fluorouracil, or etoposide. In contrast, overexpression of E2F-1 resulted in a marked increase in sensitivity to vinblastine and paclitaxel, drugs that are known to be more effective against cells in M phase. Therefore, we investigated the effect of E2F-1 overexpression on proteins regulating the G2-M transition and M phase, in particular cyclin B1 and cdc2 kinase. Cyclin B1 mRNA and protein levels increased within 24 hours of E2F1 induction together with an increase in associated cdc2 kinase activity. Overexpression of cyclin B1 also resulted in a specific increase in sensitivity to paclitaxel and an increase in the cellular growth rate. Knockdown of cyclin B1 using an RNA interference oligo resulted in a slower cellular growth rate and an increase in resistance to paclitaxel. These studies add support to recent reports that show E2F regulates genes involved in mitotic entry and exit and allow the suggestion that mitotic inhibitors may have selective effects in tumors that overexpress E2F-1.

Physalis angulata (PA) is employed in herbal medicine around the world. It is used to treat diabetes, hepatitis, asthma and malaria in Taiwan. We have evaluated PA as a cancer chemopreventive agent in vitro by studying the role of PA in regulation of proliferation, cell cycle and apoptosis in human breast cancer cell lines. PA inhibited cell proliferation and induced G2/M arrest and apoptosis in human breast cancer MAD-MB 231 and MCF-7 cell lines. In this study, under treatment with various concentrations of PA in MDA-MB 231 cell line, we checked mRNA levels for cyclin A and cyclin B1 and the protein levels of cyclin A and cyclin B1, Cdc2 (cyclin-dependent kinases), p21(waf1/cip1) and P27(Kip1) (cyclin-dependent kinase inhibitors), Cdc25C, Chk2 and Wee1 kinase (cyclin-dependent kinase relative factors) in cell cycle G2/M phase. From those results, we determined that PA arrests MDA-MB 231 cells at the G2/M phase by (i) inhibiting synthesis or stability of mRNA and their downstream protein levels of cyclin A and cyclin B1, (ii) increasing p21(waf1/cip1) and P27(kip1) levels, (iii) increasing Chk2, thus causing an increase in Cdc25C phosphorylation/inactivation and inducing a decrease in Cdc2 levels and an increase in Wee1 level. According to the results obtained, PA appears to possess anticarcinogenic properties; these results suggest that the effect of PA on the levels of phosphorylated/inactivated Cdc25C are mediated by Chk2 activation, at least in part, via p21(waf1/cip1) and P27(kip1) cyclin-dependent kinase inhibitors pathway to arrest cells at G2/M phase in breast cancer carcinoma cells.

Background: The evolutionary conserved cyclin-dependent kinase phosphatase hCdc14A has been shown to play potential roles in the regulation of mitotic exit and in the centrosome duplication cycle. We have recently shown that hCdc14A also can interact with the tumor suppressor p53 both in vitro and in vivo and specifically dephosphorylates the ser315 site of p53 in vitro. In this study we developed antibodies against hCdc14A to investigate the expression and regulation of hCdc14A in human tissues and cancer cells., Results: We show that hCdc14A is differentially expressed in human tissues and in 75 cancer cell lines examined. Treatments with the histone deacetylase inhibitor TSA, the demethylating agent 5-aza-2'-deoxycytodine or the proteasome inhibitor MG132 significantly induced expression of hCdc14A in cell lines expressing low or undetectable levels of hCdc14A. There was a strong bias for low expression of hCdc14A in cancer cell lines harboring wild-type p53, suggesting that high Cdc14A expression is not compatible with wild-type p53 expression. We present evidence for a role for hCdc14A in the dephosphorylation of the ser315 site of p53 in vivo and that hCdc14A forms a complex with Cdk1/cyclin B during interphase but not during mitosis., Conclusion: Our results that hCdc14A is differentially expressed in human cancer cells and that hCdc14A can interact with both p53 and the Cdk1/cyclin B complex may implicate that dysregulation of hCdc14A expression may play a role in carcinogenesis.

Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the ability of MG to cause DNA damage, cell cycle arrest in mimosine synchronised and the possible roles of Chk1, Chk2, Cdc2, Cdc25C, 14-3-3 and Cyclin B1 in control and MG transformed SHE cells in order to understand the differential mechanisms associated with G2/M checkpoint control. Exposure of MG to control and transformed cells causes DNA damage. Flow cytometric analysis of mimosine synchronised cells when exposed to MG showed an increase of G2/M phase in control cells whereas no such accumulation of cells at the G2/M phase was observed in response to MG in transformed cells. Western blots of phosphoactive forms of Chk1 and Chk2 cells showed opposing levels. Control cells treated with MG showed a decrease in Chk1 and increase in Chk2, whereas the transformed cells treated with MG showed an increase in Chk1 and decrease in Chk2. Also a decrease in Cdc25C, 14-3-3 and Cyclin B1 was observed in MG treated transformed cells, whereas MG treated control cells showed elevated levels. Stabilization of the proteins seems to be the possible mechanism. The present study indicates elevated phosphorylation of Chk1 and decreased phosphorylation of Chk2 and decreased levels of Cyclin B1 are the critical changes associated with abrogation of G2/M checkpoint control during transformation of SHE cells by MG.

Background: Ribosomal proteins are the components of ribosome, which also exhibit various secondary functions in DNA repair, apoptosis, drug resistance and proliferation. In our previous study of microarray, ribosomal protein L15 (RPL15) was identified as an upregulated gene in gastric cancer., Methods: We investigated the expression of ribosomal protein L15 in gastric cancer and the effect of RPL15 on proliferation of gastric cancer., Results: It was found that the expression of RPL15 was markedly up-regulated in gastric cancer tissues. RPL15 was also highly expressed in gastric cancer cell lines AGS, MKN45, MKN28, SGC7901 and KATOIII. Inhibition of RPL15 expression by siRNA vector transfection suppressed the growth of SGC7901 cells significantly, which was independent of the expression of Cyclin D1 and B1. Down-regulation of RPL15 expression inhibited SGC7901 cell growth in soft agar and its tumorigenicity in nude mice., Conclusion: RPL15 promotes cell proliferation and may be a potential target for anticancer therapy of gastric cancer.

We compared the profile of histone H1 kinase activity, reflecting Maturation Promoting Factor (MPF) activity in oocytes bisected at the germinal vesicle (GV) stage and allowed to mature as separate oocyte halves in vitro. Whereas the oocyte halves containing the nucleus exhibited the same profile of increased kinase activity as that typical for intact oocytes, the anuclear halves revealed strong inhibition of the increase in this activity soon after germinal vesicle breakdown (GVBD). In contrast, the profile of MAP kinase activity did not differ significantly between anuclear and nucleus-containing oocyte halves throughout maturation. Of the two MPF components, CDK1 and cyclin B1, the amount of the latter was significantly reduced in anuclear halves, a reduction due to low-level synthesis and not to enhanced degradation. Expression of three reporter luciferase RNAs constructed, respectively, to contain cyclin B1-specific 3'UTR, the globin-specific 3'UTR, or no 3'UTR sequence was enhanced in nuclear halves, with significantly greater enhancement for the construct containing cyclin B1-specific 3'UTR as compared to the two other RNAs. We conclude that the profile of activity of MPF during mouse oocyte maturation is controlled by an unknown GV-associated factor(s) acting via 3'UTR-dependent control of cyclin B1 synthesis. These results require the revision of the hitherto prevailing view that the control of MPF activity during mouse oocyte maturation is independent of GV-derived material.

Activation of the epidermal growth factor receptor (EGFR) and/or its family member(s) stimulates many processes of carcinogenesis, including cell invasion and the formation of new blood vessels, events that are critically involved in angiogenesis. Interference with the activation of EGFRs, therefore, represents a promising strategy for the development of novel and selective anticancer therapies. Previously, we reported that EGFR-related protein (ERRP), which we have isolated and characterized as a pan-erbB inhibitor, is a potential therapeutic agent for colorectal and other epithelial cancers. The present investigation was undertaken to determine whether ERRP would affect the invasion of colon cancer cells and formation of tubules, and the regulation of these processes. ERRP inhibited tubule formation by aortic endothelial cells and invasion of HCT-116 colon cancer cells through matrigel. These changes were associated with marked reductions in the synthesis and secretion of bFGF, VEGF and TGF-alpha by HCT-116 cells. Secretion of bFGF and VEGF by aortic endothelial cells was also inhibited by ERRP. Microarray analysis of ERRP-treated HCT-116 cells showed reduced levels of several growth regulatory proteins such as p21Rac1, Stratifin (14-3-3 Sigma), focal adhesion kinase (FAK) and mediators of the Ras-Raf-ERK pathway. ERRP treatments resulted in reduced expression of p21Rac1 and inhibited the constitutive activation of FAK and MEK2 in HCT-116 cells. Transfection of constitutively activate p21Rac1 or MEK2 into HCT-116 cells abrogated ERRP-induced inhibition of growth. In summary, it was demonstrated that ERRP not only inhibits cell growth, but also the processes of cell invasion and blood vessel formation that are critical for the development and progression of carcinogenesis.

Berberine has a wide range of biological actions that suggest it may be of use in cancer prevention. It was previously reported that berberine induced cell cycle arrest, not only at the G0/G1-phase, but also at the G2/M-phase in a dose-dependent manner. However, the mechanism of berberine-induced G2/M-phase arrest in leukemia cells is not fully understood. In the present study, the effects of the naturally occurring berberine (the major constituent of Coptis chinensis) on the cell cycle, as well as on CDK1, cyclin B1, 14-3-3sigma, Wee1 and Cdc25c expressions, were investigated in the human promyelocytic leukemia HL-60 cells and in the murine myelomonocytic leukemia WEHI-3 cells. The flow cytometry assays indicated that berberine induced G2/M-phase arrest in both examined cell lines. The berberine-induced G2/M-phase arrest in both examined cell lines was accompanied by increased levels of Wee1 and 14-3-3sigma, but decreased levels of Cdc25c, CDK1 and cyclin B1. However, CDK2 expression was not affected as revealed by Western blotting assay. Berberine induced G2/M arrest in both the examined cells via the inhibition of cyclin B1 and the promotion of Wee1.

The proliferation-specific Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) regulates expression of cell cycle genes essential for progression into DNA replication and mitosis. Expression of Foxm1 is found in a variety of distinct human cancers including hepatocellular carcinomas, intrahepatic cholangiocarcinomas, basal cell carcinomas, ductal breast carcinomas, and anaplastic astrocytomas and glioblastomas. In this study, we show that human Foxm1 protein is abundantly expressed in highly proliferative human non-small cell lung cancers (NSCLC) as well as in mouse lung tumors induced by urethane. To determine the role of Foxm1 during the development of mouse lung tumors, we used IFN-inducible Mx-Cre recombinase transgene to delete mouse Foxm1 fl/fl-targeted allele before inducing lung tumors with urethane. We show that Mx-Cre Foxm1-/- mice exhibit diminished proliferation of lung tumor cells causing a significant reduction in number and size of lung adenomas. Transient transfection experiments with A549 lung adenocarcinoma cells show that depletion of Foxm1 levels by short interfering RNA caused diminished DNA replication and mitosis and reduced anchorage-independent growth of cell colonies on soft agar. Foxm1-depleted A549 cells exhibit reduced expression of cell cycle-promoting cyclin A2 and cyclin B1 genes. These data show that Foxm1 stimulates the proliferation of tumor cells during progression of NSCLC.

The proliferation-specific Forkhead Box M1 (FoxM1 or FoxM1b) transcription factor is overexpressed in a number of aggressive human carcinomas. Mouse hepatocytes deficient in FoxM1 fail to proliferate and are highly resistant to developing carcinogen-induced liver tumors. We previously developed a transgenic (TG) mouse line in which the ubiquitous Rosa26 promoter was used to drive expression of the human FoxM1b cDNA transgene in all mouse cell types. To investigate the role of FoxM1b in prostate cancer progression, we bred Rosa26-FoxM1b mice with both TRAMP and LADY TG mouse models of prostate cancer. We show that increased expression of FoxM1b accelerated development, proliferation, and growth of prostatic tumors in both TRAMP and LADY double TG mice. Furthermore, development of prostate carcinomas in TRAMP/Rosa26-FoxM1b double TG mice required high levels of FoxM1 protein to overcome sustained expression of the alternative reading frame tumor suppressor, a potent inhibitor of FoxM1 transcriptional activity. Depletion of FoxM1 levels in prostate cancer cell lines PC-3, LNCaP, or DU-145 by small interfering RNA transfection caused significant reduction in proliferation and anchorage-independent growth on soft agar. This phenotype was associated with increased nuclear levels of the cyclin-dependent kinase inhibitor protein p27(Kip1) and diminished expression of S-phase promoting cyclin A2 and M-phase promoting cyclin B1 proteins. Finally, we show that elevated levels of FoxM1 protein correlate with high proliferation rates in human prostate adenocarcinomas. Our results suggest that the FoxM1 transcription factor regulates development and proliferation of prostate tumors, and that FoxM1 is a novel target for prostate cancer treatment.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent non-Hodgkin's lymphoma in the Western world; it is characterized by marked genetic, morphological and clinical heterogeneity. The identification of prognostic markers could help to develop risk-adapted treatment strategies. As proliferation of cells is essential for tumour growth, analysis of the cell cycle and its individual phases might give additional information on tumour progression and clinical behaviour. To investigate the prognostic value of proteins specifically related to the cell cycle phases S, G2 and M in DLBCL, the expression of cyclin A as a marker of S phase and cyclin B1 as a G2/M phase marker were analysed in combination with other clinicopathological parameters in a large cohort of patients. Expression of cyclin B1 and cyclin A were determined by immunohistochemistry using tissue microarray methodology. Immunoreactivity for cyclin B1 and cyclin A was correlated with clinical data using a two-sided Fisher's exact test. Impact on overall survival was analysed by the Kaplan-Meier method. A negative prognostic impact was found for expression of cyclin B1. Nuclear and/or cytoplasmic staining in > or = 1% of tumour cells was significantly associated with shorter overall survival in the multivariate analysis (p=0.008). Furthermore, the prognostic impact of cyclin B1 expression was independent of the tumour stage. No prognostic significance was found for expression of cyclin A. In conclusion, this study demonstrates the independent prognostic value of an expression of cyclin B1 in DLBCL and proposes its evaluation as a prognostic marker in the assessment of this entity which is easily applicable in daily routine practice.

Invasion of tumor cells into adjacent brain areas is one of the major problems in treatment of glioma patients. To identify genes that might contribute to invasion, fluorescent F98 glioma cells were allowed to invade an organotypic brain slice. Gene expression analysis revealed 5 up-regulated and 14 down-regulated genes in invasive glioma cells as compared to non-invasive glioma cells. Two gene products, ferritin and cyclin B1, were verified in human gliomas by immunohistochemistry. Ferritin exhibited high mRNA levels in migratory F98 cells and also showed higher protein expression in the infiltrating edge of human gliomas. Cyclin B1 with high mRNA expression levels in stationary F98 cells showed marked protein expression in the central portions of gliomas. These findings are compatible with the concept of tumor cells either proliferating or migrating. Our study is the first to apply brain slice cultures for the identification of differentially regulated genes in glioma invasion.

The cell commits to dividing during the G2-M transition, and timing of mitotic entry must be tightly regulated to ensure correct chromosome segregation. Identification of all proteins and molecular events that orchestrate the G2-M transition will be required for a complete understanding of the cell division cycle, and how its deregulation contributes to cell transformation. We have previously reported D53, a member of the tumor protein D52 family, to be a novel 14-3-3 partner protein in breast cancer cells. We now report that D53 expression is highly upregulated at the G2-M transition in breast cancer cell lines in which D53 is endogenously or exogenously expressed. The timing and subcellular localization of D53 expression paralleled that of cyclin B1, and D53 expression was similarly regulated at both post-transcriptional and post-translational levels. Interactions between D53 and 14-3-3, a negative regulator of the G2-M transition, were increased in synchronized populations enriched for cells in G2/M phases, compared with G1/S arrested cells. Enforced expression of two EGFP-tagged D53 isoforms and the related protein D52 produced high proportions of multinucleated MDA-MB-231 breast carcinoma cells. These results identify D53 as a cell cycle-regulated protein whose deregulated expression can adversely affect the completion of mitosis.

An immunohistochemical method for assessing cell cycle phase distribution in neurosurgical biopsies would enable such data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in glial neoplasms, without the requirement for flow cytometric analysis. Paraffin-embedded sections of intracerebral gliomas (n = 48), consisting of diffuse astrocytoma (n = 9), anaplastic astrocytoma (n = 8) and glioblastoma (n = 31), were analysed by immunohistochemistry using markers of cell cycle entry, Mcm-2 and Ki67, and putative markers of cell cycle phase, cyclins D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis). Double labelling confocal microscopy confirmed that the phase markers were infrequently coexpressed. Cell cycle estimations by immunohistochemistry were corroborated by flow cytometric analysis. There was a significant increase in Mcm-2 (P < 0.0001), Ki67 (P < 0.0001), cyclin A (P < 0.0001) and cyclin B1 (P = 0.002) expression with increasing grade from diffuse astrocytoma through anaplastic astrocytoma to glioblastoma, suggesting that any of these four markers has potential as a marker of tumour grade. In a subset of glioblastomas (n = 16) for which accurate clinical follow-up data were available, there was a suggestion that the cyclin A:Mcm-2 labelling fraction might predict a relatively favourable response to radical radiotherapy. These provisional findings, however, require confirmation by a larger study. We conclude that it is feasible to obtain detailed cell cycle data by immunohistochemical analysis of tissue biopsies. Such information may facilitate tumour grading and may enable information of prognostic value to be obtained in the routine diagnostic laboratory.

Objective: To investigate the influence of aerosols on the expression of cyclin B(1), cyclin C and proliferating cell nuclear antigen (PCNA) in wound tissue healing of burned rat models., Methods: Sprague Dawley (SD) rats were inflicted as the deep partial thickness burn models. Rats were randomly divided into experimental group and control group. The experimental group were treated with aerosols. Samples were collected in 1 approximately 10 postburn days. Immunohistochemistry and image analysis methods were conducted to examine the expression of cyclin B(1), cyclin C and PCNA in both experimental and control groups., Results: The expression of cyclin C in experimental group was detected in nucleus of skin basal cell on the second postburn day, increased evidently at the fifth days and sustained at high expression level up to the tenth days after treatment. The expression of cyclin C in experimental group was significantly higher than control group (P < 0.05). The expression of PCNA was first observed in skin basal cell nucleus and hair follicle cell nucleus in both experimental and control group on the third postburn day. The expression of PCNA increased evidently at the fifth days in experimental after treatment and that increased evidently at the seventh days in control group, which showed there were lots of active proliferation cell. And the difference of the expression of PCNA between experimental and control group was significant (P < 0.01). The expression of cyclin B(1) was detected in nucleus and cytoplasm of skin basal cell in both groups on the third postburn day, and no difference between the experimental and control group (P > 0.05)., Conclusions: Aerosols can up-regulate the expression of cyclin C and PCNA in skin basal cell nucleus. Therefore the aerosols can accelerate wound tissue healing.

Screening of cdc mutants of fission yeast for those whose cell cycle arrest is independent of the DNA damage checkpoint identified the RNA splicing-deficient cdc28 mutant. A search for mutants of cdc28 cells that enter mitosis with unspliced RNA resulted in the identification of an orb5 point mutant. The orb5+ gene, which encodes a catalytic subunit of casein kinase II, was found to be required for cell cycle arrest in other mutants with defective RNA metabolism but not for operation of the DNA replication or DNA damage checkpoints. Loss of function of wee1+ or rad24+ also suppressed the arrest of several splicing mutants. Overexpression of the major B-type cyclin Cdc13p induced cdc28 cells to enter mitosis. The abundance of Cdc13p was reduced, and the phosphorylation of Cdc2p on tyrosine 15 was maintained in splicing-defective cells. These results suggest that regulation of Cdc13p and Cdc2p is required for G2 arrest in splicing mutants.

The AT specific minor grove DNA binding ligands bisbenzimidazole derivatives like hoechst-33342 and hoechst-33258 which scavenge free radicals and stabilize macromolecular structure have been shown to afford radioprotection by reducing the induction of DNA damage. However, their ability to inhibit topoisomerases I & II, which play important roles in damage response pathways including DNA repair can enhance radiation damage under certain conditions. Since pool sizes of the topoisomerases differ not only between normal and tumor cells, but also among different tumors, it is anticipated that radiosensitization by hoechst-33342 can vary among tumors. The present studies were, therefore, undertaken to verify this proposition in human glioma (BMG-1 &U-87) and squamous carcinoma (4197 &4451) cell lines which differ in their biological behavior (ploidy, p53, cyclins, bcl, bax etc). Isotoxic concentrations of hoechst-33342 (IC50 i.e producing 50% cell kill) administered immediately following irradiation resulted in the radiosensitization of all cell lines, with a 4&7 fold increase in the cell death (loss of clonogenic cell survival) in U-87&BMG-1 and a 3 fold increase in 4197 &4451 cells. Growth inhibition and increase in cytogenetic damage (micronuclei formation) as well as delayed apoptosis observed under these conditions corroborated well with the enhanced cell death. The ligand induced a significant cell cycle delay, particularly in the late S and G2 phases of BMG-1, U-87 and 4197 cells, while no significant changes could be observed in 4451 cells. Higher endogenous levels of cyclin B1 found in both the glioma cell lines, was enhanced further by the ligand as compared to the squamous carcinoma cells. These results clearly demonstrate that the radiosensitizing effects of the ligand are indeed heterogeneous among different human tumor cell lines. The radiaosensitization is p53 independent and accompanied by enhanced mitotic death (linked to cytogenetic damage) as well as induction of cyclin B1 mediated apoptosis.

Myostatin inhibits myoblast proliferation and differentiation in developing muscle. Mounting evidence suggests that myostatin also plays a limiting role in growth/repair/regeneration of differentiated adult muscle by inhibiting satellite cell activation. We tested the hypothesis that myostatin mRNA expression would decrease after resistance loading (RL) with a blunted response in older (O) females (F) who have shown minimal hypertrophy [vs. males (M)] after long-term RL. As myostatin is thought to modulate cell cycle activity, we also studied the response of gene transcripts key to stimulation (cyclin B1 and D1) and inhibition (p21cip and p27kip) of the cell cycle, along with the muscle-specific load-sensitive mitogen mechano-growth factor (MGF). Twenty young (Y; 20-35 yr, 10 YF, 10 YM) and 18 O (60-75 yr, 9 OF, 9 OM) consented to vastus lateralis biopsy before and 24 h after a bout of RL (3 sets x 8-12 repetitions to volitional fatigue of squat, leg press, knee extension). Gene expression levels were determined by relative RT-PCR with 18S as an internal standard and analyzed by age x gender x load repeated-measures ANOVA. A load effect was found for four transcripts (P < 0.005) including myostatin, cyclin D1, p27kip, and MGF as mRNA levels decreased for myostatin (-44%) and p27kip (-16%) and increased for cyclin D1 (34%) and MGF (49%). For myostatin, age x load and gender x load interactions (P < 0.05) were driven by a lack of change in OF, while marked declines were noted in YM (-56%), YF (-48%), and OM (-40%). Higher cyclin D1 levels in OF led to a main age effect (36%, O > Y) and an age x gender interaction (66%, OF > YF vs. 10%, OM > YM; P < 0.05). An age x gender x load interaction (P < 0.05) for cyclin D1 resulted from a 48% increase in OF. Post hoc testing within groups revealed a significant increase in MGF after RL in YM only (91%, P < 0.05). Higher levels of cyclin B1 in O (27%, O > Y) led to a main age effect (P < 0.05). An age x load interaction for cyclin B1 (P < 0.05) was driven by a 26% increase in Y with no change in O after RL. No age or gender differences, or load-mediated changes, were detected in levels of p21cip mRNA expression. These data clearly demonstrate that RL downregulates myostatin expression and alters genes key to cell cycle progression. However, failure to reduce myostatin expression may play a role in limiting RL-induced hypertrophy in OF.

Background and Purpose: To elucidate the role of TP53 on differential effects of topoisomerase I inhibitor topotecan (Hycamtin on radiation sensitivity., Materials and Methods: Cell cycle distribution and protein expression of TP53, p21(WAF1/CIP1) and cyclin B was studied in CCD32 lung fibroblasts, glioblastoma cell lines U118 (mutant TP53), and U87 (wildtype TP53) after treatment with topotecan (0.05 and 1 microM) and/or ionizing radiation (2 Gy)., Results: Cell cycle effects varied with topotecan concentration, resulting in G1 arrest (1 microM), or S/G2/M arrest (0.05 microM), and was modified differentially in fibroblasts and in glioblastoma cells in combination with irradiation. Phosphorylation of TP53 and expression of p21(WAF1/CIP1) was induced by IR and/or topotecan in CCD32 cells, and in U118 cells after topotecan treatment, accompanied by cyclin B degradation. In U87 cells only 1 microM topotecan generated phosphorylation of TP53 and p21(WAF1/CIP1) expression; 0.05 microM caused stabilization of cyclin B., Conclusions: The antagonistic effect of combined topotecan/irradiation treatment in fibroblasts was most likely due to an immediate radiation induced G1 arrest, but was not observed in p53 wildtype glioblastoma cells. Thus, the impact of TP53 on the topotecan response remains indistinct, and is obviously influenced by other genomic alterations acquired by tumor cells.

Organisms living in an aerobic environment are continuously exposed to reactive oxygen species (ROS). Apoptosis of cells can be induced by ROS and cells also develop negative feedback mechanisms to limit ROS induced cell death. In this study, RAW264.7 murine macrophage cells were treated with H(2)O(2) and cDNA microarray technique was used to produce gene expression profiles. We found that H(2)O(2) treatment caused up-regulation of stress, survival and apoptosis related genes, and down-regulation of growth and cell cycle promoting genes. Numerous genes of metabolism pathways showed special expression patterns under oxidative stress: glycolysis and lipid synthesis related genes were down-regulated whereas the genes of lipid catabolism and protein synthesis were up-regulated. We also identified several signaling molecules as ROS-responsive, including p53, Akt, NF-kappa B, ERK, JNK, p38, PKC and INF-gamma . They played important roles in the process of apoptosis or cell survival. Finally, an interactive pathway involved in cellular response to oxidative stress was proposed to provide some insight into the molecular events of apoptosis induced by ROS and the feedback mechanisms involved in cell survival.

Background: To better understand the relationship between mitotic delay and the disruption of cyclin B1 and p21 in x-irradiated fibroblasts, studies were carried out to establish correlations between the downregulation of cyclin B1 by the cyclin kinase inhibitor (CKI) p21 and the induction of mitotic delay in the NIH3T3 fibroblast., Materials and Methods: Cell cycle kinetics were used to analyze mitotic delay in irradiated NIH3T3 cells and immunocytochemistry incorporated to assess the expression of cyclin B1 and p21, following 2 or 4 Gy x-irradiation., Results and Discussion: Results indicate a dose dependent increase in mitotic delay accompanied by a downregulation of cyclin B1 and corresponding upregulation of the CKI p21 in exponentially growing cultures. Data indicates that the induction of radiation-induced division delay appears to be dependent on the p21 inhibition of cyclin B1 and, furthermore, p21 and cyclin B1 expression are highly dependent on cell density.

There is accumulating evidence that Hodgkin's and Reed-Sternberg cells of classical Hodgkin's lymphomas (cHL) display multiple and concurrent alterations in different pathways and checkpoints of the cell cycle. However, the expression of cyclin D2 and its relation to other major cell cycle proteins has not been analyzed in cHL. The aim of the present study was to assess expression of cyclin D2, Ki67, cyclin A, cyclin B1, cyclin D1, cyclin D3, cyclin E, p53, Rb, p16 and p27 proteins in order to gain further insight into the proliferation profile of cHL. Overexpression of cyclin D2 in Hodgkin's and Reed-Sternberg cells was detected in 64/89 (72%) cases of cHL. This finding, in view of recent in vitro data showing that constitutive activation of nuclear factor (NF)-kB could upregulate cyclin D2 expression in part via signal transducer and activator of transcription (STAT)-5a, suggests that induction of cyclin D2 expression may support the proliferation of Hodgkin's and Reed-Sternberg cells. In addition, the present study showed that (1) increased p27 expression status was significantly correlated with higher levels of cyclin A expression (P=0.048) and (2) increased p53 expression status was significantly correlated with higher levels of cyclin A (P<0.001) and cyclin B1 (P=0.040) expression. The association between increased p27 and p53 expression status and higher expression levels of G2/M cyclins suggests that the impairment of the growth inhibitory activity of the p27 and p53 tumor suppressor pathways may promote the proliferation of Hodgkin's and Reed-Sternberg cells.

A total of 111 pulmonary neuroendocrine tumors comprising 13 typical carcinoids, five atypical carcinoids, 44 large-cell neuroendocrine carcinomas and 49 small-cell carcinomas were immunohistochemically studied for dysregulated cyclin B1 expression and disruption of the Rb/p16/cyclin D1 pathway (Rb pathway), and the results were correlated with tumor proliferation activity and clinical outcome. Overexpression of cyclins B1 and D1, respectively, was detected in no and 15% typical carcinoids, 20 and 20% atypical carcinoids, 84 and 32% large-cell neuroendocrine carcinomas, 84 and 10% small-cell carcinomas. Loss of Rb and p16 expression, respectively, was observed in no and 14% typical carcinoids, no and 40% atypical carcinoids, 49 and 18% large-cell neuroendocrine carcinomas, 84 and 8% small-cell carcinomas. In summary, 29% typical carcinoids, 20% atypical carcinoids, 78% large-cell neuroendocrine carcinomas and 93% small-cell carcinomas had Rb pathway aberrations. Rb pathway aberration was mostly attributed to Rb loss in small-cell carcinomas, while p16 loss and/or cyclin D1 overexpression besides Rb loss also played an important role in large-cell neuroendocrine carcinomas, while cyclin D1 overexpression was the only cause of Rb pathway aberration in carcinoid tumors. Thus, both cyclin B1-associated G2/M arrest and Rb-mediated G1 arrest are consistently compromised in high-grade large-cell neuroendocrine carcinoma and small-cell carcinoma, but are generally intact or occasionally altered in carcinoid tumor; the mechanisms involved in Rb pathway aberration among the tumor categories are different, reflecting a genetic divergence among the individual tumor categories. Cyclin B1 expression closely correlated with the Ki-67 labeling index either in the individual tumor categories or overall tumors (P < 0.0001, r = 0.742), suggesting that cyclin B1 is one of the key factors regulating cell proliferation in pulmonary neuroendocrine tumors. Neither cyclins B1 and D1, Rb, p16, nor Ki-67 correlated with patient survival in individual tumor categories, suggesting that the prognostic significance of these factors is tumor-type specific.

Because proliferation of eukaryotic cells requires cell cycle-regulated chromatid separation by the mitotic spindle, it is subject to regulation by mitotic checkpoints. To determine the mechanism of the antiproliferative activity of the flavoprotein-specific inhibitor diphenyleneiodonium (DPI), I have examined its effect on the cell cycle and mitosis. Similar to paclitaxel, exposure to DPI causes an accumulation of cells with a 4N DNA content. However, unlike the paclitaxel-mediated mitotic block, DPI-treated cells are arrested in the cell cycle prior to mitosis. Although DPI-treated cells can arrest with fully separated centrosomes at opposite sides of the nucleus, these centrosomes fail to assemble mitotic spindle microtubules and they do not accumulate the Thr(288) phosphorylated Aurora-A kinase marker of centrosome maturation. In contrast with paclitaxel-arrested cells, DPI impairs cyclin B1 accumulation. Release from DPI permits an accumulation of cyclin B1 and progression of the cells into mitosis. Conversely, exposure of paclitaxel-arrested mitotic cells to DPI causes a precipitous drop in cyclin B and Thr(288) phosphorylated Aurora-A levels and leads to mitotic catastrophe in a range of cancerous and noncancerous cells. Hence, the antiproliferative activity of DPI reflects a novel inhibitory mechanism of cell cycle progression that can reverse spindle checkpoint-mediated cell cycle arrest.

Background: To understand the molecular response of tumor cells to therapeutic ionizing radiation (IR), we previously reported that human breast cancer cells derived following chronic exposure to fractionated ionizing radiation (MCF+FIR) showed a transient radioresistance. MCF+FIR cells also demonstrated increased activity of NF-kappaB, increased expression of the mitochondrial antioxidant enzyme (MnSOD), and increased expression of a cell cycle regulatory protein (Cyclin B1). The present studies were designed to determine the relationship of NF-kappaB, MnSOD and Cyclin B1 expression in cellular adaptive responses to ionizing radiation., Materials and Methods: The first intron of the cyclin B1 gene with a putative NF-kappaB element was cloned into the pGL3 luciferase reporter (pGL3CB1EI1). PGL3CB1EI1 and control NF-kappaB luciferase activities were determined in MCF-7 and MCF+FIR cells treated with a single dose of radiation, over expression of the dominant negative mutant IkB (mIkB) or over expression of the SOD2 gene., Results: MCF+FIR cells derived from fractionated IR demonstrated increased transactivation of the pGL3CB1EI1 and NF-kappaB controlled reporter activities, relative to the parental cell line. Transfection of dominant negative mutant IkB that inhibits NF-kappaB nuclear translocation, inhibited pGL3CB1EI1 and NF-kappaB activity, indicating the NF-kappaB dependence of pGL3CB1EI1 mediated transcription. In addition, over expression of the human SOD2 gene (MnSOD) inhibited NF-kappaB and pGL3CB1EI1 activity, indicating that superoxide or some species derived from superoxide may have participated in the up-regulation of reporter activity in response to chronic exposure to fractionated ionizing radiation. These results provide evidence suggesting that a signaling pathway involving NF-kappaB and Cyclin B1 may contribute to adaptive radioresistance induced by chronic exposure to fractionated IR and support the conclusion that MnSOD appears to be a negative regulator of this pathway.