Cells In Mitotic Phase Research Articles

Human breast tumor derived endothelial cells exhibit distinct biological properties.

Excessive angiogenesis characterized by leaky, tortuous, and chaotic vasculature is one of the hallmarks of cancers and is significantly correlated to poor prognosis. Disorganized angiogenesis leads to poor perfusion of anti-cancer drugs and limits access to immune cells. Hence, impeding angiogenesis is one of the attractive therapeutic targets to inhibit progression and metastasis in several solid tumors including breast. We have developed a robust and reproducible method for isolating and ex vivo culture of endothelial cells (EC) derived from non-malignant (Endo-N) and malignant (Endo-T) part from clinically characterized human breast tumors. RT-PCR and immunoblotting analysis indicated that these cells exhibited expression of endothelial specific genes such as PECAM-1 (CD31), Endoglin (CD105), eNOS, VE-cadherin, VCAM1, and MCAM. Vasculogenic mimicry and contamination of progenitor EC recruited in tumors was ruled out by absence of CD133 expression and normal karyotype. Both the cell types showed stable expression of CD31 and CD105 up to seven passages. Furthermore, compared to Endo-N cells, Endo-T cells showed (a) constitutively increased proliferation marked by nearly 36% of cells in mitotic phase, (b) requirement of glutamine for cell survival, (c) pro-migratory phenotype, (d) produced increased number of sprouts in 3D cultures, and (e) resistance to sorafenib. Tumor derived EC showed distinct biological properties compared to normal breast EC. Our method for isolating endothelial cell types from human breast tumors may be explored to (a) understand cellular and molecular mechanisms, (b) screen anti-angiogenic molecules, and (c) formulate organoid cultures to develop personalized medicine facilitating better clinical management of breast cancers.

The effects of different stimulated macrophages on HaCaT proliferation and secretion.

Background-As an important cell of innate immunity, macrophages secrete a large number of inflammatory cytokines, including TNF-α, which may play crucial role in the initiation stage of psoriatic inflammation. Keratinocytes are the effector cells of psoriasis and may produce circulatory stimulation networks with other immune cells in the lesions. Understanding the effect of macrophages induce keratinocytes’ biological function is necessary to understand the mechanisms of psoriasis. Objectives-To evaluate the effects of different polarized macrophages on the proliferation, differentiation, apoptosis and secretion of immortalized human keratinocytes (HaCaT). Materials and Methods-Establish co-culture system of macrophages and HaCaT in vitro to mimic macrophage infiltration microenvironment of psoriasis skin. The proliferation of HaCaT was detectd by CCK8, Cell cycle. Apoptosis of HaCaT was analysed by flow cytometry. The secretion of IL-1β, TNF and IL-6 by HaCaT was analysed by ELISA. Results-The results showed a decreased proliferation of HaCaT when co-cultured with LPS-induced macrophage and upregulated IL-6, IL-1β and TNF-α. While more HaCaT cells were blocked in the G0/G1 phase, the number of mitotic phase cells decreased and apoptotic cells increased. On the contrast, HaCaT showed increased proliferation when co-cultured with IL-4-induced macrophage, while no difference in apotosis of HaCaT. Moreover, proinflammatory cytokine TNF-α and IL-12 production by LPS-induced macrophage were significantly increased. Conclusion-Considering the changes in the biological function of HaCaT cells caused by the infiltration microenvironment of macrophages, these findings indicate that the polarization process of macrophage is a crucial mechanism in the development of psoriasis, and the underlying immunological mechanisms should be understood.

The Novel Nature Microtubule Inhibitor Ivalin Induces G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma SMMC-7721 Cells In Vitro.

Background and Objectives: Microtubules are an attractive target for cancer chemotherapy. Previously, we reported that Ivalin exhibited excellent anti-migration and anti-invasion activities in human breast cancer cells. Here, we examined the microtubule inhibition effect of Ivalin in human hepatocellular carcinoma SMMC-7721 cells. Materials and Methods: We used the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the cell proliferation effect of Ivalin and flow cytometry analysis to detect the apoptotic and cell cycle arrest effects of Ivalin. Immunofluorescence staining was used to measure the effect of Ivalin on the cytoskeleton network, and Western blotting was used to detect the expression levels of Bax, Bcl-2, Cdc2, phosphor-Cdc2, Cdc25A, Cyclin B1, and tubulin. Results: Ivalin induced cell cycle G2/M arrest and subsequent triggered apoptosis in human hepatocellular carcinoma SMMC-7721 cells. Furthermore, microtubules were shown to be involved in Ivalin-meditated apoptosis. In this connection, Ivalin treatment suppressed cellular microtubule network formation by regulating microtubule depolymerization. Moreover, Western blotting revealed Cdc25A and Cyclin B1 were upregulated in Ivalin-meditated cell cycle arrest. Subsequently, the induction of Bax (a proapoptotic protein) and reduction of Bcl-2 (an anti-apoptotic protein) expression were observed in Ivalin-treated SMMC-7721 cells. Conclusion: Ivalin induced microtubule depolymerization, then blocked cells in mitotic phase, and eventually resulted in apoptosis in SMMC-7721 cells. Collectively, these data indicate that Ivalin, acting as a novel inhibitor of microtubules, could be considered as a promising lead in anticancer drug development.

Cytological and phytochemical studies on Mesembryanthemum nodiflorum L.

Mesembryanthemum nodiflorum L. (Aizoaceae) has long been used as food and in traditional medicine. This study was intended to explore the active groups within M. nodiflorum ethanolic extract and to study the cytological effects of M.nodiflorum water extract on Allium cepa L. root tips. Phytochemical analysis of M.nodiflorum ethanolic extract indicated the presence of alkaloids and triterpenes. Tannins, flavonoids and saponins did not found in M.nodiflorum alcoholic extract. Mitotic indices and distribution of cells in mitotic phases of A.cepa root tips were clearly changed after treatment with three different concentration of M.nodiflorum water extract (0.1, 1 and 3%) for 3h, 6h, and 12h. The maximum value of mitotic index 2.68% was observed after treatment with 3% for 6h, while the minimum value 1.35% was scored after 12h exposure to the same concentration. Different types of chromosomal aberrations were noticed. The present study revealed that M.nodiflorum water extract has a potent inhibitory effect on the mitotic activity of A.cepa root tip cells.

DNA-cytometric characteristics of recurrent soft tissue sarcomas

Aim. To determine the content of deoxyribonucleic acid (DNA) and distribution of cells in mitotic phases in patients with recurrent soft tissue sarcomas for the assessment of malignancy of the process. Methods. Tumor tissues of patients with recurrent soft tissue sarcomas were studied. Research methods included histological, DNA-cytometric and statistical methods. Results. Proliferative activity and proliferative index of recurrent sarcomas differed depending on the tumor grade and stage. Differences in the number of diploid, aneuploid and polyploid cells were determined in each group and between the groups depending on the cell cycle phases. Cell cycle parameters were as following: 100% of G1 (well-differentiated) cancer were diploid, as well as 33.3% of G2 (moderately differentiated) and 15% of G3 (poorly differentiated) tumors. Aneuploid tumors prevailed in G2 and G3, the ratio of which was 66.7 and 85%, respectively. The analysis of kinetic parameters of the cell cycle allowed establishing a decrease in the number of cells in G1/G0 phase of the cell cycle from G1 to G3, which was accompanied by a statistically significant increase in the proportion of cells in S-phase (p ˂0.05). Conclusion. The DNA-cytometric study of cell cycle parameters showed high biological potential of recurrent soft tissue sarcomas, which was determined by two indices - the proportion of cells in G2+M-phase and the cell loss factor; 100% of well-differentiated (G1) tumors, 33.3% of moderately differentiated (G2) and 15% of poorly differentiated (G3) tumors were diploid; aneuploid tumors prevailed in G2 and G3.

Effects of miR-126 on the STAT3 signaling pathway and the regulation of malignant behavior in lung cancer cells.

The functions of miR-126-mediated signal transducers and activators of the transcription 3 (STAT3) signal pathway were investigated in regulating the behavior of cells in non-small cell lung cancer (NSCLC). Cultured NSCLC A549 cells were transfected with empty, miR-126 overexpression or miR-126 knocked-down expression plasmids. After transfection efficiency verification by reverse transcription polymerase chain reaction (RT-PCR) and culture for 24 h, methyl thiazolyl tetrazolium (MTT) was applied to detect cell proliferation rate, migration distance was measured in scratch assays, cell cycle was determined through flow cytometry, the mRNA expression level of caspase-3 in cells was detected using RT-PCR and protein expression levels of STAT3 were detected using western blotting. Our results showed the cell proliferation rate was significantly higher in cells of the overexpression group than that in those of the control group (p<0.05) and the rate in the cells of the low-expression group was the lowest among the three groups (p<0.05). The migration distance of the overexpression group cells was significantly longer than that in the control group cells and the shortest migration distance was found in the low-expression group cells (p<0.05). The amount of cells in mitotic phase in the overexpression group was significantly higher than that in the control group and the same amount in the low-expression group was the lowest (p<0.05). The mRNA expression level of caspase-3 of cells in the overexpression group was significantly lower than that of cells in the control group and the highest expression level was found in the low-expression group (p<0.05). Finally, the protein expression levels of STAT3 in cells in the overexpression group were significantly lower than those in the control group and the highest expression levels were identified in the low-expression group (p<0.05). Based on our findings, the cancer-promoting miR-126 can mediate the activation of the STAT3 signal pathway to regulate the malignant biological behavior of NSCLC cells affecting their proliferation, migration, cycle and apoptosis susceptibility.

Synthesis, structural studies, and cytotoxic evaluation of novel ursolic acid hybrids with capabilities to arrest breast cancer cells in mitosis

Some novel chemically modified frameworks of ursolic acid have been designed and synthesized. The key step was the cycloaddition of azidopropyl-3β-hydroxy-urs-12-en-28-oate with the appropriate C28 propargyl esters of ursolic, corosolic, asiatic, oleanolic, and betulinic acid under Click reaction conditions, and the products were obtained in 74–84% yields. In view of their intriguing structural diversity, they have been subjected to detailed 1D and 2D NMR studies and their structures are thoroughly assigned. The synthesized compounds were screened for their anticancer potential against two human breast cancer cell lines (MCF-7 & MDA-MB-231) using sulforhodamine B cell proliferation assay. The GI50 data revealed that the synthesized compounds exhibit highly potent activities against the two tested cell lines. Interestingly, the synthesized compounds showed selectivity and higher activity against MDA-MB-231 cell line than MCF-7. Among the tested compounds, compound 17 is the most potent one with GI50 value of 1.4 ± 0.1 μM and showed 2.9 times more activity than the standard doxorubicin against MDA-MB-231. In addition, 17 arrests cells in mitotic phase of cell cycle, resulting in a change in cell phenotype. In view of the selective and highly promising activity against breast cancer cell lines, these compounds can serve as promising leads for further development.

A re-evaluation of anti-NA-14 antibodies in patients with primary Sjögren’s syndrome: Significant role of interferon-γ in the production of autoantibodies against NA-14

Novel autoantibodies against nuclear antigen of 14 kDa (NA-14)/Sjögren’s syndrome nuclear antigen-1 (SSNA-1) are predominantly recognized in sera of patients with primary Sjögren’s syndrome (pSS). However, the detailed characteristics of the anti-NA-14 antibody remain unknown. Here, we sought to clarify the characteristics of anti-SSNA-1/NA-14 antibodies and the mechanisms of autoantibody production using sera from patients with connective tissue diseases (including pSS), autoimmune sera reacting with standard autoantigens (SS-A/Ro and/or SS-B/La, ds DNA, Scl-70 and Jo-1), and normal healthy controls (NHCs). Anti-NA-14 antibodies were predominantly recognized in sera from patients with pSS and in autoimmune sera reacting with thSS-A/Ro and/or -SS-B/Lo. Indirect immunofluorescence analysis showed that NA-14 was strongly expressed in mitotic-phase cells. Patients with pSS having anti-NA-14 antibodies exhibited significant elevation of serum IP-10 and BAFF compared to that in patients with pSS without anti-NA-14 antibodies and NHCs. Thus, our data demonstrated that anti-NA-14 antibodies could be classified as novel autoantibodies reacting with mitosis-related autoantigens predominantly recognized in pSS. Moreover, interferon-γ played an important role in the production of anti-NA-14 autoantibodies as patients with pSS having anti-NA-14 antibodies exhibited increased serum levels of IP-10 and BAFF.

In vitroandin vivogenotoxicity and molecular response of silver nanoparticles on different biological model systems

AbstractIn vitro and in vivo experiments on human cell lines (breast adenocarcinoma, MCF-7, and liver hepatocellular carcinoma, HepG2), plants (Allium cepa) and bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) displayed cytotoxic and genotoxic impacts of silver nanoparticles (AgNPs). The cell viability assays indicated that AgNPs had inhibitory effects on MCF-7 cell line more effectively than HepG2 cell line. Mitotic indices and distribution of cells in mitotic phases of A. cepa were clearly changed after treatment with three different weight ratios of silver metal (1, 3 and 5%) for 3 and 12 h. With increasing concentration of the nanoparticles a decrease in the mitotic index was detected (from 4.19 to 0.79 after 3 h and from 4.56 to 0.25 after 12 h) and different types of chromosomal aberrations were noticed. Protein electrophoresis results of S. aureus and P. aeruginosa demonstrated the change in the protein profiles of the two bacterial strains and it was more noticeable in P. aeruginosa tha...

Gastric-and-Intestinal Mixed Intestinal Metaplasia Is Irreversible Point with Eradication of &amp;lt;i&amp;gt;Helicobacter pylori&amp;lt;/i&amp;gt;

Helicobacter pylori (H. pylori) represents an important factor in the development of atrophic gastritis, intestinal metaplasia (IM), and gastric cancer. Eradication of H. pylori has been reported to prevent gastric cancer only in cases without atrophy or IM. However, histological changes with eradication have yet to be fully clarified. We evaluated 38 H. pylori-positive cases before and after eradication at the gland level; pyloric glands were classified as showing gastric proper (G) and IM gland types, with the latter including gastric-and-intestinal mixed IM (GI-IM) and solely intestinal IM (I-IM), depending on the remaining gastric phenotypes. On eradication, acute and chronic inflammation attenuated rapidly and gradually, respectively, whereas levels of MUC5AC and MUC6 expression were not markedly altered. Gland width, size of nuclei and cytoplasm and their ratio in surface foveolar epithelium, the number of Ki-67-positive cells and the length of the proliferating zone in each gland were significantly decreased in G glands after eradication compared with those in GI-IM and I-IM. The number of mitotic phase cells, positive for phosphorylated histone H3 at serine 28, was increased in both types of IM compared to that in G glands in the H. pylori-infected state, but unexpectedly remained unchanged with eradication. These results suggest that GI-IM, as the beginning of IM, could represent a histological irreversible point with eradication and be considered as a “histological point of no return”.

Ansamitocin P3 Depolymerizes Microtubules and Induces Apoptosis by Binding to Tubulin at the Vinblastine Site

Maytansinoid conjugates are currently under different phases of clinical trials and have been showing promising activity for various types of cancers. In this study, we have elucidated the mechanism of action of ansamitocin P3, a structural analogue of maytansine for its anticancer activity. Ansamitocin P3 potently inhibited the proliferation of MCF-7, HeLa, EMT-6/AR1 and MDA-MB-231 cells in culture with a half-maximal inhibitory concentration of 20±3, 50±0.5, 140±17, and 150±1.1 pM, respectively. Ansamitocin P3 strongly depolymerized both interphase and mitotic microtubules and perturbed chromosome segregation at its proliferation inhibitory concentration range. Treatment of ansamitocin P3 activated spindle checkpoint surveillance proteins, Mad2 and BubR1 and blocked the cells in mitotic phase of the cell cycle. Subsequently, cells underwent apoptosis via p53 mediated apoptotic pathway. Further, ansamitocin P3 was found to bind to purified tubulin in vitro with a dissociation constant (Kd) of 1.3±0.7 µM. The binding of ansamitocin P3 induced conformational changes in tubulin. A docking analysis suggested that ansamitocin P3 may bind partially to vinblastine binding site on tubulin in two different positions. The analysis indicated that the binding of ansamitocin P3 to tubulin is stabilized by hydrogen bonds. In addition, weak interactions such as halogen-oxygen interactions may also contribute to the binding of ansamitocin P3 to tubulin. The study provided a significant insight in understanding the antiproliferative mechanism of action of ansamitocin P3.

Design, Synthesis, and Biological Evaluation of (E)-N-Aryl-2-arylethenesulfonamide Analogues as Potent and Orally Bioavailable Microtubule-Targeted Anticancer Agents

A series of novel (E)-N-aryl-2-arylethenesulfonamides (6) were synthesized and evaluated for their anticancer activity. Some of the compounds in this series showed potent cytotoxicity against a wide spectrum of cancer cell-lines (IC50 values ranging from 5 to 10 nM) including all drug resistant cell-lines. Nude mice xenograft assays with compound (E)-N-(3-amino-4-methoxyphenyl)-2-(2',4',6'-trimethoxyphenyl)ethenesulfonamide (6t) showed dramatic reduction in tumor size, indicating their in vivo potential as anticancer agents. A preliminary drug development study with compound 6t is predicted to have increased blood-brain barrier permeability relative to many clinically used antimitotic agents. Mechanistic studies indicate that 6t and some other analogues disrupted microtubule formation, formation of mitotic spindles, and arrest of cells in mitotic phase. Compound 6t inhibited purified tubulin polymerization in vitro and in vivo and circumvented drug resistance mediated by P-glycoprotein. Compound 6t specifically competed with colchicine binding to tubulin and with similar avidity as podophylltoxin, indicating its binding site on tubulin.

Cortical rigidity of round cells in mitotic phase and suspended state

This paper describes the results of the analysis of cortical rigidity in two round cell states: mitotic round cells and detached round cells after trypsinization using atomic force microscopy (AFM). These two states are primary cell events with dynamic morphological alterations in vitro. The trypsinized detached cells were fixed on the substrate of membrane anchoring oleyl surface. Fluorescent images taken by confocal laser scanning microscopy revealed diverse cell surface protrusions and cortical actin development in the round cells under different conditions. Although the cortical actin of these cells seemed to develop similarly, cortical rigidity of the trypsinized round cells showed greater stiffness than that of mitotic round cells. The elasticity measurements by AFM may detect invisible information about the maturation or strength of F-actin structures and such measurements may indicate that the strength of the actomyosin cortex would be higher in trypsinized round cells compared to mitotic cells. The mechanical properties can help provide better insights into the characteristics of the actin cytoskeleton network in vicinity of cell surface during dynamic morphological alterations.

Cell-cycle control as a target for calcium, hormonal and developmental signals: the role of phosphorylation in the retinoblastoma-centred pathway

During the life cycle of plants, both embryogenic and post-embryogenic growth are essentially based on cell division and cell expansion that are under the control of inherited developmental programmes modified by hormonal and environmental stimuli. Considering either stimulation or inhibition of plant growth, the key role of plant hormones in the modification of cell division activities or in the initiation of differentiation is well supported by experimental data. At the same time there is only limited insight into the molecular events that provide linkage between the regulation of cell-cycle progression and hormonal and developmental control. Studies indicate that there are several alternative ways by which hormonal signalling networks can influence cell division parameters and establish functional links between regulatory pathways of cell-cycle progression and genes and protein complexes involved in organ development. An overview is given here of key components in plant cell division control as acceptors of hormonal and developmental signals during organ formation and growth. Selected examples are presented to highlight the potential role of Ca(2+)-signalling, the complex actions of auxin and cytokinins, regulation by transcription factors and alteration of retinoblastoma-related proteins by phosphorylation. Auxins and abscisic acid can directly influence expression of cyclin, cyclin-dependent kinase (CDK) genes and activities of CDK complexes. D-type cyclins are primary targets for cytokinins and over-expression of CyclinD3;1 can enhance auxin responses in roots. A set of auxin-activated genes (AXR1-ARGOS-ANT) controls cell number and organ size through modification of CyclinD3;1 gene expression. The SHORT ROOT (SHR) and SCARECROW (SCR) transcriptional factors determine root patterning by activation of the CYCD6;1 gene. Over-expression of the EBP1 gene (plant homologue of the ErbB-3 epidermal growth factor receptor-binding protein) increased biomass by auxin-dependent activation of both D- and B-type cyclins. The direct involvement of auxin-binding protein (ABP1) in the entry into the cell cycle and the regulation of leaf size and morphology is based on the transcriptional control of D-cyclins and retinoblastoma-related protein (RBR) interacting with inhibitory E2FC transcriptional factor. The central role of RBRs in cell-cycle progression is well documented by a variety of experimental approaches. Their function is phosphorylation-dependent and both RBR and phospho-RBR proteins are present in interphase and mitotic phase cells. Immunolocalization studies showed the presence of phospho-RBR protein in spots of interphase nuclei or granules in mitotic prophase cells. The Ca(2+)-dependent phosphorylation events can be accomplished by the calcium-dependent, calmodulin-independent or calmodulin-like domain protein kinases (CDPKs/CPKs) phosphorylating the CDK inhibitor protein (KRP). Dephosphorylation of the phospho-RBR protein by PP2A phosphatase is regulated by a Ca(2+)-binding subunit.

Genotoxicity of silver nanoparticles in Allium cepa

Potential health and environmental effects of nanoparticles need to be thoroughly assessed before their widespread commercialization. Though there are few studies on cytotoxicity of nanoparticles on mammalian and human cell lines, there are hardly any reports on genotoxic and cytotoxic behavior of nanoparticles in plant cells. This study aims to investigate cytotoxic and genotoxic impacts of silver nanoparticles using root tip cells of Allium cepa as an indicator organism. A. cepa root tip cells were treated with four different concentrations (25, 20, 75, and 100 ppm) of engineered silver nanoparticles (below 100 nm size) dispersion, to study endpoints like mitotic index, distribution of cells in mitotic phases, different types of chromosomal aberrations, disturbed metaphase, sticky chromosome, cell wall disintegration, and breaks. For each concentration five sets of microscopic observations were carried out. No chromosomal aberration was observed in the control (untreated onion root tips) and the mitotic index (MI) value was 60.3%. With increasing concentration of the nanoparticles decrease in the mitotic index was noticed (60.30% to 27.62%). The different cytological effects including the chromosomal aberrations were studied in detail for the treated cells as well as control. We infer from this study that silver nanoparticles could penetrate plant system and may impair stages of cell division causing chromatin bridge, stickiness, disturbed metaphase, multiple chromosomal breaks and cell disintegration. The findings also suggest that plants as an important component of the ecosystems need to be included when evaluating the overall toxicological impact of the nanoparticles in the environment.

Localization of p53(301-393) mutant and its effect on mitosis

To observe the localization of p53(301-393)(residues 301-393) in p53 positive/negative cells and its effect on cell mitosis. The protein expression of p53-GFP and p53(301-393)-GFP was checked by immunoblotting after transfection. Immunofluorescence staining was performed to detect the localization of wide type and mutant in Hela cells (p53 positive) and H1299 cells (p53 negative). The cell morphology of H1299 cells transfected of p53(301-393)-GFP and the cells in mitotic phase were observed. Cell cycle was analyzed by flow cytometry and p53 protein level in HeLa cells was evaluated by Western blot after transfection of p53-GFP and p53(301-393)-GFP. The protein expression of p53-GFP and p53(301-393)-GFP was verified, p53-GFP was about 90 kMr and p53(301-393)-GFP about 40 kMr. Immunofluorescence microscopy demonstrated that both proteins were diffusely located in the nuclei of HeLa cells and H1299 cells. But different from the p53-GFP, the p53(301-393)-GFP was distributed in the nucleolus of HeLa cells. After transfection of the two plasmids, mitosis was inhibited in H1299 cells and some cells underwent apoptosis. G2/M progression of HeLa cells could be blocked by transfection of p53(301-393)-GFP, but endogenous p53 protein level was not changed. p53(301-393)has a different localization in the p53 positive and p53 negative cells and could inhibit mitosis and cause the cell cycle arrest in G2/M.

Cytotoxic activity and cytostatic mechanism of novel 2-arylbenzo[b]furans

The aims of this study were to screen cytotoxic compounds from 14 newly-synthesized 2-arylbenzo[b]furans and explore their mechanisms of action. Cytotoxicity was determined by the MTT method. Cell-cycle distribution was detected by flow cytometry. Wright-Giemsa staining was performed to demonstrate the morphological features of cells in mitotic phase. Polymerization of tubulin was detected by tubulin assembly assay, and the cellular microtubule network was observed by immunocytochemical study. Among the 14 compounds screened, 4-formyl-2-(4-hydroxy-3-methoxyphenyl)-5-(2-methoxycarbonyethyl)-7-methoxy-benzo[b]furan (ERJT-12) showed significant cytotoxicity. Our results demonstrated that ERJT-12 exhibited anti-cancer activity in a variety of tumour cell lines with an IC50 value (concentration resulting in 50% inhibition of cell growth) of 5.75 approximately 17.29 microM. Cell cycle analysis showed a concentration-dependent accumulation of tumour cells in G2/M phase after treatment with ERJT-12. Further investigation indicated that ERJT-12 blocked the cell cycle in M phase, with separation and dispersion of chromosomes. ERJT-12 inhibited tubulin polymerization in-vitro. Changes of the cellular microtubule network caused by ERJT-12 were also detected, which were similar to the changes caused by colchicine. These results suggested that the anti-cancer activity of ERJT-12 is worth further investigation.

Skp2 Contains a Novel Cyclin A Binding Domain That Directly Protects Cyclin A from Inhibition by p27Kip1

Skp2 is well known as the F-box protein of the SCF(Skp2) x Roc1 complex targeting p27 for ubiquitylation. Skp2 also forms complexes with cyclin A, which is particularly abundant in cancer cells due to frequent Skp2 overexpression, but the mechanism and significance of this interaction remain unknown. Here, we report that Skp2-cyclin A interaction is mediated by novel interaction sequences on both Skp2 and cyclin A, distinguishing it from the well known RXL-hydrophobic patch interaction between cyclins and cyclin-binding proteins. Furthermore, a short peptide derived from the mapped cyclin A binding sequences of Skp2 can block Skp2-cyclin A interaction but not p27-cyclin A interaction, whereas a previously identified RXL peptide can block p27-cyclin A interaction but not Skp2-cyclin A interaction. Functionally, Skp2-cyclin A interaction is separable from Skp2 ability to mediate p27 ubiquitylation. Rather, Skp2-cyclin A interaction serves to directly protect cyclin A-Cdk2 from inhibition by p27 through competitive binding. Finally, we show that disruption of cyclin A binding with point mutations in the cyclin A binding domain of Skp2 compromises the ability of overexpressed Skp2 to counter cell cycle arrest by a p53/p21-mediated cell cycle checkpoint without affecting its ability to cause degradation of cellular p27 and p21. These findings reveal a new functional mechanism of Skp2 and a new regulatory mechanism of cyclin A.

Quantitative aspects of endocytic activity in lipid-mediated transfections

Variation in transfection efficiency observed in different cell-types is poorly understood. To investigate the influence of endocytic activity on lipid-mediated transfections, we have monitored both the processes in 12 different cell-types. The endocytic activity shows a strong positive correlation ( P < 0.01), with transfection efficiency. Treatment with wortmannin resulted in cell-type-dependent inhibition of transfection. Studies on M-phase cells by confocal microscopy show that compared to interphase cells, uptake of cationic liposomes was substantially reduced. In addition, transfection efficiency of cells in mitotic phase was inhibited by >70% compared to controls. Our study based on several cell-types demonstrates for the first time that quantitative aspects of endocytosis have decisive influence on the overall process of lipid-mediated transgene expression.

72 EFFECT OF CELL CYCLE PHASE OF GENE-MANIPULATED FETAL FIBROBLASTS ON THE DEVELOPMENT OF CLONED BOVINE EMBRYOS

Transgenic bovine fetuses and offspring can be produced by using gene-modified somatic cells and clones of these cells. In this study, we examined the effects of specific cell cycle (early G1 phase) of donor cell (gene-manipulated fibroblasts) on the development of the nuclear transfer (NT) embryos into blastocysts and on the fetus production after embryo transfer. The gene-manipulated (tg; targeting of one or both alleles of gene encoding α-1,3-galactosyltransferase) or non-manipulated (control) bovine fetal fibroblasts were used for NT. The fibroblasts transfected with the targeting vector were selected with 0.4 mg mL−1 G418. The G418-resistant cells were monitored by PCR and Southern blot analysis. The cells (tg cells) in which homologous recombination occurred were used for NT. For NT, both tg cells and control cells were cultured in DMEM with 10% FCS. Early G1 cells were prepared by choosing pairs of bridged cells derived from mitotic phase cells (Urakawa M et al. 2004 Theriogenology 62, 714–728), and non-synchronized cells were obtained from a culture plate that had reached 60–80% confluence. Each donor cell was inserted into an enucleated, in vitro-matured (19 h) oocyte. Oocyte-cell couples were electrofused and activated with calcium ionophore and cycloheximide. The NT embryos were then co-cultured with bovine oviduct epithelial cells in CR1aa with 5% CS. The blastocyst rates were determined at 6 days after NT. The blastocysts were nonsurgically transferred to recipient heifers, and the developmental rate to the normal fetus was examined by the recovery of fetus or by using ultrasonography at Days 35–42. Data were analyzed by ANOVA. The developmental rate to the blastocyst stage did not differ significantly between tg (28.4%, 128/425) and control (25.4%, 181/739) cell groups. In the control group, the blastocyst rate of embryos constructed from early G1 phase fibroblasts (25.7%, 80/311) was not significantly different from that of embryos constructed with non-synchronized fibroblasts (23.6%, 101/428). In contrast, the blastocyst rate of tg cell derived-embryos was lower (P &lt; 0.05) in early G1 phase (23.5%, 71/302) than in non-synchronized cell phase (46.3%, 57/123). The rate of development to a normal fetus in the tg group (15.4%, 4/26) was significant lower than that in the control group (62.5%, 25/40). For both the tg group and the control group, the rate of development to fetus tended to be higher (P &gt; 0.05) for blastocysts derived from cells at the early G1 phase than for blastocysts derived from non-synchronized cells (tg group, 25.0%, 3/12 v. 7.1%, 1/14; control group, 90.0%, 9/10 v. 53.3%, 16/30). These results demonstrate that gene modification of fetal fibroblasts affects the development of NT embryos to fetuses. In addition, the synchronization of genetically modified donor cells to the early G1 phase may increase the potential to develop to a normal fetus.