Bromodeoxyuridine induces p53-dependent and -independent cell cycle arrests in human gastric carcinoma cell lines.

OBJECTIVES: This study was designed to clarify the effects of bromodeoxyuridine (BrdU) on cell cycle progression and induction of apoptosis, and to demonstrate the role of P53 in these processes. METHODS: We continuously exposed four human gastric carcinoma cell lines with different P53 status (P53 wild-type AGS and MKN-45, P53-mutated MKN-28 and P53-deleted KATO-III) to BrdU in asynchronous and synchronous culture conditions, and analyzed DNA histograms of apoptotic and nonapoptotic cells determined by static DNA cytofluorometry. RESULTS: Continuous exposure to 20 microM BrdU after synchronization with hydroxyurea resulted in S phase delay and G1 arrest in MKN-45 and an increase of apoptosis in the first S/G(2) phase in AGS and MKN-45. In the second S phase, a delay of 3-6 h was observed in all the four cell lines. In asynchronous cultures, continuous exposures to 20 and 200 microM BrdU for 72 h or more caused growth suppression with G(1) and G(2) arrests, respectively, in all the cell lines. CONCLUSIONS: These data suggested that the BrdU-induced growth suppression of the cell lines examined was mainly caused by cell cycle arrest rather than cell death, and that the cell cycle arrests in the first S and G(1) phases (elicited by BrdU in the single DNA strand) and those in the second S, G(2) and G(1) phases (elicited by BrdU in the double DNA strands) were mediated by p53-dependent and -independent pathways, respectively.     

The action of epidermal growth factor (EGF) is limited to specific phases of the cell cycle in an EGF dependent colonic cell line

In the presence of epidermal growth factor (EGF) a human colon cell line, LIM 1215, proliferates in serum-free medium. Under these culture conditions the cells are dependent on the presence of EGF for both proliferation and survival. In order to study the action of growth factors at different stages of the LIM 1215 cell cycle, pure populations of G1, S and G2/M cells were obtained by cell sorting after supravital staining of the DNA with Hoechst 33342. Conditions were established for Hoechst 33342 staining which produced satisfactory DNA histograms and greater than 80% survival of cells. The kinetics of passage for sorted S or G2/M cells into G1 were not affected by EGF or fetal calf serum. After sorting there appeared to be a 4 h delay before the cells proceeded in the cell cycle. Sorted S cells entered G2 over an 8 h period and maintained this same transition period from G2 into G1. If EGF or serum was present, these cells then re-entered the cell cycle after a variable delay and in an asynchronous manner. EGF was applied to S phase and G2/M phase LIM 1215 cells for periods of 2-10 h at various times after replating in serum-free conditions. Cells in S phase only responded to EGF as they passed from G2/M into G1. Exposure to EGF in S phase resulted in little growth stimulus once the cells returned to G1. For cells in G2/M phase, EGF was required immediately to give the maximum stimulus for re-entering the cell cycle. If the EGF was delayed for more than 8 h, the cells did not re-enter the cycle within the following 20 h. Exposure to EGF for less than 2 h failed to stimulate proliferation. These results indicate that EGF must be present as cells enter G1 from mitosis. Once the cells have entered G1, EGF is required for a 10 h period for a large number of cells to re-enter the cycle from G1.     

异氟醚对膀胱癌 5637 细胞恶性生物学行为及化疗敏感性的影响

目的 探究异氟醚对膀胱癌 5637 细胞恶性生物学行为及化疗敏感性的影响。 方法 单独或联合 给予异氟醚及顺铂处理膀胱癌 5637 细胞, 将细胞随机分为 4 组: 对照组、 异氟醚组、 顺铂组和异氟醚 + 顺 铂联合处理组。 BrdU 染色检测 BrdU 阳性细胞数; 流式细胞仪检测细胞凋亡率和细胞周期分布; Transwell 检测侵袭细胞数; 划痕愈合实验检测划痕愈合率; Western 印迹检测 Ki67、 PCNA、 Bax、 Bcl-2、 cleaved caspase-3 和 caspase-3 蛋白表达; 结果 与顺铂组相比较, 异氟醚 + 顺铂联合处理组 BrdU 阳性细胞数和 Ki67、 PCNA 蛋白表达降低, G0 / G1期比率和 S 期细胞数降低, G2 / M 期比率升高, 细胞凋亡率和 Bax / Bcl-2、 cleaved caspase-3 / caspase-3 比值升高, 侵袭细胞数、 划痕愈合率降低, 均有显著性差异 (P< 0. 05)。 结论 异氟醚可通过抑制细胞增殖、 侵袭、 迁移, 诱导细胞凋亡, 提高 5637 细胞对顺铂的敏感性。     

Presenilin Overexpression Arrests Cells in the G1 Phase of the Cell Cycle : Arrest Potentiated by the Alzheimer’s Disease PS2(N141I) Mutant

To investigate the mechanism by which presenilin (PS) overexpression induces apoptosis, we studied the effects of these proteins on cell cycle progression. Transiently transfected HeLa cells were bromodeoxyuridine (BrdU) labeled to visualize DNA synthesis by immunofluorescence and stained with propidium iodide to measure DNA content by fluorescence-activated cell sorting (FACS). BrdU labeling was decreased in cells expressing presenilin-1 (PS1), presenilin-2 (PS2), an Alzheimer's disease-associated missense mutation PS2(N141I), and the carboxyl-terminally deleted PS2 construct PS2(166aa), compared with mock and neurofilament-light (NF-L) transfected cells. Analysis of BrdU incorporation in mitotically synchronized HeLa cells suggested that cells were arresting in the G1 phase of the cell cycle, and this was confirmed by FACS analysis. Interestingly, cell cycle progression was more inhibited by the expression of PS2(N141I) compared with wild-type PS2. In addition, ATM, the gene product mutated in ataxia-telangiectasia, does not appear to be a downstream effector of PS-induced cell cycle arrest as transfection of PS constructs into an ataxia-telangiectasia cell line also resulted in cell cycle inhibition. Quantitative immunoblotting of whole-cell lysates from PS-transfected cells did not reveal increases or decreases in the steady-state levels of p21, p27, p53, pRb, or c-myc, suggesting that the presenilins mediate cell cycle arrest by mechanisms other than simple changes in the steady-state levels of these cell-cycle-related proteins.     

Preferential S phase entry and apoptosis of CD4(+) T lymphocytes of HIV-1-infected patients after in vitro cultivation

We have studied the relationship between spontaneous apoptosis and cell cycle perturbations in circulating peripheral blood lymphocytes of HIV-1-infected patients and healthy controls. PBMC obtained from HIV-1-infected patients and healthy controls were incubated in culture medium for 48 h. Cells were separated into CD4(+) and CD8(+) populations using immunomagnetic beads. Apoptosis and cell cycle phases were measured by propidium iodide staining and bromodeoxyuridine (BrdU) incorporation followed by flow cytometric analyses. In experiments using cells obtained from HIV-1-infected patients, spontaneous apoptosis was more frequent in CD4(+) T lymphocytes than in CD8(+) T lymphocytes (17.6% vs 9.5%, P < 0.005). Among healthy controls, spontaneous apoptosis in CD4(+) and CD8(+) T lymphocytes was comparable (4.5% vs 5.1%). Lymphocytes obtained from patients were more frequently in S phase than healthy controls' cells (2.2 +/- 0.9% vs 0.5 +/- 0.2%, P < 0.002) and patients' CD4(+) cells tended to enter S phase more frequently than controls' CD4(+) cells (4.2% +/- 3.5% vs 1.8% +/- 0.5% P < 0.04), whereas the frequency of S phase CD8(+) T cells was not different among patients (2.8% +/- 2.9%) and controls (1.8% +/- 0.5%) (P > 0.4). Kinetic analyses using BrdU and PI staining revealed that S phase cells were more likely to become apoptotic than resting (G(0)-G(1)) cells (28.4% +/- 10.3% vs 11.3% +/- 9.9% in patients, P < 0.04, and 15.3% +/- 2.8% vs 1.8% +/- 0.5% in controls, P < 0.003). Lymphocytes obtained from HIV-1-infected persons are activated in vivo to enter S phase and to undergo spontaneous apoptosis after brief in vitro cultivation. The present studies indicate that most apoptotic cells in this system are CD4(+) and kinetic analyses reveal that S phase cells are more likely to undergo spontaneous apoptosis than G(0)-G(1) cells. Accelerated cell death in HIV-1 disease may contribute to the failure of lymphocyte responsiveness to appropriate T cell receptor stimulation.     

人巨细胞病毒感染对宿主细胞周期进程影响的实验研究

目的观察人巨细胞病毒（HCMV）感染对宿主细胞DNA合成及细胞周期蛋白（Cyclins）表达的影响。方法用HCMV感染同步化于G0／G1期的人胚肺成纤维细胞（HEL），分别于感染后0、3．6、12、24、48、72、96h终止培养。用流式细胞术测定HCMV感染后细胞周期进程及DNA含量。用免疫蛋白印迹法（Western Blot）检测CyclinE、Cyclin A、Cyclin D1蛋白的表达。结果HCMV感染细胞后24h-96h，S期细胞明显增多，G2／M期细胞减少，至感染后96h，未发现有G2／M期细胞。感染后24h细胞保持2N DNA含量，全部感染细胞DNA含量在48h内开始升高，感染后72h多数细胞的DNA含量大于2N DNA含量。在正常对照细胞DNA含量没有增加。HCMV＋PAA组没有检测到DNA含量增加。HCMV感染接触抑制细胞12h时CyclinE蛋白被诱导，感染后24h出现Cyclin E峰值；HCMV不能诱导Cyclin A蛋白表达；CyclinD1在感染后24h开始下降。结论HCMV感染同步化于G0／G1期的细胞后，诱导CyclinE蛋白明显升高，激活CyclinE／Cdk2激酶，使细胞周期越过G1／S限制点，将细胞周期阻止于晚G1期。病毒感染未能激活细胞DNA合成，病毒感染后总DNA的增加由病毒DNA复制造成。     

Herpes Simplex Virus Gene Products Required for Viral Inhibition of Expression of G1-Phase Functions

HSV infection blocks G1 events in the cell cycle and arrests host cell growth in the G1 phase. To further define the mechanism of the effect and determine the viral gene product(s) responsible, we examined various mutant viruses for their effects on cell cycle regulatory proteins (pRb, cyclin D1, and cdk4) and on cell cycle progression into S phase. Unlike the wild-type virus, the ICP27 mutant virus was defective for blocking the phosphorylation of pRb proteins, and the normal pRb pattern was restored in cells infected with a rescued virus. The virion host shutoff (vhs) function, DNA replication, and late gene functions were not required for the virus-induced effects on pRb protein. BrdU incorporation in synchronized HSV-infected cells showed that ICP27 was required for blocking the cell cycle in the G1 phase. Furthermore, ICP27, ICP4, ICP0, and vhs were required for blocking the induction of the G1 cell cycle regulators cyclin D1 and cdk4 in HSV-infected cells. Both ICP27 and the vhs function contributed to the reduction of cyclin D1 mRNA levels in HSV-infected cells. These results provide evidence that HSV-1 ICP27 protein is essential for viral inhibition of G1-phase functions and that certain other HSV proteins are required for some of the viral effects on the cell cycle. Finally, these results show that HSV-1 ICP27 and vhs act jointly to reduce host mRNA levels in infected cells.     

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine(BrdU) on cell cycle progression and micronucleus inductionwere studied in different mammalian cell cultures. Simultaneousflow cytometric measurements of DNA content and side scatterof nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependenttemporary block in the G₂/M phase of the first cell cycle. NTH3T3 cells and human amniotic fluid fibroblast-like cells, onthe contrary, did not show any cell cycle disturbances in thepresence of BrdU. Micronucleus frequency increased as soon asCHE cells started to divide and reached a plateau when all cellshave divided. The height of this plateau was almost equal for60 and 100 µM BrdU. This saturation of micronucleus inductionwas due to a saturation of BrdU incorporation into DNA alreadyat a dosis of 60 µM as shown by the BrdU/Hoechst quenchingtechnique. Indirect immunofluorescent staining of kinetochoreswith CREST antibodies revealed that nearly all BrdU-inducedmicronuclei were kinetochore-negative suggesting the presenceof acentric chromosome fragments in these micronuclei. DNA distributionsof micronuclei measured by flow cytometry showed several peaksrepresenting micronuclei which contain DNA fragments of definedsizes induced by non-random breakage of chromosomes 1 and Xas verified by flow karyotyping and C-banding.     

Topoisomerase II-alpha expression in different cell cycle phases in fresh human breast carcinomas.

Topoisomerase II-alpha (topo II alpha) is the key target enzyme for the topoisomerase inhibitor class of anti-cancer drugs. In normal cells, topo II alpha is expressed predominantly in the S/G2/M phase of the cell cycle. In malignant cells, in vitro studies have indicated that the expression of topo II alpha is both higher and less dependent on proliferation state in the cell. We studied fresh specimens from 50 cases of primary breast cancer. The expression of topo II alpha in different cell cycle phases was analyzed with two-parameter flow cytometry using the monoclonal antibody SWT3D1 and propidium iodide staining. The expression of topo II alpha was significantly higher in the S/G2/M phase of the cell cycle than in the G0/G1 phase in both DNA diploid and DNA non-diploid tumors. In 18 of 21 diploid tumors, and in 25 of 29 non-diploid tumors, >50% of the topo II alpha-positive cells were in the G0/G1 phase. This significant expression of topo II alpha in the G0/G1 phase of the cell cycle may have clinically important implications for treatment efficacy of topoisomerase II inhibitors.     

Pseudolaric Acid B Induced Cell Cycle Arrest,Autophagy and Senescence in Murine Fibrosarcoma L929 Cell

Objective: PAB induced various cancer cell apoptosis, cell cycle arrest and senescence. But in cell line murine fibrosarcoma L929, PAB did not induce apoptosis, but autophagy, therefore it was thought by us as a good model to research the relationship of cell cycle arrest, autophagy and senescence bypass apoptosis.Methods: Inhibitory ratio was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Phase contrast microscopy visualized cell morphology. Hoechst 33258 staining for nuclear change, propidium iodode (PI) staining for cell cycle, monodansylcadaverine (MDC) staining for autophagy, and rodanmine 123 staining for mitochondrial membrane potential (MMP) were measured by fluorescence microscopy or flowcytometry. Apoptosis was determined by DNA ladder test. Protein kinase C (PKC) activity was detected by PKC assay kit. SA-β-galactosidase assay was used to detect senescence. Protein expression was examined by western blot.Results: PAB inhibited L929 cell growth in time-and dose-dependent manner. At 12 h, 80 μmol/L PAB induced obvious mitotic arrest; at 24 h, PAB began to induce autophagy; at 36 h, cell-treated with PAB slip into G1 cell cycle; and 3 d PAB induced senescence. In time sequence PAB induced firstly cell cycle arrest, then autophagy, then slippage into G1 phase, lastly senescence. Senescent cells had high level of autophagy, inhibiting autophagy led to apoptosis, and no senescence. PAB activated PKC activity to induce cell cycle arrest, autophagy and senescence, inhibiting PKC activity suppressed cell cycle arrest, autophagy and senescence.Conclusion: PAB induced cell cycle arrest, autophagy and senescence in murine fibrosarcoma L929 cell through PKC.     

HIV-1 Vpr蛋白对C8166细胞毒性研究

目的 利用腺病毒系统研究HIV-1 Vpr蛋白在T细胞来源的C8166细胞内的高表达以及细胞内该蛋白对T细胞毒性.方法 将表达目的 蛋白Vpr的重组腺病毒rAd-vpr和空白载体病毒rAd-vector分别感染对数生长期的C8166细胞,流式细胞术(FCM)检测细胞周期分布、凋亡和坏死.用Hoechst-PI荧光染色观察细胞的凋亡和坏死,JC-1荧光染色测定线粒体膜电势.结果 Annexin V-PI染色及Hoechst-PI染色一致显示HIV-1 Vpr能显著诱导C8166细胞凋亡和坏死;PI细胞周期结果表明HIV-1 Vpr能阻滞C8166细胞于G2期;JC-1荧光染色法测定HIV-1 Vpr致C8166线粒体膜电势下降.结论 细胞内HIV-1 Vpr介导的T细胞毒性包括线粒体功能障碍、细胞周期的G2期阻滞和细胞的死亡.     

Dlx2基因过表达与前成骨细胞系MC3T3-E1成骨分化过程中的细胞凋亡和周期调控*****◆

背景：Dlx2基因在颅神经嵴细胞迁徙进入第一鳃弓过程和颅颌面骨骼发育中起重要作用,但Dlx2基因对成骨细胞分化过程中细胞凋亡和细胞周期调控的影响尚未见报道。 目的：观察Dlx2基因过表达对前成骨细胞系MC3T3-E1成骨分化过程中细胞凋亡和细胞周期调控的影响。 方法：构建反转录病毒pMSCV-puro-Dlx2并转染矿化诱导液培养下的MC3T3-E1细胞,构建稳定过表达Dlx2基因的细胞系MC3T3-E1-Dlx2。RT-PCR和Western blot验证Dlx2基因过表达细胞系的建立。Annexin V/PI双染色后流式细胞分选检测细胞凋亡,PI/RNase双染色后流式细胞分选检测细胞周期变化。 结果与结论：实验成功构建稳定过表达Dlx2基因的细胞系MC3T3-E1-Dlx2。发现Dlx2基因过表达促进细胞凋亡(P < 0.05),同时阻滞细胞周期于G1/G0期(P < 0.05),减低细胞增殖性,促进细胞分化行使功能。 关键词：成骨细胞分化;Dlx2基因;稳定过表达;细胞凋亡;细胞周期 doi:10.3969/j.issn.1673-8225.2012.10.022     

两种不同染色法在流式细胞术中检测细胞周期的探讨

目的比较碘化丙啶（PI）和4,6-联脒-2苯基吲哚（DAPI）两种不同染色方法在流式细胞术中对细胞周期检测的影响。方法 A549细胞分别采用PI和DAPI染色法进行染色,于染色后0和24h进行流式细胞仪检测细胞G0/G1期、S期和G2/M期的DNA含量,比较两种方法的测定结果,同时进行细胞计数,观察细胞浓度变化。结果PI和DAPI法染色后0h上机测定,结果分别为：CV值（7.72±0.19）、（5.92±0.09）,G0/G1期含量（69.63±1.16）%、（69.87±1.28）%,S期含量（24.53±0.47）%、（24.43±0.86）%,G2/M期含量（5.85±1.04）%、（5.72±0.65）%,两种方法检测细胞周期的结果基本一致（P〉0.05）;染色后24h重新测定,结果分别为：CV值（8.82±0.05）、（6.09±0.30）,G0/G1期含量（58.50±0.90）%、（70.47±0.81）%,S期含量（31.73±0.75）%、（23.67±0.45）%,G2/M期含量（9.51±0.47）%、（5.86±0.46）%,两种方法检测细胞周期的结果差异有统计学意义（P〈0.05）。DAPI法染色后0和24h测定结果基本一致（P〉0.05）,而PI法检测结果差异有统计学意义（P〈0.05）。细胞计数结果显示,放置24hPI法细胞损耗为63.14%,DAPI法细胞损耗为12.50%,两种方法结果差异有统计学意义（P〈0.05）。染色24h后PI法细胞开始崩解,镜下见较多的细胞碎片,而DAPI法细胞形态良好。结论应用流式细胞术检测细胞周期,DAPI染色法优于PI染色法。     

"Unusual SCD" explained by differences in BrdU incorporation during subsequent cell cycles

In the course of three consecutive cell cycles, V79 cells were labeled with BrdU by different labeling protocols. Cells treated for three cycles with bromodeoxyuridine (BrdU) showed third division metaphases, with the typical appearance after fluorescent plus Giemsa (FPG) staining (i.e., 75% of the chromatids showed light staining, 25% showed dark staining). The same staining pattern is achieved by the second labeling protocol, during which the cells have replicated for two cycles in the presence of BrdU and, during the last cycle, in the absence of BrdU. Cells that have replicated only for one cell cycle in BrdU-containing medium, and the following two cycles in normal medium, depict just the opposite staining pattern (i.e., 75% dark, 25% light). These experiments explain how the variation of BrdU substitution in the DNA leads to altered FPG staining. Unexpected staining patterns ("unusual SCD") are also observed after seemingly permanent BrdU substitution. This phenomenon, which has been found in cancer cells, is due to the decrease in BrdU concentration and not to a peculiarity of the cells investigated.     

赫赛汀诱导乳腺癌细胞凋亡及对其细胞周期的影响

目的研究免疫靶向治疗药物赫赛汀(Herceptin)对HER-2过表达的乳腺癌细胞凋亡及细胞周期的影响。方法Herceptin处理体外培养的乳腺癌SKBR3细胞系,经MTT试验筛选最佳药物处理浓度和时间的组合,应用荧光显微镜、激光共聚焦显微镜、扫描电镜、透射电镜及流式细胞仪检测乳腺癌细胞凋亡的特征、凋亡细胞百分率及细胞周期的变化。结果在Herceptin作用下,荧光显微镜、激光共聚焦显微镜、扫描电镜、透射电镜观察SKBR3细胞均出现凋亡特征。Annexin/PI染色流式仪测定,药物处理组早期凋亡百分率较对照组显著增加(P<0.01)。流式仪细胞周期分析显示,S期细胞含量下降,而G2期细胞含量上升。结论免疫靶向治疗药物Herceptin可特异性地诱导HER-2过表达的乳腺癌细胞发生凋亡,并可使其生长受阻于G2期。诱导凋亡可能是Herceptin抗肿瘤作用的重要机制之一。     

Analysis of cell division among subpopulations of lymphoid cells in sheep. I. Thymocytes

The number, distribution and surface phenotype of dividing cells in the thymus, and differences between the cell cycle status of thymocyte subpopulations, were studied in fetal and post-natal lambs using double-labelling techniques. Dividing cells were labelled in vivo for various periods with 5-bromo-2-deoxyuridine (BrdU). The proportions of constituent thymocyte subpopulations that had synthesized DNA during the labelling period were measured by flow cytometry or immunohistochemistry using a panel of monoclonal antibodies (mAbs) specific for sheep lymphocyte differentiation antigens and MHC class I and class II antigens in conjunction with an anti-BrdU mAb. The proportion of thymocytes that incorporated BrdU during a 1-hr labelling period varied with age, and levels of 30%, 13% and 9% were measured, respectively, in 40- and 125-day-old fetuses and 8-week-old lambs. Eight percent of the thymocytes in lambs were synthesizing DNA, with 4% entering the G2 phase per hour, and a substantial number of thymocytes (21%) had a G2 + M phase DNA content. A small subset of thymocytes (1-3%) recognized by mAb E-79 localized to the subcapsular region of the cortex and displayed the highest level of BrdU incorporation. Cortical-type thymocytes (CD1+) comprised 50-70% of thymocytes; however, few of these incorporated BrdU and the proportion in the G2 + M phase of the cell cycle was higher than for other thymocyte subpopulations. The 197+CD4-CD8- T cells also showed no evidence of in vivo division.     

Determination of key aspects of precursor cell proliferation, cell cycle length and kinetics in the adult mouse subgranular zone

Neurogenesis studies on the adult mouse hippocampal subgranular zone (SGZ) typically report increases or decreases in proliferation. However, key information is lacking about these proliferating SGZ precursors, from the fundamental—what dose of bromodeoxyuridine (BrdU) is appropriate for labeling all S phase cells?—to the detailed—what are the kinetics of BrdU-labeled cells and their progeny? To address these questions, adult C57BL/6J mice were injected with BrdU and BrdU-immunoreactive (IR) cells were quantified. Initial experiments with a range of BrdU doses (25–500 mg/kg) suggested that 150 mg/kg labels all actively dividing precursors in the mouse SGZ. Experiments using a saturating dose of BrdU suggested BrdU bioavailability is less than 15 min, notably shorter than in the developing mouse brain. We next explored precursor division and maturation by tracking the number of BrdU-IR cells and colabeling of BrdU with other cell cycle proteins from 15 min to 30 days after BrdU. We found that BrdU and the Gap2 and mitosis (G₂/M) phase protein pHisH3 maximally colocalized 8 h after BrdU, indicating that the mouse SGZ precursor cell cycle length is 14 h. In addition, triple labeling with BrdU and proliferating cell nuclear antigen (PCNA) and Ki-67 showed that BrdU-IR precursors and/or their progeny express these endogenous cell cycle proteins up to 4 days after BrdU injection. However, the proportion of BrdU/Ki-67-IR cells declined at a greater rate than the proportion of BrdU/PCNA-IR cells. This suggests that PCNA protein is detectable long after cell cycle exit, and that reliance on PCNA may overestimate the length of time a cell remains in the cell cycle. These findings will be critical for future studies examining the regulation of SGZ precursor kinetics in adult mice, and hopefully will encourage the field to move beyond counting BrdU-IR cells to a more mechanistic analysis of adult neurogenesis.     

ICRF-193, a catalytic inhibitor of DNA topoisomerase II, inhibits re-entry into the cell division cycle from quiescent state in mammalian cells

BACKGROUND: To describe the requirement of DNA topoisomerase II (topo II) during transition from the quiescent state (G0 phase) to the cell division cycle in mammalian cells, we examined the influence of ICRF-193, a catalytic inhibitor of topo II, on re-entry into the cell division cycle of quiescent cells in response to appropriate growth stimuli. RESULTS: The re-entry into the S phase of cultured cell lines arrested at the quiescent (G0) phase by serum-starvation was sensitive to 10 microm ICRF-193. DNA syntheses induced by lipopolysaccharide in murine spleen cells or by release from contact-inhibition were also inhibited by ICRF-193. The cell lines with a high-level of resistance toward ICRF-193 due to a point mutation in the topo IIalpha gene entered into the S phase from quiescence in the presence of ICRF-193. The drug did not inhibit entry into the S phase in cultured cells released from arrest at the metaphase or G1 phase. CONCLUSION: There is an ICRF-193-sensitive step during re-entry of quiescent mammalian cells into the cell division cycle upon growth stimulation and the drug targets topo IIalpha during the process.     

The promotion of neural progenitor cells proliferation by aligned and randomly oriented collagen nanofibers through β1 integrin/MAPK signaling pathway

In regenerative medicine, accumulating evidence demonstrates that the property of substrates monitors neural stem cells behavior. However, how stem cells sense and interpret biochemical and topographical cues remains elusive. This study aimed to explore the mechanism how nanofibrous scaffold modulated stem cells behavior. Spinal cord derived neural progenitor cells (NPCs) were cultured on electrospun aligned and randomly oriented collagen nanofibrous scaffolds. A 30% increase in proliferation and an elevation of BrdU incorporation were observed in NPCs on collagen nanofibers, compared to that on collagen-coated surface. In particular, NPCs expanded faster on aligned nanofibers in comparison with that on randomly oriented nanofibers. Moreover, an alteration in cell cycle progression with a reduced percentage of cells in G0/G1 phase and increased cell proliferation index (S phase plus G2/M phase) was also detected in NPCs cultured on collagen nanofibers. Incubating NPCs with anti-β1 integrin antibody or U1026 (an inhibitor of mitogen-activated protein kinase kinase, MEK) eliminated the altered cell cycle dynamics and BrdU incorporation induced by collagen nanofibers. In addition, cyclin D1 and cyclin dependent kinase 2 (CDK2), downstream genes of β1 integrin/mitogen-activated protein kinase (MAPK) pathway that control G1/S phase transition, were correspondingly regulated by nanofibers. Collectively, these data suggested that the property of substrate modulated NPCs proliferation by promoting cell cycle through β1 integrin/MAPK pathway. Our findings provide a better understanding of the interaction between NPCs and the substrate and therefore will pave way for regenerative medicine.     

Anticancer effect of salidroside on colon cancer through inhibiting JAK2/STAT3 signaling pathway

Salidroside is considered to have anti-tumor properties. We investigate its effects on colon carcinoma SW1116 cells. Cell viability was assessed by CCK-8. Propidium iodide (PI) staining was used to determine the cell cycle by flow cytometry. The migration and invasion were detected by Transwell. Western blot was used to detect the expression of STAT3 signal related proteins. As the result, high concentrations of salidroside (10, 20. 50 μg/ml) significantly inhibited proliferation of SW1116 cells in a parallelly, cell cycle arrest was increased at the G0/G1 phase after salidroside treatment. Furthermore, salidroside inhibited migration and invasion of SW1116 cells. Salidroside treatment decreased proteins expression of phosphorylation levels in JAK2/STAT3 signaling, while MMP-2 and MMP-9 proteins levels were decreased and protein expression of VEGF and VEGFR-2 were down-regulated. In Conclusion, salidroside inhibited proliferation, decreased the migration and invasion of SW1116 cells in JAK2/STAT3-dependent pathway, the specific mechanisms need further study.