Kaiso通过抑制cyclin D1转录影响肺癌细胞增殖和周期

目的研究Kaiso对cyclin D1转录抑制作用,探讨Kaiso抑制肺癌细胞增殖的分子机制。方法MTT和流式细胞仪检测A549和H1299两种肺腺癌细胞增殖和周期改变;Western blot和RT-PCR检测cyclin D1表达改变;生物信息学方法预测并设计cyclin D1启动子区Kaiso结合位点(KBS)的引物探针,ChIP证实Kaiso与cyclinD1启动子区结合。结果 Kaiso能够直接结合cyclin D1启动子区KBS,抑制cyclinD1转录,导致细胞增殖下降,G/G期比例升高,S期比例下降。结论 Kaiso通过下调cyclin D1转录抑制肿瘤细胞增殖和周期进程。     

Immunohistochemical expression of cyclin D1, E2F-1, and Ki-67 in benign and malignant thyroid lesions

Cyclin D1 and E2F-1 proteins are essential for the regulation of the G1/S transition through the cell cycle. Cyclin D1, a product of the bcl-1 gene, phosphorylates the retinoblastoma protein, releasing E2F-1, which in turn activates genes involved in DNA synthesis. Expression patterns of E2F-1 protein in thyroid proliferations have not been reported. This study used monoclonal antibodies for cyclin D1 and E2F-1 proteins to immunostain sections of normal thyroid, hyperplastic (cellular) nodules, follicular adenomas, follicular carcinomas, and papillary carcinomas. The proliferation rate was examined using an antibody specific for the Ki-67 antigen. Fluorescence in situ hybridization (FISH) methods and chromosome 11-specific probes were also employed to determine chromosome copy number and to assess for evidence of amplification at the 11q13 locus in papillary and follicular carcinomas with cyclin D1 overexpression. Concurrent overexpression of Ki-67, cyclin D1, and E2F-1 was found in the majority of benign and malignant thyroid lesions, compared with normal thyroid tissue. Cyclin D1 up-regulation was not due to extra copies of chromosome 11, or bcl-1 gene amplification. Malignant tumours showed the highest expression for all three markers, particularly papillary carcinomas. E2F-1 was detected at the same or slightly lower levels than cyclin D1. It was only found when cyclin D1 was overexpressed. Because cyclin D1 normally activates E2F-1, up-regulation of cyclin D1 may lead to E2F-1 overexpression in benign and malignant thyroid lesions.     

Expression and prognostic significance of cyclin B1 and cyclin A in non-small cell lung cancer

Aims:  Aberrant expression of cell cycle regulators has been implicated in the pathogenesis of many neoplasms, including non-small cell lung cancer (NSCLC). The aim was to examine the expression and prognostic value of cyclin B1 and cyclin A, key regulators of the G₂/M checkpoint of the cell cycle, in NSCLC and bronchial precursor lesions.
Methods and results:  Immunohistochemical expression of cyclin B1 and A was examined in 90 cases of stage I–II primary NSCLC and bronchial precursor lesions using tissue microarrays. Increased cyclin B1 and A expression was found in 40.9 and 58.9% of NSCLC cases, respectively, and was significantly higher in primary NSCLC, lymph node metastases and some bronchial precursor lesions compared with normal bronchial epithelium. Increased expression of cyclin A and cyclin B1 correlated with tumour type, poorly differentiated tumours and male gender. A significant association was found between increased cyclin B1 expression and reduced survival using Kaplan–Meier survival analysis. On multivariate analysis, cyclin B1 was not an independent prognostic factor ( P  = 0.067). Cyclin A expression was not associated with survival.
Conclusions:  Cyclin B1 and cyclin A are aberrantly expressed in NSCLC and some precursor lesions. Cyclin B1, but not cyclin A, shows some promise as a potential prognostic marker in NSCLC.     

The eleven stages of the cell cycle,with emphasis on the changes in chromosomes and nucleoli during interphase and mitosis

Since we had subdivided the cell cycle into 11 stages—four for mitosis and seven for the interphase—and since we had experience in detecting DNA in the electron microscope (EN) by the osmium-amine procedure of Cogliati and Gauthier (Compt. Rend. Acad. Sci., 1973;276:3041–3044), we combined the two approaches for the analysis of DNA-containing structures at all stages of the cell cycle. Thin Epon sections of formaldehyde-fixed mouse duodenum were stained by osmium-amine for electron microscopic examination of the stages in the 12.3-hr long cell cycle of mouse duodenal crypt columnar cells. In addition, semi-thin Lowicryl sections of mouse duodenal crypts and cultured rat kidney cells were stained with the DNA-specific Hoechst 33258 dye and examined in the fluorescence microscope. The DNA detected by osmium-amine is in the form of nucleofilaments, seen at high magnification as long rows of 11 nm-wide rings (consisting of stained DNA encircling unstained histones). At all stages of the cycle as well as in nondividing cells, nucleofilaments are of three types: ‘free,’ ‘attached’ to chromatin accumulations, and ‘compacted’ in all chromatin accumulations, the form of dense spirals within. At stage I of the cycle, besides free and attached nucleofilaments, compacted ones are observed in the three heterochromatin forms (peripheral, nucleolus-associated, clumped). Soon after the S phase begins, chromatin ‘aggregates’ appear, which are small at stage II, mid-sized at stage III, and large at stage IV. Chromatin ‘bulges’ also appear at stage III and enlarge at stage IV, while heterochromatins disappear. At stage V, aggregates and bulges accrete into ‘chromomeres,’ a process responsible for the apparent chromosome condensation observed at prophase. The chromomeres gradually line up in rows and, at stage VIa (prometaphase), approach one another within each row and coalesce to build up the metaphase chromosomes which are fully formed at stage VIb (metaphase). Daughter chromosomes arising at stage VII (anaphase) are eventually packed into a chromosomal mass at each pole of the cell. During stage VIII (telophase), the chromosomal mass is split into large chunks. In the course of the G1 phase, the chunks thin out to give rise to irregular ‘bands’ at stage IX, the bands are then cleaved into central and peripheral fragments at stage X, and finally the central fragments are replaced by free nucleofilaments and clumps at stage XI, while the peripheral fragments are replaced by peripheral heterochromatin. The “nucleoli” at stages I–III are associated with stained heterochromatin but otherwise appear as unstained lucent areas, except for weakly stained patches composed of histone-free DNA filaments. During stage IV, nucleoli lose patches and associated heterochromatin, while weakly lucent, pale vesicles appear within nucleoli and in the nucleoplasm. By the end of substage VIa, nucleoli generally disappear, while pale vesicles persist around the chromosomes appearing at substage VIb. At stages VIII and IX, the vesicles seem to become strongly lucent and, at stages IX and X, they associate and fuse to yield homogeneous lucent areas, the ‘prenucleolar bodies,’ which include histone-free DNA patches. During stage XI, groups of these bodies associate to give rise to nucleoli. In conclusion, the cell cycle DNA changes can be classified into 4 broad periods (Fig. 6): 1) Stage I is a 2-hr long interphase “pause,” during which the stained DNA shows no signs of either chromosome condensation or decondensation, while the overall nuclear pattern is similar to that in nondividing cell nuclei. Nucleoli are fully developed. 2) From stage II to VIa, the “chromosome condensation” period extends over about 7 hr, during which the events are interpreted as follows. Throughout the S phase (stages II–IV), newly-synthesized segments of nucleofilaments approach one another, adhere and thus build aggregates and later bulges on nuclear matrix sites. The process continues until, soon after S ends, that is in G2, every segment of nucleofilament has become part of aggregates and bulges which thus include wholly new nucleofilaments. Then, the bonds tying aggregates and bulges to the matrix are broken, leaving them free at prophase (stage V) to approach one another, adhere, and thus build chromomeres. At prometaphase (substage VIa), a last adhesion process unites the chromomeres of a row which thus build metaphase chromosomes. Meanwhile, nucleoli lose the histone free-DNA patches at stage IV and disappear at the end of stage VIa. 3) Stages VIb and VII constitute the “climax” of the cell cycle, a 20-min period during which metaphase chromosomes at the equatorial plate split into daughter chromosomes. 4) From stage VIII to XI, the “chromosome decondensation” period extends over about 2 hr during which progressive dissociation of the stage VIII chromosomal mass eventually yields free and attached nucleofilaments as well as the three heterochromatins. Meanwhile lucent vesicles fuse into prenucleolar bodies which finally coalesce into nucleoli. Anat. Rec. 252:426–443, 1998. © 1998 Wiley-Liss, Inc.     

cyclin D3与淋巴瘤细胞增殖相关性的初步研究

目的：初步探讨cyclin D3与淋巴瘤细胞增殖活性的相关性。方法：运用荧光免疫细胞化学法结合激光共聚焦显微镜观察，分析淋巴瘤细胞株Raij细胞内cyclin D3与Ki67表达、定位的关系。通过转染pDsRed-cyclin D3和pSuPER-cyclin D3 siRNA质粒，分析改变eyelin D3表达水平对肿瘤细胞增殖能力的影响。结果：在分裂增殖旺盛的Raji细胞中，Ki67与cyclin D3共定位于细胞核内。导入外源性cyclin D3引起相应细胞核Ki67的表达增加，阻断内源性cyclin D3基因表达，导致同一细胞相同部位Ki67表达水平下降。结论：cyclin D3的表达水平与NHL细胞增殖活性密切相关，提示其可能在淋巴瘤的发生、发展过程中发挥重要作用。     

Bim regulates BCR-induced entry of B cells into the cell cycle

BH3-only Bcl-2 homologs are key regulators of the intrinsic apoptotic pathway. In particular, Bim, is critical for mediating apoptosis of hematopoietic cells including B cells. While studies using Bcl-2 Tg mice have defined an important role for Bcl-2 in cell cycle control, the role of BH3-only proteins is less clear. Using Bim KO mice, we show that Bim is required for B cells to enter the cell cycle normally. Bim KO B cells had reduced cell division compared to WT B cells in response to BCR, TLR3 or TLR4 signaling, whereas Bim deficiency did not affect TLR9-induced B cell division. Cell cycle progression in BCR- and LPS-stimulated Bim KO B cells was blocked at the G0-G1 stage. BCR-induced p130 degradation and pRb hyperphosphorylation on Ser807/811, which are critical for G1 entry, were reduced in Bim KO compared to WT B cells. Likewise, BCR-induced p27(Kip1) degradation was decreased in Bim KO compared to WT B cells. These defects in BCR-induced cell cycle entry correlated with a proximal defect in BCR-mediated intracellular calcium release in Bim KO B cells. Our results suggest that the balance of pro- and anti-apoptotic Bcl-2 family proteins is critical for controlling both cell cycle progression and apoptosis in B cells.     

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.     

HMGB2对肺腺癌细胞周期和增殖的影响

目的:探讨高迁移率族蛋白B2(HMGB2)对肺腺癌细胞周期和增殖的影响。方法:从Cancer RNA-Seq Nexus (CRN)数据库分析肺腺癌组织HMGB2表达情况;从OncoLnc数据库分析HMGB2与肺腺癌患者预后的相关性;从肿瘤单细胞数据库(CancerSEA)分析HMGB2与肺腺癌细胞14种功能状态的相关性;利用siRNA技术下调人肺腺癌A549细胞中HMGB2表达,通过real-time PCR和Western blot验证沉默效果,CCK8和EdU实验检测细胞的增殖。结果:HMGB2在肺腺癌中高表达;HMGB2高表达组肺腺癌患者的总生存期明显低于HMGB2低表达患者(log-rank检验P=0.017 3);HMGB2表达与肺腺癌细胞周期和增殖呈正相关;敲减HMGB2表达后A549细胞的活力和增殖能力显著降低(P<0.05)。结论:HMGB2的表达与肺腺癌细胞周期和增殖显著正相关,可以作为潜在的评估肺腺癌患者预后的标志物和治疗靶标。     

Zebrafish cdc25a is expressed during early development and limiting for post-blastoderm cell cycle progression.

Cdc25 phosphatases are required for eukaryotic cell cycle progression. To investigate mechanisms governing spatiotemporal dynamics of cell cycle progression during vertebrate development, we isolated two cdc25 genes from the zebrafish, Danio rerio, cdc25a, and cdc25d. We propose that Zebrafish cdc25a is the zebrafish orthologue of the tetrapod Cdc25A genes, while cdc25d is of indeterminate origin. We show that both genes have proliferation promoting activity, but that only cdc25d can complement a Schizosaccharomyces pombe loss of function cdc25 mutation. We present expression data demonstrating that cdc25d expression is very limited during early development, while cdc25a is widely expressed and consistent with the mitotic activity in previously identified mitotic domains of the post-blastoderm zebrafish embryo. Finally, we show that cdc25a can accelerate the entry of post-blastoderm cells into mitosis, suggesting that levels of cdc25a are rate limiting for cell cycle progression during gastrulation.     

IGFBP-2对人胶质瘤细胞的恶性增殖和细胞周期进程的影响

目的研究IGFBP-2的表达水平对胶质瘤细胞系增殖和细胞周期进程的作用。方法采用免疫印迹筛选IGFBP-2高表达的胶质瘤细胞系,设计并合成IGFBP-2特异的siRNA,将其导入筛选出的胶质瘤细胞系,采用实时定量PCR和免疫印迹验证IGFBP-2的表达下调情况,随后采用MTT实验的流式细胞技术检测IGFBP-2的下调对细胞增殖和细胞周期进程的影响情况。结果合成的IGFBP-2特异siRNA能够显著降低胶质瘤细胞系IGFBP-2的表达,siRNA介导的IGFBP-2的表达下调能够显著抑制胶质瘤细胞的增殖和细胞周期的推进(0.01)。结论 IGFBP-2对胶质瘤细胞的恶性表型具有促进作用,为进一步探讨IGFBP-2影响胶质瘤细胞行为学的机制奠定了基础。     

Cyclin D3 coordinates the cell cycle during differentiation to regulate erythrocyte size and number

Genome-wide association studies (GWASs) have identified a genetic variant of moderate effect size at 6p21.1 associated with erythrocyte traits in humans. We show that this variant affects an erythroid-specific enhancer of CCND3. A Ccnd3 knockout mouse phenocopies these erythroid phenotypes, with a dramatic increase in erythrocyte size and a concomitant decrease in erythrocyte number. By examining human and mouse primary erythroid cells, we demonstrate that the CCND3 gene product cyclin D3 regulates the number of cell divisions that erythroid precursors undergo during terminal differentiation, thereby controlling erythrocyte size and number. We illustrate how cell type-specific specialization can occur for general cell cycle components—a finding resulting from the biological follow-up of unbiased human genetic studies.     

碘酸钾对人甲状腺癌细胞系周期的影响

目的探讨碘酸钾(KIO3)对人甲状腺鳞癌细胞系SW579细胞周期/增殖、细胞周期素D1(cyclin D1)及增殖细胞核抗原Ki67 mRNA/蛋白水平的影响。方法 CCK8法检测不同浓度(0、10-6、10-5、10-4、10-3、10-2和10-1mol/L)KIO3对细胞增殖的影响;0、10-6或10-2mol/L KIO3处理细胞48 h后,用碘化丙啶单染流式细胞术检测细胞周期;用实时定量PCR和Western blot分别检测cyclin D1和Ki67的mRNA及蛋白水平。结果 10-6mol/L或10-2mol/L KIO3分别促进或抑制SW579细胞增殖(P0.05);10-6mol/L组细胞G1期比例下降(P0.05),S期比例增加(P0.05),而10-2mol/L组各期细胞比例发生相应变化(P0.05);同时,10-6mol/L KIO3显著上调细胞内的cyclin D1(P0.05)和Ki67(P0.05)mRNA水平;而10-2mol/L KIO3显著下调细胞内的cyclin D1和Ki67 mRNA及蛋白水平(P0.05)。结论 KIO3影响SW579细胞增殖及周期可能与其对cyclin D1和Ki67的调节作用及剂量有关。     

Langerhans cell histiocytosis: an evaluation of histopathological parameters,demonstration of proliferation by Ki-67 and mitotic bodies

PURPOSE: Langerhans cell histiocytosis (LCH) is a disease with a variable clinical manifestation, being localised (SS) or disseminated (MS). The etiology and pathogenesis of LCH is unknown. It is a proliferative disorder of monoclonal origin, but not necessarily neoplastic. In our study we evaluated histopathological parameters and proliferative activity in LCH. MATERIALS AND METHODS: Infiltrates from 43 patients with LCH were investigated (nSS=32, nMS=11). We evaluated different histopathological parameters semiquantitatively, demonstrating proliferation using immunohistochemistry for Ki-67. RESULTS: Overall, the histopathological picture of LCH was heterogeneous. The degree of eosinophilia and presence of necroses was significantly higher in SS-infiltrates compared to MS-infiltrates. Mitotic figures were detected in more than half the infiltrates. The Langerhans origin was verified by CD1a. Ki-67 was highly expressed in all but one infiltrate. CONCLUSIONS: The presence of necrosis and the degree of eosinophilia are related to SS-disease in our study. Ki-67 expression and the presence of mitotic figures indicate that local proliferation contributes to the accumulation of Langerhans cells. Supported by the histopathological appearance of the lesions and a level of Ki-67 expression lower than that of neoplastic tissue, we suggest that LCH is a reactive condition, possibly induced by immunostimulation caused by unknown agents.     

Cyclin D2 is essential for BCR-mediated proliferation and CD5 B cell development

Progression into G(1) in B lymphocytes is regulated by cyclins D2 and D3, components of the cell cycle machinery currently believed to have overlapping and potentially redundant roles in cell cycle control. To study the specific role of cyclin D2 in B lymphocyte proliferation, we examined B cells from cyclin D2(-/-) mice and demonstrate a specific requirement for cyclin D2 in BCR- but not CD40- or lipopolysaccharide-induced proliferation. Furthermore, conventional B cell development proceeds normally in the mutant mice; however, the CD5 B cell compartment is dramatically reduced, suggesting that cyclin D2 is important in CD5 B cell development as well as antigen-dependent B cell clonal expansion.     

红景天苷对小鼠MSCs的增殖及其细胞周期的影响

目的本研究探讨了不同浓度红景天苷通过ERK信号通路对小鼠骨髓间充质干细胞增殖和细胞周期变化的影响及机制。方法实验分为对照组(D-MEM/F-12完全培养液)、不同浓度红景天苷(5~100μg/m L)诱导组和阻断组(5~100μg/m L+30μg/L PD98059),利用CCK8法、流式细胞术及Western blot分别检测ERK信号通路阻断前后细胞抑制率、细胞周期及其相关蛋白的表达。结果 10μg/m L诱导组和阻断组均能抑制细胞增殖,且5μg/m L(P0.01)、25μg/m L(P0.05)和100μg/m L(P0.05)诱导组对细胞抑制显著高于阻断组。25μg/m L诱导组和阻断组G_0/G_1期细胞比例均增加,细胞周期阻滞于G0/G1期。诱导组和阻断组cyclin A和cyclin D1蛋白的表达与对照组比较均下调,P21蛋白的表达上调。阻断组cyclin A蛋白的表达与诱导组比较明显下调(P0.01);50和100μg/m L诱导组与阻断组相比cyclin D1蛋白表达显著下调,P21蛋白的表达显著上调(P0.01)。结论红景天苷能通过调节细胞周期相关蛋白的表达抑制小鼠MSCs细胞的增殖和细胞周期的改变。