Recombinant cyclin E expression activates proliferation and obviates surface attachment of chinese hamster ovary (CHO) cells in protein-free medium

Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. (c) 1995 John Wiley & Sons Inc.     

Janus faces of ras: anti or pro-apoptotic?

Cell population homeostasis is a balance between cell proliferation on one hand and the rate of cell loss on the other hand. Normal tissue homeostasis requires the physiological deletion of cells by activation of apoptosis, a genetically determined program of autonomous cell death. ras is most probably the most important oncogene in human cancer. It is mutated in 30% of all tumors especially those of the gastrointestinal tract. In regulating apoptosis ras has many faces. it has both negative and positive effects depending on the stimulation and cell type. The responses of cells to ras signaling depends on the level of Ras expression, the activity of various pathways, and which of the cell cycle check points are functioning. New farnesyl transferase inhibitors and non-steroidal anti-inflammatory compounds may provide a new strategy for novel therapeutic modalities in the treatment of human cancer. The first clinical trials have been initiated, preliminary results are promising, although no firm results are yet available.     

Differences in degradation lead to asynchronous expression of cyclin E1 and cyclin E2 in cancer cells

Cyclin E1 is expressed at the G₁/S phase transition of the cell cycle to drive the initiation of DNA replication and is degraded during S/G₂M. Deregulation of its periodic degradation is observed in cancer and is associated with increased proliferation and genomic instability. We identify that in cancer cells, unlike normal cells, the closely related protein cyclin E2 is expressed predominantly in S phase, concurrent with DNA replication. This occurs at least in part because the ubiquitin ligase component that is responsible for cyclin E1 downregulation in S phase, Fbw7, fails to effectively target cyclin E2 for proteosomal degradation. The distinct cell cycle expression of the two E-type cyclins in cancer cells has implications for their roles in genomic instability and proliferation and may explain their associations with different signatures of disease.     

In Vitro transformation of LW13 rat liver epithelial cells

A rat liver epithelial cell line designated LW 13 was established using a sequential sedimentation method.The cell line retained many normal proerties of liver epithelial cells and showed some structural and functional features resembling those of liver parenchymal cells,LW13 cells became malignant after the intrduction of exogenous transforming EJ Ha ras gene,Tumors produced by inoculation of the transformed cells into baby rats contained areas of poorly differentialted hepatocellular carcinoma,In situ hybridization analysis confirmed the random rather than specific integration of exogenous ras gene into host chromosomes.Furthermore,an at least tenfold increase in the expression of the endogenous c mys gene was detected among transformed cell lines,suggesting the involvement of the c myc proto oncogene in the in vitro transformation of rat liver epithelial cells by EJ Ha ras oncogene.     

糖原合酶激酶3β以细胞周期蛋白D1依赖性方式引发人肺腺癌细胞A549细胞周期阻滞

对糖原合酶激酶3β（glycogen synthase kinase 3β,6SK3β）在细胞增殖中的作用研究,在不同细胞系和不同刺激因素作用下得出了不同结论,本文旨在探讨GSK3β在人肺腺癌细胞系A549细胞生长中的直接作用。A549细胞瞬时转染持续激活型S9A-GSK3β以及显性负突变型KM-GSK3β两种GSK3β突变型质粒,改变GSK3β活性。24 h后,分别进行细胞计数,流式细胞术及Western blot检测。结果显示,增强GSK3β活性可导致细胞数量下降,G．期细胞百分比升高。细胞周期蛋白D1表达水平被GSK3β下调。结果提示,GSK3β可能以细胞周期蛋白D1依赖性方式引发A549细胞的G,期阻滞,从而发挥生长抑制效应。     

Mitotic and mitogenic Wnt signalling

Canonical Wnt signalling plays an important role in development, tissue homeostasis, and cancer. At the cellular level, canonical Wnt signalling acts by regulating cell fate, cell growth, and cell proliferation. With regard to proliferation, there is increasing evidence for a complex interaction between canonical Wnt signalling and the cell cycle. Mitogenic Wnt signalling regulates cell proliferation by promoting G1 phase. In mitosis, components of the Wnt signalling cascade function directly in spindle formation. Moreover, Wnt signalling is strongly activated in mitosis, suggesting that 'mitotic Wnt signalling' plays an important role to orchestrate a cell division program. Here, we review the complex interplay between Wnt signalling and the cell cycle.     

慢性缺氧大鼠肺内(肺血管壁)细胞增殖、凋亡及c-myc、p53基因表达研究

目的和方法：应用免疫组织化学、原位末端标记技术及Ｎｏｒｔｈｅｒｎ杂交等方法检测慢性缺氧大鼠肺内特别是肺血管壁细胞增殖、凋亡及相关基因ｃｍｙｃ、ｐ５３表达。结果：正常及慢性缺氧大鼠肺内检出一定比率的增殖、凋亡阳性细胞,两类细胞在肺内呈不均匀散在分布。在缺氧大鼠肺内,增殖阳性细胞绝大部分是肺小血管壁细胞,凋亡性染色细胞在肺小血管壁上较对照组少见。缺氧１、２周组大鼠肺内细胞增殖指数显著增高而凋亡指数显著减少,细胞增殖凋亡比值分别约为对照组３与３５倍。ｃｍｙｃ及ｐ５３是细胞增殖、凋亡密切相关的两种癌（抑癌）基因,前者在缺氧大鼠肺内表达显著增加,而后者（野生型）表达显著减少。结论：可能由于ｃｍｙｃ及ｐ５３基因异常表达所致的细胞增殖、凋亡失衡参与了慢性缺氧性肺血管结构改建的调节。     

Growth of myoblasts in lipoprotein-supplemented,serum-free medium: Regulation of proliferation by acidic and basic fibroblast growth factor

Summary BC₃H1 myoblast cells seeded at low density on gelatin-coated dishes and exposed to a 1∶1 (vol/vol) mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, insulin, and basic or acidic fibroblast growth factor (FGF). This serum-free medium combination supported cell multiplication at a rate equal to that of serum-supplemented medium, and at low cell input (10³ cells/35-mm dish). It also allowed serial transfer of the cultures under serum-free conditions. HDL seems to promote cell survival and to act as progression factor allowing cells to divide when exposed to either basic or acidic FGF. When the potency of basic and acidic FGF were compared, acidic FGF was 20-fold less potent than basic FGF.     

Ankyrin Repeat-Rich Membrane Spanning (ARMS)/Kidins220 Scaffold Protein Regulates Neuroblastoma Cell Proliferation through p21

Cell proliferation is tightly controlled by the cell-cycle regulatory proteins, primarily by cyclins and cyclin-dependent kinases (CDKs) in the G₁ phase. The ankyrin repeat-rich membrane spanning (ARMS) scaffold protein, also known as kinase D-interacting substrate of 220 kDa (Kidins 220), has been previously identified as a prominent downstream target of neurotrophin and ephrin receptors. Many studies have reported that ARMS/Kidins220 acts as a major signaling platform in organizing the signaling complex to regulate various cellular responses in the nervous and vascular systems. However, the role of ARMS/Kidins220 in cell proliferation and cell-cycle progression has never been investigated. Here we report that knockdown of ARMS/Kidins220 inhibits mouse neuroblastoma cell proliferation by inducing slowdown of cell cycle in the G₁ phase. This effect is mediated by the upregulation of a CDK inhibitor p21, which causes the decrease in cyclin D1 and CDK4 protein levels and subsequent reduction of pRb hyperphosphorylation. Our results suggest a new role of ARMS/Kidins220 as a signaling platform to regulate tumor cell proliferation in response to the extracellular stimuli.     

In vitro growth properties and PCR ras gene analysis of a murine embryonal carcinoma cell line harboring an amplified c-Ki-ras gene

Ras proto-oncogenes are thought to be involved in both proliferation and malignant processes. We examined some growth properties of a murine embryonal carcinoma cell line (ECC), PCC4, that have been shown previously to be amplified for the c-KI-ras gene. Our results show that doubling time and plating efficiency are not significantly affected by such amplification. To examine the possible link between malignant behavior and c-Ki-ras alteration, we investigated activating mutations in this PCC4 cell line as well as in other ECC. Analysis of the in vitro amplified Ki-ras gene by PCR technology has not revealed point mutations in any of the ECC examined.     

抗酶1基因转染对HeLa细胞增殖及细胞周期的抑制作用

研究抗酶(antizyme)1对人宫颈癌HeLa细胞增殖与细胞周期的影响,并分析抗酶1对细胞周期蛋白D1(cyclin D1)的表达影响.采用定点突变技术,将抗酶1的frameshift位点缺失,随后将突变基因重组至真核表达载体pEGFP-N1中,鉴定后转染HeLa细胞.通过MTT法检测细胞增殖变化,流式细胞术分析抗酶1对细胞周期的影响.RT-PCR和Western印迹检测抗酶1转染对细胞周期蛋白 D1基因表达的影响.酶切结果显示,抗酶1突变基因成功克隆至pEGFP-N1中.成功转染HeLa细胞后,检测结果显示,抗酶1能够减慢HeLa细胞增殖速度,并使细胞停滞于G₀/G₁期,细胞周期蛋白D1基因的表达同时受到抑制.实验说明,抗酶1基因能够抑制HeLa细胞增殖,通过降低细胞周期蛋白D1的表达阻滞细胞周期.     

Insulin but not IGF-I is required for the maintenance of the adipose phenotype in the adipogenic cell line 1246

Summary The mouse adipogenic cell line 1246 which possesses both insulin and insulin-like growth factor I (IGF-I) receptors was used to investigate the role of IGF-I and insulin on the proliferation of adipocyte precursors and their differentiation into mature adipocytes. Results indicate that both insulin and IGF-I stimulate the proliferation of the 1246 adipocyte precursors with IGF-I being slightly more potent than insulin. Dose-response studies indicated that both polypeptides acted at physiological concentrations corresponding to binding to their own receptors. In contrast, comparison of insulin and IGF-I capacity to stimulate terminal adipose differentiation indicated that only insulin was active when added at physiological concentrations. IGF-I could not stimulate adipocyte differentiation except at supraphysiological concentrations (100 ng/ml and above) permitting its binding to the insulin receptors on 1246 cells. Time course study of expression of early and late markers of adipose differentiation indicated that the induction of markers such as adipose differentiation-related protein (ADRP), lipoprotein lipase (LPL) and fatty acid binding protein (FAB) took place even in the absence of insulin. However, the level of early and late differentiation markers decreased to a level below the one found in undifferentiated cells when cells had been maintained in the absence of insulin after differentiation had been initiated. These data indicate that although insulin is not necessary for the early onset of the adipose differentiation program, it is stringently required for the maintenance of the adipocyte phenotype and cannot be substituted by IGF-I.     

锂和三尖杉酯碱对HL—60细胞增殖,分化和c—myc表达的影响

本研究利用细胞培养技术观察了氯化锂和三尖杉酯碱（ＨＴ）对ＨＬ－６０细胞增殖的影响,不同浓度的氯化锂对ＨＬ－６０细胞的集落形成和３Ｈ－ＴｄＲ参入均呈剂量依赖式抑制;三尖杉酯碱亦有类似的作用。在培养体系中加氯化锂和三尖杉酯碱时,对ＨＬ－６０细胞数及集落形成抑制作用与单用二者相比较有明显增加。用ＮＢＴ还原试验,氯化锂和三尖杉酯碱均促进ＨＬ－６０细胞的分化,小剂量氯化锂还能加强三尖杉酯碱对ＨＬ－６０细胞诱导分化作用。从氯化锂和三尖杉酯碱处理的ＨＬ－６０细胞中提取总ＲＮＡ,应用ＲＴ／ＰＣＲ检测ｃ－ｍｙｃ的表达,结果表明经氯化锂和三尖杉酯碱处理的ＨＬ－６０细胞ｃ－ｍｙｃ表达均降低,与未处理的ＨＬ－６０细胞ｃ－ｍｙｃ比较,说明氯化锂和三尖杉酯碱均能抑制ｃ－ｍｙｃ的表达,提示ｃ－ｍｙｃ很可能在白血病细胞增殖、分化中起调控作用。     

p27Kip1 enforces maintenance of quiescence in the mammalian ear and the pituitary gland

Comment on: Oesterle EC, et al. Cell Cycle 2011; 10:1237-48.