豆类凝集素FRIL通过调控细胞周期分子的表达维持造血干/祖细胞的静息

目的从细胞周期角度探讨豆类凝集素 FRIL(Flt3 receptor-interacting lectin)体外维持造血干/祖细胞静息的分子机制。方法以添加 FRIL、Flt3配体(FL)和不添加因子的培养基分别培养脐血 CD34~ 细胞,采用 RT-PCR 和 Western blot 法分别从 mRNA 和蛋白水平检测细胞周期相关分子的表达。结果新分离的 CD34~ 细胞中未检测到 G_0/G_1期相关 Cyclin 和 CDK 蛋白的表达,培养3d 时FRIL 组 CyclinD3、CDK6蛋白相对表达量(分别为483±63、553±39)低于两个对照组(FL 组:2437±52、3209±98;空白对照组:914±105、1497±55);培养3 d 时 FRIL 组 P27蛋白相对表达量(0.312±0.030)低于对照组(FL 组:0.787±0.024;空白对照组:0.616±0.029),但表达较高的 P53蛋白(FRIL组、FL 组、空白对照组分别为4.476±0.159、0.581±0.099、2.167±0.114)。FRIL 组细胞周期正向调节因子的 mRNA 相对表达量低于或相当于 FL 组和空白对照组。结论 FRIL 通过抑制参与造血细胞周期调控的 CyclinD3和 CDK6的表达,延迟了造血干/祖细胞的细胞周期,P27在 FRIL 抑制造血干/祖细胞分化中发挥了重要作用,P53也参与了 FRIL 对造血干/祖细胞的维持作用。     

Activation and growth of colony-stimulating factor-dependent cell lines is cell cycle stage dependent

Hematopoietic cell development is regulated by a series of growth factors that are progressively restricted in their biological activity. IL-3 is a multi-lineage growth factor that supports the growth and differentiation of progenitor cells belonging to multiple lineages. However, the mechanism by which IL-3 induces proliferation and differentiation of these cells is not completely understood. In this report, we have used two IL-3-dependent cell lines, FDC-P1 (a myeloid progenitor) and F15.12 (a lymphoid progenitor) to investigate IL-3-mediated growth and differentiation. When either FDC-P1 or FL5.12 cells are deprived of IL-3, greater than 90% of all cells accumulate in the G0 phase of the cell cycle. Upon readdition of IL-3, the cells will reenter the active phases of the cell cycle. Therefore, IL-3 can act as both a competence (G0----G1) factor, and a progression (G1----M) factor for hematopoietic precursor clones. FDC-P1 cells can also proliferate in response to granulocyte/macrophage colony-stimulating factor (G/M-CSF) and IL-4 (B cell stimulatory factor 1 [BSF-1]). However, resting (G0) FDC-P1 cells have lost their ability to grow in response to both G/M-CSF and IL-4, even though both factors can induce a G0----G1 transition. Therefore, G/M-CSF or IL-4 behave as progression factors among certain IL-3-responsive clones, and in those cases only in defined points in the cell cycle. Both IL-4 and G/M-CSF can maintain long-term growth of FDC-P1 cells. Upon removal of factor for 24 h, these clones accumulate in the G1 phase of the cell cycle and do not appear to enter G0 even after 36 h of factor deprivation. Therefore, cells maintained in G/M-CSF or IL-4 have altered growth requirements compared with the IL-3-dependent lines from which they were derived. The ability of various hematopoietic growth factors to regulate cell cycle progression in IL-3-dependent cell lines is dependent not only upon the lineage from which these cells were derived, but also the phase of the cell cycle in which those cells reside. The consequences of these interactions dictate the manner by which various clones will respond to CSFs and whether the cells will grow and/or differentiate.     

Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death

Expression of the bcl-2 gene has been shown to effectively confer resistance to programmed cell death under a variety of circumstances. However, despite a wealth of literature describing this phenomenon, very little is known about the mechanism of resistance. In the experiments described here, we show that bcl-2 gene expression can result in an inhibition of cell division cycle progression. These findings are based upon the analysis of cell cycle distribution, cell cycle kinetics, and relative phosphorylation of the retinoblastoma tumor suppressor protein, using primary tissues in vivo, ex vivo, and in vitro, as well as continuous cell lines. The effects of bcl-2 expression on cell cycle progression appear to be focused at the G1 to S phase transition, which is a critical control point in the decision between continued cell cycle progression or the induction programmed cell death. In all systems tested, bcl-2 expression resulted in a substantial 30-60% increase in the length of G1 phase; such an increase is very substantial in the context of other regulators of cell cycle progression. Based upon our findings, and the related findings of others, we propose a mechanism by which bcl-2 expression might exert its well known inhibition of programmed cell death by regulating the kinetics of cell cycle progression at a critical control point.     

丁酸钠联合全反式维甲酸对MDS细胞株SKM-1的诱导分化作用及其机制初步探讨

本研究探讨丁酸钠(NaB)抑制MDS细胞株SKM-1细胞生长、诱导其分化的分子机制，并研究其与全反式维甲酸(ATRA)的协同作用。用台盼蓝拒染实验观察药物对细胞生长曲线的影响；四氮唑盐还原试验和细胞表面分化抗原检测观察药物对细胞的分化作用；流式细胞术分析细胞周期；RT-PCR检察D型细胞周期蛋白、CDK和P21在mRNA水平的表达。结果表明：NaB和(或)ATRA均可抑制SKM-1细胞的生长，诱导细胞分化，将细胞周期阻滞于G0／G1期；ATRA下调CDK6、CDK4、cyclin D3和cyclin D1 mRNA的水平；NaB下调CDK2、cyclin D2和cyclin D1 mRNA的水平；两药联用下调CDK6、CDK4、CDK2、cyclin D1、cyclin D2和cyclin D3 mRNA的水平；ATRA和(或)NaB均上调P21 mRNA的水平。结论:NaB诱导SKM-1的分化可能是通过上调P21 mRNA的水平和抑制cycLin D-CDK复合体的形成完成的，NaB与ATRA对SKM-1细胞株的分化有协同作用。     

Identification of genes highly expressed in G2-arrested Chinese hamster ovary cells by differential display analysis

Abnormal cell cycle regulation is believed to be an important step in tumorigenesis. In mammalian cells, DNA damage commonly leads to cell cycle arrest in G2; however, little is known about the detailed biochemical mechanisms underlying the DNA damage-induced G2 arrest. In order to identify genes differentially expressed in association with G2 arrest, differential display analysis was performed between exponentially growing Chinese hamster ovary (CHO) cells and G2-arrested CHO cells induced by etoposide, SN-38, or X-radiation. We identified five cDNA clones whose expression was up-regulated in G2-arrested CHO cells. Sequence analysis revealed that three clones were homologous to known genes: isogene I of translation initiation factor eIF-4A, ribosomal protein L13, and translation repressor NAT1. The remaining two clones showed no homology to known genes. These results indicate that DNA damage can alter the expression of multiple genes, including translational regulators.     

新维甲类化合物对早幼粒细胞白血病和正常造血增殖分化作用的…

发掘维甲１号、维甲２号、维甲３号和维甲４号应用价值。在体外研究其对人急性早幼粒细胞白血病细胞株ＮＢ４和原代ＡＰＬ细胞诱导分化和克隆增殖的抑制及其对正常造血细胞的促增殖作用。研制的新维甲类化合物对ＡＰＬ细胞具有诱导分化并对其克隆生长有抑制作用，且能促进正常造血，其中以维甲２号分化作用为突出进一步研究开发它的临床应用有一定的价值。     

Use of merocyanine 540 for the isolation of quiescent, primitive human bone marrow hematopoietic progenitor cells.

Merocyanine 540 (MC540) is a membrane probe that inserts preferentially into loosely packed domains in the phospholipid bilayer of intact cells. Previous experiments have demonstrated that MC540 will bind to human bone marrow (BM) hematopoietic progenitor cells (HPC). Fractions of mononuclear BM cells expressing high MC540 fluorescence have been shown to be enriched for myeloid progenitors and cells residing in the S/G2 + M phases of the cell cycle. We rationalized that MC540 uptake could be used to distinguish between quiescent and metabolically active cells and, therefore, to fractionate normal and leukemic BM cells and normal mobilized peripheral blood (MPB) cells into functionally distinct groups of progenitors. BM and MPB cells were separated into fractions ranging in fluorescence from MC540Bright to MC540Dim. Cell cycle analysis of these fractions revealed that the MC540Dim fraction of normal and CML BM CD34+ cells constituted the most quiescent fraction, and the MC540Bright fractions from these cell types contained the most actively cycling cells. However, no differences in the percentage of cells in G/G1 were observed between MC540Bright and MC540Dim fractions of MPB CD34+ cells. To investigate if these cell cycle status differences translated into distinct functional properties, the hematopoietic potential of BM CD34+MC540Bright and CD34+MC540Dim cell fractions was analyzed in vitro in long-term BM cultures and limiting dilution analysis (LDA) assays. CD34+MC540Dim cells produced more total and committed progenitor cells in long-term cultures than did the CD34+MC540Bright fraction. The CD34+MC540Dim fraction also contained a 2-fold higher number of long-term hematopoietic culture-initiating cells (LTHCIC) than the CD34+MC540Bright fraction, as defined by LDA assays. These data demonstrate that MC540 can be a useful probe for the isolation of primitive HPC from some hematopoietic tissues and may assist in monitoring structural changes in the phospholipid bilayer during proliferation and differentiation of HPC.     

Pharmacologic inhibition of cyclin-dependent kinase 4/6 activity arrests proliferation in myoblasts and rhabdomyosarcoma-derived cells

Myoblast cell cycle exit and differentiation are mediated in part by down-regulation of cyclin D1 and associated cyclin-dependent kinase (Cdk) activity. Because rhabdomyosarcoma may represent a malignant tumor composed of myoblast-like cells failing to exit the cell cycle and differentiate, we considered whether excess Cdk activity might contribute to this biology. Cyclin D-dependent Cdk4 and Cdk6 were expressed in most of a panel of six human rhabdomyosarcoma-derived cell lines. Cdk4 was expressed in 73% of alveolar and embryonal rhabdomyosarcoma tumors evaluated using a human tissue microarray. When challenged to differentiate by mitogen deprivation in vitro, mouse C2C12 myoblasts arrested in G(1) phase of the cell cycle, whereas four in the panel of rhabdomyosarcoma cell lines failed to do so. C2C12 myoblasts maintained in mitogen-rich media and exposed to a Cdk4/Cdk6 inhibitor PD 0332991 accumulated in G(1) cell cycle phase. Similar treatment of rhabdomyosarcoma cell lines caused G(1) arrest and prevented cell accumulation in vitro, and it delayed growth of rhabdomyosarcoma xenografts in vivo. Consistent with a role for Cdk4/Cdk6 activity as a regulator of myogenic differentiation, we observed that PD 0332991 exposure promoted morphologic changes and enhanced the expression of muscle-specific proteins in cultured myoblasts and in the Rh30 cell line. Our findings support the concept that pharmacologic inhibition of Cdk4/Cdk6 may represent a useful therapeutic strategy to control cell proliferation and possibly promote myogenic differentiation in rhabdomyosarcoma.     

新维甲类化合物对早幼粒细胞白血病和正常造血增殖分化作用的体外研究

目的：发掘维甲１号、维甲２号、维甲３号和维甲４号（与全反式维甲酸结构不同的新维甲类化合物）应用价值。方法：在体外研究其对人急性早幼粒细胞白血病（ＡＰＬ）细胞株ＮＢ４和原代ＡＰＬ细胞诱导分化和克隆增殖的抑制及其对正常造血细胞的促增殖作用。结果：细胞形态学、ＮＢＴ反应、细胞周期动力学和ＣＦＵ－Ｌ克隆形成抑制率等多参数研究表明，新维甲类化合物具有诱导ＮＢ４细胞株和原代ＡＰＬ细胞分化成熟及抑制ＣＦＵ－Ｌ克隆生长的作用，作用强度与结构有关，维甲２号活性较强，明显高于全反式维甲酸及其它新维甲类化合物（Ｐ＜０．０１）。半固体造血祖细胞集落培养研究表明，新维甲类化合物能促进ＣＦＵ－ＧＭ，ＢＦＵ－Ｅ和ＣＦＵ－Ｍｅｇ增殖。结论：研制的新维甲类化合物对ＡＰＬ细胞具有诱导分化并对其克隆生长有抑制作用，且能促进正常造血，其中以维甲２号分化作用为突出，进一步研究开发它的临床应用有一定的价值。     

Ex vivo expansion and differentiation of hematopoietic stem cells.

Hematopoietic stem cells are phenotypically very heterogeneous, probably reflecting the degree of activation and/or differentiation. This cell population is capable of high-level proliferative activity and multilineage differentiation. Despite its potential for self-renewal, the hematopoietic stem cell exists in a quiescent state for prolonged periods of time. The mechanism(s) involved in triggering these cells to enter the cell cycle is/are not totally clear; however, cytokines (both positive and negative regulators) are implicated. Most, if not all known cytokines that interact at the stem cell level do so not only by inducing proliferation but also differentiation. The ability to maintain a population of truly primitive stem cells for extended periods of time in vitro is currently under investigation by many research groups.     

Distinct effects of dinuclear ruthenium(III) complexes on cell proliferation and on cell cycle regulation in human and murine tumor cell lines

We have examined the biological and antitumor activity of a series of dinuclear ruthenium complexes. The aim of this study was to compare the in vitro effects of these new compounds on cell proliferation, cell distribution among cell cycle phases, and the expression of some proteins involved in cell cycle regulation. Results obtained show a mild cytotoxic activity against human and murine cell lines, more evident after prolonged exposure of cell challenge. Two of the eight dinuclear complexes [namely, compounds D3 (Na(2)[(RuCl(4)(dmso-S))(2)(mu-bipy)]) and D7 ([NH(4)][(RuCl(4)(dmso-S))(mu-pyz)(RuCl(3)(dmso-S)(dmso-O))]) modify cell cycle distribution similarly to imidazolium trans-imidazoledimethylsulfoxidetetrachlororuthenate (NAMI-A), whereas the others have a low or negligible effect on this parameter. If we correlate the induction of cell cycle modifications with ruthenium uptake by tumor cells and with the modulation of proteins regulating cell cycle, we may stress that the induction of G(2)-M cell cycle arrest is related to the achievement of a threshold concentration of ruthenium inside the cells, which is dependent on the cell line being used, and that only cyclin B, among cell cycle regulating proteins examined by immunoblotting assays, appears to be significantly modified. This in vitro study shows that dinuclear ruthenium complexes may have a behavior similar to that of the monomer NAMI-A. These results encourage the future experimentation of their pharmacological properties in in vivo models.     

转录因子GATA—2在早期造身过程中的作用

转录因子ＧＡＴＡ－２是ＧＡＴＡ家族的一成员,以其锌指结构结合于「（Ｔ／Ａ（ＧＡＴＡ）Ａ／Ｇ」的共同ＤＮＡ序列结构。近期,通过敲除小鼠ＧＡＴＡ－２基因的实验证明了ＧＡＴＡ－２在造血细胞发育中的重要地位。ＧＡＴＡ－２基因断裂导致全部造血祖细胞的减少;相反,强制表达ＧＡＴＡ－２则阻止正常造血。ＧＡＴＡ－２的调节作用发挥于早期胚胎时期并与其它的ＧＡＴＡ转录因子共同作用于粒系、红系、巨核系和肥大细胞系等的增     

Nicotine activates nuclear factor of activated T cells c2 (NFATc2) and prevents cell cycle entry in T cells

We used primary peripheral blood T cells, a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously, to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly, T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that, in the face of chronic nicotine exposure, selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases.     

MIP-1α和血小板第4因子对造血干细胞化疗药物损伤的保护作用

目的 研究巨噬细胞炎性蛋白（ＭＩＰ－１α）、血小板第４因子（ＰＦ４）及二联合对造血干细胞化疗药物损伤的保护效应及其分子机制。方法 将骨髓、脐血单个核细胞及白血病细胞株ＨＬ－６０分别予ＭＩＰ－１α、ＰＦ４、ＭＩＰ－１α＋ＰＦ４、ＰＢＳ预处理４８ｈ,再经柔红霉（ＤＮＲ）孵育２４ｈ后,用锥虫蓝９台盼蓝）拒染法测细胞活性,用流式细胞仪测细胞仪测细胞周期及ＣＤ３４＾＋、ＣＤ３８＾－细胞含量,细胞集落培养、     

Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates

Knowledge of the molecular networks controlling the proliferation and fate of hematopoietic stem cells (HSC) is essential to understand their function in maintaining blood cell production during normal hematopoiesis and upon clinical transplantation. Using highly purified stem and progenitor cell populations, we define the proliferation index and status of the cell cycle machinery at discrete stages of hematopoietic differentiation and during cytokine-mediated HSC mobilization. We identify distinct sets of cell cycle proteins that specifically associate with differentiation, self-renewal, and maintenance of quiescence in HSC and progenitor cells. Moreover, we describe a striking inequality of function among in vivo cycling and quiescent HSC by demonstrating that their long-term engraftment potential resides predominantly in the G(0) fraction. These data provide a direct link between HSC proliferation and function and identify discrete molecular targets in regulating HSC cell fate decisions that could have implications for both the therapeutic use of HSC and the understanding of leukemic transformation.     

胆固醇合成抑制剂洛伐他汀对NB4细胞体外作用的研究

目的 研究胆固醇合成抑制剂洛代他汀（Lovastain,LOV)对NB4白血病细胞增殖、凋亡、分化的影响。方法 以NB4白血病细胞为模型,用MTT比色法、锥虫蓝拒染法首先观察LOV对细胞增殖及活力的影响。通过细胞形态学观察、NBT还原试验、DNA凝胶电泳、流式细胞术细胞周期分析、TUNEL、RT-PCR半定量测定bcl-2 mRNA水平,系统观察LOV对NB4体外诱导分化和凋亡的情况。最后利用RT-PCR半定量测定H、K、N-ras基因表达水平,结合流式细胞术进行胞膜P21^Ras蛋白测定,探讨LOV作用机制。结果 （1）LOV抑制NB4细胞增殖,IC50为12.59μmol/L。（2）LOV诱导NB4细胞凋亡,影响细胞周期进程,细胞阻滞于G1/S期。LOV在诱导细胞凋亡过程中随作用时间延长,bcl-2表达水平逐渐下降。（3）LOV不影响NB4细胞分化。（4）LOV作用于NB4细胞,H、K、N-ras基因转录水平无变化,但膜表面P21^Ras蛋白水平随时间进行性下降。结论 LOV抑制NB4细胞增殖并诱导凋亡,使细胞周期进程阻滞于G1/S期。bcl-2参与了LOV诱导NB4细胞凋亡的基因调控。LOV对NB4细胞分化无影响。p21^Ras蛋白异戊二烯化受抑而阻滞p21^Ras蛋白定位细胞膜上可能是LOV影响NB4细胞的主要机制。     

Peroxisome proliferator-activated receptor ligands affect growth-related gene expression in human leukemic cells

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear receptors. Three subtypes of PPARs (alpha, beta, and gamma) have been identified in different tissues. PPAR alpha and PPAR gamma ligands inhibit cell proliferation and induce differentiation in several human cell models. We demonstrated that both PPAR alpha (clofibrate and ciprofibrate) and PPAR gamma ligands (troglitazone and 15 deoxy-prostaglandin J2, 15d-PGJ2) inhibited growth, induced the onset of monocytic-like differentiation, and increased the proportion of G0/G1 cells in the HL-60 leukemic cell line. Moreover, 3 days after the treatment with 2.5 microM 15d-PGJ2, an increase in sub-G0/G1 population occurred, compatible with an induction of programmed cell death. To clarify the mechanisms involved in HL-60 growth inhibition due to the effects of PPAR ligands, we investigated their action on the expression of some genes involved in the control of cell proliferation, differentiation, and cell cycle progression such as c-myc, c-myb, and cyclin D1 and D2. Clofibrate (50 microM), ciprofibrate (50 microM), and 15d-PGJ2 (2.5 microM) inhibited c-myb and cyclin D2 expression, whereas they did not affect c-myc and cyclin D1 expression. Only troglitazone (5 microM) decreased c-myc mRNA and protein levels, besides decreasing c-myb and cyclin D2. The down-regulations of c-myb and cyclin D2 expression represent the first evidence of the inhibitory effect exerted by PPAR ligands on these genes. Moreover, the inhibition of c-myc expression by troglitazone may depend on a PPAR-independent mechanism.