间期和有丝分裂期HeLa细胞中核仁蛋白B23的分布和含量的差异

Ｂ２３蛋白是真核细胞核仁的两种主要蛋白成份之一。已往的工作表明,细胞内Ｂ２３蛋白的分布与ｒＲＮＡ合成速率和细胞的生长状况密切相关。本工作利用抗Ｂ２３蛋白单克隆抗体,研究了被两种作用于微管的药物秋水仙酰胺和紫极酚阻断的有丝分裂期和间期ＨｅＬａ细胞内Ｂ２３蛋白的分布和含量的差异。结果发现有丝分裂期细胞内Ｂ２３蛋白含量明显高于间期细胞,而且Ｂ２３蛋白在两类细胞中的分布也有明显的不同。     

NuMA 的分布和功能

核有丝分裂器蛋白(Nuclear Mitotic Apparatus Protein,NuMA)是一种在间期细胞核内有大量表达的大分子蛋白。NuMA是微管聚合因子,能使微管锚定于纺锤体极。在细胞有丝分裂,减数分裂过程中对纺锤体的形成和形态的维持发挥重要作用。     

多头绒泡菌类cyclin B1蛋白在细胞周期中的变化

以自然同步化的多头绒泡菌(Physarum polycephalum L.)为材料,经抗cyclin B1抗体的免疫印迹和免疫电镜实验观察结果表明,多头绒泡菌中含有类cyclin B1蛋白,该蛋白的含量和细胞内位置在细胞周期进程中存在着动态变化:类cyclin B1蛋白在S期开始合成并在细胞质中积累,G2晚期开始进入细胞核,该蛋白在细胞质和细胞核中含量逐渐增加,有丝分裂中期时达最大值,后末期时骤然消失.在G2晚期到有丝分裂中期期间,类cyclin B1蛋白既是细胞核蛋白又是细胞质蛋白,细胞质是类cyclin B1蛋白的主要存在区域,细胞核中的类cyclin B1蛋白主要结合于染色体和核仁区域.     

层粘连蛋白对晚G1期人胃癌BGC-823细胞生长的促进作用

为研究层粘连蛋白（laminin,LN）促进肿瘤细胞生长作用,采用脉冲标记计数有丝分裂百分率(percentage labeling mitosis,PLM)法测得体外培养人胃癌 (BGC 823) 细胞周期时间为41 h,其中G1期时间为24.5 h. 分裂细胞脱离法获取分裂期细胞,继续培养23 h,在细胞运行进入G1晚期时,将其置于LN 0、0.11、0.55、1.10 μmol/L基质上孵育4 h; 细胞荧光光度计检测晚G1期细胞内Ca2+浓度、钙调蛋白、DNA含量. 结果显示,LN与其膜上受体结合后引起细胞内Ca2+浓度、钙调蛋白、DNA含量增加,尤以在0.55 μmol/L LN作用显著（P<0. 001）.蛋白质免疫印迹分析证明,cPKC α呈现表达,提示 LN与其受体结合可增强其细胞cPKC-α的活性;分析G1期细胞周期蛋白E(cyclinE)、细胞周期蛋白依赖性激酶CDK2表达水平,呈现逐渐增强的趋势; LN可诱导c-Myc蛋白呈现高表达,提示 LN与其受体结合增强与细胞增殖密切相关的基因表达;在LN作用前后的BGC 823细胞均未检测到Bax蛋白表达.结果提示,在人胃癌 (BGC 823)细胞G1/S期交界处,层粘连蛋白与其膜上受体结合引起细胞内Ca2+浓度升高,诱导钙调蛋白的释放,其含量增加,增强蛋白激酶C的活化,导致细胞内DNA含量增加、G1/S期细胞周期蛋白与CDK表达增强、诱导原癌基因c-Myc呈持续表达状态,而凋亡基因Bax不表达.     

NuMA的分布和功能

核有丝分裂器蛋白（Nuclear Mitotic Apparatus Protein,NuMA）是一种在间期细胞核内有大量表达的大分子蛋白。NuMA是微管聚合因子,能使微管锚定于纺锤体极。在细胞有丝分裂,减数分裂过程中对纺锤体的形成和形态的雏持发挥重要作用。     

急性低氧和间断低氧习服对HepG2细胞内血管内皮细胞生长因子基因表达的影响

目的:观察急性低氧和间断低氧习服对人HepG2细胞内血管内皮细胞生长因子(VEGF)及转录因子低氧诱导因子-1α(HIF-1α)的mRNA和蛋白含量的影响及其可能的生物学意义.方法:HepG2细胞随机分为常氧对照组,急性低氧组和间断低氧习服组.采用Northern blot和Western blot分别检测不同组别HepG2细胞内VEGF和HIF-1α mRNA表达和蛋白含量的变化.结果:急性低氧诱导HepG2细胞内VEGF和HIF-1α基因的转录,增加两种蛋白在细胞内的含量.间断低氧习服组的细胞内VEGF和HIF-1α的mRNA含量分别为常氧对照组细胞的(108.6±17.7)%和(116.74±19.8)%,与常氧对照组相比无显著差异(P＞0.05);而其蛋白表达的含量分别为对照组细胞的1.4和2.7倍,都明显低于急性低氧组细胞内两种蛋白的含量(P＜0.05).结论:HepG2细胞达到低氧习服状态后,抑制急性低氧对HepG2细胞内VEGF基因表达的促进作用,其中HIF-1α可能起着重要的调节作用.     

培养的正常细胞和肿瘤细胞内微管变化的研究:1.正常细胞内微管的免疫荧光细胞化学观察

本工作用我们自制的兔抗管蛋白血清经过间接免疫荧光染色检查,效价达到1:32,显示出我国小儿包皮成纤维细胞(正常二倍体细胞)内微管的特异分布。我们观察到在间期细胞内的微管(CMTC)系由核附近的微管组织中心(MTOC)发出到达细胞的边沿,或终止于质膜下方,或弯曲沿细胞表面平行分布;胞质内的微管纤维有的紧绕核周,有的伸入细胞突起内与主轴方向平行排列。当细胞进入分裂期(M期)时,不仅CMTC解聚,同时有丝分裂器纺锤体微管出现,细胞从扁平形状变成圆的外形。当有丝分裂完成后,纺锤体微管荧光消失,CMTC又逐渐代替而出现,细胞也恢复成为扁平形。CMTC在早G_1期开始出现,此时的子细胞之间由于极间微管束的残迹而呈现出荧光染色的阳性反应,是为中体。用秋水仙胺(0.06微克/毫升)在37℃下处理培养细胞,2小时后,CMTC解聚,变为弥散于细胞质内的管蛋白荧光,细胞外形变成更近似圆形或不规则的外形。洗去秋水仙胺,细胞在37℃的新鲜培养基内保温1.6小时后,CMTC又可复现,同时细胞外形又恢复到处理前的成纤维细胞形状。细胞在低温(0—4℃)处理1小时后,CMTC消失;当细胞再放回到37℃下保温16分钟后,CMTC开始恢复,30分钟后完全恢复。本实验用未免疫的同一家兔血清做对照染色,结果为阴性。本工作改用冷氯仿:甲醇(2:1)液固定细胞,在染色和洗涤时采用非离子性去污剂Triton X-100处理法,微管的荧光染色效果好,背景的非特异荧光减少。     

培养的正常细胞和肿瘤细胞内微管变化的研究——Ⅱ.肿瘤细胞内微管的免疫荧光细胞化学观察

本实验用管蛋白抗体间接免疫荧光细胞化学方法,观察了我国建株的人胃低分化粘液腺癌MGc 80-3,人胃腺癌SGC-7901,人鼻咽癌上皮样细胞CNE,人食管癌上皮细胞ECa-109,人肺鳞癌LTEP-78,人啼腺癌LTEP-a_1,人肺小细胞癌LTEP-p七株癌细胞和HeLa细胞,小鼠S_(180)-V肉瘤细胞的微管形态。与人的正常包皮成纤维细胞和食管上皮细胞内精细的CMTC结构对比,肿瘤细胞间期的胞质微管普遍有减少或缺如的现象。参考Brin-kley对微管免疫荧光染色图形的分型方法,我们将观察的各种微管染色图形归纳为四种类型,比较各种细胞群体内微管类型的分布。肿瘤细胞群体内多数为微管缺如型和稀疏型,未见典型的丰满型,而正常细胞群体内都是丰满型。同时,肿瘤细胞的MTOC区面积明显增大。分裂期的肿瘤细胞内,有丝分裂器纺锤体微管荧光形态与正常细胞的没有差别。本文对肿瘤细胞间期胞质微管减少和缺如以及MTOC区明显增大的现象及其可能的意义进行了讨论,认为这是癌变机制研究中值得深入探讨的重要课题之一。     

类Cyclin A蛋白在多头绒泡菌细胞周期中的定位研究

采用免疫电镜技术对多头绒泡菌(Physarum polycephalum)是否含有类CyclinA蛋白以及该蛋白在有丝分裂周期各时相的定位进行了研究;并以抗CyclinA抗体封闭细胞内源类CyclinA蛋白的方法,探讨类CyclinA蛋白在多头绒泡菌细胞周期中的作用。免疫电镜结果表明,经抗CyclinA抗体标记的实验组细胞中的金颗粒密度明显高于对照组,说明多头绒泡菌细胞中含有类CyclinA蛋白。实验组样品中,细胞核的金颗粒密度很高,而细胞质的金颗粒密度与对照组的相仿,说明多头绒泡菌细胞中的类CyclinA蛋白是核蛋白。细胞核的金颗粒密度在S期最高,G2期的次之,早中期时明显降低,中期和中期以后与对照组的相近。这种金颗粒密度的变化反映了类CyclinA蛋白在细胞周期中的含量变化。以抗CyclinA抗体分别处理S期和G2期的多头绒泡菌细胞,处理后的细胞分别停滞在原来的时相,细胞核形态变得不规则,核内有空洞现象。处于有丝分裂前期的多头绒泡菌细胞经抗CyclinA抗体处理后,细胞核出现畸变。抗体处理结果说明类CyclinA蛋白是参与多头绒泡菌细胞周期多个转换过程调控的种重要蛋白,主要在S期／G2期和G2期／M期的转换以及走出有丝分裂期的进程中发挥作用。     

Stathmin调控及与疾病的关系

Stathmin是细胞内重要的细胞周期相关的微管作用蛋白。在有丝分裂间期,Stathmin以有活性的去磷酸化形式存在,抑制微管聚合。当细胞进人分裂期,Stathmin被磷酸化失活,微管聚合形成纺锤体,进而染色体分离、细胞分裂。Stathmin的表达和活性受多种转录因子和激酶／磷酸化酶的调控,其表达和活性异常与细胞增殖、神经系统发育及肿瘤等病理生理过程密切相关。     

RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

PROTEIN SYNTHESIS AND RNA SYNTHESIS DURING MITOSIS IN ANIMAL CELLS

Protein synthesis and RNA synthesis during mitosis were studied by autoradiography on mammalian tissue culture cells. Protein synthesis was followed by incubating hamster epithelial and human amnion cells for 10 or 15 minutes with phenylalanine-C¹⁴. To study RNA synthesis the hamster cells were incubated for 10 minutes with uridine-C¹⁴. Comparisons of the synthetic capacity of the interphase and mitotic cells were then made using whole cell grain counts. The rate of RNA synthesis decreased during prophase and reached a low of 13 to 16 per cent of the average interphase rate during metaphase-anaphase. Protein synthesis in the hamster cells showed a 42 per cent increase during prophase with a subsequent return to the average interphase value during metaphase-anaphase. The human amnion cells showed no significant change at prophase but there was a 52 to 56 per cent drop in phenylalanine incorporation at metaphase-anaphase as compared to the average interphase rate. Colcemide was used on the hamster cells to study the effect of a prolonged mitotic condition on protein and RNA synthesis. Under this condition, uridine incorporation was extremely low whereas phenylalanine incorporation was still relatively high. The drastic reduction of RNA synthesis observed under mitotic conditions is believed to be due to the coiled condition of the chromosomes. The lack of a comparable reduction in protein synthesis during mitosis is interpreted as evidence for the presence in these cells of a relatively stable messenger RNA.     

Involvement of the 24-kDa cap-binding protein in regulation of protein synthesis in mitosis

The rate of protein synthesis in metaphase-arrested cells is reduced as compared to interphase cells. The reduction occurs at the translation initiation step. Here, we show that, whereas poliovirus RNA translation is not affected by the mitotic translational block, the translation of vesicular stomatitis virus mRNAs is. In an attempt to elucidate the mechanism by which initiation of protein synthesis is reduced in mitotic cells, we found that the interaction of the mRNA 24-kDa cap-binding protein (CBP) with the mRNA 5' cap structure is reduced in mitotic cell extracts, consistent with their lower translational efficiency. Addition of cap-binding protein complex stimulated the translation of endogenous mRNA in extracts from mitotic but not interphase cells. In addition, we found that the 24-kDa CBP from mitotic cells was metabolically labeled with 32P to a lesser extent than the protein purified from interphase cells. These results are consistent with a hypothesis that the 24-kDa CBP is implicated in the inhibition of protein synthesis in metaphase-arrested cells. Possible mechanisms for this inhibition are offered.     

New patterns of nuclear lamina induced by cell fusion

During the eukaryote cell cycle the nuclear envelope displays a series of major morphogenetic changes, the most significant of which include its breakdown and reconstitution as cells move up to, pass through and emerge from division. The three polypeptides, lamins A, B and C, are major components of the nuclear pore complex-lamina fraction of the nuclear envelope and their association with the nuclear membrane or their dispersal in the cytoplasm reflects the existing balance between polymerization and depolymerization in the envelope. We have perturbed the lamina polymerization cycle by means of cell fusion between mitotic and interphase cells, following the redistribution of nuclear lamina protein by means of immunofluorescence techniques. In these heterophasic heterokaryons changes in the distribution of lamina occur as a function of (1) the time elapsed after fusion; (2) the ratio of mitotic to interphase elements in the cell, and (3) the stage in the cell cycle occupied by the interphase partner at the time of fusion. Depolymerization of nuclear lamina occurs most rapidly in cells with high ratios of mitotic to interphase elements, and especially in G1 rather than S-phase nuclei. While lamina depolymerization predominates early after fusion, at later times lamina is deposited around both the original metaphase and interphase nuclear masses and this is associated with the resumption of interphase activity in the form of limited DNA synthesis. These observations lead us to conclude that lamina depolymerization is under positive control mediated by diffusible factors in the cytoplasm of the metaphase partner. Repolymerization is likely to be associated with the inactivation of these factors as the heterokaryons age and, as a result, pass into an interphase-like state.     

Ki-67: more than a proliferation marker

Ki-67 protein has been widely used as a proliferation marker for human tumor cells for decades. In recent studies, multiple molecular functions of this large protein have become better understood. Ki-67 has roles in both interphase and mitotic cells, and its cellular distribution dramatically changes during cell cycle progression. These localizations correlate with distinct functions. For example, during interphase, Ki-67 is required for normal cellular distribution of heterochromatin antigens and for the nucleolar association of heterochromatin. During mitosis, Ki-67 is essential for formation of the perichromosomal layer (PCL), a ribonucleoprotein sheath coating the condensed chromosomes. In this structure, Ki-67 acts to prevent aggregation of mitotic chromosomes. Here, we present an overview of functional roles of Ki-67 across the cell cycle and also describe recent experiments that clarify its role in regulating cell cycle progression in human cells.     

Cell cycle dependent resistance to staphylococcal delta toxin-induced lysis of cultured cells

Staphylococcal delta toxin is a protein capable of rapidly disrupting cell membranes. Synchronized populations of 3T3 mouse fibroblasts in mitosis and early G₁ phases of the cell cycle exhibit resistance to delta toxin at concentrations cytolytic to interphase cells. Similar results were obtained with HeLa cells grown attached or in suspension culture. Increased resistance appears to result from structural or biochemical features other than cell rounding or detachment. Delta toxin stimulated significantly less cellular phospholipase A₂ (a potentially lytic enzyme activity) in mitotic 3T3 cells than in interphase cells.     

Cellular compartmentalization of protein kinase activity during the cell cycle

The quantities and types of protein kinases found in the cytoplasmic and nuclear or chromosomal compartments of interphase and mitotic human culture cells were compared. Using histone as substrate, the total quantity of kinases recovered from cytoplasmic and chromosomal fractions of mitotic cells was several times greater than from cytoplasmic and nuclear fractions of interphase cells. In both mitotic and interphase cells, more activity was recovered from cytoplasmic fractions than from chromosomal or nuclear fractions, respectively. When activity against various substrates was examined, mitotic chromosomal extracts were found to display the greatest preference for the H1 fraction of histones. Neither cytoplasmic nor chromosomal fractions from mitotic cells exhibited enhanced activity in the presence of cAMP, whereas the activity of both cytoplasmic and nuclear fractions of interphase cells was enhanced. Protein kinases, previously identified by nondenaturing polyacrylamide gel electrophoresis as present in the cytoplasmic fraction of mitotic but not interphase cells, were also present in chromosomal fractions of mitotic cells; only one of these kinases may be present in nuclear extracts of interphase cells. In addition, the profiles of nuclear extracts of interphase cells differ from their cytoplasmic fractions. These results indicate that there are protein kinases which are restricted to the mitotic phase of the cell cycle and that they apparently partition between the cytoplasmic and chromosomal compartments of cells in mitosis.