Induction of apoptosis by the marine sponge (Mycale) metabolites, mycalamide A and pateamine

The marine sponge metabolites mycalamide A (myca-lamide) and pateamine are extremely cytotoxic. While mycalamide has been shown to inhibit protein synthesis, the mechanism by which these compounds induce cell death is unknown. Using DNA laddering, Annexin-V staining, and morphological analysis, we demonstrate that both metabolites induce apoptosis in several different cell lines. Furthermore, both mycalamide and pateamine were more potent inducers of apoptosis in the 32D myeloid cell line after transformation with either the ras or bcr-abl oncogenes. This increased sensitivity was also observed in response to the protein synthesis inhibitors cycloheximide and puromycin, and cytosine--D-arabinofurano-side (Ara-C), an inducer of DNA damage. We propose, therefore, that in 32D cells where Ras signalling has been altered either by constitutive expression of oncogenic ras or by Bcr/abl-mediated perturbation of upstream signalling events, increased susceptibility to apoptosis by a range of stimuli is conferred.     

Programmed cell death: alive and well in the new millennium

Research performed over the past decade has transformed apoptosis from a distinctive form of cell death known only by its characteristic morphology and genomic destruction to an increasingly well understood cellular disassembly pathway remarkable for its complex and multifaceted regulation. Here, we summarize current understanding of apoptotic events, note recent advances in this field and identify questions that might help guide research in the coming years.     

RNAi干扰Shp2基因对K562细胞增殖的抑制效应

通过RNA干扰技术沉默蛋白酪氨酸磷酸酶跏2基因,构建重纽质粒,采用实时荧光定量PCR（Real-timePCR）法、Westernblot、MTT法、流式细胞术（FCM）分别检测转染后K562细胞中bcr／abl融合基因、bcr／abl融合蛋白的表达水平、细胞生长增殖变化及细胞凋亡率,探索该基因的沉默表达对K562N胞的抑制作用。结果表明,该实验成功构建出能明显下调Shp2基因及其蛋白表达的重组质粒,转染K562细胞后,其bcr／abl融合基因及融合蛋白水平均明显降低、K562细胞增殖活力被抑制（P〈0．05）、细胞凋亡水平上升（P〈0．05）。与对照组相比,其差异具有统计学意义。提示,重组质粒可显著降低bcr／abl基因及蛋白的表达,抑制K562细胞的生物学效应,表明在细胞水平沉默Shp2有可能成为治疗慢性粒细胞白血病的有效靶点。     

bcr／abl反义RNA片段对Ph＋人白血病细胞系分化的影响

为探讨Ph 白血病细胞中bcr/ab1表达的降低对细胞分化的影响,本文利用脂质体介导的方法将表达bcr/ab1融合区反义RNA片段的重组质粒导入K562和BV173细胞系,以Southern和Northern杂交以及筑巢式逆转录酶-聚合酶链反应技术证实外源DNA已在靶细胞内整合与表达,且bcr/ab1反义RNA片段的表达使内源性bcr/ab1 mRNA表达水平降低。未观察到表达bcr/ab1反义RNA片段的K562细胞出现粒单系或红系分化,重组质粒转染前后BV173细胞表面的CD10和CD34抗原以及K562细胞表面的CD13和CD33抗原表达无明显变化,提示bcr/ab1反义RNA片段抑制增殖的同时未引发分化与成熟。     

莪术醇诱导慢性粒细胞白血病K562细胞分化的研究

目的:以人慢性粒细胞白血病细胞株K562细胞为对象,研究莪术醇(curcumenol)诱导K562细胞分化作用,并同莪术油做了比较研究。方法:通过测定细胞内酶变化判定莪术醇等诱导K562细胞的分化;用RT-PCR测定莪术醇等作用后K562细胞bcr/abl mRNA表达量的变化,同时测定莪术醇等对K562细胞微核影响,探讨诱导K562细胞的分化机理。结果:莪术醇能够诱导K562细胞向成熟分化,30μg/mL莪术醇作用72h后,Gimsa染色后可见K562细胞向终末细胞分化,出现杆状及分叶核细胞,细胞内酶学指标也呈分化表现;同时,莪术醇作用后bcr/abl融合基因的表达降低,K562细胞的微核率减少。结论:莪术醇对畸变的K562细胞染色体有作用,影响bcr/abl融合基因的表达,使K562细胞向成熟分化。     

Microbial community analysis at crude oil‐contaminated soils targeting the 16S ribosomal RNA,xylM, C23O,and bcr genes

Aims: The analyses targeting multiple functional genes were performed on the samples of crude oil‐contaminated soil, to investigate community structures of organisms involved in monoaromatic hydrocarbon degradation. Methods and Results: Environmental samples were obtained from two sites that were contaminated with different components of crude oil. The analysis on 16S rRNA gene revealed that bacterial community structures were clearly different between the two sites. The cloning analyses were performed by using primers specific for the catabolic genes involved in the aerobic or anaerobic degradation of monoaromatic hydrocarbons, i.e. xylene monooxygenase (xylM), catechol 2,3‐dioxygenase (C23O), and benzoyl‐CoA reductase (bcr) genes. From the result of xylM gene, it was suggested that there are lineages specific to the respective sites, reflecting the differences of sampling sites. In the analysis of the C23O gene, the results obtained with two primer sets were distinct from each other. A comparison of these suggested that catabolic types of major bacteria carrying this gene were different between the two sites. As for the bcr gene, no amplicon was obtained from one sample. Phylogenetic analysis revealed that the sequences obtained from the other sample were distinct from the known sequences. Conclusions: The differences between the two sites were demonstrated in the analyses of all tested genes. As for aerobic cleavage of the aromatic ring, it was also suggested that analysis using two primer sets provide more detailed information about microbial communities in the contaminated site. Significance and Impact of the Study: The present study demonstrated that analysis targeting multiple functional genes as molecular markers is practical to examine microbial community in crude oil‐contaminated environments.     

bcr/abl嵌合基因与白血病分子机制

bcr/abl嵌合基因是白血病ph染色体的分子基础,其表达的融合蛋白具有活跃的酪氨酸激酶活性,它与细胞信号传导功能有关的蛋白作用干扰细胞正常信号的传导,影响细胞生长或凋亡。bcr/abl在白血病中的作用机制的进一步阐明有助于白血病的治疗。     

Comparison of triple helical COMBO-FISH and standard FISH by means of quantitative microscopic image analysis of abl/bcr positions in cell nuclei

In this study, a novel DNA fluorescence labelling technique, called triple helical COMBO-FISH (Combinatorial Oligo Fluorescence In Situ Hybridisation), was compared to the standard FISH (Fluorescence In Situ Hybridisation by means of commercially available probe kits) by quantitative evaluation of the nuclear position of the hybridisation signals of the Abelson murine leukaemia (abl) region and the breakpoint cluster region (bcr) in 3D-conserved cell nuclei of lymphocytes and CML blood cells. Two sets of 31 homopyrimidine oligonucleotides each, corresponding to co-localising sequences in the abl region of chromosome 9 and in the bcr region of chromosome 22 were synthesised. Probe types and sizes (in bases) as well as the binding mechanisms of both FISH techniques were completely different. In accordance to established findings that cell type specific radial positioning of chromosomes and sub-chromosomal elements is evolutionarily conserved, no significant difference was found between the two FISH techniques for the radial localisation of the barycentre of the analysed genomic loci. Thermal denaturation and hypotonic treatment of cell nuclei subjected to standard FISH, however, led to different absolute radii and volumes of the cell nuclei, in comparison to the quantities determined for the triple helical COMBO-FISH technique; the chromatin appears to shrink in laterally enlarged, flat nuclei. Consequently, the absolute distances of the homologous labelled sites shifted to greater values. For precise quantitative microscopic analysis of genomic loci, fluorescence labelling procedures are recommended that well maintain the native chromatin topology. Triple helical COMBO-FISH may offer such an approach.     

BCR3/ABL2核酶的设计、构建及对K562细胞增殖与集落形成的抑制作用和凋亡诱导作用

利用计算机模拟设计合成了针对 K5 62细胞致癌融合 bcr3/abl2 m RNA的锤头状核酶 .该核酶以融合点附近 UUC为识别切割三联体 ,在核酶的 3′端增加一段 T7噬菌体终止子序列 .用基因克隆结合体外转录的方法 ,肯定了核酶的体外切割活性 .进而将核酶基因克隆到 p CEP4真核细胞高效表达载体上 ,利用脂质体 Lipofectin AMINE介导的转染技术将核酶与核酶基因导入靶细胞 ,从抑制靶细胞 K5 62的增殖与集落形成及引起靶细胞凋亡等方面验证了核酶在细胞水平上对融合基因 bcr3/abl2 m RNA的特异切割作用 ,并观察到了 T7噬菌体终止子序列对核酶切割效率的增强影响 .     

Bcr3/abl2和VEGF基因反义寡核苷酸联用增强对K562细胞的生长抑制作用

目的:研究BCRABL和VEGF反义寡核苷酸联用对K562细胞株的作用及其相互作用的影响。方法:设计针对bcr3/abl2和VEGF的反义寡核苷酸(ASODNs),应用脂质体Oligofectamine作为转染载体。在转染后72h进行台盼蓝染色细胞计数;建立裸鼠K562移植瘤动物模型,瘤内注射ASODNs,观察肿瘤体积生长变化,组织学检测肿瘤血管密度和肿瘤细胞凋亡情况。结果:转染后72h,各实验组与空白组相比,细胞增殖抑制率分别为13.47%(ASOB3/A2组),12.79%(ASOVEGF组)和41.55%(半量联合治疗组)。经过4次治疗后,与对照组相比,肿瘤生长抑制率分别为23.18%(ASOB3/A2组),17.28%(ASOVEGF组)和57.83%(半量联合治疗组)。联合治疗组肿瘤生长速率显著低于单一治疗组,伴随明显的肿瘤细胞凋亡增加和肿瘤血管密度减少。结论:双基因反义寡核苷酸联合应用协同抑制K562细胞增殖,抗肿瘤作用明显优于单一治疗组,可为CML基因治疗提供一项新策略。