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1.
报道了一种基于发夹型荧光探针的甲基化酶活性的分析方法, 甲基化酶和相应的限制性内切酶的识别位点被设计在发夹型探针的茎部, 四甲基罗丹明(TAMRA)被连接在探针的5'端, 其荧光被连在3'端的熄灭基团4-(4'-二甲基对胺基偶氮苯)苯甲酸(DABCYL)所熄灭. 限制性内切酶可切割未发生甲基化修饰的探针, 导致探针的发夹结构遭到破坏, 引起TAMRA荧光信号的恢复. 根据荧光信号的恢复程度可实现对甲基化酶活性的分析. 在此基础上, 建立了一种简便、快速分析抗肿瘤药物对DNA甲基化酶活性的影响的方法, 为筛选针对基因甲基化异常引起的恶性肿瘤的治疗药物提供了一种新的思路和方法.  相似文献   

2.
A solid leukemia sarcoma has been successfully developed after subcutaneous inoculation of the cultured human promyelocytic leukemia cells (HL-60 cells) into unde mice. The solid leukemia sarcoma is a more plantiful source than the cultured cells for enzymatic study and its growing environment is closer to that of the human body than the cultured cells.We establish an efficient procedure of purifying HL-60 cells DNA methylase which includes: disruption of HL-60 cells by homogenization and sonication, removing the cell fragments and cellular particles by centrifuge and ultracentrifuge (105.000 g); removing endogenous DNA by streptomycin sulfate, salting out by (NH4)2SO4, ion exchange chromatography on DEAE-cellulose (DE-52), gel filtration over Sephadex G-100 column.The DNA methylase from HL-60 cells has been purified 204 fold by this procedure. The purified enzyme shows a single-band on PG-PAGE. A 479-kD molecular weight of this enzyme is measured by PG-PAGE. The enzyme properties of HL-60 DNA methylase  相似文献   

3.
Human 8-oxo-G-DNA glycosylase 1 (hOGG1) is a DNA glycosylase to cleave 8-oxo-7,8-dihydroguanine (8-oxo-G), a mutagenic DNA adduct formed by oxidant stresses. Here, we examined hOGG1 protein expression and repair activity to nick a DNA strand at the site of 8-oxo-G during differentiation of hematopoietic cells using HL-60 cells. Overall expression of hOGG1 protein was increased during granulocytic differentiation of HL-60 cells induced by DMSO and monocytic differentiation by vitamine D(3). Greater level of hOGG1 protein was expressed in DMSO-treated cells. However, change in the DNA nicking activity was not in parallel with the change in hOGG1 protein expression, especially in PMA-treated cells. In PMA- treated cells, the level of hOGG1 protein was lowered, even though the DNA nicking activity was elevated, in a manner similar to the changes in serum- deprived HL-60 cells. These results indicate that hOGG1 expression change during differentiation of hematopoietic stem cells for adaptation to new environments. And the DNA cleaving activity may require additional factor(s) other than expressed hOGG1 protein, especially in apoptotic cell death.  相似文献   

4.
The epigenetic control of genes by the methylation of cytosine resulting in 5‐methylcytosine (5mC) has fundamental implications for human development and disease. Analysis of alterations in DNA methylation patterns is an emerging tool for cancer diagnostics and prognostics. Here we report that two thermostable DNA polymerases, namely the DNA polymerase KlenTaq derived from Thermus aquaticus and the KOD DNA polymerase from Thermococcus kodakaraensis, are able to extend 3′‐mismatched primer strands more efficiently from 5 mC than from unmethylated C. This feature was advanced by generating a DNA polymerase mutant with further improved 5mC/C discrimination properties and its successful application in a novel methylation‐specific PCR approach directly from untreated human genomic DNA.  相似文献   

5.
Juan HF  Lin JY  Chang WH  Wu CY  Pan TL  Tseng MJ  Khoo KH  Chen ST 《Electrophoresis》2002,23(15):2490-2504
A biomic approach by integrating three independent methods, DNA microarray, proteomics and bioinformatics, is used to study the differentiation of human myeloid leukemia cell line HL-60 into macrophages when induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Analysis of gene expression changes at the RNA level using cDNA against an array of 6033 human genes showed that 5950 (98.6%) of the genes were expressed in the HL-60 cells. A total of 624 genes (10.5%) were found to be regulated during HL-60 cell differentiation. Most of these genes have not been previously associated with HL-60 cells and include genes encoded for secreted proteins as well as genes involved in cell adhesion, signaling transduction, and metabolism. Protein analysis using two-dimensional gel electrophoresis showed a total of 682 distinct protein spots; 136 spots (19.9%) exhibited quantitative changes between HL-60 control and macrophages. These differentially expressed proteins were identified by mass spectrometry. We developed a bioinformatics program, the Bulk Gene Search System (BGSS, http://www.sinica.edu.tw:8900/perl/genequery.pl) to search for the functions of genes and proteins identified by cDNA microarrays and proteomics. The identified regulated proteins and genes were classified into seven groups according to subcellular locations and functions. This powerful holistic biomic approach using cDNA microarray, proteomics coupled to bioinformatics can provide in-depth information on the impact and importance of the regulated genes and proteins for HL-60 differentiation.  相似文献   

6.
微量热法研究药物对肿瘤细胞的诱导分化作用   总被引:4,自引:0,他引:4  
Sp-Octyl-(8-Chloroadenosine)-3',5'-Cyclophosphate (OCC), a newlysynthesized cAMP analog, strongly induces growth inhibition and differentiation in human promyelocytic leukemia HL-60 cells. The effect of OCC is dose-time dependent. By using the LKB2277 Bioactivity Monitor, we determined the heat production rate of HL-60 cells treated by OCC at different concentrations. It was found that the heat production rate of HL-60 cell treated by OCC gradually decreased to that of normal human neutrophiles.  相似文献   

7.
N-烷基-1,10-菲咯啉2-甲胺La(Ⅲ)配合物的合成及抗癌活性   总被引:4,自引:0,他引:4  
合成和表征了甲基、乙基、丙基、丁基和苄基N-取代1,10-菲咯啉2-甲胺衍生配体及其镧(Ⅲ)配合物. 研究了配合物对HL60人白血病、PC-3MIE8人前列腺癌、BGC-823人胃癌、MDA-MB-435人乳腺癌、Bel-7402人肝癌、Hela人宫颈癌共6种瘤株的体外抗肿瘤活性及其与DNA的作用方式. 结果表明, 该系列化合物对实验的6种瘤株均具有不同程度的生长抑制作用, 其中配合物L5LaL5对MDA-MB-435人乳腺癌和Bel-7402人肝癌的抑制效果较好, 对Bel-7402人肝癌和Hela人宫颈癌的抑制效果优于顺铂. 其作用机理可能是配合物以部分插入方式同时伴随共价和静电与DNA发生作用, 影响其基因调控与表达, 进而抑制肿瘤细胞的生长, 最终导致癌细胞凋亡.  相似文献   

8.
Azacytidines (AzaC and AzadC) are clinically relevant pharmaceuticals that operate at the epigenetic level. They are integrated into the genome as antimetabolites to block DNA methylation events. This leads to a reduction of the 5‐methyl‐2′‐deoxycytidine (m5dC) level in the genome, which can activate epigenetically silenced genes. Because of the inherent chemical instability of Aza(d)Cs, their incorporation levels in DNA and RNA are difficult to determine, which hinders correlation of therapeutic effects with incorporation and removal processes. Existing methods involve radioactive labeling and are therefore unsuitable to monitor levels from patients. We report here a new direct chemical method that allows absolute quantification of the levels of incorporated AzaC and AzadC in both RNA and DNA. Furthermore, it clarifies that Aza(d)C accumulates to high levels (up to 12.9 million bases per genome). Although RNA‐based antimetabolites are often 2′‐deoxygenated in vivo and incorporated into DNA, for AzaC we see only limited incorporation into DNA. It accumulates predominantly in RNA where it, however, only leads to insignificant demethylation.  相似文献   

9.
To provide more information on the 5-aminolaevulinic acid (ALA)-based photodynamic effect (PDE) on nuclei and nucleoli of individual leukemic cells, these structures were studied in cultured HL-60 cells which originated from leukemic highly immature and less differentiated precursors of granulocytes. The nuclear morphology was visualized by panoptic May-Grünwald/Giemsa staining and cytochemical method for DNA, nucleoli were visualized by cytochemical methods for the demonstration of RNA and silver stainable proteins including those of interphase silver stained nucleolus organizer regions (AgNORs). In most cells ALA-based photodynamic treatment (PDT) produced marked alterations such as formation of apoptotic bodies, and large condensation of nuclear chromatin structure but without nuclear segmentation. Such changes are in harmony with the apoptotic process induced in these cells but without previous terminal differentiation. In nucleoli ALA-based PDT produced the reduction and disappearance of nucleolar silver stainable particles (SSPs) representing AgNORs which apparently reflected the alteration of the nucleolar biosynthetic activity and cell proliferation. The latter is also reflected by the disappearance of mitotic divisions. On the other hand, a small subpopulation of cells was less sensitive or resistant to the ALA-based PDE since they did not show mentioned nuclear and nucleolar alterations.  相似文献   

10.
Methylation of bone morphogenetic protein 3 (BMP3) in stool DNA is an effective biomarker for non-invasive screening of colorectal cancer. However, a highly sensitive and specific detection method is required. Here, a quantification method for BMP3 methylation was developed by combining real-time polymerase chain reaction (PCR) with invader assay using Beta-actin (ACTB) as a reference. Amplification efficiencies of BMP3 and ACTB were close to 100% after optimizing the concentration of detection probes, FEN1 enzyme and Taq polymerase, and the relative quantification of BMP3 methylation was achieved accurately by ΔCT algorithms. Ten copies and 0.01% of BMP3 methylation level could be successfully detected and non-specific signal was generated from non-methylated template, indicating that the method was highly sensitive and specific. The method was successfully applied to detect BMP3 methylation in fecal DNA from 16 colorectal cancer patients, 7 adenoma patients and 19 healthy volunteers. The results indicated that BMP3 methylation occurred in 5 of 16 cancer patients and 2 of 7 adenoma patients, but was not observed in 19 of healthy volunteers. Therefore, this method could be used to quantify methylation of gene in stool samples, providing an effective technique for non-invasive screening of colorectal cancer.  相似文献   

11.
DNA methylation is an epigenetic modification that represses gene expression. In cancer cells, alterations of the DNA methylation state in promoter regions and repetitive DNA sequences are observed; therefore, DNA methyltransferase inhibitors have been the focus of interest as potential anticancer drugs. We previously reported a simple global DNA methylation level-sensing assay using methyl-CpG binding domain (MBD) fused to luciferase (MBD-luciferase). In the assay, the MBD-luciferase binds to methyl-CpG sites on genomic DNA. Subsequently, bioluminescence resonance energy transfer (BRET) between the luciferase and a fluorescent DNA intercalating dye generates a signal that is dependent on DNA methylation level. In this study, we investigated whether global DNA hypomethylation induced by a DNA methyltransferase inhibitor or nutrient can be monitored by the BRET assay. 5-Aza-2′-deoxycytidine and folic acid were utilized as the DNA-methyltransferase inhibitor and nutrient that affect DNA methylation in cells. The HeLa cells were cultured with the inhibitor or in folic acid-deficient medium and their global DNA methylation levels measured. Both time- and concentration-dependent hypomethylation were detected by the BRET assay. These results demonstrate that global DNA hypomethylation can be monitored by the BRET assay, indicating that the assay is applicable to cell-based screening of DNA-methyltransferase inhibitors.  相似文献   

12.
Resveratrol has been shown to possess antioxidant and anticancer activities, but little is known on the effect of resveratrol derivatives. Recently we have isolated resveratrol and its dimers and trimers from peony (Paeonia lactiflora) seeds, and reported their strong antioxidant and cytotoxic activity. In the present study, we have evaluated cellular effects of resveratrol derivatives; viniferin, gnetin H, and suffruticosol B on the proliferation and apoptosis in HL-60 cells in vitro. All resveratrol and its derivatives reduced viability of HL-60 cells in a dose-dependent manner with their IC(50) values of 20-90 microM. Ascending orders of IC(50) values were suffruticosol B, gnetin H, viniferin and resveratrol respectively. HL-60 cells treated with the four stilbenes exhibited the distinct morphological changes characteristics of cell apoptosis such as chromatin condensation, apoptotic bodies, and DNA fragmentations. A time-dependent histogram of the cellular DNA analyzed by flow cytometry revealed a rapid increase in subdiploid cells and a concomitant decrease in diploid cells exposed to 100 microM resveratrol for 0-24 h. Cells treated with 25 microM of resveratrol, viniferin, gnetin H, and suffruticosol B for 24 h resulted in increment of sub-G1 population by 51, 5, 11 and 59%, respectively. Treatment of cells with 0-20 microM resveratrol for 5 h produced a concentration-dependent decrease in cytochrome P450 (CYP) 1B1 mRNA levels. Suffruticosol B also suppressed CYP1B1 gene expression. These results demonstrated that resveratrol oligomers also strongly suppressed HL-60 cell proliferation, and induced DNA damage. In addition, CYP1B1 gene supression may suggest an involvement in the resveratrol-induced apoptosis in HL-60 cells.  相似文献   

13.
Bis (Bag-3, CAIR), a Bcl-2-interacting protein, promotes the anti-apoptotic activity of Bcl-2 and increased levels of Bis have been observed in several disease models. The involvement of Bcl-2 and some Bcl-2-binding proteins in differentiation has recently been reported. However, the relevance of Bis to cellular differentiation remains unknown. The findings herein show that Bis expression is up-regulated during the differentiation of HL-60 cells. To investigate the effect of Bis expression on differentiation, we established Bis-overexpressing HL-60 cells (HL-60-bis). HL-60-bis cells have a low nuclear: cytoplasmic ratio and indented nucleus in Wright- Giemsa staining, and an increased expression of CD11b in immunofluorescence study, indicating the promotion of differentiation. The overexpression of Bis also resulted in a retarded cell growth rate, accompanied by the accumulation of HL-60 cells at the G0/G1 phase of the cell cycle, which was sustained during the differentiation process. Western blot analysis revealed that the expression of p27, a representative inducer of cell cycle arrest at the G1 phase, was increased 2.5-fold in HL-60-bis cells compared to HL-60-neo cells. These results suggest that the Bis induced growth inhibition of HL-60 cells promotes G0/G1 phase arrest via up-regulation of p27, which seems to be a prerequisite for differentiation. Further studies will be required to define the exact roles of Bis on cellular differentiation more precisely.  相似文献   

14.
p33ING1作为一种重要的抑癌基因,在乳腺癌及胃癌等恶性肿瘤细胞中的mRNA表达水平显著低于癌旁正常组织细胞中的表达,并导致这些肿瘤细胞的侵袭转移能力增强,对化疗药物及r射线和紫外线处理的敏感性降低。  相似文献   

15.
To provide more information on the 5-aminolaevulinic acid (ALA)-induced photodynamic effect on nucleoli, morphologically expressed nucleolar asynchrony (the presence of 'active' large nucleoli with an uniform distribution of RNA and 'resting' ring-shaped nucleoli in one and the same nucleus) was studied in cultured HL-60 leukemic granulocytic precursors using a simple cytochemical procedure for the demonstration of RNA. Nucleolar asynchrony was mainly expressed in cells which were apparently resistant to ALA-based photodynamic treatment (PDT) since most of them (about 75%) exhibited this phenomenon.  相似文献   

16.
Studies on differentiation-inducing activities of triterpenes.   总被引:5,自引:0,他引:5  
Differentiation-inducing activity of over 180 extracts of crude drugs and plants was tested using mouse myeloid leukemia cell line (M1). The methanol extracts of clove (Syzygium aromaticum Merrill et Perry, Myrtaceae) showed remarkable induction of differentiation of M1 cells into macrophage-like cells. From the extract, oleanolic acid (1) and crategolic acid (2) were isolated as the active components. We also tested other triterpenes, such as oleananes, ursanes and dammaranes, to investigate the structure-activity relationship. Some triterpene aglycones showed differentiation-inducing activity, but triterpene glycosides showed little activity. Furthermore, the differentiation-inducing activity of these triterpene compounds was tested against human acute promyelocytic leukemia cell line (HL-60).  相似文献   

17.
A cDNA for human TNF-alpha (615bp) was isolated by means of polymerase chain reaction (PCR) using first strand cDNA from PMA-induced HL-60 cells as template. The result from sequencing the 615 bp cDNA fragment indicated that it corresponded to the entire sequence of mature human TNF coding region. Direct expression of mature human TNF was achieved using a plasmid pHT-1 constructed by ligation of the cDNA and a synthetic DNA. The IPTG-induced bacterial product (hTNF) showed cytotoxicity to mouse L-929 cells. The TNF activity was further identified by neutralization of a specific monoclonal antibody against human TNF-alpha. Approximately 80,000 units of activity were detected per ml of culture at A600 = 2.  相似文献   

18.
Various analogs of known immunomodulatory derivatives of thalidomide (1) (IMiDs: 3, 5) were synthesized, focusing on cell-differentiation-inducing, cyclooxygenase-inhibitory and anti-angiogenesis activities. Among the prepared compounds, NIDO-33 (14) showed cell differentiation-inducing activity on HL-60 cells and anti-angiogenic activity on human umbilical vein endothelial cells (HUVEC). AIDO-00 (7) also showed anti-angiogenic activity. NIDO-11 (8) showed an enhancing effect on all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation, and AIDO-30 (13) exhibited cyclooxygenase (COX)-inhibitory activity.  相似文献   

19.
Many different types of mammalian cells accumulate fluorescing and photosensitizing concentrations of protoporphyrin IX (PpIX) when exposed to exogenous 5-aminolevulinic acid (ALA) in vivo or in vitro. Most types of malignant cells accumulate substantially more ALA-induced PpIX than do the normal cells from which they arose. Most types of malignant cells also are less differentiated than their normal counterparts. We therefore considered the possibility that malignant cells demonstrate a malignant ALA phenotype (accumulate abnormally large amounts of PpIX when exposed to exogenous ALA) as a direct consequence of their less differentiated state. Human promyelocyte cell line HL-60 and mouse preadipocyte cell line 3T3 L1 were induced to differentiate by exposing them to inducing agents in vitro. The HL-60 cells accumulated less ALA-induced PpIX when differentiated, but the 3T3 L1 cells accumulated more. It appears then that changes in the ALA phenotype with changes in the state of differentiation are cell-type specific. The decreased accumulation of ALA-induced PpIX that accompanied differentiation of the promyelocytic leukemia cells may have clinical application for rapid quantitation of the response of myelocytic leukemia patients to differentiation therapy.  相似文献   

20.
Twenty-six 5-azacytidine analogues have been synthesized, including 4-amino- 6-alkyl-1-pyranosyl/ribofuranosyl-1,3,5-triazin-2(1H)-ones 1a-j, 6-amino-4-alkyl/aryl-1- pyranosyl/ribofuranosyl-1,3,5-triazin-2(1H)-ones 2a-f and 4-amino-6-alkyl-1,3,5-triazin-2- yl-1-thio-pyranosides/ribofuranosides 3a-j. The antiproliferative activities of these synthetic analogues were investigated in human leukemia HL-60 cells. Ribofuranosyl S-nucleoside 3a, a bioisostere of 5-azacytidine, had a similar antiproliferative ability as that of the latter. Introduction of a methyl at the 6 position of 5-azacytidine and/or replacement of the ribofuranosyl moiety with pyranosyl sugars or disaccharides significantly decreased the antiproliferative activities of the 5-azacytidine derivatives. Several compounds with the replacement of pyranosyl sugars enhanced all-trans retinoic acid-induced differentiation ability in human leukemia HL-60 cells.  相似文献   

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