ODC,AdoMetDC双反义腺病毒对肺癌增殖和侵袭制作用的体外和体内研究

鸟氨酸脱羧酶(ODC)和S-甲硫氨酸脱羧酶(AdoMetDC)是多胺体内合成的2个关键酶.研究腺病毒Ad-ODC-AdoMetDCas介导的ODC和AdoMetDC反义RNA对肺癌多胺合成,细胞增殖以及侵袭的抑制作用.用活细胞计数和流式细胞术分别检测Ad-ODCas和Ad-ODC-AdoMetDCas对肺癌A-549细胞增殖的影响,蛋白质印迹和HPLC方法分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,TUNEL标记检测法观察Ad-ODC-AdoMetDCas对肺癌细胞凋亡的影响,Matrigel侵袭实验分析腺病毒对肺癌A-549细胞侵袭活性的改变,裸鼠皮下移植瘤模型研究Ad-ODC-AdoMetDCas对体内肺癌生长的抑制作用.实验结果显示,Ad-ODC-AdoMetDCas明显抑制肺癌A-549细胞的增殖,导致细胞凋亡,显著降低肺癌A-549细胞的体外侵袭能力,肺癌A-549细胞感染Ad-ODC-AdoMetDCas后细胞内3种多胺含量都明显降低,Ad-ODC-AdoMetDCas对已形成的裸鼠皮下移植瘤具有明显的抑制作用.实验表明,ODC和AdoMetDC双反义腺病毒具有显著抑制肺癌增殖和侵袭的作用,对于肺癌的防治研究具有一定的前景.     

ODC, AdoMetDC双反义腺病毒对肺癌增殖和侵袭抑制作用的体外和体内研究

鸟氨酸脱羧酶(ODC)和S-甲硫氨酸脱羧酶(AdoMetDC)是多胺体内合成的2个关键酶.研究腺病毒Ad-ODC-AdoMetDCas介导的ODC和AdoMetDC反义RNA对肺癌多胺合成,细胞增殖以及侵袭的抑制作用.用活细胞计数和流式细胞术分别检测Ad-ODCas和Ad-ODC-AdoMetDCas对肺癌A-549细胞增殖的影响,蛋白质印迹和HPLC方法分别检测腺病毒对肺癌A-549细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,TUNEL标记检测法观察Ad-ODC-AdoMetDCas对肺癌细胞凋亡的影响,Matrigel侵袭实验分析腺病毒对肺癌A-549细胞侵袭活性的改变,裸鼠皮下移植瘤模型研究Ad-ODC-AdoMetDCas对体内肺癌生长的抑制作用.实验结果显示,Ad-ODC-AdoMetDCas明显抑制肺癌A-549细胞的增殖,导致细胞凋亡,显著降低肺癌A-549细胞的体外侵袭能力,肺癌A-549细胞感染Ad-ODC-AdoMetDCas后细胞内3种多胺含量都明显降低,Ad-ODC-AdoMetDCas对已形成的裸鼠皮下移植瘤具有明显的抑制作用.实验表明,ODC和AdoMetDC双反义腺病毒具有显著抑制肺癌增殖和侵袭的作用,对于肺癌的防治研究具有一定的前景.     

Adenovirus-mediated expression of both antisense ornithine decarboxylase and S-adenosylmethionine decarboxylase inhibits lung cancer cell growth

Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC). Antisense sequences of ODC and AdoMetDC genes were cloned into an adenoviral vector (named Ad-ODC-AdoMetDCas). To evaluate the effects of recombinant adenovirus Ad-ODC-AdoMetDCas that can simultaneously express both antisense ODC and AdoMetDC, the human lung cancer cell line A-549 was infected with Ad-ODC-AdoMetDCas or the control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were carried out to assess the properties of tumor growth and invasiveness. Our study showed that adenovirus-mediated antisense ODC and AdoMetDC expression inhibits tumor cell growth through blocking the polyamine synthesis pathway. Tumor cells were arrested at the G_1 phase after gene transfer and the invasiveness was reduced. It suggested that the recombinant adenovirus Ad-ODC-AdoMetDCas might be a new anticancer reagent in the treatment of lung cancers.     

ODC和AdoMetDC双反义腺病毒载体对食管癌细胞凋亡影响的研究

为探讨ODC和AdoMetDC双反义腺病毒载体(Ad-ODC-AdoMetDCas)对食管癌Eca109细胞凋亡作用的影响,应用MTT法观察Ad-ODC-AdoMetDCas对食管癌Eca109细胞生长增殖的影响,采用Western blot和HPLC的方法分别检测腺病毒载体对食管癌Eca109细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,同时应用原位末端标记(TUNEL) 法观察Ad-ODC-AdoMetDCas对食管癌Eca109细胞凋亡作用的影响, 透射电镜进一步观察细胞超微结构的改变． 实验结果显示,应用MTT法观察发现Ad-ODC-AdoMetDCas对食管癌Eca109细胞生长增殖有显著抑制作用． 以Ad-ODC- AdoMetDCas感染食管癌Eca109细胞,可明显抑制食管癌Eca109细胞中ODC和AdoMetDC基因表达． HPLC结果显示,食管癌Eca109细胞感染Ad-ODC-AdoMetDCas后,细胞内3种多胺含量都明显降低． TUNEL标记检测结果显示Ad-ODC-AdoMetDCas可明显引起食管癌Eca109细胞凋亡．透射电镜观察到典型的细胞凋亡特征(表现细胞体积缩小,核皱缩、碎裂,染色质呈块状边集等)． 实验表明,ODC和AdoMetDC双反义腺病毒载体(Ad-ODC-AdoMetDCas)具有显著抑制食管癌细胞生长增殖,降低细胞多胺合成,促进细胞凋亡,为探讨食管癌基因治疗的可行性提供实验依据．     

Adenovirus-mediated expression of both antisense ornithine decarboxylase and s-adenosylmethionine decarboxylase induces G1 arrest in HT-29 cells

To evaluated the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC) on cell cycle distribution in colorectal cancer cell and investigated underlying regulatory responses, human colorectal cancer cells HT-29 were cultured in RPMI 1640 medium and infected with Ad-ODC-AdoMetDCas. Cell cycle progression was detected by flow cytometry analysis. The expression levels of cell cycle regulated proteins were measured by Western blot analysis. The mRNA level of cyclin D1 was measured by RT-PCR. And a luciferase reporter plasmid of cyclin D1 promoter was constructed to observe the effect of Ad-ODC-AdoMetDCas on cyclin D1 promoter activity. The results showed that recombinant adenovirus Ad-ODC-AdoMetDCas significantly induced G1 arrest, decreased levels of cyclin D1 protein and mRNA and suppressed the promoter activity. Ad-ODC-AdoMetDCas also inhibited nuclear translocation of beta-catenin. In conclusion, downregulation of ODC and AdoMetDC mediated by Ad-ODC-AdoMetDCas transfection induces G(1) arrest in HT-29 cells and the arrest was associated with suppression of cyclin D1 expression and inhibition of beta-catenin nuclear translocation. As a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of colorectal cancers.     

Downregulation of ornithine decarboxylase by pcDNA-ODCr inhibits gastric cancer cell growth in vitro

Ornithine decarboxylase (ODC), the first rate-limiting enzyme of polyamine biosynthesis, was found to be associated with cell growth, proliferation and transformation. ODC gene expression in gastric cancer was increased and its level was positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. In order to evaluate the therapeutic effects of antisense RNA of ODC on gastric cancer, an antisense RNA of ODC expressing plasmid pcDNA-ODCr which delivered a 120 bp fragment complementary to the initiation codon of ODC gene was constructed and transfected to gastric cancer cells SGC7901 and MGC803. Expression of ODC in gastric cancer cells was determined by western blot. Cell proliferation was assessed by MTS assay. Cell cycle was analyzed by flow cytometry and Matrigel assay was performed to assess the ability of gastric cancer cell invasiveness. The results showed that the ODC gene expression in gastric cancer cells transfected with the pcDNA-ODCr was downregulated efficiently. Tumor cell proliferation was suppressed significantly, and cell cycle was arrested at G1 phase. Gastric cancer cells had reduced invasiveness after gene transfer. Our study suggested that antisense RNA of ODC expressing plasmid pcDNA-ODCr had antitumor activity by inhibiting the expression of ODC, and downregulation of ODC expression using a gene therapy approach might be a novel therapeutic strategy for gastric cancer.     

Concomitant Changes in Polyamine Pools and DNA Methylation during Growth Inhibition of Human Colonic Cancer Cells

The effects of CGP 48664 and DFMO, selective inhibitors of the key enzymes of polyamine biosynthesis, namely, ofS-adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), were investigated on growth, polyamine metabolism, and DNA methylation in the Caco-2 cell line. Both inhibitors caused growth inhibition and affected similarly the initial expression of the differentiation marker sucrase. In the presence of the AdoMetDC inhibitor, ODC activity and the intracellular pool of putrescine were enhanced, whereas the spermidine and spermine pools were decreased. In the presence of the ODC inhibitor, the AdoMetDC activity was enhanced and the intracellular pools of putrescine and spermidine were decreased. With both compounds, the degree of global DNA methylation was increased. Spermine and spermidine (but not putrescine) selectively inhibited cytosine–DNA methyltransferase activity. Our observations suggest that spermidine (and to a lesser extent spermine) controls DNA methylation and may represent a crucial step in the regulation of Caco-2 cell growth and differentiation.     

评价农作物蛋白质优劣的一种排序方法──灰色关联度法

依据灰色系统理论中关联度分析法，提出一种对农作物蛋白质氨基酸构成品质进行考察排序的方法，结果与现行的化学评价法具有较好的一致性，且可避免因某一信息的缺乏而使分离结果偏离实际较大的不足。本法也可根据研究目的不同，选取不同的指标总数作为参考数列对待评价样品进行不同角度的评价考察。     

Role of ornithine decarboxylase in breast cancer

Ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis that decarboxylates ornithine to putrescine, has become a promising target for cancer research. The aim of this study is to investigate the role of ODC in breast cancer. We detected expression of ODC in breast cancer tissues and four breast cancer cell lines, and transfected breast cancer cells with an adenoviral vector carrying antisense ODC (rAd-ODC/Ex3as) and examined their growth and migration. ODC was overexpressed in breast cancer tissues and cell lines compared with non-tumor tissues and normal breast epithelial cells, and there was a positive correlation between the level of ODC mRNA and the staging of tumors. The expression of ODC correlated with cyclin D1, a cell cycle protein, in synchronized breast cancer MDA-MB-231 cells. Gene transfection of rAd-ODC/Ex3as markedly down-regulated expression of ODC and cyclin D1, resulting in suppression of proliferation and cell cycle arrest at G₀–G₁ phase, and the inhibition of colony formation, an anchorage-independent growth pattern, and the migratory ability of MDA-MB-231 cells. rAd-ODC/Ex3as also markedly reduced the concentration of putrescine, but not spermidine or spermine, in MDA-MB-231 cells. The results suggested that the ODC gene might act as a prognostic factor for breast cancer and it could be a promising therapeutic target.     

Translational regulation of ornithine decarboxylase and other enzymes of the polyamine pathway

It has long been known that polyamines play an essential role in the proliferation of mammalian cells, and the polyamine biosynthetic pathway may provide an important target for the development of agents that inhibit carcinogenesis and tumor growth. The rate-limiting enzymes of the polyamine pathway, ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), are highly regulated in the cell, and much of this regulation occurs at the level of translation. Although the 5' leader sequences of ODC and AdoMetDC are both highly structured and contain small internal open reading frames (ORFs), the regulation of their translation appears to be quite different. The translational regulation of ODC is more dependent on secondary structure, and therefore responds to the intracellular availability of active eIF-4E, the cap-binding subunit of the eIF-4F complex, which mediates translation initiations. Cell-specific translation of AdoMetDC appears to be regulated exclusively through the internal ORF, which causes ribosome stalling that is independent of eIF-4E levels and decreases the efficiency with which the downstream ORF encoding AdoMetDC protein is translated. The translation of both ODC and AdoMetDC is negatively regulated by intracellular changes in the polyamines spermidine and spermine. Thus, when polyamine levels are low, the synthesis of both ODC and AdoMetDC is increased, and an increase in polyamine content causes a corresponding decrease in protein synthesis. However, an increase in active eIF-4E may allow for the synthesis of ODC even in the presence of polyamine levels that repress ODC translation in cells with lower levels of the initiation factor. In contrast, the amino acid sequence that is encoded by the upstream ORF is critical for polyamine regulation of AdoMetDC synthesis and polyamines may affect synthesis by interaction with the putative peptide, MAGDIS.     

抗酶的研究进展

抗酶（antizyme）是当细胞内多胺水平升高时刺激机体合成的一种小分子量调节蛋白,能特异性地与鸟氨酸脱羧酶（omithine decarboxylase,ODC）结合,经泛素非依赖途径被26S蛋白酶体降解,从而使多胺合成减少;抗酶还可以调节多胺转运,以稳定细胞内多胺水平。近年来随着生物技术的不断发展,对抗酶的认识也逐步深入,本文综述了抗酶家族、合成、作用及定位等方面的研究进展。     

Decarboxylases involved in polyamine biosynthesis and their inactivation by nitric oxide

Polyamines are ubiquitous cellular components that are involved in normal and neoplastic growth. Polyamine biosynthesis is very highly regulated in mammalian cells by the activities of two key decarboxylases acting on ornithine and S-adenosylmethionine. Recent studies, which include crystallographic analysis of the recombinant human proteins, have provided a detailed knowledge of their structure and function. Ornithine decarboxylase is a PLP-requiring decarboxylase, whereas S-adenosylmethionine decarboxylase (AdoMetDC) contains a covalently bound pyruvate prosthetic group. Both enzymes have a key cysteine residue, which is involved in protonation of the Schiff base intermediate C(alpha) to form the product. These residues, Cys360 in ornithine decarboxylase (ODC) and Cys82 in AdoMetDC, react readily with nitric oxide (NO), which is therefore a potent inactivator of polyamine synthesis. The inactivation of these enzymes may mediate some of the antiproliferative actions of NO.     

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides

Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5-AAAGCT GCTCATGGTTCT-3 which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5-TGCAGCTTCCATCACCGT-3. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from – 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation.Abbreviations ODC ornithine decarboxylase - AdoMetDC S-adenosylmethionine decarboxylase - DFMO difluoromethylornithine     

Antitumor effect of antisense ornithine decarboxylase adenovirus on human lung cancer cells

Ornithine decarboxylase (ODC),the first enzyme of polyamine biosynthesis,was found toincrease in cancer cells,especially lung cancer cells.Some chemotherapeutic agents aimed at decreasingODC gene expression showed inhibitory effects on cancer cells,ha this study,we examined the effects ofadenoviral transduced antisense ODC on lung cancer cells.An adenovirus carrying antisense ODC(rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549.The 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to analyze the effect on cell growth.Expression of ODC and concentrationof polyamines in cells were determined by Western blot analysis and high performance liquid chromatography.Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cellapoptosis.The expression of ODC in A-549 cells was reduced to 54%,and that of three polyamines wasalso decreased through the rAd-ODC/Ex3as treatment.Consequently,cell growth was substantially inhibitedand terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3ascould lead to cell apoptosis,with apoptosis index of 46%.This study suggests that rAd-ODC/Ex3as has anantitumor effect on the human lung cancer cells.     

Regulation and function of polyamines in African trypanosomes

The polyamine biosynthetic pathway is an important drug target for the treatment of human African trypanosomiasis (HAT), raising interest in understanding polyamine function and their mechanism of regulation. Polyamine levels are tightly controlled in mammalian cells, but similar regulatory mechanisms appear absent in trypanosomes. Instead trypanosomatid S-adenosylmethionine decarboxylase (AdoMetDC), which catalyzes a key step in the biosynthesis of the polyamine spermidine, is activated by dimerization with an inducible protein termed prozyme. Prozyme is an inactive paralog of the active AdoMetDC enzyme that evolved by gene duplication and is found only in the trypanosomatids. In Trypanosoma brucei, AdoMetDC activity appears to be controlled by regulation of prozyme protein levels, potentially at the translational level.