ODC反义RNA转染细胞的肿瘤相关基因表达及其致瘤性

鸟氨酸脱羧酶(ODC)是多胺生物合成途径中的限速酶，其活性的异常升高和继之引起的多胺堆积与肿瘤的发生、发展及转移密切相关。我们曾自行构建人ODC反义RNA真核表达质粒，并转染人肺鳞癌细胞，观察到稳定转染细胞株的生长速度减慢；大分子生物合成受到抑制；ODC mRNA表达减少和ODC活性降低等。     

ODC反义寡核苷酸对K562细胞的抑瘤作用

鸟氨酸脱羧酶(ODC)是催化多胺生物合成的限速酶.多胺在维持肿瘤恶性表型中起着重要作用,本室多年的研究表明,控制多胺生物合成可以抑制肿瘤细胞生长,诱导分化,增强对化疗药物的敏感性.近年,我们以ODC为靶基因构建了ODC反义RNA表达载体,以此转染人肺鳞癌细胞,成功地使其恶性表型得到了逆转.进一步说明抑制多胺生物合成     

ODC反义寡核苷酸对K562细胞生长抑制作用

鸟氨酸脱羧酶(ornithine decarboxylase,ODC)是催化多胺生物合成的限速酶.过去的研究表明多胺在维持肿瘤恶性表型中起着重要作用.用ODC不可逆抑制剂抑制多胺生物合成不仅能抑制肿瘤细胞的增殖而且还诱导细胞分化.近年我们采用反义技术,以ODC反义RNA转染肿瘤细胞,通过对ODC表达的封闭,成功地使其恶性表型得到了逆转.提示抑制肿瘤细胞的多胺合成可能是抗癌的有效途径.本文用ODC反义寡核苷酸控制肿瘤细胞的多胺生物合成,观察了其对K562细胞生长特性的影响,以期为ODC反义寡核苷酸在肿瘤临床的应用提供实验依据.     

RAd-ODC／Ex3as对肺癌A-549细胞的抑制作用

鸟氨酸脱羧酶（ornithine decarboxylase，ODC）是多胺生物合成途径中的第一个限速酶。据报道，ODC的含量在许多肿瘤中增高，并与肿瘤的复发有一定的关系。为探讨以ODC为靶标进行反义基因治疗的可行性，本实验利用表达ODC第三外显子反义RNA的腺病毒介导的rAd-ODC／Ex3as，将腺病毒介导的ODC反义RNA转入肺癌A-549细胞中，观察其体外抑制肺癌细胞的作用。     

双反义腺病毒载体Ad—ODC—AdoMetDOas对食管癌细胞凋亡及抑制细胞生长影响的研究

目的：探讨双反义腺病毒载体（Ad—ODC—AdoMetDCas）对食管癌Eca109细胞凋亡作用及抑制细胞生长的影响。方法：应用M1Tr法测定不同MOI的腺病毒对肺癌Eca109细胞的基因转染效率，观察Ad—ODC—AdoMetDCas对食管癌Eca109细胞生长增殖的影响；采用Western印迹和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，采用流式细胞术检测腺病毒介导的鸟氨酸脱羧酶反义RNA（Ad—ODCas）和Ad—ODC—AdoMetDCas对食管癌Eca109细胞周期分布的影响，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。结果：MTT法实验表明，Ad—ODC—AdoMetDCas对食管癌Eca109细胞生长增殖有显著抑制作用（P〈0．05）。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（P〈0．05）。流式细胞术结果显示感染了Ad—ODC—AdoMetDCas的食管癌Eca109细胞周期阻滞在G。期。HPLC结果显示，食管癌Eca109细胞感染Ad—ODC—AdoMetDCas后，细胞内3种多胺含量均明显降低（P〈0．05）。TUNEL标记检测结果显示，Ad—ODC—AdoMet—DCas可明显引起食管癌Eca109细胞凋亡（P〈0．05）。结论：Ad—ODC—AdoMetDCas能显著抑制食管癌细胞生长，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。     

鸟氨酸脱羧酶反义RNA对人肺鳞癌细胞恶性表型的逆转

在已获两株有鸟氨酸脱羧酶（ＯＤＣ）反义序列稳定整合的细胞克隆的基础上，进一步研究发现，反义转染细胞的Ｇ０／Ｇ１期细胞数增多，Ｓ期细胞数相应减少；其软琼脂集落形成能力及裸鼠致瘤能力均显著下降。核酸杂交结果显示，在反义转染细胞中有ＯＤＣ反义ＲＮＡ表达，ＯＤＣｍＲＮＡ含量明显减少。结果表明，人肺鳞癌细胞恶性表型的逆转与ＯＤＣ反义ＲＮＡ有效控制多胺生物合成有关。     

人核糖体蛋白S13(RPS13)编码基因的克隆及其正反义核酸转染胃癌细胞的初步研究

目的：扩增人核糖体蛋白S13（RPS13）编码基因序列，构建其正，反义真核表达载体，分别转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，以进一步研究RPS13基因与胃癌多药耐药的关系。方法：采用RT-PCR法扩增RPS13cDNA片段编码区全长序列，利用DNA重组技术的建正，反义真核表达载体，经脂质体介导转染胃癌细胞SGC7901及胃癌耐药细胞SGC7901/VCR，筛选正，反义基因稳定转染细胞，RNA斑点杂交法检测正，反义稳定转染细胞mRNA水平的变化。结果：从高表达RPS13的SGC7901/VCR耐药细胞中提取细胞总RNA，RT-PCR法成功扩增了RPS13cDNA片段编码区全长序列，并经测序证实；目的基因因按正，反两个方向亚克隆至真核表达载体pcDNA3.1( ),并经酶切鉴定证实；经脂质体介导将正义真核表达载体转染SGC7901细胞，反义真核表达载体转染SGC7901/VCR细胞，G418筛选获得稳定转染细胞；RNA斑点杂交试验证实；正义稳定转染细胞RPS13mRNA水平上调，反义稳定转染细胞RPS13mRNA水平下调。结论：成功克隆了人RPS13编码基因序列，并构建了其正，反义真核表达载体，在胃癌细胞系SGC7901及长春新碱耐药细胞SGC7901/VCR中得到稳定转染细胞，正义转染细胞中RPS13mRNA表达明显增加，反义转染细胞中表达明显受抑。     

鸟氨酸脱羧酶基因沉默抑制宫颈癌细胞生长的研究

目的：探讨鸟氨酸脱羧酶（ODC）基因作为宫颈癌治疗靶基因的可行性。方法：采用慢病毒介导的RNA干扰技术,沉默Hela细胞ODC基因,建立ODC稳定沉默的细胞系Hela／ODC,用定量RT—PCR和免疫印迹检测ODC的表达水平。用MTT实验、克隆形成实验、细胞倍增时间和细胞周期分析评价ODC基因沉默对Hela细胞生长的影响。结果：Hela／ODC的ODCmRNA和蛋白质表达水平均明显下降,mRNA水平下调93．4％。Hela／ODC的增殖受到明显抑制,细胞倍增时间从22．3h延长到32．0h,细胞周期分析S期细胞增加,G0／G1期和G2／M期细胞显著减少。结论：慢病毒载体介导RNA干扰能够稳定沉默ODC基因表达,从而成功抑制肿瘤细胞的生长,表明以ODC基因为靶基因进行宫颈癌的基因治疗是可行的。     

RNA干扰细胞黏附分子L1表达逆转胶质瘤U251细胞多药耐药

背景与目的：细胞黏附分子L1（cell adhesionmoleculeL1,L1CAM）是一种跨膜黏附蛋白,在神经系统发育及肿瘤发生中发挥重要作用。本研究运用RNA干扰（RNAinterference,RNAi）技术抑制L1CAM表达,并探讨其对胶质瘤U251细胞多药耐药的逆转作用及相关分子机制。方法：将针对L1CAM的小干扰RNA（siLl）和阴性对照siRNAfsiCon）转染人胶质瘤U251细胞。实验分3组：空白对照组（胶质瘤U251细胞）、阴性对照组（转染siCon的胶质瘤U251细胞）、实验组f转染siLl的胶质瘤U251细胞）。蛋白质印迹法（Westernblotting）~U251细胞中L1CAM、MRPl、pAKT及pERK1／2等蛋白表达。细胞增殖实验检测L1CAM对顺铂（cisplatin）和P13K／AKT抑制剂LY294002抑制U251细胞增殖的影响。免疫荧光染色法检测抑制LICAM表达对U251细胞系中AKT磷酸化情况的影响。结果：实验组L1CAM、pAKT．pERKl／2蛋白表达量明显低于阴性对照组和空白对照组（P〈0．01）,但实验组多药耐药蛋白MRPl表达量以及Bcl-2cdBax与阴性对照组和空白对照组相比无明显变化。实验组顺铂和LY294002对U251细胞增殖的抑制率高于阴性对照组和空白对照组,提示抑制LICAM表达后细胞对顺铂和LY294002的敏感性增加。免疫荧光结果显示,与阴性对照组和空白对照组相比,实验组细胞内AKT磷酸化信号明显降低。结论：RNAi抑制L1CAM表达能增强顺铂和LY294002对U251细胞增殖的抑制作用,并可抑制P13K／AKT和MAPK信号激活．一定程度上可逆转胶质瘤细胞的多药耐药性。     

增殖细胞核抗原基因反义RNA表达质粒的构建及对人胃癌细胞的抑瘤效应

目的观察增殖细胞核抗原（ＰＣＮＡ）基因反义ＲＮＡ表达质粒对人胃癌细胞生长特性的影响，探讨其抗肿瘤作用。方法用ＤＮＡ重组方法将人ＰＣＮＡ基因反向克隆到真核表达质粒ｐＤＯＲｎｅｏ中，构建成人ＰＣＮＡ基因真核表达质粒ｐＤＲＰＣＮＡ，用脂质体介导转染人胃癌细胞系ＳＧＣ７９０１。结果经Ｇ４１８筛选获得转染细胞ＳＧＣ／ＰＣＮＡ，与亲本细胞相比，其生长速度减慢，ＲＮＡ、蛋白质的生物合成受到抑制，Ｓ期、Ｇ２／Ｍ期ＤＮＡ含量降低。结论ＰＣＮＡ基因反义ＲＮＡ对胃癌细胞的生长具有明显的抑制作用。     

Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells

Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ER??) in human breast cancer cells. However, the mechanism underlying the potential regulation of ER?? expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ER??-positive MCF7 and T47D and ER??-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N ¹-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ER??. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ER?? expression, suggesting that ODC plays an important role in regulation of ER?? expression. Decrease of ER?? expression by ODC siRNA altered the mRNA expression of a subset of ER?? response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ER?? minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ER?? minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer.     

APC-dependent changes in expression of genes influencing polyamine metabolism, and consequences for gastrointestinal carcinogenesis, in the Min mouse.

The colorectal mucosa of pre-symptomatic individuals with familial adenomatous polyposis (FAP) contains elevated levels of the proliferation-associated polyamines. The Min mouse, like humans with FAP, expresses an abnormal genotype for the APC tumor suppressor gene. In order to determine how APC mutation influences intestinal tissue polyamine content, we measured steady-state RNA levels of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, antizyme (AZ), a protein which negatively regulates ODC, and the spermidine/spermine N(1)-acetyltransferase (SSAT), the first enzyme in polyamine catabolism. RNA content was increased 6- to 8-fold in both the small intestine and colon for ODC, decreased significantly in the small intestine but not the colon for AZ and was not statistically different in either intestinal tissue for SSAT in Min mice compared with normal littermates. Consistent with the changes in ODC and AZ gene expression, small intestinal, but not colonic, polyamine content was elevated in Min mice compared with normal littermates. Treatment of Min mice with the specific ODC inhibitor difluoromethylornithine (DFMO) suppressed small intestinal, but not colonic, polyamine content and tumor number. These data indicate that small intestinal tissue polyamine content is elevated in Min mice by a mechanism involving APC-dependent changes in ODC and AZ RNA. Further, ODC enzyme activity, which is influenced by both ODC and AZ RNA levels and inhibited by DFMO, is consequential for small intestinal tumorigenesis in this model. In the FAP population, DFMO may be of value in the chemoprevention of small intestinal adenocarcinoma that remains a risk following colectomy.     

Ornithine decarboxylase activity and polyamine content in adenocarcinomas of human stomach and large intestine

Ornithine decarboxylase (ODC) activity and differential polyamine composition have been studied in macroscopically normal mucosa, adjacent mucosa, and neoplastic tissue of 95 patients with adenocarcinomas of stomach and large intestine. In tumors, we found increased ODC activity and polyamine content as compared with surrounding mucosa. ODC activity in macroscopically normal tissue of patients with tumors of stomach and large intestine increased as the disease progressed. An inverse relationship was observed between ODC activity in adenocarcinomas and differentiation.     

REVERSION OF MALIGNANT PHENOTYPES OF HUMAN LUNG SQUAMOUS CARCINOMA CELLS BY ORNITHINE DECARBOXYLASE ANTISENSE RNA

ＲＥＶＥＲＳＩＯＮＯＦＭＡＬＩＧＮＡＮＴＰＨＥＮＯＴＹＰＥＳＯＦＨＵＭＡＮＬＵＮＧＳＱＵＡＭＯＵＳＣＡＲＣＩＮＯＭＡＣＥＬＬＳＢＹＯＲＮＩＴＨＩＮＥＤＥＣＡＲＢＯＸＹＬＡＳＥＡＮＴＩＳＥＮＳＥＲＮＡＧｕａｎＪｕｎ关钧，ＦａｎＭｕｚｈｅｎ范慕贞，Ｃａｏ...     

Regulation of ornithine decarboxylase gene expression in MCF-7 breast cancer cells by antiestrogens

Ornithine decarboxylase (ODC) is an enzyme intimately related to cell growth regulation. The metabolic products of ODC, the polyamines, are known to play a vital role in the structure and function of biological macromolecules including nucleic acids and proteins. The activity of ODC is stimulated by estrogens in their target cells. In order to gain insight into the molecular mechanism of action of antiestrogens in human breast cancer, we studied the effect of tamoxifen and 4-hydroxytamoxifen on the concentration of ODC mRNA, ODC activity, and the polyamine levels in a hormone-responsive breast cancer cell line, MCF-7. ODC mRNA concentration was reduced to 40% of the controls after 6 h of treatment of the cells with 100 nM 4-hydroxytamoxifen, but tamoxifen had no significant effect on ODC mRNA after treating with even 1 microM concentration for 36 h. ODC activity was, however, reduced to 40 and 75% of the controls after 24 h of treatment with 4-hydroxytamoxifen and tamoxifen, respectively. There was a significant reduction in the concentration of putrescine to 63% of control in tamoxifen-treated cells, but spermidine and spermine levels were not affected. With 4-hydroxytamoxifen, putrescine, spermidine, and spermine levels were reduced to 41, 62, and 79% of the control, respectively. In addition, exogenous putrescine was able to reverse the growth inhibitory effects of 4-hydroxytamoxifen. Overall, these results indicate that ODC and polyamine levels in MCF-7 cells are controlled by antiestrogens, and that suppression of polyamine biosynthesis plays a critical role in the growth inhibitory effects of antiestrogens.     

Elevated ornithine decarboxylase levels activate ataxia telangiectasia mutated-DNA damage signaling in normal keratinocytes

We examined the effect of increased expression of ornithine decarboxylase (ODC), a key rate-limiting enzyme in polyamine biosynthesis, on cell survival in primary cultures of keratinocytes isolated from the skin of K6/ODC transgenic mice (Ker/ODC) and their normal littermates (Ker/Norm). Although elevated levels of ODC and polyamines stimulate proliferation of keratinocytes, Ker/ODC undergo apoptotic cell death within days of primary culture unlike Ker/Norm that continue to proliferate. Phosphorylation of ataxia telangiectasia mutated (ATM) and its substrate p53 are significantly induced both in Ker/ODC and in K6/ODC transgenic skin. Chromatin immunoprecipitation analyses show that the increased level of p53 in Ker/ODC is accompanied by increased recruitment of p53 to the Bax proximal promoter. ATM activation is polyamine dependent because alpha-difluoromethylornithine, a specific inhibitor of ODC activity, blocks its phosphorylation. Ker/ODC also displays increased generation of H(2)O(2), acrolein-lysine conjugates, and protein oxidation products as well as polyamine-dependent DNA damage, as measured by the comet assay and the expression of the phosphorylated form of the histone variant gamma H2AX. Both reactive oxygen species generation and apoptotic cell death of Ker/ODC may, at least in part, be due to induction of a polyamine catabolic pathway that generates both H(2)O(2) and cytotoxic aldehydes, because spermine oxidase (SMO) levels are induced in Ker/ODC. In addition, treatment with MDL 72,527, an inhibitor of SMO, blocks the production of H(2)O(2) and increases the survival of Ker/ODC. These results show a novel activation of the ATM-DNA damage signaling pathway in response to increased ODC activity in nontumorigenic keratinocytes.     

Prognostic value of ornithine decarboxylase and polyamines in human breast cancer: correlation with clinicopathologic parameters.

The polyamines putrescine, spermidine, and spermine and ornithine decarboxylase (ODC), the rate-limiting enzyme in their biosynthetic pathway, play an important role in cell proliferation, differentiation, and transformation. In the present study, we have analyzed polyamine concentrations and ODC activity in samples from benign breast diseases (n = 36), benign breast tissue adjacent to the primary carcinoma (n = 19), and breast carcinoma (n = 104). ODC activity in primary carcinoma was significantly higher (2.42 +/- 0.22 nmol CO2/h g; P < 0.001) than that found in benign breast (0.62 +/- 0.15 nmol CO2/h g) or in breast tissue adjacent to the primary carcinoma (0.52 +/- 0.16 nmol CO2/h g). The total polyamine content of breast cancer tissues was higher than in benign breast diseases (704.3 +/- 38.3 nmol/g wet weight versus 295.8 +/- 27.4 nmol/g wet weight) and correlated well with ODC activity (Pearson, r = 0.42; P < 0.001). ODC activity correlated with histological grade, peritumoral lymphatic or blood vessel invasion, S-phase fraction, and cathepsin D. Total polyamine concentration increased with S-phase fraction, cathepsin D, and aneuploidy. No significant correlation was found between ODC or polyamines and tumor size, lymph node involvement, or steroid receptor status. A major finding in our study was that ODC activity was an independent prognostic factor for recurrence and death. The results indicate that the estimation of ODC activity and polyamines in human breast carcinoma might be useful to determine tumor aggressiveness and suggest that ODC may have a potential value as both a prognostic factor and a chemoprevention target in human breast cancer.