Bcl-2反义寡核苷酸对环已亚胺诱导U251细胞凋亡的影响

目的 探讨Bcl-2反义寡核苷酸对环已亚胺（CHX）诱导胶质瘤细胞U251凋亡的影响。方法在光镜下观察Bcl-2反义寡核苷酸、CHX及二者联合应用对U251凋亡的形态学改变;应用MTT法检测各组U251细胞的抑制率;通过DNA电泳检测CHX诱导U251细胞凋亡的情况。结果 Bcl-2反义寡核苷酸可明显促进CHX诱导的U251凋亡,对照组、SODN组与CHX组、AODN组、SODN＋CHX组及AODN＋CHX组的细胞抑制率相比P均〈0．01,AODN组、SODN＋CHX组和AODN＋CHX组的细胞抑制率相比P均〈0．01;Bcl-2反义寡核苷酸可促进细胞凋亡,呈现出特征性的DNA梯状带。结论Bcl-2反义寡核苷酸和CHX均可诱导胶质瘤细胞U251发生凋亡,且Bcl-2反义寡核苷酸可明显促进CHX诱导的胶质瘤细胞U251凋亡,提高胶质瘤细胞对CHX的敏感性。     

Dysregulated Fas and Bcl-2 expression leading to enhanced apoptosis in T cells of multiple myeloma patients

We have previously reported the presence of activated (HLA-DR+) T cells in multiple myeloma (MM) patients. These cells produce high amounts of interleukin (IL)-2 and interferon (IFN)-gamma and generate a potent antiplasma cell activity after appropriate in vitro stimulation, but they are unable in vivo to hold in check the disease. Activated T cells are highly susceptible to apoptosis, a form of programmed cell death involved in the modulation of immune responses and regulated by molecules such as Fas (CD95) and bcl-2. The aim of this study was to determine the expression of Fas and bcl-2 antigens and the susceptibility to apoptosis in T cells of MM patients. Fas+ cells were significantly higher, whereas bcl-2+ cells were significantly lower in MM patients than in the controls. MM patients with the highest number of HLA-DR+ T cells showed the highest Fas and the lowest bcl-2 expression. Two-color cytofluorometric analysis confirmed in individual cells that HLA-DR+ T cells coexpressed Fas and lacked bcl-2. Susceptibility to apoptosis was then investigated to evaluate the consequence of dysregulated Fas and bcl-2 expression. The percentage of apoptotic cells after incubation in medium alone (spontaneous apoptosis) or in the presence of methylprednisolone (MP) or anti-Fas monoclonal antibody (triggered apoptosis) was significantly higher in MM and mainly restricted to HLA-DR+ T cells. Spontaneous apoptotosis was reverted by exogenous IL-2. In conclusion, MM T cells have a dysregulated expression of Fas and bcl-2 antigens that is associated with an enhanced susceptibility to apoptosis. These data may unravel a novel mechanism by which activated MM T cells are weakened in their ability to exert an effective antitumor activity in vivo.     

Bcl-2 antisense oligodeoxyribonucleotide increases apoptosis of lung carcinoma cells induced by cisplatin]

OBJECTIVE: To study the effect of antisense oligodeoxyribonucleotide (ODN) to apoptotic suppress gene bcl-2 on apoptosis of lung carcinoma cells induced by cisplatin. METHODS: The lung carcinoma cells expressing bcl-2 were chosen to participate in this experiment. Cultured cells were divided into 7 groups: ODN, nonsense, ODN + cisplatin, nonsense + cisplatin, cisplatin, lipofectin and control. Bcl-2 antisense or nonsense mixed with lipofectin was added into above corresponding cultured cells. After cultured for 6 hours, cisplatin was added into corresponding groups. The cells were cultured again for 16 hours. And then, the cells were smeared on slides. Apoptotic cells were labeled with TdT-mediated dUTP nick end labeling (TUNEL) method on cell smears. Apoptotic index (AI) was counted to show the percentage of apoptotic cancer cells. The immunocytochemistry was used to detect the expression of bcl-2 in carcinoma cells. RESULTS: The bcl-2 expression of cancer cell in ODN group was significantly decreased compared to the control and nonsense groups. The AI of ODN + cisplatin group was 16.4 +/- 1.7, cisplatin group 4.1 +/- 0.8, antisense group 5.9 +/- 0.2, nonsense group 3.3 +/- 0.7, nonsense + cisplatin 7.6 +/- 1.1, lipofectin 5.1 +/- 0.9, control group 3.6 +/- 0.6. The AI of antisense + cisplatin group was significantly higher than that of other groups. CONCLUSION: Antisense oligodeoxyribonucleotide to bcl-2 can inhibit significantly the expression of bcl-2 of lung cancer cells and increase apoptosis of cancer cells induced by cisplatin.     

Apoptosis of tumoral and nontumoral lymphoid cells is induced by both mdm2 and p53 antisense oligodeoxynucleotides

Following stress signals, the p53 tumor suppressor protein plays a critical role in regulation of cell proliferation, mainly through induction of growth arrest or apoptosis. Therefore, this protein needs to be strictly regulated and numerous studies have shown that the MDM2 protein is an essential element for p53 regulation in normal cells and, most importantly, that overexpression of MDM2 is responsible for p53 inactivation in various types of tumors. A previous study showed that this is the case in some Burkitt lymphoma (BL) cell lines, where enhanced translation of mdm2 messenger RNA results in overexpression of the protein that complexes and inactivates wild-type p53. To further investigate the role of the p53/MDM2 complex in these BL cells, as well as in other lymphoid cells that do not overexpress MDM2, this study used antisense oligodeoxynucleotides directed either against mdm2 or against p53. Results show that the mdm2 antisense oligodeoxynucleotide induces apoptosis of cells that express a high or low level of MDM2 protein, only if they contain wild-type p53. Moreover, apoptosis is independent of the accumulation of p53 following mdm2 antisense treatment. Finally, the p53 antisense oligodeoxynucleotide, which inhibits the expression of wild-type p53, also induces a decrease of the MDM2 level in cells, whether or not they overexpress this protein, and causes apoptosis of these cells. These results indicate that decreasing the MDM2 protein level by directly or indirectly targeting its biosynthesis is a potent tool for the induction of apoptosis.     

Bcl-2家族在冬凌草甲素诱导人多发性骨髓瘤细胞凋亡中的作用

目的探讨部分Bcl-2家族成员基因表达变化在冬凌草甲素（Ori）诱导人多发性骨髓瘤ARH-77细胞凋亡过程中的作用。方法采用MTT法和流式细胞术检测0ri作用后ARH-77细胞的活力和凋亡情况,相差显微镜观察细胞形态改变,RT-PCR法检测Bcl-2家族成员的基因表达变化。结果Ori能明显抑制ARH-77细胞的生长,且呈时间一剂量依赖性。10μmol／LOri作用于ARH-77细胞24h后可见细胞变小、胞质中出现空泡,并可见凋亡小体。流式细胞仪检测显示,细胞凋亡率随Ori作用时间的延长而逐渐增加（P〈0．05）。Ori诱导ARH-77细胞凋亡与Bcl-2、Bcl-xl、BaxmRNA水平改变有关（P〈0．05）。结论Ori能通过调节ARH-77细胞Bcl-2家族成员的表达而诱导其发生凋亡。     

Bcl-2反义核酸的设计及对K562细胞凋亡的研究

目的：探讨bcl-2不同靶点的反义寡核苷酸（ASODN）对白血病细胞株K562细胞凋亡的影响。方法：用RNA二级结构预测程序预测bcl-2mRNA的二级结构；免疫荧光标记观察细胞bcl-2蛋白水平；细胞记数观察细胞的生存情况；用流式细胞仪观察细胞凋亡。结果：在5个初选的反义序列中，靶向bcl-2mRNA蛋白编码区和翻译起始区的ASODN能明显地下调bcl-2蛋白的表达，并且两个不同靶点的ASODN能有效地抑制K562细胞的生长活性、促进细胞凋亡；靶向bcl-2mRNA蛋白编码区的ASODN降低细胞bcl-2蛋白、促进细胞凋亡的作用较靶向翻译起始区的ASODN强。随机的无诳寡核苷酸对K562细胞的生长活性、bcl-2蛋白水平及细胞凋亡率均无影响。结论：分别在bcl-2mRNA翻译起始区和蛋白编码区发现了两个有效的反义作用靶点，针对这两个靶点的ASODN能特异性促进K562细胞的凋亡。     

胰腺癌Bcl-2,P53蛋白表达和细胞凋亡

目的　探讨ｂｃｌ２ ,ｐ５３ 基因和细胞凋亡在胰腺癌发病机制中的作用以及它们之间相互关系．方法　应用ＡＢＣ 免疫组化技术检测５０ 例胰腺癌中Ｂｃｌ２ 和Ｐ５３ 蛋白表达,运用原位末端标记法观察肿瘤中细胞凋亡数量．结果　Ｐ５３ 蛋白表达阳性率为５４ ％ ,临床Ⅰ期阳性率（２６－７ ％ ）却显著低于Ⅱ期（６１－１ ％ ） 和Ⅲ＋ Ⅳ期（７０－６ ％ ,Ｐ＜ ０－０５） ;Ｂｃｌ２蛋白表达阳性率为６４ ％ ,临床Ⅰ期阳性率（９３－３ ％ ） ,显著高于Ⅱ期（５５－６ ％ ） 和Ⅲ＋ Ⅳ期（４７－１ ％ ,Ｐ＜ ０－０５） ;组织学Ⅲ级癌细胞中凋亡指数明显高于Ⅰ,Ⅱ级（ Ｐ＜ ０－０５） ,Ｂｃｌ２ 蛋白阴性病例中凋亡指数明显高于Ｂｃｌ２ 阳性者（ Ｐ＜ ０－０１） ．结论　Ｂｃｌ２ 是通过抑制细胞凋亡参与肿瘤的生长过程,Ｂｃｌ２和Ｐ５３ 蛋白表达之间 存在密切负相关（τ＝ － ０－１７４７ ,Ｐ＜ ０－０５） ．     

反义端粒酶基因与顺铂诱导原代胃癌细胞凋亡作用的研究

目的　探讨端粒酶反义寡核苷酸 (PS- ASODN)与顺铂 (DDP)联合应用对原代胃癌细胞凋亡作用的影响。方法　常规组织块培养法进行胃癌细胞原代纯化培养 ,取第 3代对数生长期细胞分六组进行实验。其中四组在培养 2 4小时及 4 8小时分别加入相同剂量的培养液 ,终浓度为 PS- ASODN3μM,N- ASODN3μM,DDP2 .0μg/ ml。作用 2 4小时后分别在 PS- ASODN组及 N- ASODN组加入终浓度为 2 .0 μg/ ml的 DDP;分别于培养后 2 4、4 8、72及 96小时收集各组细胞。以台盼蓝拒染法计算各组细胞生长抑制率 ,观察 PS- ASODN联合 DDP对原代胃癌细胞生长的影响 ;流式细胞学观察细胞凋亡率及细胞周期变化。结果　终浓度为 3μM的 PS- ASODN作用于原代胃癌细胞 2 4小时后加入 DDP 2 .0μg/ m l,能明显抑制胃癌细胞增殖 ,流式细胞学可检测到凋亡峰 ,细胞受阻于G0 / G1 期 ,作用 4 8及 72小时的凋亡细胞百分率 (35 .1%、4 5 .7% )明显高于 N- ASODN组、PS- ASODN组、DDP组及 N- ASODN+DDP组 ,差异有显著性 (P<0 .0 5 )。其作用呈时间依赖性及序列特异性。结论　以端粒酶 RNA模板区为靶点的 PS- ASODN可促进 DDP诱导的胃癌细胞凋亡 ,对胃癌具有治疗价值     

Apo2L/TRAIL and Bcl-2-related proteins regulate type I interferon-induced apoptosis in multiple myeloma

It has been reported that interferons (IFNs) may have antitumor activity in multiple myeloma (MM). The mechanism for their effect on MM, however, remains elusive. This study shows that IFN-alpha and -beta, but not -gamma, induce apoptosis characterized by Annexin V positivity, nuclear fragmentation and condensation, and loss of clonogenicity in 3 MM cell lines (U266, RPMI-8266, and NCI-H929), and in plasma cells from 10 patients with MM. Apo2 ligand (Apo2L, also TRAIL) induction was one of the earliest events following IFN administration in U266 cells. Treatment of these cells with TRAIL, but not with Fas agonistic antibodies, induces apoptosis. Cell death induced by IFNs and Apo2L in U266 cells was partially blocked by a dominant-negative Apo2L receptor, DR5, demonstrating the functional significance of Apo2L induction. This study shows that IFNs activate caspases and the mitochondrial-dependent apoptotic pathway, possibly mediated by Apo2L production. Thus, IFN-alpha and -beta induce cytochrome c release from mitochondria starting at 12 hours, with an amplified release seen at 48 hours. Moreover, Bid cleavage precedes the initial cytochrome c release, whereas the late, amplified cytochrome c release coincides with changes in levels of Bcl-2, Bcl-X(L), and reduction of mitochondrial membrane potential. These results link the Apo2L induction and modulation of Bcl-2 family proteins to mitochondrial dysfunction. Furthermore, IFNs and Apo2L induce cell death of CD38(+)/CD45(-/dim) plasma cells, without significant effect on nonplasma blood cells, in a caspase and Bcl-2 cleavage-dependent manner. These results warrant further clinical studies with IFNs and Apo2L in MM.     

LivinmRNA反义寡核苷酸对人肝癌细胞株HepG-2体外增殖及凋亡的影响

目的 探讨LivinmRNA反义寡核苷酸（ASODN）进行肿瘤基因治疗的可行性。方法设计合成嵌合骨架型ASODN及其对照错义寡核苷酸（MSODN）,脂质体转染到HepG-2细胞（观察1组和观察2组）,同时设未转染者为对照组。采用MTr法检测HepG-2细胞增殖抑制率（IR）,电镜、流式细胞仪与吖啶橙／溴化乙锭（AO／EB）细胞染色法检测细胞凋亡水平和形态学变化。结果ASODN在终浓度200nmol／L作用72h时,观察1组HepG-2细胞IR及细胞凋亡数明显高于其他两组.结论以Livin为靶点的ASODN能够促使肿瘤细胞凋亡,具有靶特异性和低毒性,有可能成为抗肿瘤药物开发的新工具。     

Selective apoptosis of multiple myeloma cells in primary samples induced by arsenic trioxide

Objectives

Currently, multiple myeloma (MM) is an incurable disease. Despite the fact that arsenic trioxide (ATO) shows promising results in vitro, data from treatment of patients with MM are disappointing. Due to these discrepancies, we compared the efficacy and selectivity of ATO at two different concentrations in samples from MM patients.

Methods

The extent of apoptosis induced by 2 and 5 µM ATO was evaluated by flow cytometry using annexin V. 34 diagnostic bone marrow samples obtained from MM patients were analysed.

Results

5 µM ATO efficiently induced apoptosis in primary samples. Besides efficacy, also selectivity of action on MM cells in comparison to remaining haematopoietic cells was demonstrated for 5 µM ATO but not for 2 µM ATO.

Discussion

Our study on primary samples confirmed that ATO has a potential role in therapeutic management of MM. Further controlled studies on MM patients are needed.     

MicroRNA-15b 下调Bcl-2促进耐药肺癌细胞凋亡

目的 探讨微小RNA-15b （microRNA-15b,miR-15b）对人肺癌耐药细胞A549/DDP凋亡的影响.方法 采用体外转染法将MicroRNA-15b瞬时转染到A549/DDP细胞后,应用Real-time PCR检测A549/DDP细胞中MicroRNA-15b的表达情况;流式细胞仪（FCM）检测细胞凋亡变化;并用Western印迹法（Western blot）检测细胞中Bcl-2表达.结果 转染后miR-15b组的miR-15b表达水平显著增加（P〈0.05）;miR-15b组细胞凋亡率为：32.4%±5.1%,与Mock组（5.73%±1.2%）和miR-15b-Cont（6.24%±2.4%）比较,差异具有统计学意义（P〈0.05）;miR-15b组的Bcl-2表达量较对照组明显增加.结论 miR-15b可能通过下调Bcl-2的表达从而诱导A549/DDP细胞的凋亡,这可能为肺癌耐药的治疗提供新靶点.