腺病毒介导人血管抑素抑制乳腺癌生长的研究

目的：研究腺病毒介导的重组人血管抑制Plasminogen K5基因对乳腺癌的治疗作用。方法：通过PCR将人血纤维蛋白酶原的信号肽基因加至Plasminogen的K5结构域基因，克隆到质粒pCA13，并在293细胞中同源重组，得到腺病毒AdK5。将Ad-K5体外感染乳腺癌细胞B－Cap-37和血管内皮细胞ECV304，观察其对细胞生长的影响，同时在乳腺癌的裸鼠动物模型上，检测其对肿瘤生长的抑制作用。结果：在感染Ad-K5的B－Cap-37细胞中检测到K5基因mRNA的表达。Ad-K5对血管内皮细胞ECV304有抑制作用，但对癌细胞B-Cap-37没有直接抑制作用。动物实验表明Ad-K5能显著抑制肿瘤的生长。结论：K5基因能抑制乳腺癌实体瘤的生长。     

Induction of Apoptosis in Resistant Glioma Cells by Synthetic Caspase-Activation

The detailed mechanisms behind the resistance of malignant gliomas to therapy are not known. Inherent resistance to apoptosis is, however, one plausible explanation. In the present study we tried to delineate the molecular defects and to induce apoptosis by inducible caspases in three apparently apoptosis resistant glioma cell lines. U-105 MG, U-251 MG, and SF-767 were resistant to Fas-induced apoptosis as shown by the lack of Fas-induced cell death, morphological changes, annexin-V reactivity, Parp cleavage, caspase-3 cleavage, and caspase-3 activation. The glioma cells showed no consistent down-regulation of the pro-apoptotic proteins Fas, Fadd, caspase-3, caspase-8, caspase-9, Apaf-1, Bid, Bad, or Bax, and no consistent up-regulation of the anti-apoptotic proteins Bcl-x or Bcl-2. In U-105 MG, Fas was, however, not detected at the cell surface indicating intracellular retention. To assess if the apoptotic blocks could be by-passed, we introduced the so-called artificial death switches, i.e., inducible caspases and Fadd, into the glioma cells. Synthetic activation of inducible caspase-3, but not of caspase-8, resulted in apoptosis in the three glioma cell lines and inducible Fadd induced apoptosis in SF-767. The results were consistent with a block in the apoptotic signaling pathways of glioma cells between caspase-8 and caspase-3 activation, and that inducible Fadd could induce caspase-8 independent apoptosis in some cells. Apparently resistant glioma cells could thus be induced to undergo apoptosis by activation of appropriate death switches. This might have implications for the design of future therapeutic strategies.     

逆转录病毒介导的双自杀基因对淋巴瘤细胞的杀伤作用研究

目的 观察逆转录病毒介导的双自杀基因对淋巴瘤细胞的杀伤作用，探讨淋巴瘤的基因治疗方法。方法 在脂质体的介导下将含有双自杀基因的逆转录病毒载体pWZLneoCDglytk导入包装细胞PA317，经G418筛选后大量培养产病毒的阳性克隆PA317／CD tk细胞株，收集病毒上清，浓缩后转染Raji淋巴瘤细胞，再次经G418筛选，获得稳定表达双自杀基因的Raji/CD tk细胞株。采用RT-PCR法检测双自杀基因的表达。MTT法测定转基因组及末转基因组Raji细胞的存活率。结果 双自杀基因在Raji细胞中可稳定表达，联合使用5-氟胞咳暖(5-FC)和无环鸟苷(QCV)对细胞增殖的杀伤作用及旁杀伤效应高于单独使用5-FC或GCV。结论 逆转录病毒介导双自杀基因较单一自杀基因具有更强的杀伤作用。     

逆转录病毒介导的双自杀基因对K562细胞杀伤作用的实验研究

目的 :观察逆转录病毒介导的双自杀基因对K5 6 2细胞的杀伤作用 ,探讨慢性粒细胞白血病的基因治疗方法。方法 :通过脂质体将含有双自杀基因的逆转录病毒载体PWZLneoCDglytk导入包装细胞PA317,经G4 18筛选后大量培养产病毒的阳性克隆PA317 CD +tk细胞株 ,收集病毒上清 ,浓缩后转染K5 6 2细胞 ,再次经G4 18筛选 ,获得稳定表达双自杀基因的K5 6 2 CD +tk细胞株。用RT PCR检测双自杀基因的表达。给予前体药物 5 氟胞嘧啶 (5 flourocytosine ,5 FC)和 或无环鸟苷 (Ganciciovir,GCV)后MTT法测定转基因组及未转基因组K5 6 2细胞的存活率。结果 :双自杀基因在K5 6 2细胞中可稳定表达 ,联合使用 5 FC和GCV对细胞增殖的杀伤作用及旁杀伤效应高于单独使用 5 FC或GCV。结论 :逆转录病毒介导自杀基因可有效杀死K5 6 2细胞 ,双自杀基因较单一自杀基因具有更强的抗肿瘤作用     

Induction of Apoptosis in Glioma Cells by Molecules Released from Activated Macrophages

Macrophages play an important role in the regulation of malignant tumors. Although glioma contains abundance of macrophages, their role in apoptosis of glioma is not known. We stimulated macrophages with lipopolysaccharide and culture supernatants of activated macrophages were collected to treat glioma cells. The results showed that molecules released from activated macrophages significantly increased apoptosis of glioma via Fas/FasL and caspase-3 pathways. The level of soluble Fas did not appear to be involved in the mechanism responsible for apoptosis seen in this study, as its level was barely detected in both experimental and control groups. Two cytokines, TNF and IFN, were significantly elevated in the supernatant obtained from the activated macrophages. Considering an important role of these two molecules in the induction of apoptosis mediated by the Fas/FasL system, the present data suggested that TNF and IFN were the main molecules to trigger the cascade of apoptotic reactions in glioma cells. In conclusion, the present study indicates that molecules released from the activated macrophages provide significant signals to stimulate the expression of Fas/FasL and caspase-3, which function to induce apoptosis in glioma cells.     

腺病毒介导的肝癌自杀基因疗法的实验研究

本文观察了胞嘧啶脱氨酶(CD)基因/5-氟胞嘧啶(5FC)自杀基因疗法对肝癌模型的治疗作用。以CMV启动子调控CD基因的重组腺病毒载体AdexCMV.CD在体外能有效转染人肝癌细胞株SMMC-7721和HepG2,转染后的细胞体外生长能力无明显变化,对5FC的敏感性明显增高。当以AFP上游调控序列驱动CD基因的重组腺病毒载体AdexAFP.CD分别转染SMMC-7721和HepG2时,CD基因能在HepG2中表达,使HepG2对5FC的敏感性增高,但不能使SMMC-7721的生长受到5FC的抑制。[~3H]TdR掺入法观察体外旁观者效应时发现,当细胞总数中转染细胞数超过20％时,其生长明显被5FC抑制。将AdexCMV.CD直接注射入裸鼠接种SMMC-7721细胞形成的皮下肿瘤中,并全身应用5FC后,与对照组相比,肿瘤大小在开始治疗后第8天约缩小3倍,第18天约缩小4倍,以上结果表明,腺病毒介导的CD/5FC自杀基因系统能在体内、外有效地抑制肝癌的生长。以AFP上游调控序列驱动CD基因的腺病毒载体介导的基因转染能在AFP阳性的肝癌细胞中特异表达。     

hTERT启动子调控的融合自杀基因CD:UPRT载体的构建及其应用

目的 构建hTERT启动子调控的融合自杀基因CD:UPRT表达载体,研究其对人胃癌细胞SGC7901的体外靶向性杀伤作用。方法 PCR扩增hTERT核心启动子片段,克隆入荧光素酶报告基因质粒pGL3 Basic,检测hTERT启动子在人胃癌细胞SGC7901和人成纤维细胞HLF中的转录活性。构建hTERT启动子调控的CD:UPRT基因表达载体hTERT CD:UPRT,将其和CMV启动子调控的CD:UPRT基因表达载体pcDNA3.1 CD:UPRT用脂质体转染法分别转染入SGC7901和HLF细胞,筛选稳定表达细胞系,用RT PCR和Western blot方法检测CD基因的表达,用MTT法检测5 FC对转染细胞的杀伤作用。结果 成功克隆hTERT核心启动子;荧光素酶活性检测显示,hTERT启动子在SGC7901细胞中的转录活性为阳性对照的(21.50±2.15)%,而在HLF细胞中仅有背景活性。成功构建hTERT启动子调控的CD:UPRT基因表达载体,转染pcDNA3.1 CD:UPRT的SGC7901和HLF细胞以及转染hTERT CD:UPRT的SGC7901细胞在mRNA和蛋白质水平均可检测到CD基因的表达,且对5 FC敏感;而转染hTERT CD:UPRT的HLF细胞未检测到CD基因的表达,对5 FC不敏感。结论 构建的hTERT启动子调控的融合自杀基因系统CD:UPRT/5 FC能在体外靶向性杀伤SGC7901细胞。     

Hyperfractionated and Hypofractionated Radiation Therapy for Human Malignant Glioma Xenograft in Nude Mice

Xenografts of a human malignant glioma subcutaneously transplanted into nude mice were irradiated with graded single doses (2, 5, 10 or 20 Gy) or five types of fractionation schedules in two weeks: conventional [20 Gy in 10 fractions (fr)], hyperfractionated [24 Gy in 20 fr (two fractions per day)], and hypofractionated-1, 2, 3 [20 Gy, 18 Gy, 16 Gy in 4 fr]. All of the fractionated irradiation groups showed tumor regression. The hypofractionation-1 group (20 Gy in 4 fr) demonstrated the most prominent tumor regression, while the hyperfractionation group (24 Gy in 20 fr) showed the least effect. The hypofractionation-2 group (18 Gy in 4 fr) showed similar regression to the conventional fractionation group (20 Gy in 10 fr). Histologically, tumors in the control groups consisted of a homogenous population of small anaplastic cells, and only a small number of tumor cells were glial fibrillary acidic protein (GFAP)-positive. Following irradiation, the population of small anaplastic cells decreased and the percentage of GFAP-positive cells increased. Cellular pleomorphism became much more prominent after irradiation in all of the fractionated irradiation groups as compared with the graded single dose irradiation groups. In this study, hyperfractionation was not effective against human glioma xenografts compared with conventional fractionation and hypofractionation. This indicates that care is needed in applying hyperfractionation regimens to human malignant gliomas.     

绿茶儿茶素对于人肝癌细胞的凋亡诱导作用

目的：探讨绿茶儿茶素（GTC）对BEL－7402人肝癌细胞的凋亡诱导作用。方法：应用透射电镜、流式细胞术、TUNEL和免疫组化等方法检测受100或200μg／ml GTC作用4天后的BEL－7402细胞的凋亡情况及其PCNA、bcl－2、c－myc、p53蛋白表达情况。结果：受GTC作用4天后的BEL－7402细胞的凋亡显著性增加,其PCNA表达显著性下降,而与凋亡相关的癌基因bcl－2、c－myc、p53蛋白表达未见明显改变。结论：GTC对于BEL－7402细胞的生长抑制作用可能与诱导细胞凋亡有关,也可能与抑制癌细胞DNA合成有关。本研究未发现GTC诱导BEL－7402细胞凋亡与bcl－2、c－myc、p53蛋白表达的相关性。     

Liposome-mediated Induction of Apoptosis of Human Hepatoma Cells by C-Myc Antisense Phosphorothioate Oligodeoxynucleotide and 5-Fluorouracil

Background: The aim of this study was to investigate the effect of a c-myc antisense oligodeoxynucleotideand 5-fluorouracil on the expression of c-myc, invasion and proliferation of HEPG-2 liver cancer cells. Materialsand Methods: HEPG-2 cells were treated with lipiosome-mediated c-myc ADSON and 5-fluorouracil. Theproliferation inhibition rate and invasion were measured by MTT and invasion assay, respectively. Cell apoptosiswas detected by flow cytometry and expression of c-myc by RT-PCR and immunohistochemistry. Results: Theproliferation inhibition rate was significantly higher in the antisense oligodeoxynucleotide added-5-fluorouracilgroup than single antisense oligodeoxynucleotide or 5-fluorouracil group (p<0.05). G0/G1 cells in the antisenseoligodeoxynucleotide group and S cells in the 5-fluorouracil groups were significantly increased than that in thecontrol group, respectively (P<0.01). The amplification strips of PCR products in 5-FU, ASODN and combinationgroups were significantly weaker than that in the control group (P<0.01). The percentage of c-myc-proteinpositivecells were significantly lower in antisense oligodeoxynucleotide, 5-fluorouracil and combination groupsthan that in the control group (P<0.01). Conclusions: A liposome-mediated c-myc antisense oligodeoxynucleotideand 5-fluorouracil can inhibit the proliferation and invasion of liver cancer cells by reducing the expression ofc-myc. A c-myc antisense oligodeoxynucleotide can increase the sensitivity of liver cancer cells to 5-fluorouraciland decrease the dosage of the agent necessary for efficacy, providing an experimental basis for the clinicaltherapy of liver cancer.     

In vitro Growth Suppression of Human Glioma Cells by a 16-mer Oligopeptide: A Potential New Treatment Modality for Malignant Glioma

Journal of Neuro-Oncology -     

Efficient Retrovirus-mediated Cytokine-gene Transduction of Primary-cultured Human Glioma Cells for Tumor Vaccination Therapy

In order to realize a novel vaccination treatment for malignant gliomas using tumor cells genetically modified to express certain cytokines, it is essential to achieve an efficient gene transduction into primary cultured cells. We investigated the feasibility of preparing a glioma vaccine through retro-virus-mediated gene transduction. Glioma cells were cultured primarily from surgically resected tumor tissues of six patients. We obtained more than 1000-fold proliferation of cultures within eight weeks in all six cases. In vitro infection with a recombinant retrovirns GKlacZ carrying an Escherichia coli β -galsictosidase marker gene resulted in over 65% gene transfer to the primary cultured glioma cells. Further enrichment (∼90%) of transduced cells was possible by employing repeated infections or using vectors with neo marker gene. Two cytokine genes, granulocyte-macrophage colony-stimulating factor and interleukin-4, were introduced into glioma cells by sequential transduction with two single-expression GK vectors. The transduced glioma cells produced high levels of both cytokines. We also evaluated simultaneous introduction of two genes with double-expression GK vectors containing internal ribosomal entry site (IRES) or internal promoter (PGK). Although the cells transduced with double-expression vectors secreted both cytokines, the level of the gene product following IRES or PGK was 10 times lower than that of the upstream gene product. The transduced cells retained cytokine secretion in vitro for 14 days after 100 Gy irradiation. Our data indicate the feasibility of retrovirus-mediated preparation of gene-modified tumor vaccines from clinical glioma materials, which could be useful for potentiating antitumor immunity in glioma patients.     

Induction of Apoptosis in Human Pancreatic Carcinoma Cells by a Synthetic Bleomycin-like Ligand

Histidine-pyridine-histidine-3 (HPH-3) is an oxygen-activating ligand based on the structure of bleomycin. HPH-3 induced the death of human pancreatic adenocarcinoma AsPC-1 cells in 24 h, causing apoptotic morphology and internucleosomal degradation of DNA. HPH-3-induced cell death was not inhibited by antioxidants such as reduced glutathione and N -acetylcysteine, whereas hydrogen peroxide-induced cell death was inhibited by them, indicating that hydrogen peroxide is not involved in the induction of apoptosis by HPH-3. Induction of apoptosis by HPH-3 was inhibited by zinc and copper ions, indicating that chelation with ferrous ion is responsible for induction of apoptosis, as is the case in chelation by bleomycin to cleave DNA. Bleomycin A₂ and its fragment having no DNA-binding region, glycopeptide-3, did not induce apoptosis in AsPC-1 cells. Bleomycin A₂ induced G2/M block in flow-cytometric analysis, but HPH-3 did not and instead induced an apoptotic pre-G1 peak. Thus, HPH-3 induced apoptosis in human pancreatic carcinoma cells, which is a unique characteristic among bleomycin-related compounds.     

重组腺病毒介导KDRP-CD/TK基因对大肠癌细胞增殖与凋亡的影响

目的探讨腺病毒介导KDR启动子驱动的融合基因体系对人大肠癌细胞LOVO的增殖活性及凋亡的影响。方法以重组腺病毒AdEasy-KDR-CDglyTK体外感染表达KDR的LOVO细胞株和对照组不表达KDR的LS174T细胞株,观察该体系对LOVO细胞的杀伤效应;并以流式细胞仪检测细胞周期的变化,电镜观察细胞的病变。结果重组腺病毒对LOVO细胞及LS174T细胞的感染率相似,其感染率随腺病毒滴度的递增而增加,当MOI为200时,所有细胞株均达约100％感染。以MOI为100的重组体分别感染各细胞株表现出对前药的不同敏感性：表达KDR的LOVO细胞对前药的具有较高的敏感性,与前者相比,不表达KDR的LS174T细胞对前药不敏感（P均〈0．001）。融合基因的疗效优于任一单自杀基因（P均〈0．001）。将感染腺病毒的细胞与未感染细胞以不同混合培养,观察到该体系明显的旁观者效应。流式细胞术检测表明该体系抑制LOVO细胞DNA的合成,表现为S期阻止。同时,电镜下可见LOVO有凋亡和坏死改变。结论KDR基因启动子可调控融合基因体系选择性杀伤人大肠癌LOVO细胞,其机制与细胞周期阻滞、凋亡及坏死有关。     

5-氟尿嘧啶诱导人卵巢癌细胞凋亡的实验研究

 用不同剂量的5-氟尿嘧啶（5—Fu）处理人卵巢癌AO10／17细胞株，观察药物作用后细胞形态学，DNA片段化和细胞内钙离子浓度的变化。结果表明受药物影响的人卵巢癌细胞内ca⁺⁺升高，出现了细胞凋亡的形态改变。最后探讨了5—Fu抑制卵巢肿瘤细胞的作用机理。     

依托泊甙（VP—16）诱导人肺腺癌细胞凋亡

目的探讨ＶＰ－１６对肺腺癌细胞凋亡的诱导作用。方法采用光镜、电镜、流式细胞仪、凝胶电泳和ＴｄＴ原位末端标记法检测ＶＰ－１６诱导人肺腺癌ＡＧＺＹ－８３－α细胞凋亡规律,并观察放线菌酮对凋亡的抑制程度。结果ＶＰ－１６在显著抑制细胞增殖的同时,能够诱导细胞凋亡,将细胞阻滞于Ｇ２期。放线菌酮对凋亡有显著的抑制作用。结论ＶＰ－１６诱导细胞凋亡,是其杀死肿瘤细胞的重要机制之一。该凋亡具有明显的时间和剂量依赖性。     

Protein Kinase C Inhibition by UCN-01 Induces Apoptosis in Human Glioma Cells in a Time-dependent Fashion

Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly. we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3–6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2–4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.     

Inhibition of Cellular Proliferation and Induction of Apoptosis by Curcumin in Human Malignant Astrocytoma Cell Lines

Summary Nuclear factor (NF)-κB is known to control cellular proliferation and apoptosis. In malignant astrocytoma cells, it was reported that NF-κB was activated aberrantly and promoted their proliferation. Thus, inhibition of NF-κB activity is considered to be a promising therapeutic strategy for malignant astrocytoma. Recently, curcumin, the major constituent of turmeric, was reported to inhibit NF-κB activity. In this study, we investigated inhibitory effects of curcumin on NF-κB activity and cellular proliferation, and induction of apoptosis by curcumin in human malignant astrocytoma cell lines. Alteration of NF-κB activity in NP-2 human malignant astrocytoma cell line after treatment with curcumin was examined using electrophoretic mobility shift assay. Alterations of DNA synthesis and cellular growth in five human malignant astrocytoma cell lines after treatment with curcumin were examined using [³H]thymidine incorporation assay and the trypan blue dye exclusion method, respectively. Induction of apoptosis by curcumin in NP-2 and NP-3 human malignant astrocytoma cell lines was examined by DNA-fragmentation analysis and morphological observation. We found that the NF-κB activity in NP-2 was significantly reduced by curcumin. The DNA synthesis and the cellular growth were inhibited by curcumin in dose-dependent manner in all the five malignant astrocytoma cell lines. Nuclear condensation and fragmentation, and DNA fragmentation were observed in both NP-2 and NP-3 after the treatment with curcumin. These results indicate that curcumin inhibits the cellular proliferation and induces apoptosis in human malignant astrocytoma cell lines. These results are considered to be resulted from the inhibition of NF-κB activity by curcumin.