腺病毒载体AdING4 对MCF-7乳腺癌细胞的增殖抑制及化疗增敏作用

目的:研究腺病毒载体AdING4对人MCF-7乳腺癌细胞的生长抑制及化疗增敏作用。方法:将搭载有ING-4基因的重组腺病毒载体AdING4感染人MCF-7乳腺癌细胞,用荧光显微镜观察感染后的MCF-7细胞形态学变化;RT-PCR和Western-Blot法检测ING-4基因在MCF-7细胞中的转录和表达;RT-PCR法检测凋亡相关基因在MCF-7细胞中的表达;CCK法测定Ad-ING4感染MCF-7乳腺癌细胞后所发挥的细胞增殖抑制作用。流式细胞技术检测ING-4对MCF-7乳腺癌细胞的促凋亡作用。CCK-8法分别测定病毒感染前后的MCF-7乳腺癌细胞的药物半数抑制浓度IC50,并观察Ad-ING4与化疗药物合用后对MCF-7细胞增殖抑制和化疗增敏现象。结果:MCF-7细胞在转染ING-4基因后,明显出现变圆、脱落、皱缩、聚集等现象;外源性ING-4基因在MCF-7细胞中获得成功表达;外源性ING-4基因作用下MCF-7细胞的增殖受到了明显抑制,凋亡率有所升高,凋亡相关基因Bax的表达水平明显上调,Bcl-2、Survivin的表达水平明显下调。ING-4基因感染MCF-7细胞后,使MCF-7细胞对相关化疗药物的敏感度更高;ING-4基因与化疗药物合用后对MCF-7细胞的增殖抑制作用,较之单用化疗药物更为明显。结论:MCF-7细胞在转染ING4基因后其增殖受到了明显抑制并更易凋亡,该现象可能是通过改变Bax,Bcl-2及Survivin表达水平来实现的,且对化疗药物的敏感性更高。     

Reduction of thymocyte numbers in transgenic mice expressing viral FLICE-inhibitory protein in a Fas-independent manner

A viral FLIP (FLICE/caspase-8-Inhibitory Protein), equine herpesvirus type 2 E8 protein, has been shown to inhibit Death receptor-induced apoptosis by suppressing the activation of FLICE/caspase-8. We generated transgenic mice specifically expressing E8 in thymocytes under the control of lck-proximal promoter. Although E8-expressing thymocytes were resistant to Fas-mediated apoptosis, the total number of thymocytes in 4-8-week-old E8 transgenic mice was more than 3-fold less than that in control littermates. This reduction was also observed in E8 transgenic mice with a Fas-/- background suggesting the reduction to be independent of Fas. The thymocytes of the transgenic mice, however, could similarly respond to CD3-mediated stimulation, indicating that the reduction of thymocyte numbers might be independent of T cell receptor complex-mediated stimulation. Thus, the Death receptor-mediated signaling pathway is too complex to be regarded as only an executor for apoptosis.     

Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage

A number of clinically useful anticancer drugs, including etoposide (VP-16), target DNA topoisomerase (topo) II. These drugs, referred to as topo II poisons, stabilize cleavable complexes, thereby generating DNA double-strand breaks. Bis-2,6-dioxopiperazines such as ICRF-193 also inhibit topo II by inducing a distinct type of DNA damage, termed topo II clamps, which has been believed to be devoid of double-strand breaks. Despite the biological and clinical importance, the molecular mechanisms for the repair of topo II-mediated DNA damage remain largely unknown. Here, we perform genetic analyses using the chicken DT40 cell line to investigate how DNA lesions caused by topo II inhibitors are repaired. Notably, we show that LIG4-/- and KU70-/- cells, which are defective in nonhomologous DNA end-joining (NHEJ), are extremely sensitive to both VP-16 and ICRF-193. In contrast, RAD54-/- cells (defective in homologous recombination) are much less hypersensitive to VP-16 than the NHEJ mutants and, more importantly, are not hypersensitive to ICRF-193. Our results provide the first evidence that NHEJ is the predominant pathway for the repair of topo II-mediated DNA damage; that is, cleavable complexes and topo II clamps. The outstandingly increased cytotoxicity of topo II inhibitors in the absence of NHEJ suggests that simultaneous inhibition of topo II and NHEJ would provide a powerful protocol in cancer chemotherapy involving topo II inhibitors.     

The glucocorticoid receptor is increased in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, and its nuclear translocation counteracts c-myc expression

We have previously demonstrated that spontaneous DNA synthesis in immature thymocytes of Atm-/- mice is elevated, and that treatment with the glucocorticoid dexamethasone (Dex) attenuates this increased DNA synthesis and prevents the development of thymic lymphomas. Deregulation of c-myc may drive the uncontrolled proliferation of Atm-/- thymocytes, since upregulation of c-myc parallels the elevated DNA synthesis in the cells. In this study, we show that the glucocorticoid receptor (GR) is expressed at high levels in Atm-/- thymocytes and in Atm-/- thymic lymphoma cells, although serum glucocorticoid (GC) levels in Atm-/- mice are similar to those in Atm+/+ mice. In cultured Atm-/- thymic lymphoma cells treated with Dex, GR nuclear translocation occurs, resulting in suppression of DNA synthesis and c-myc expression at both the mRNA and protein levels. Interestingly, the GR antagonist RU486 also causes GR nuclear translocation, but does not affect DNA synthesis and c-myc expression in Atm-/- thymic lymphoma cells. As expected, RU486 reverses the suppressive effects of Dex on DNA synthesis and c-myc expression. Administration of Dex to Atm-/- mice decreases the elevated c-Myc protein levels in their thymocytes. These findings suggest that GC/GR signaling plays an important role in regulating c-myc expression in Atm-/- thymocytes and thymic lymphoma cells.     

Increased dexamethasone-induced apoptosis of thymocytes from mice exposed to long-term extremely low frequency magnetic fields

To address the effect of extremely low frequency electromagnetic fields on programmed cell death we assessed both the spontaneous and dexamethasone (Dex)-induced apoptosis of thymocytes and spleen cells from mice submitted to a long-term continuous exposure of a 0.4–1.0 μT 60 Hz magnetic field or an 8–20 μT direct current (DC) magnetic field. Dex-induced apoptosis but not spontaneous apoptosis was substantially increased in thymocytes from 0.4 to 1.0 μT 60 Hz field-exposed animals. Spontaneous apoptosis and Dex-induced apoptosis of spleen cells were not affected by the 0.4–1.0 μT 60 Hz field exposure. In addition, spontaneous apoptosis and Dex-induced apoptosis of thymocytes and spleen cells from mice exposed to an 8–20 μT DC field were similar to the controls. These findings represent the first demonstration that thymocytes from mice exposed to a long-term 0.4–1.0 μT 60 Hz field may show abnormal response to Dex apoptotic stimuli. Bioelectromagnetics 19:131–135, 1998. © 1998 Wiley-Liss, Inc.     

BCL-2 protects peroxynitrite-treated thymocytes from poly(ADP-ribose) synthase (PARS)-independent apoptotic but not from PARS-mediated necrotic cell death

In thymocytes, peroxynitrite induces poly(ADP-ribose) synthetase (PARS) activation, which results in necrotic cell death. In the absence of PARS, however, peroxynitrite-treated thymocytes die by apoptosis. Because Bcl-2 has been reported to inhibit not only apoptotic but also some forms of necrotic cell death, here we have investigated how Bcl-2 regulates the peroxynitrite-induced apoptotic and necrotic cell death. We have found that Bcl-2 did not provide protection against peroxynitrite-induced necrotic death, as characterized by propidium iodide uptake, mitochondrial membrane potential decrease, secondary superoxide production, and cardiolipin loss. In the presence of a PARS inhibitor, peroxynitrite-treated thymocytes from Bcl-2 transgenic mice showed no caspase activation or DNA fragmentation and displayed smaller mitochondrial membrane potential decrease. These data show that Bcl-2 protects thymocytes from peroxynitrite-induced apoptosis at a step proximal to mitochondrial alterations but fails to prevent PARS-mediated necrotic cell death. Activation of tissue transglutaminase (tTG) occurs in various forms of apoptosis. Peroxynitrite did not induce transglutaminase activity in thymocytes and did not have a direct inhibitory effect on the purified tTG. Basal tTG was not different in Bcl-2 transgenic and wild type cells.     

Cytotoxic and apoptosis-inducing properties of auriculoside A in tumor cells

The C21-steroidal glycoside auriculoside A (1), recently isolated from the roots of Cynanchum auriculatum, was found to inhibit the growth of several human tumor cell lines and to induce apoptosis in human breast cancer (MCF-7) cells. Compound 1 was evaluated for its in vitro cytotoxicity against MCF-7, HO-8910, and Bel-7402 cells, and for its in vivo antitumor effects on implanted sarcoma-180 (S180) tumors in mice. It showed significant, concentration-dependent inhibition of the cancer cells, both in vitro and in vivo. MCF-7 Cells exposed to 1 displayed typical morphological apoptosis characteristics such as cytoplasm contraction and nuclear-chromatin condensation. Flow-cytometric analysis showed that the MCF-7 cell cycle was arrested at the G0/G1 phase. When treated with 40 microg/ml of 1 for 24, 48, and 72 h, respectively, the apoptotic rates of the cells were ca. 5, 8, and 18.5%, respectively.     

Beta-catenin expression results in p53-independent DNA damage and oncogene-induced senescence in prelymphomagenic thymocytes in vivo

The expression of β-catenin, a potent oncogene, is causally linked to tumorigenesis. Therefore, it was surprising that the transgenic expression of oncogenic β-catenin in thymocytes resulted in thymic involution instead of lymphomagenesis. In this report, we demonstrate that this is because the expression of oncogenic β-catenin induces DNA damage, growth arrest, oncogene-induced senescence (OIS), and apoptosis of immature thymocytes. In p53-deficient mice, the expression of oncogenic β-catenin still results in DNA damage and OIS, but the thymocytes survive and eventually progress to thymic lymphoma. β-Catenin-induced thymic lymphomas are distinct from lymphomas that arise in p53^−/− mice. They are CD4⁻ CD8⁻, while p53-dependent lymphomas are largely CD4⁺ CD8⁺, and they develop at an earlier age and in the absence of c-Myc expression or Notch1 signaling. Thus, we report that oncogenic β-catenin-induced, p53-independent growth arrest and OIS and p53-dependent apoptosis protect developing thymocytes from transformation by oncogenic β-catenin.     

Thymic nurse cell multicellular complexes in HY-TCR transgenic mice demonstrate their association with MHC restriction

This study examines thymic nurse cell (TNC) function during T-cell development. It has been suggested that TNCs function in the removal of nonfunctional and/or apoptotic thymocytes and do not participate in major histocompatibility complex restriction. We analyzed TNCs isolated from both normal C57BL/6 mice and C57BL/6 TgN (TCRHY) mice (HY-TCR transgenic mice). Using confocal microscopic analyses of TNCs isolated from C57BL/6 animals, we showed that 75%-78% of the enclosed thymocyte subset was viable, and 87%-90% of these cells expressed both CD4 and CD8. CD4 and CD8 also were expressed on TNC thymocytes isolated from both male and female HY-TCR transgenic mice. The transgenic female thymus was shown to have 17 times more TNCs per milligram of thymus than the transgenic male thymus. TNCs from HY-TCR transgenic females were 8-10 microm larger than transgenic male TNCs, and the female TNCs contained five times more thymocytes within intracytoplasmic vacuoles, with less than 4% apoptosis. However, more than 42% of the thymocytes within transgenic male TNCs were apoptotic. The large number and size of TNCs containing viable thymocytes in the female transgenic thymus suggest that TNC function is not limited to the removal of apoptotic thymocytes. We believe that the selective uptake of viable double-positive thymocytes by TNCs in C57BL/6 and HY-TCR transgenic female mice provides evidence that this interaction occurs during the process of major histocompatibility complex restriction.     

肝细胞生长因子基因转染对人淋巴瘤细胞凋亡的影响

探讨肝细胞生长因子（HGF）基因转染人淋巴瘤细胞系Raji细胞后,拮抗足叶乙甙（VP－16）诱导细胞凋亡的研究。将三种细胞：未转染Raji细胞、空载体pVITR02－mcs转染细胞和HGF基因转染细胞,分成正常对照组和经vP－16处理的药物组。采用Westernblot法验证HGF蛋白的表达：CCK－8法检测诱导Raji细胞凋亡的药物浓度;通过透射电镜、流式细胞术、吖啶橙（A0）染色、苏木精咿红（HE）染色等方法观察Raji细胞的凋亡情况,并进行相关分析。结果显示：Westernblot法验证了HGF蛋白质的表达;CCK．8法显示100μg／mL足叶乙甙可明显抑制Raii细胞增殖;透射电镜下可发现典型的凋亡细胞;流式检测结果表明：给药组与正常组相比,三组细胞的凋亡率明显升高（P〈0．01）,提示VP－16具有诱导细胞凋亡的作用：但给药组间：HGF基因转染组凋亡率明显低于未转染组（P＜0．05）和空载体pVITR02．mcs转染组（P〈0．05）,提示嬲F基因转染可明显抑制VP-16诱导的Raji细胞的凋亡,AO染色和HE染色结果也同样提示HGF具有拮抗VP－16诱导的细胞凋亡效应。     

Staurosporine-induced death of MCF-7 human breast cancer cells: a distinction between caspase-3-dependent steps of apoptosis and the critical lethal lesions

To test the role of caspase 3 in apoptosis and in overall cell lethality caused by the protein kinase inhibitor staurosporine, we compared the responses of MCF-7c3 cells that express a stably transfected CASP-3 gene to parental MCF-7:WS8 cells transfected with vector alone and lacking procaspase-3 (MCF-7v). Cells were exposed to increasing doses (0.15-1 microM) of staurosporine for periods up to 19 h. Apoptosis was efficiently induced in MCF-7c3 cells, as demonstrated by cytochrome c release, processing of procaspase-3, procaspase-8, and Bid, increase in caspase-3-like DEVDase activity, cleavage of the enzyme poly(ADP-ribose) polymerase, DNA fragmentation, changes in nuclear morphology, and TUNEL assay and flow cytometry. For all of these measures except cytochrome c release, little or no activity was detected in MCF-7v cells, confirming that caspase-3 is essential for efficient induction of apoptosis by staurosporine, but not for mitochondrial steps that occur earlier in the pathway. MCF-7c3 cells were more sensitive to staurosporine than MCF-7v cells when assayed for loss of viability by reduction of a tetrazolium dye. However, the two cell lines were equally sensitive to killing by staurosporine when evaluated by a clonogenic assay. A similar distinction between apoptosis and loss of clonogenicity was observed for the cancer chemotherapeutic agent VP-16. These results support our previous conclusions with photodynamic therapy: (a) assessing overall reproductive death of cancer cells requires a proliferation-based assay, such as clonogenicity; and (b) the critical staurosporine-induced lethal event is independent of those mediated by caspase-3.     

NK4拮抗HGF促进肿瘤细胞凋亡的生物学效应

观察NK4通过拮抗肝细胞生长因子（HGF）诱导不同肿瘤细胞凋亡,研究其生物学作用及分子机制.以足叶乙甙（VP-16）诱导凋亡,分别或经HGF蛋白、NK4蛋白处理5种肿瘤细胞（B细胞淋巴瘤细胞系Raji、人急性粒细胞白血病细胞系HL-60、宫颈癌细胞系HeLa、前列腺癌细胞系PC-3、非小细胞肺癌细胞系A549）,采用流式细胞术（FCM）、吖啶橙 (AO) 染色法、苏木素 伊红（HE）染色法定量观察5种肿瘤细胞的凋亡情况,并进行相关分析. FCM发现,经VP-16处理5种肿瘤细胞凋亡率均显著高于对照组(P<0.001),而HGF+VP-16组凋亡率明显下降(P<0.01),HGF+NK4+VP-16组细胞凋亡率均明显高于HGF+VP-16组(P<0.05). AO染色和HE染色结果也证实,5种肿瘤细胞经VP-16处理后凋亡率均显著增高 (P<0.001,P<0.001),而HGF+VP-16组细胞凋亡率均明显低于VP-16组(P<0.001,P<0.01), HGF+NK4+VP-16组细胞凋亡率均明显高于HGF+VP-16组(P<0.001,P<0.05).此外,发现NK4+VP-16组、HGF+ NK4+VP-16组、VP-16组等3组间凋亡率无统计学差异(P>0.05). 以上结果提示,HGF蛋白可抵抗凋亡诱导剂VP-16的作用, 明显降低细胞凋亡;NK4通过竞争性抑制HGF从而促进肿瘤细胞的凋亡,具有潜在的肿瘤治疗价值.     

格列卫与多西紫杉醇联合对人乳腺癌细胞株MCF-7及其裸鼠移植瘤的作用

目的：探讨分子靶向药物格列卫与多西紫杉醇联合对人乳腺癌细胞株MCF-7的凋亡及其裸鼠皮下移植瘤生长的影响。方法：采用流式细胞检测仪检测MCF-7细胞在格列卫与多西紫杉醇单独处理及共同处理条件下的凋亡率;建立人乳腺癌细胞株MCF-7裸鼠皮下移植瘤模型,观察格列卫与多西紫杉醇单独治疗组及联合治疗组移植瘤的生长,计算抑瘤率。结果：MCF-7细胞在格列卫与多西紫杉醇共同处理条件下的凋亡率（50.86%）远高于多西紫杉醇单独处理（22.06%）及同剂量格列卫组单独处理（8.13%）条件下的凋亡率;高剂量多西紫杉醇与格列卫联合组肿瘤质量与单独多西紫杉醇组比较,差异虽然没有显著性,但联合组抑瘤率高达99.55%,高于多西紫杉醇组（97.43%）,q值为1.014;中剂量多西紫杉醇与格列卫联合组肿瘤质量与单独多西紫杉醇组比较,差异有高度显著性,联合组抑瘤率达96.53%,高于多西紫杉醇组（92.01%）,q值为1.02;低剂量多西紫杉醇与格列卫联合组肿瘤质量与单独多西紫杉醇组比较,差异有高度显著性,联合组抑瘤率达68.20%,高于多西紫杉醇组（58.40%）,q值为1.004。结论：格列卫与凋亡诱导剂多西紫杉醇共同处理MCF—7细胞能达到协同诱导凋亡的效果;高、中、低剂量的多西紫杉醇与格列卫联合对人乳腺癌细胞株MCF-7裸鼠移植瘤增殖的抑制具有相加作用。     

Glutathione peroxidase-1 protects from CD95-induced apoptosis

Through the induction of apoptosis, CD95 plays a crucial role in the immune response and the elimination of cancer cells. Ligation of CD95 receptor activates a complex signaling network that appears to implicate the generation of reactive oxygen species (ROS). This study investigated the place of ROS production in CD95-mediated apoptosis and the role of the antioxidant enzyme glutathione peroxidase-1 (GPx1). Anti-CD95 antibodies triggered an early generation of ROS in human breast cancer T47D cells that was blocked by overexpression of GPx1 and inhibition of initiator caspase activation. Enforced expression of GPx1 also resulted in inhibition of CD95-induced effector caspase activation, DNA fragmentation, and apoptotic cell death. Resistance to CD95-mediated apoptosis was not due to an increased expression of anti-apoptotic molecules and could be reversed by glutathione-depleting agents. In addition, whereas the anti-apoptotic protein Bcl-xL prevented CD95-induced apoptosis in MCF-7 cells, it did not inhibit the early ROS production. Moreover, Bcl-xL but not GPx1 overexpression could suppress the staurosporine-induced late generation of ROS and subsequent cell death. Altogether, these findings suggest that GPx1 functions upstream of the mitochondrial events to inhibit the early ROS production and apoptosis induced by CD95 ligation. Finally, transgenic mice overexpressing GPx1 were partially protected from the lethal effect of anti-CD95, underlying the importance of peroxide formation (and GPx1) in CD95-triggered apoptosis.     

Impairment of T-cell functions with the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin

It has been observed that the progressive ascitic growth of a transplantable T-cell lymphoma of spontaneous origin, designated Dalton's lymphoma (DL), in a murine host induces inhibition of various immune responses and is associated with an involution of thymus accompanied by a massive depletion of the cortical region and alteration in the distribution of thymocytes caused by tumour serum-dependent induction of apoptosis with a decrease of CD4(+)CD8(+), CD4(+)CD8(-) and CD4(-)CD8(+) thymocytes. Here, we report that thymocytes of DL-bearing mice are defective in their proliferative ability and in their response to non-specific mitogenic stimulus in vitro. Also, antigen-specific T-cell proliferative ability representing the fundamental T(H) function declines under DL-bearing conditions and upon treatment with serum of DL-bearing mice. Moreover, a significant inhibition of T-cell cytolytic activity with a decreased ability to produce interferon gamma is shown by the T cells of DL-bearing mice and by the T cells treated with DL-ascitic fluid, DL-conditioned medium or serum of DL-bearing mice. Further, addition of interleukin-2 and anti-interleukin-10 to the cultures of thymocytes treated with serum of DL-bearing mice is found to inhibit the induction of apoptosis in thymocytes, a phenomenon associated with the progression of DL growth. Analysis of the results indicates an immune deviation with the predominance of a T(H2)-type response with the progression of tumour. We further discuss the possible mechanisms that may explain the observed tumour-induced diminution of T-cell immunity.