肺癌细胞株中GEM和CDDP药物敏感性相关基因的研究

背景与目的 筛选小细胞肺癌(small cell lung cancer,SCLC)和非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞株中与健择(gemcitabine hydrochloride,GEM)和顺铂(cisplatin,CDDP)药物敏感性相关的基因,有助于进一步阐明抗癌药物作用机制,为克服抗癌药物的耐药性、研制开发新的抗癌药物提供新线索,同时也将为临床的个体化治疗提供理论依据.方法 采用MTT比色分析法测定4株SCLC细胞株和6株NSCLC细胞株对CDDP和GEM的药物敏感性,同时应用cDNA macroarray技术检测10株肺癌细胞株中1 291个药物敏感性相关基因的表达状态,分析二者之间的相关性.结果 与GEM药物敏感性呈明显正相关(r≥0.632,P<0.05)的基因有6个;与CDDP药物敏感性关联的基因共有45个;与GEM、CDDP敏感性关联(r≥±0.4)的基因有41个;肺癌细胞系中与GEM和CDDP两类药物敏感性呈明显相关的基因足Metallothinein(信号转导分子)、Cathepsin B(组织蛋白酶B)和TIMP1(生长因子);肺癌细胞系中与GEM、CDDP药物敏感性相关联的基因主要分布于Metallothinein、CathepsinB、牛长因子TIMP1等类别.结论 SCLC和NSCLC细胞株中GEM、CDDP存在药物敏感性相关基因,其中Metallothinein、Cathepsin B和TIMP1基因与GEM药物敏感性呈正相关,与CDDP药物敏感性呈负相关.     

人肺癌细胞中去甲长春新碱和泰索帝药物敏感性基因的比较分析

目的筛选人肺癌细胞株中与去甲长春新碱（NVB）、泰索帝（Doc）药物敏感性相关的基因。方法采用MTT比色分析法,测定4株小细胞肺癌（SCLC）细胞株和6株非小细胞肺癌（NSCLC）细胞株对NVB、Doc的药物敏感性;应用cDNA macroarray技术检测10株肺癌细胞株中1291个药物敏感性关联基因的表达状态,聚类分析二者间的相关性。结果（1）NVB对10株肺癌细胞株的作用效果明显优于Doc。（2）10株肺癌细胞株中,与Doc呈正相关的药物敏感性关联基因多于NVB,而与NVB呈负相关的多于Doc;而在6株NSCLC中,与Doc和NVB呈正相关、负相关一致的基因较多。（3）10株肺癌细胞株中,有51个基因与Doc、NVB药物敏感性相关联,占全部候选基因的3．95％（51／1291）。与NVB药物敏感性呈显著相关的基因27个,其中负相关24个,正相关3个;与Doc药物敏感性关联的基因24个,其中负相关13个,正相关11个。6株NSCLC株中,有67个基因与药物敏感性相关,占全部实验基因的5．19％（67／1291）。与NVB药物敏感性呈显著相关的基因29个,其中负相关25个,正相关4个;与Doc药物敏感性关联的基因38个,其中负相关34个．正相关4个。（4）Rab 1、Rab 3、Rho B、Rho C、Rac 1、Rac 2、Gho GDI B、CD44、Integrin α5、Integrin α6、Integrin β5、Vinculin等细胞骨架关联基因在SCLC、NSCLC中的表达存在差异。结论SCLC和NSCLC细胞株中,NVB、Doc药物敏感性相关联基因表达存在明显差异。     

肺癌细胞株中NVB和Doc药物敏感性关联基因的研究

目的:筛选小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)细胞株中与诺维苯(NVB)、泰索帝(Doc)药物感受性相关的基因.方法:采用MTT比色分析法测定4株SCLC细胞株和6株NSCLC细胞株对NVB、Doc的药物感受性,同时应用cDNA macroarray技术检测10株肺癌细胞株中1291个药物感受性关联基因的表达状态,分析二者之间的相关性.结果:与NVB和Doc药物敏感性关联(r≥±0.4)的基因共有56个.1)与NVB药物敏感性相关联的基因有36个,SCLC NSCLC组与NSCLC组共同表达的基因有20个,其中,SCLC NSCLC组表达的基因有27个,特异表达的有7个,NSCLC组为29个基因,9个基因特异表达.2)与Doc药物敏感性相关联的基因有50个,其中,SCLC NSCLC组与NSCLC组共同表达的基因有12个,SCLC NSCLC组表达的基因有24个,12个基因特异表达,NSCLC组为38个基因,26个基因特异表达.3)肺癌细胞株中与NVB、Doc药物感受性相关联基因的分类主要分布于:信号转导分子、代谢酶类及其抑制剂、细胞因子和转录因子等4类.结论:SCLC和NSCLC细胞株中NVB、Doc药物敏感性相关联基因存在明显差异,但分类基本相同.     

肺癌细胞株中顺铂药物敏感性的相关基因

目的 筛选小细胞肺癌(SCLC)和非小细胞肺癌(NSCLC)细胞株中与顺铂(DDP)药物敏感性相关的基因.方法 采用二苯基溴化四氮唑蓝(MTT)比色分析法,测定4株SCLC细胞株和6株NSCLC细胞株对DDP的药物敏感性;应用cDNA微阵列技术检测肺癌细胞株中1291个药物敏感性相关基因的表达状态,并分析基因表达与DDP敏感性之间的相关性.结果 Metallothionein、Cathepsin B、TIMP1、TNF-R1、TGFβ-induced 68000、Cathepsin L、Galectin-1、Annexin 11、PAI-1、IGFBP4、UPAR、Jagged、CD13、α1 A-AR、EphA2(Eek)、APC、RhoC、Fibromodulin、GATA-6、HSC 70共20个基因,在SCLC和NSCLC细胞株中均与DDP的药物敏感性呈负相关,只有Procoagula和MDM2与DDP的敏感性呈正相关.VHL、MMP-7、Elongin A、GSK-3β、SLC、Galectin-3、Integrinβ5、Moesin、IKKβ和ETV 1等10个基因,只在SCLC细胞株中与DDP的药物敏感性呈负相关,而AT2与DDP敏感性呈正相关.Clusterin、FG FR-2、Thrombospondin 1、HSP 32、Lactate dehydrogenase A、P300、Thymosinβ10、CD81、C/EBPγ和Rak等10个基因,只在NSCLC细胞株中与DDP的药物敏感性呈负相关,而只有CaMKK和TPA与DDP的敏感性呈正相关.结论 与DDP药物敏感性相关的基因共有45个,其中共同表达于SCLC和NSCLC细胞株中的基因有22个,在SCLC细胞株中特异表达的基因有11个,在NSCLC细胞株中特异表达的基因有12个.     

人大细胞肺癌高低转移株差异表达基因消减cDNA文库的构建和初步筛选

背景与目的 筛选肺癌转移相关基因有助于阐明肺癌侵袭转移的分子机理.为了筛选不同转移潜能肺癌的转移相关差异表达基因,本研究构建不同转移潜能的人大细胞肺癌细胞株NL9980和L9981间差异表达基因的消减cDNA文库,并对消减文库进行初步筛选.方法 应用抑制消减杂交技术构建人大细胞肺癌高低转移株NL9980与L9981间差异表达基因的正向消减cDNA文库和反向消减cDNA文库,利用α互补原理,经蓝白菌落初步筛选获得阳性克隆,应用斑点杂交的方法对正向和反向消减cDNA文库进行杂交筛选.结果 成功构建了人大细胞肺癌高低转移株差异表达基因的正向消减cDNA文库和反向消减cDNA文库.经蓝白菌落筛选和斑点杂交,正向消减文库获得了307个阳性克隆,反向消减文库获得了78个阳性克隆.结论 抑制消减杂交是克隆差异表达基因的有效方法,利用此方法可以成功构建人大细胞肺癌高低转移株差异表达基因消减cDNA文库.不同转移潜能人大细胞肺癌细胞株中存在可能与转移相关的差异表达基因.     

nm23-H1基因转染前后人大细胞肺癌细胞株抑制消减cDNA文库的构建

背景与目的 已有的研究表明,nm23-H1基因是一个重要的肺癌转移抑制基因,为了筛选与该基因表达相关的差异表达基因.本研究拟构建nm23-H1基因转染前后人大细胞肺癌细胞株(L9981和L9981-nm23-H1)差异表达基因的抑制消减cDNA文库,为进一步筛选、克隆与nm23-H1转移相关的基因奠定基础.方法利用抑制消减杂交(suppressive subtractive hybridization,SSH)技术构建nm23-H1基因转染前后人大细胞肺癌细胞株(L9981和L9981-nm23-H1)间差异表达基因的正向和反向消减cDNA文库,经蓝白菌落筛选克隆,并PCR反应鉴定.结果 成功构建了该两株细胞株差异表达基因的正向和反向消减cDNA文库.经蓝白菌落筛选,正向消减文库总共获得约300个白斑克隆,反向消减文库总共获得约400个白斑克隆,从正反向消减文库中各挑选96个克隆进行PCR扩增检测是否有插入片段,结果显示在挑选的正向文库中有84个克隆有插入片段,反向文库中有83个克隆有插入片段.其片段大小范围为(300-750)bp.结论 抑制消减杂交是克隆差异表达基因的有效方法,我们应用SSH法和T/A克隆技术成功建立了nm23-H1基因转染前后人大细胞肺癌细胞株(L9981和L9981-nm23-H1)差异表达基因的抑制消减cDNA文库.nm23-H1基因在肺癌细胞中的表达可能影响某些转移相关基因的差异表达.     

肺癌淋巴结转移相关基因差异表达分析

目的:应用人类14K cDNA基因表达谱芯片筛选肺癌淋巴结转移相关基因,探讨肺癌淋巴结转移发生过程中相关基因群的表达及初步功能.方法:分别抽取肺癌组织和淋巴结转移组织的总RNA,采用逆转录方法,制成cDNA链,并以2种荧光Cy5和Cy3标记做探针,与含有14 784条人类14K cDNA基因表达谱芯片进行杂交.以Agilent荧光扫描仪扫描芯片上2种荧光信号,进行计算机处理和分析,筛选出肺癌淋巴结转移组织中差异表达基因,并用RT-PCR实验对部分差异表达基因进行验证.结果:在14 784条基因中,肺癌组织和淋巴结转移组织间存在差异表达的基因共40条,有21条基因出现显著表达上调,19条基因出现显著表达下调.经RT-PCR验证,差异表达趋势与基因芯片技术检测的结果一致.结论:肺癌淋巴结转移发生过程中有多基因的参与,肺癌组织与淋巴结转移组织共同差异表达的40条基因可能与淋巴结转移的发生、发展有关.     

具有转移能力鼻咽癌细胞株候选抗药与多药耐药相关基因的筛选

目的 筛选具有转移能力的鼻咽癌细胞株中候选抗药与多药耐药相关基因.方法 利用8000点的基因芯片比较5-8F和6-10B细胞株之间的差异表达基因,并利用在线MILANO程序分析,筛选抗药与多药耐药相关基因.半定量RT-PCR验证差异表达基因.结果 分析5-8F和6-10B细胞株之间基因表达谱后,共找到283个差异表达基因,其中表达上调基因185个,下调基因98个.MILANO程序分析后,在5-8F细胞株中找到4个可能与抗药和多药耐药相关的高表达基因:UGT1A9(15.85倍)、MVP(6.77倍)、CAV1(2.49倍)和HIF1A(2.67倍).半定量RT-PCR验证4个基因筛选结果的可靠性.结论 经在线MILANO程序分析鉴定的鼻咽癌5-8F和6-10B细胞株之间的差异表达基因可能与具有转移能力鼻咽癌细胞株的抗药和多药耐药有关.     

胶质瘤耐药细胞株的建立及其继发性耐药相关基因的筛查

目的:构建胶质瘤耐替尼泊苷(teniposide,又称VM-26)细胞株,利用基因芯片筛查该细胞株继发性耐药相关基因并证实其表达。方法:选用人胶质瘤细胞系SHG44为靶细胞,从低剂量起始逐步递增用药量(每4 000个细胞VM-26剂量由0.135 ng至4.5 ng)诱导建立对VM-26耐药的细胞株,绘制亲代细胞系和耐药细胞株增殖曲线比较两者倍增时间,采用细胞毒性实验计算半数抑制浓度(IC_(50))比较两者耐药程度的差异。应用人类cDNA表达谱芯片检测耐药和敏感细胞基因表达谱的变化,筛查出与耐药有关的基因并经半定量RT-PCR验证。结果:经过72代诱导培养,建立了稳定耐药的细胞株SHG44/VM-26,耐药性是亲代细胞的52.6倍;耐药细胞倍增时间明显长于亲代细胞(25.6 vs 48.9 h);cDNA芯片筛选出11个基因表达上调,42个基因表达下调;半定量RT-PCR证实基因MDR1、NGFR、HSP22、CX IX、CDKN3和NADE的表达与基因芯片结果基本一致。结论:成功建立胶质瘤耐替尼泊苷细胞株SHG44/VM-26,该细胞株的继发性耐药与基因MDR1、NGFR、HSP22的高表达和CX IX、CDKN3和NADE的低表达相关。     

基因芯片技术筛选非小细胞肺癌A549细胞顺铂耐药相关基因的研究

目的:采用基因芯片技术筛选人非小细胞肺癌(NSCLC)耐顺铂(DDP)A549细胞与A549细胞之间的差异表达基因,为进一步研究其耐DDP相关分子机制提供资料。方法:分别抽取耐DDPA549细胞与A549细胞的总RNA,采用逆转录的方法,制成cDNA链,并以2种荧光Cy5和Cy3标记后作为探针,与含有17101条人类16KcDNA基因表达谱芯片进行杂交,扫描荧光强度,并用计算机进行分析,寻找两组差异表达基因。结果:在17101条基因中,3株A549/DDP和3株A549细胞共同差异表达基因24条,其中上调基因12条,下调基因12条。结论:A549细胞发生DDP耐药过程中有多个基因参与,两者共同差异表达的24条基因可能参与其耐DDP分子机制。     

The NCI60 human tumour cell line anticancer drug screen

The US National Cancer Institute (NCI) 60 human tumour cell line anticancer drug screen (NCI60) was developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumours in anticancer drug screening. This screening model was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumour-cell kill. Recently, its role has changed to that of a service screen supporting the cancer research community. Here I review the development, use and productivity of the screen, highlighting several outcomes that have contributed to advances in cancer chemotherapy.     

Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy

The success of some chemo- and radiotherapeutic regimens relies on the induction of immunogenic tumor cell death and on the induction of an anticancer immune response. Cells succumbing to immunogenic cell death undergo specific changes in their surface characteristics and release pro-immunogenic factors according to a defined spatiotemporal pattern. This stimulates antigen presenting cells such as dendritic cells to efficiently take up tumor antigens, process them, and cross-prime cytotoxic T lymphocytes, thus eliciting a tumor-specific cognate immune response. Such a response can also target therapy-resistant tumor (stem) cells, thereby leading, at least in some instances, to tumor eradication. In this review, we shed some light on the molecular identity of the factors that are required for cell death to be perceived as immunogenic. We discuss the intriguing observations that the most abundant endoplasmic reticulum protein, calreticulin, the most abundant intracellular metabolite, ATP, and the most abundant non-histone chromatin-binding protein, HMGB1, can determine whether cell death is immunogenic as they appear on the surface or in the microenvironment of dying cells.     

Differential effects of RhoA signaling on anticancer agent-induced cell death

Substantial evidence exists to support a role for RhoA signaling in adhesion and cytoskeletal reorganization, while relatively less is known about the participation of RhoA on cell survival. We provide evidence that RhoA functions as a differential modulator of apoptosis induced by anticancer agents. Specifically, both RhoA and caRhoA induce statistically significant resistance to statin, etoposide, 5-FU and taxol while increasing sensitivity to vincristine (all p<0.001). The IC50 values for statin, etoposide, 5-fluorouracil (5-FU) and taxol in caRhoA transfectant were 8.70+/-0.74, 4.08+/-0.12, 4.12+/-0.12 microg/ml and 3.84+/-0.16 ng/ml, respectively, whereas the respective IC50 values in the mock-transfected control were 3.40+/-0.21, 1.12+/-0.06, 1.21+/-0.06 microg/ml and 2.84+/-0.15 ng/ml. This represented a 2.6-, 3.5-, 3.2- and 1.4-fold resistance to statin, etoposide, 5-FU and taxol, respectively. In contrast, caRhoA increased sensitivity to vincristine, decreasing IC50 values from 4.61+/-0.46 to 3.73+/-0.44 ng/ml (p<0.001). Western blot analysis demonstrated that RhoA mediates induction of E2F-1, Cdk2 and PCNA, accompanying concurrent reduction in p21 and p27. However, cleavage assays of poly (ADP-ribose) polymerase, BID, caspase-8 and caspase-3 indicate that the cell growth modulation mediated by RhoA in response to these anticancer agents occurs through the inhibition of apoptosis. Taken together, these results indicate that RhoA differentially modulates cancer cell death depending on the anticancer agent.     

The cell membrane and cell signals: new targets for novel anticancer drugs.

In the concluding Discussion session, emphasis focussed on the potential for interfering selectively with cell membranes and cell signalling in tumour as against normal tissues. There could be no doubt that tremendous advances are being made in our understanding of the molecular changes associated with malignancy and that the information available for the rational design of inhibitors of particular signalling pathways is increasingly sophisticated. There was a consensus that we need more information on the qualitative and quantitative differences in the structure and function of membranes and the signalling machinery in various normal tissues as compared to their cancerous counterparts. Ideally we will develop drug against, for example, specific forms of, let us say, protein kinase C or tyrosine kinase which are found to be predominantly active in neoplastic cells. This may well prove possible, at least in some instances, in which case a safe therapeutic margin will be assured. But differences may in other situations turn out to be in the level of expression rather than purely qualitative in nature, and the scale of the disparate expression may not always be great. Even in such situations, adequate therapeutic selectivity may still be achieved. This may derive from a "damping down" of signalling in the hyperactive tumour. Although there are legitimate concerns regarding the possible toxic effects of administering signal-wrecking molecules in man, we should not be pessimistic as there are clear precedents elsewhere in medicine for drugs acting on membrane signals proving to be safe and effective against expectation informed by hindsight. There may also be concerns about new forms of drug resistance. But this will be so for any new agent or novel target. And with mechanism of action clearly to the fore we should be able to predict resistance pathways in advance and devise appropriate circumvention strategies or targeted second line therapies. There was a palpable buzz at the meeting that this is a valid, different and above all rational approach. Not only that, but the new therapeutic molecules which we discover will themselves prove to be valuable tools with which to probe further into the mechanisms of malignancy and signal transduction. We had expected to see a bewildering amount of new information from the basic sciences of molecularbiology and cell physiology, and we got it. But it was also impressive to witness the number of new compounds coming through which look like real drugs or at least exciting lead compounds. The membrane-active ether lipids are in clinical trial. Bryostatin 1 will shortly join them.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxicity of the anticancer agents cisplatin and taxol during cell proliferation and the cell cycle

The overt effects of the anti-cancer drugs cisplatin (cis-DDP) and taxol appear to be DNA modification and microtubule stabilization respectively, yet the mechanisms by which these drugs elicit tumor cell death are not well understood. In this report cell sensitivities to cis-DDP and taxol were accurately determined as a function of cell proliferation and cell cycle stage. Quiescent fibroblasts restimulated to synchronously enter the cell cycle become maximally sensitive to cis-DDP immediately preceding DNA synthesis, and resistance increases with onset of DNA synthesis. Mid-log proliferating cells were separated into progressive stages of the cell cycle by centrifugal elutriation or by double thymidine (dThd) block. Cells staged by either method are maximally sensitive to cis-DDP in G₁, just prior to the onset of DNA synthesis and minimally sensitive in peak DNA synthesis, with entry into S phase resulting in a 2-fold decrease in sensitivity. Cells that remained blocked at the G₁/S phase boundary during cis-DDP treatment remain maximally sensitive after release. Sensitivity to taxol increases at 2 points: transiently during transition of normal cells from quiescence to proliferation and steadily as proliferating cells progress from early G₁ to late G₂. This 3-fold increase in taxol sensitivity through the cell cycle is rapidly reversed upon cell division. Synchronous cells treated with either drug at points of maximum sensitivity initiate apoptotic DNA fragmentation 12-14 hr post-exposure to drug. © 1994 Wiley-Liss, Inc.     

Interaction between novel anticancer agents and radiation in non-small cell lung cancer cell lines.

Integration of chemotherapy and radiation is the standard practice in the management of locally advanced inoperable NSCLC. To assess the biological interaction between third generation chemotherapeutic agents and radiation in non-small cell lung cancer (NSCLC) in vitro, we tested a number of different drugs (paclitaxel, docetaxel, gemcitabine, topotecan, SN-38 and cisplatin) combined with radiation, in lung cancer cell lines. Cellular chemosensitivity was determined, using the semi-automated colorimetric MTT assay, after 48, 72 and 96 h of exposure to increasing drug concentrations, (0.001-100 microM) and radiation doses (100-400 cGy). Cell lines used were the adenocarcinoma (ADK), A-549, and the squamous-cell carcinoma (SCC), LX-1. Cells were pre-treated with anticancer agents at 24, 12 and 0 h before irradiation. Cytofluorimetric cell cycle analysis was performed. A significant S-phase block or a G(2)/M block was seen with gemcitabine and topotecan or paclitaxel pre-treatment, respectively. Apoptosis was seen only after paclitaxel exposure in the A-549 cell line. Despite a similar pattern of cell-kinetic changes induced by chemotherapy pre-treatment in all cell lines, the adenocarcinoma A-549 cell line was not radiosensitized by any of the anticancer agents tested, whereas synergism was observed in the LX-1 squamous carcinoma cell line, when exposed to gemcitabine, SN-38, topotecan and cisplatin. Paclitaxel, despite a favourable cell cycle effect, was not found to be synergistic with radiotherapy in our experimental model. In conclusion, the observed synergism appears to be dose- and timing-independent and seems to be related to the histological subtype being present in SCC only. Favourable perturbation of the cell cycle is evident with all the new agents tested in both cell types, but was not sufficient to produce synergism with radiation.     

Design of new anticancer therapies targeting cell cycle checkpoint pathways.

The mammalian cell cycle is exquisitely controlled by the cyclin-dependent kinases, which regulate cell cycle progression. Cell cycle transitions are, in turn, controlled by checkpoints that monitor the integrity and replication status of the genetic material before cells commit to either replicate or segregate their DNA. On activation, checkpoints interface with cyclin-Cdk complexes to block the cell cycle. Pharmacologic compounds that exploit our current knowledge of cell cycle and checkpoint pathway regulation offer insights into the development of novel therapeutic strategies.     

Recent advances in understanding the cell death pathways activated by anticancer therapy

Over the past two decades, the role of apoptosis in the cytotoxicity of anticancer drugs has become clear. Apoptosis may occur via a death receptor-dependent (extrinsic) or independent (intrinsic or mitochondrial) pathway. Mitochondria play a central role in cell death in response to DNA damage, and mediate the interaction(s) of various cytoplasmic organelles, including the endoplasmic reticulum, Golgi apparatus, and lysosomes. The mitochondrial pathway of cell death is mediated by Bcl-2 family proteins, a group of antiapoptotic and proapoptotic proteins that regulate the passage of small molecules, such as cytochrome c, Smac/Diablo, and apoptosis-inducing factor, which activates caspase cascades, through the mitochondrial transition pore. In addition, apoptosis can induce autophagic cell death via crosstalk between the two pathways upon treatment with anticancer drugs. The current review focused on recent advances surrounding the mechanism(s) of cell death induced by anticancer agents and discussed potential molecular targets for enhancing the chemotherapeutic effect(s) of anticancer agents.