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Pointsoft Pro ATC X 自己专用的 ATC
插件名称: Pro ATC X
插件出版公司: Pointsoft
硬盘空间: 225 MB
安装方法: 特殊安装方法（安装方法在贴内已标明）
FSX 自带的 空管功能太简易了，只能适合初学者。当你使用过一段时间后，会发现里面缺很多功能。比如副驾驶协助操作，等候，复飞和 SID/STAR 程序。PRO-ATC/X 的空管功能替代了 FSX 自带的功能。可以单独使用起飞和降落机场数据计算出完整的航线。PRO-ATC/X 可以全程控制所有使用 IFR 形式飞行的飞机。PRO-ATC/X 中可以操作 12 种检查单，兼容标准仪表起飞和降落，课可以选择飞行高度。虚拟副驾驶可以在飞行中设置自动驾驶，起落架，襟翼，扰流板，灯具和电台。所有功能打开后，几乎没有什么其他的事要做。PRO-ATC/X 的空管功能会自动检测 FSX 的 AI 飞机交通。PRO-ATC/X 在下达命令时会注意到 AI 飞机交通，这代表其他飞机使用跑道时不会下达起飞的命令。
主要特点：
PRO-ATC/X 在 FSX 后台通过 SimConnect 运行，不会影响 FSX 功能和频数。从出发机场停机位一直到目的地机场停机位的完整的空管系统。
完整的导航数据库，包括所有飞机场数据，VOR，NDB，交叉口，SID 和 STAR 航线。兼容 Navigraph AIRAC 导航数据。会自动添加 第三方飞机场插件跑道 SID，STAR 数据。SID，STAR 航线的选择会根据天气状况变化。可以选择其他 SID 航线。自动识别空中的 AI 飞机交通。航线可以转换。可以输出和输入各种格式的飞行计划文件。自动计算飞行高度。可以编辑飞行航线。兼容 PMDG 737 NGX，Airbus X Extended。虚拟副驾驶可以在飞行中设置自动驾驶，起落架，襟翼，扰流板，灯具和电台。可以操作 12 种检查单。可以独自添加检查单。FSX 生成的 GPS 飞行计划可以直接导入。PMDG 和 AXE 生成的 GPS 飞行计划只能通过 FMC 导入。驾驶员，副驾驶和空管声音效果。多种国家的空管语音效果。等候，复飞程序地图上可以看到所有需要的信息。
下载地址：FTP：/FSX/工具类/Pro ATC X(Pointsoft)/
关键是不知道怎么用。& &
期待试用报告
终于有这样的软件了
我来试试，默认ATC太弱了
这个看起来很强大
这个可以有
前排试用。
这个怎么用啊？功能挺强的
LZ强大啊，这几天正在四处找这个呢
卖的死贵，前期骂声一片，实在不敢直接下手啊
据说现在改进的不错了，先下来试试
期待测试和 指导教程~
坚决不做第一个吃螃蟹的人
&&等等你们的试用报告
木有截图？
求测试报告！
有米有使用教程啊
没有教程，估计装了也并不会用
多种国家语言难不成还有天朝。。。
有中文语音不？
小试了一下，跟AXE结合着用的，还是喜欢AXE自带的checklist和FO，只是用这个的FO控制电台和AP，
只飞到巡航阶段，明显能感受到一点是爬升控制的合理的很多，不会像默认ATC那样都进入改平了才批准下一个高度；
另外看到选项里面可以选择直接到的导航点了，这个不错；
那PMDG不能用吗？&
感谢试用报告&
airbus X extended&
AXE是什么？&
赞一个，先下载试用
請問如何更新導航數據??
cks 发表于
請問如何更新導航數據??
我直接把navigraph的aerosoft airbus extended的数据放在了它要求的文件夹下，
更新成功了
前排小白鼠试用，
falconluan 发表于
我直接把navigraph的aerosoft airbus extended的数据放在了它要求的文件夹下，
更新成功了 ...
我也曾如此試過，不過放入指定路徑後是個資料夾，從ㄒdata處import還是停留在1210.
另外是不是沒sid變無法儲存。
我放的是文件夹里面的内容，你试下看看？&
论坛的首页改版了今天已打开给人耳目一新的感觉
有木有测试+截图
真的很强大，期待试试
表示会做一回白耗子去摸索
论坛公告 /1
飞行者俱乐部模拟舱等LOGO征集令，悬赏金2000元Wiley Online Library will be unavailable on Saturday 14th May 11:00-14:00 BST / 06:00-09:00 EDT / 18:00-21:00 SGT for essential maintenance.Apologies for the inconvenience.
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SPECIAL FEATURES
Eva Hernando1,&, Irene Orlow1,&, Vasco Liberal2, Gloria Nohales1, Robert Benezra2 andCarlos Cordon-Cardo1,&D,*DOI:&10.15(:4&223::AID-IJC.CO;2-L
International Journal of Cancer pages 223&227, Author Information1Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA2Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, NY, USAEmail: Carlos Cordon-Cardo (cordon-c@mskcc.org)*Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, USAPublication HistoryIssue published online: 18 APR 2001Article first published online: 18 APR 2001Manuscript Accepted: 2 FEB 2001Manuscript Revised: 15 JAN 2001Manuscript Received: 22 NOV 2000
ARTICLE TOOLS
hsMAD2;hBUB1;hBUB3During the metaphase&anaphase transition, the spindle checkpoint prevents segregation of chromosomes if the spindle assembly is perturbed. Critical components of this checkpoint are the MAD and BUB families of proteins, which prevent the proteolysis of Pds1 and B cyclins, producing mitotic arrest. In the present study, we first intended to resolve the role of the hsMAD2 gene in human cancer by determining the potential presence of hsMAD2 mutations in 44 primary bladder tumors, 42 soft-tissue sarcomas and 10 hepatocellular carcinomas. The entire coding region of the hsMAD2 gene was analyzed using PCR-SSCP and sequencing. One of the bladder tumor samples showed a point mutation consisting of a transition, ATCGTC (IleVal) in codon 190 of hsMAD2. However, no differences were found in the mitotic arrest between cells transfected with mutant and wild-type MAD2 cDNA. We also identified mobility shifts in hsMAD2 in both normal and tumor DNA in 3 bladder tumors, 3 soft-tissue sarcomas and 1 hepatocellular carcinoma, consistent with a polymorphism at codon 143, CCACCG (ProPro). Another polymorphism was identified in a hepatocellular carcinoma case at codon 22, GAGGAA (GluGlu). In addition, a subgroup of 67 primary tumors was analyzed by Southern blot hybridization. No deletion or visible re-arrangements were detected by comparing tumor and normal DNA band signals. Two other important components of the spindle mitotic checkpoint, hBUB1 and hBUB3, were also screened for mutations: hBUB1 in 43 bladder tumors and 9 bladder cell lines and hBUB3 only in the cell lines. Two polymorphisms were found in hBUB1 at positions 144, CAGCAA (GlnGln) in 1 primary tumor and 1 bladder cell line, and 913 (ATCATT, IleIle) in 1 primary tumor. We did not find sequence alterations in hBUB3. These results suggest that mutations of the hsMAD2, hBUB1 and hBUB3 genes are very rare in bladder tumors and that hsMAD2 alterations are also infrequent in soft-tissue sarcomas and hepatocellular carcinomas. & 2001 Wiley-Liss, Inc.A wide range of structural and numerical chromosomal changes are frequently observed in cancer,,
to the extent that in certain tumor types cytogenetic analysis has been used as a predictive parameter of the disease. Tumor cells elude checkpoint mechanisms that normally regulate cell-cycle progression, compromising the accuracy of DNA duplication and of chromosomal segregation.,
In normal cells, one of these controls occurs during the metaphase&anaphase transition. The spindle checkpoint prevents the actual segregation of chromosomes unless assembly of the mitotic spindle and bipolar attachment of chromosomes to microtubules have occurred.,
Ubiquitin-mediated proteolysis of certain molecules plays a critical role in regulating the exit from mitosis. More specifically, sister chromatid separation and cytokinesis require proteolysis of Pds1 and B-type cyclins, respectively.,
This protein destruction depends, in turn, on a ubiquitin protein ligase known as the cyclosome or anaphase-promoting complex (APC). Pds1 destruction occurs shortly before the onset of anaphase and depends on the activation of APC/cyclosome by cdc20, termed p55Cdc in vertebrates.Budding yeasts with mitotic checkpoint defects have been utilized in the identification of critical regulatory genes in the spindle checkpoint. As a result, several genes have been cloned, including the MAD (mitotic arrest-deficient) and BUB (budding uninhibited by benzimidazole) gene families. In response to microtubule-unattached kinetochores, Bub1p binds and phosphorylates Bub3p. Bub1p&Bub3p active complexes and Mps1 kinase (TTK in humans) are required for Mad1 phosphorylation. This step precedes Mad2 activation, which establishes Mad2&cdc20&APC complexes, inactive in terms of Pds1 degradation. Thus, chromosomal segregation and cell division are inhibited., Mad2 appears to be essential for the timing of anaphase onset as mammalian cells microinjected with anti-mad2 antibody enter anaphase prematurely. The human homologue of MAD2, termed hsMAD2, has been reported to be required for the response to nocodazole treatment in HeLa cells. Defects in hsMAD2 may contribute to the sensitivity of certain tumors to mitotic spindle inhibitors. For example, the human cancer cell line T47D has decreased expression of mad2 and fails to arrest in mitosis after nocodazole treatment. Moreover, in colorectal cancers displaying chromosomal instability, loss of the spindle checkpoint was associated with mutational inactivation of the hBUB1 gene.In view of the fact that aneuploidy is commonly observed in many cancers and that the mitotic checkpoint plays an important role in regulating chromosomal segregation, the present study was undertaken to investigate whether mutational defects in the hsMAD2, hBUB1 and hBUB3 genes are relevant in primary tumors.Cell linesNine bladder tumor&derived cell lines were used: 5637, J82, SCaBER, TCCSUP, UMUC3, T24, RT4, HT1376 and HT1197. All were obtained from the ATCC (Manassas, VA). 5637 cells were grown in RPMI medium supplemented with 10% FCS. J82, SCaBER and TCCSUP were cultured in Eagle's minimum essential medium (MEM) in Earle's BSS with non-essential amino acids (NEAAs), 1 mM sodium pyruvate and 10% FCS. T24 and RT4 were grown in McCoy's medium supplemented with 10% FCS. HT1376 was maintained in MEM with NEAAs and 10% FCS. HT1197 cells were cultured in MEM with NEAAs, 1 mM sodium pyruvate, glutamine and 1.5 g/l sodium bicarbonate. UMUC3 cells were cultured in in MEM with 1 mM sodium pyruvate and 10% FCS. In addition, we used 293T cells, derived from primary human kidney, to perform the transfection and functional assays (see below). 293T cells were cultured in MEM with 1.5 g/l sodium bicarbonate, 0.1 mM NEAAs, 1 mM sodium pyruvate and 10% FCS.Tissue sampleshsMAD2 genetic analyses were performed in a group of 96 human primary tumors comprising 44 transitional-cell carcinomas (TCCs) of the bladder, 42 adult soft-tissue sarcomas and 10 hepatocellular carcinomas. hBUB1 was screened for mutations in 43 primary bladder TCCs. Tissue samples were embedded in a cryopreservative solution (OCT C Miles, Elkhart, IN), snap-frozen in isopentane pre-cooled in liquid nitrogen and stored at &70&C. Representative hematoxylin and eosin&stained sections of each frozen block were examined microscopically to confirm the presence of tumor, and only lesions with &70% neoplastic cells were included.DNA and RNA isolationDNA from tumor was extracted by a non-organic method developed by Oncor (Gaithersburg, MD). Normal DNA from the same patient, when available, was used as a paired control. Total RNA from the bladder cell lines was extracted using the RNeasy Mini Kit (Qiagen, Valencia, CA), following the manufacturer's instructions.Deletion analysesFor Southern blot analysis, a 660 bp cDNA fragment containing the complete human MAD2 sequence was used as a probe, to determine potential deletions or re-arrangements. A cDNA fragment containing glyceraldehyde phosphate dehydrogenase (GAPDH) sequences was used as control. In general, Southern blot analysis was performed as described previously. Briefly, extracted DNA (7.5 &g aliquots) was digested with EcoRI restriction endonuclease, and digested DNA was subjected to electrophoresis in 0.7% agarose gels and blotted onto nylon membranes. Membranes were pre-hybridized with Hybrisol I (Oncor, Purchase, NY) at 43&C for at least 1 hr and then incubated overnight at the same temperature with probes labeled to high specific activity using &[32P]dCTP (Dupont NEN, Boston, MA). Hybridized membranes were washed with 0.1 & SSC/0.1% SDS at 45& to 50&C (hsMAD2) or 60&C (GAPDH) and autoradiographed using intensifying screens at &70&C for 24 to 72 hr. Densitometric evaluation of autoradiographic band intensities was performed using an Ultrascan XL Laser Densitometer (Pharmacia, Piscataway, NJ) and the FUJIX Bio-Imaging Analyser (Fuji, Tokyo, Japan). Relative amounts of hsMAD2 were determined by comparing gene-specific hybridization signals with those obtained using the control probe.Mutational analysesPCR single-stranded conformational polymorphism (PCR-SSCP) assays were conducted as previously described. PCR amplification was performed using primers specific to sequences surrounding exons 1 to 5 of the hsMAD2 gene: exon 1 forward, 5&-GTGGAAGCGCGTGCTTTTGTTTG-3&, and reverse, 5&-GGCCTGCGCGAGAACTTACAGAAG-3& (146 bp); exon 2 forward, 5&-GCTTGCAGCATTCGGCATC-3&, and reverse, 5&-GACTGCTTAAGATGCTAGAAAAC-3& (187 bp); exon 3 forward, 5&-GTGTTGCAGATTGGTTATACAT-3&, and reverse, 5&-GTAATTCCTATTTACCTGTCATC-3& (156 bp); exon 4 forward, 5&-TGACAGTGCACC CAGAGAAAAG-3&, and reverse, 5&-GCCAATCAATTTAATTCAACAAGAAG-3& (151 bp); exon 5 forward, 5&-GTCCCCAATTTAGGTTCATTTG-3&, and reverse, 5&-CATGTCATCC TCAGTCATTGAC-3& (219 bp).PCRs were performed in 10 &l volumes containing 80 to 100 ng of template DNA, 2.2 &Ci &[33P]dCTP (Amersham, Arlington Heights, IL), 1.5 to 3.25 mM MgCl2, 160 &M deoxynucleoside triphosphates, 5 pmol of each primer, 5% DMSO (exons 1, 3, 4), 0.5 U TaqI polymerase and 1 & PCR buffer (Promega, Madison, WI). DNA was amplified with 30 cycles of PCR using a thermal cycler (Perkin-Elmer Cetus, Foster City, CA). Annealing temperatures ranged from 55& to 71&C. PCR products were denatured and loaded onto a non-denaturing 0.6&0.8& mutation detection enhancement (MDE) gel that contained 8% glycerol. Electrophoresis was performed at room temperature for 16 to 20 hr at 8 to 10 W. Gels were dried and exposed to X-ray film. In cases where mobility shifts were identified by PCR-SSCP, DNA was amplified in a separate reaction and sequenced using the ABI Prism 377 DNA Sequencer (Perkin-Elmer, Norwalk CT).The hBUB1 gene was similarly analyzed by PCR-SSCP in both primary tumors and cell lines. Primers targeting CD1 (exons 2&5) were as follows: exon 2 forward, 5&-CTAGGATGCTTGAAGCCCAC-3&, and reverse, 5&-CTTCTACCCACTGTATGTATCT-3& (160 bp); exon 3 forward, 5&-TTCTAGATACATACAGTGGGTAG-3&, and reverse, 5&-TGATTATACATCTTAAGTATACTTACA-3& (165 bp); exon 4 forward, 5&-TTCGTGTAGGCTGAGTACAACAACAG-3&, and reverse, 5&-GTTTGACTTTGTAACTACCTGTATTG-3& (225 bp); exon 5 forward, 5&-CTCTAACAGCTATGTACTGTAAG-3&, and reverse, 5&-CCATGCCCAGCCGGGTTTG-3& (205 bp). Primers targeting NLS (exons 8 to 9) were as follows: exon 8 forward, 5&-GTTGTAAATAATGTATTTTCCCC-3&, and reverse, 5&-CTTGCAGTCTTTCAACATAGTC-3& (320 bp); exon 9 forward, 5&-CCTGGATTTAAATTTTGCATGC-3&, and reverse, 5&-CTTGGGCACAGGATGTGTC-3& (240 bp). Primers targeting CD2 (exons 21&25) were as follows: exon 21 forward, 5&-CGAGATTGGATATGAATTTCAC-3&, and reverse, 5&-CCAAAAATCCATTTTAAATTCATG-3& (240 bp); exon 22 forward, 5&-GGATTCCACTTTCCATTGTTCC-3&, and reverse, 5&-GAAAGGCTGCTATGGAAATAGG-3& (240 bp); exon 23 forward, 5&-GTGCTCATGCAGCCTGGCT-3&, and reverse, 5&-TCCCTCACTTTAGTTTTATACC-3& (230 bp); exon 24 forward, 5&-GGCATCTTGATATAGTCATGTG-3&, and reverse, 5&-GCACCAATGCTAATACTCACC-3& (200 bp); exon 25 forward, 5&-GGTTATCCTGCATATTGAAGG-3&, and reverse, 5&-GCAGATTCATATTTACAGTGTG-3& (300 bp).In addition to standard PCR conditions, certain exons required specific conditions: exons 3 and 4: buffer pH 8.8, 3.5 mM Mg2+, 75 mM KCl, (NH4)2SO4 15 mM; exon 5: buffer pH 9.2, 3.5 mM Mg2+, 75 mM KCl; exon 22: 15% glycerol, 0.5% DMSO.Direct sequencing was performed for all cell lines and selected primary tumor samples that showed band shifts or suspicion of band shifts. An independent DNA amplification was performed in all of these cases, to repeat and confirm results.RT-PCR and sequencinghBUB3 cDNAs from bladder cell lines were obtained and amplified in overlapping fragments using SuperScript One-Step RT-PCR with Platinum Taq (GIBCO BRL Life Technologies, Gaithersburg, MD).cDNA synthesis was performed at 54&C for 30 min, and PCR amplification consisted of initial denaturing at 94&C, followed by 40 cycles of 15 sec at 94&C, 30 sec at the adequate annealing temperature according to each primer couple and 40 sec at 72&C. The reaction was concluded by 7 min at 72&C.hBUB3 primer sequences were as follows: BUB3-50 forward 5&-TAACGAGTTCAAGCTGAACC-3&, and BUB3-697 reverse, 5&-GCATACTTCTTCTTCTGTACC-3& (647 bp); BUB3-189 forward, 5&-CTCAAGTACCAGCACCC-3&, and BUB3-809 reverse, 5&-AAAGCCATCAGAACCACC-3& (620 bp); BUB3-792 forward, 5&-GGTGGTTCTGATGGCTTTG-3&, and BUB3-1089 reverse, 5&-CACCATTGGGGAGTACGAAT-3& (297 bp); BUB3- 1076 forward, 5&-ACTCCCCAATGGTGGATTTA-3&, and BUB3- 1521 reverse, 5&-GAAAATAATGGACGGCCAAG-3& (445 bp); BUB3-1505 forward, 5&-GGCCGTCCATTATTTTCTGA-3&, and BUB3-2004 reverse, 5&-GGCCAAGCTAAGTTCGTGTT-3& (499 bp); BUB3-1796 forward, 5&-CAGGCATCTATTTGGACCTG-3&, and BUB3-2294 reverse, 5&-GAAGCAAACAAAGCTTTCCA-3& (498 bp); BUB3-2225 forward, 5&-TTTTCTTCATTGCAGGTCCA-3&, and BUB3-2553 reverse, 5&-GGGTTCTGCATCTCGTTTATT-3& (328 bp).Mad2 functional assayhsMAD2 point mutations found by PCR-SSCP and sequencing were introduced in the MAD2 cDNA cloned into pFLAG-CMV-2 vector by site-directed mutagenesis, using the overlap extension method. Mutant and wild-type constructs were transfected into 293T cells using lipofectamine reagent (GIBCO BRL). After 48 hr, cells were harvested and analyzed for mitotic arrest by flow cytometry.Mutation screeningNine bladder tumor&derived cell lines and a group of 43 primary bladder TCCs were screened for hBUB1 mutations. Two polymorphisms were found, at position 144 (codon 5), CAGCAA (GlnGln) in 1 primary tumor and 1 bladder cell line (J82), and at position 913 (codon 22), ATCATT (IleIle) in 1 primary tumor (Fig. ). The hBUB3 gene was also sequenced in the same panel of cell lines, and no sequence variations were detected.Figure&1. Mutational analysis of the hBUB1 gene by PCR-SSCP and sequencing. Left panel illustrates representative band patterns obtained in primary tumors (26, 54, 70) and normal DNA. An abnormal band pattern was identified in a tumor sample (asterisk, case 70). Sequencing analysis of DNA that corresponded to the shifted, re-amplified bands is shown in the right panels. Note the nucleotide change at codon 144 (GA, arrow) that corresponds to a polymorphism (GlnGln) in sample 70. (-), Negative control (H2O); Nl, normal DNA.Ninety-six primary tumors, including 44 TCCs, 42 soft-tissue sarcomas and 10 hepatocellular carcinomas, were analyzed for the presence of hsMAD2 point mutations. All cases were studied using PCR-SSCP as a mutation screening method. The area screened spanned the entire coding region of the hsMAD2 gene. One of the bladder tumors evaluated by PCR-SSCP showed extra bands, or mobility band shifts, not present in the normal control tissue (Fig. a). Direct sequencing of normal and shifted bands excised from the gels demonstrated a point mutation of the hsMAD2 gene. The change consisted of a transition, ATCGTC (IleVal), that affected codon 190 (Fig. b). We also identified mobility shifts in hsMAD2 in both normal and tumor DNA in 3 bladder primary tumors and 3 soft-tissue sarcomas, consistent with a polymorphism at codon 143 (CCACCG, ProPro). In addition, 2 hepatocellular carcinomas presented a silent mutation, one at codon 22 (GAGGAA, GluGlu) and the other at codon 143 (CCACCG, ProPro).Figure&2. Mutational analysis of the hsMAD2 gene by PCR-SSCP and sequencing. (a) Abnormal hsMAD2 band pattern in a bladder tumor DNA identified by PCR-SSCP (asterisk, case 63). (b) Direct sequencing of PCR products obtained from tumor DNA from the same case compared to those obtained from normal DNA. The single-base substitution (AG, arrow) produces the mutation (IleVal), as indicated in the forward sequence. Nl, normal DNA; Tm, tumor DNA.Deletion analysesThe hsMAD2 gene maps to chromosome 4, in the 4q27 region. Deletions and translocations involving chromosome 4q occur non-randomly in certain tumor types. Therefore, we also analyzed a subgroup of 67 primary tumors (29 TCCs and 38 soft-tissue sarcomas) using Southern blot hybridization with a specific hsMAD2 probe. Normal genomic DNA samples digested with the restriction enzyme EcoRI showed 2 strong bands corresponding to fragments of 5 to 6 kb, as well as 2 bands corresponding to fragments of approximately 2 to 3 kb (Fig. ). No deletion or visible gene re-arrangements were detected by comparing tumor and normal DNA band signals, despite the fact that the majority of tumors analyzed corresponded to high-grade lesions.Figure&3. Southern blot analysis of the hsMAD2 gene. Genomic DNA was digested with the restriction enzyme EcoRI. Southern blotting was performed using a specific hsMAD2 cDNA fragment probe, as described in . A control probe (GAPDH) for DNA loading was used in the analysis. The hsMAD2-specific signals shown correspond to bands of 2 to 6 kb. The control probe signal shown corresponds to 4 to 6 kb bands. 51, N 52, 148 and 150, b 261, 374, 455 and 459, soft-ti Nl, normal DNA.Mad2 functional analysisThe point mutation identified at codon 190 (ATCGTC, IleVal) was introduced into MAD2 cDNA by site-directed mutagenesis. Mutant and wild-type constructs were transfected into 293T cells. After 48 hr, cells were harvested and analyzed for mitotic arrest. As for cells transfected with the wild-type construct, cells transfected with mutant MAD2 cDNA arrested in mitosis with double DNA content (data not shown).Chromosomal instability is thought to contribute to tumorigenesis in diverse tumor types. Advances in elucidating the molecular components of the spindle checkpoint, which contribute to the maintenance of chromosomal stability, have identified spindle checkpoint defects in neoplastic cells.,
Specifically, a breast tumor cell line sensitive to paclitaxel (Taxol) and nocodazole had reduced mad2 expression and failed to arrest in mitosis after nocodazole treatment. In addition, 2 colorectal tumor cell lines were reported to harbor mutant alleles of the hBUB1 gene, found to be functional in a dominant negative mode. The present study was undertaken to evaluate the importance of the spindle components hsMAD2, hBUB1 and hBUB3 in primary tumors of different origins. No significant genetic alterations were observed in the 3 genes analyzed (Table ). Only 1 case harbored a sense mutation in hsMAD2; however, the functional activity of this allele did not appear to be affected since the mitotic checkpoint was not impaired. In accordance with our results, others have confirmed the absence of mutations in hsMAD2 and hBUB3 in colorectal carcinomas and lung cancer,,
as well as in hBUB1 in head-and-neck squamous-cell carcinomas, lung cancer cell lines and breast cancer cell lines.Table&I.&Sequence Alterations Found in the hBUB1, hBUB3 and hsMAD2 Genes in the Group of Tumors and Cell Lines AnalyzedGeneSequence positionCodonFrequencyCell linesTumor sampleshBUB3No variants found0/9&hBUB1Codon 5(nt. 144)CAG (Gln)
CAA (Gln)1/91/43Codon 22(nt. 913)ATC (Ile)
ATT (Ile)0/91/43hsMAD2Codon 190ATC (Ile)
GTC (Val)&1/96Codon 143CCA (Pro)
CCG (Pro)&7/96Codon 22GAG (Glu)
GAA (Glu)&1/96The lack of deletions, re-arrangements or point mutations in the tumors and cell lines analyzed suggests that other mechanisms could underlie chromosomal instability in these cases, e.g., deregulation of these genes at the expression level. This phenomenon has been described for other cell-cycle regulators, such as p27/Kip1.,
Genetic evidence of the functional relevance of this group of genes comes from the reported phenotypes of the MAD2 and BUB3 knockout murine models. Mice null for either MAD2 or BUB3 are not viable, and the embryonic cells undergo chromosomal missegregation and apoptosis.,
Due to their critical role, reduced levels of expression of these genes could disrupt their function, leading to aberrant chromosomal segregation and accumulation of other tumor-specific mutations. Alternatively, other genes involved in the control of the G2&M transition, either identified but yet not analyzed or that remain to be discovered, could be the target of mutational events in these tumors.Eva Hernando is the recipient of a fellowship from Caja Madrid, Spain-CN10, Spain-MSKCC.1
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