Cloning tumor suppressor genes (TSG) from human chromoso

从人类染色体中克隆肿瘤抑制基因（TSG）

• 批准号: 6559009
• 负责人: MICHAEL LERMAN
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6559009
• 关键词: Von Hippel Lindau syndrome; carcinogenesis; chromosomes; cytogenetics; differential display technique; genetic mapping; genetically modified animals; human genetic material tag; human tissue; laboratory mouse; lung neoplasms; molecular cloning; molecular oncology; neoplasm /cancer genetics; nucleic acid sequence; transfection; tumor suppressor genes

VHL TSG (3p25) To analyze the function(s) of the VHL gene and its carcinogenic pathway(s) we obtained the entire genomic sequence of the gene including the promoter, introns, and flanks, and constructed a set of VHL minigenes (wild type and mutant) and a complete intronless VHL gene driven by the VHL promoter. We then set out to discover target genes controlled by pVHL. The differential display technology was employed to discover these genes using the UMRC6 and 786-0 cells stably transfected with wt and mutant VHL minigenes. To date (September 2001) six down regulated genes were identified, namely, NOTCH2 and DEC1, that specify cell fate determination and may have oncogenic potential, two transmembrane type carbonic anhydrases, CA9 and CA12, and two new unknown genes. The CA9 and CA12 genes are overexpressed in many tumor types due to hypoxia causing the loss of functional pVHL.The CAIX/XII enzymes could sense the intracellular pH and control the acidity (extracellular pH) of the miliew surrounding the cancer cells and thus create a microenvironment conducive to tumor growth and spread.They also play fundamental roles in normal physiology such as production of eye humor, brain and kidney functions etc. Analysis of the methylation of the VHL promoter in renal carcinoma cells carrying a methylated VHL endogene by monochromosome gene transfer, cell fusion, and VHL gene transfections showed that the meth+ phenotype is dominant in the UOK 21 cells probably resulting from changes in cis-acting elements of the VHL locus.We then created a mouse transgenic model expressing the human genomic VHL locus and demonstrated that human VHL methylation pattern was reproduced during mouse development and was very similar to that of the mouse VHL gene. This model would allow studying the local methylation protection mechanisms in the VHL locus and the effect of chromosomal context on de novo methylation of various elements of the VHL locus, such as repetitive sequences and the VHL CpG promoter. The future work will be focused (i) on the role of carbonic anhydrases (CAs) in the regulation of tumor pH and its impact on cancer growth, (ii) discovery new specific inhibitors of these enzymes to treat cancer, (iii) deveop and test CAIX/XII cDNA based vaccines to treat cancer, and (iv) the nature of the cis-acting elements in the VHL locus involved in de novo aberrant methylation. The 3p21.3 TSG We used overlapping and nested homozygous deletions, contig building, genomic sequencing, physical, and transcript mapping to further define a ~630-kb lung cancer homozygous deletion region harboring one or more tumor suppressor gene(s) (TSGs) on chromosome 3p21.3. This location was identified through somatic genetic mapping in cancers, cancer cell lines and pre-malignant lesions of the lung and breast including the discovery of several homozygous deletions. The combination of molecular manual methods and computational predictions permitted us to detect, isolate, characterize and annotate a set of 25 genes which likely constitute the complete set of protein-coding genes residing in this ~630-kb sequence. A subset of 19 of these genes were found within the deleted overlap region of ~370-kb. This region was further subdivided by a nesting 200-kb breast cancer homozygous deletion into two gene sets: 8 genes lying in the proximal ~120-kb segment and 11 genes lying in the distal ~250-kb segment. These 19 genes were analyzed extensively by computational methods and were tested by manual methods for loss of expression and mutations in lung cancers to identify candidate TSGs from within this group. Several genes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (NSCLC) (CACNA2D2/ (a2d-2), SEMA3B (formerly SEMA(V),) BLU, RASSF1/A (formerly 123F2), and HYAL1) or small cell lung cancer (SCLC) (SEMA3B, BLU, RASSF1/A (formerly 123F2), and HYAL1) cell lines. We found six of the genes to have 2 or more amino acid sequence altering mutations including: BLU, NPRL2/Gene21, FUS1, HYAL1, FUS2, and SEMA3B. However, none of the 19 genes tested for mutation showed a frequent (>10%) mutation rate in lung cancer samples. This led us to exclude several of the genes in the region as classical tumor suppressors for sporadic lung cancer. On the other hand, the putative lung cancer TSG in this location may either be inactivated by tumor acquired promoter hypermethylation or belong to the novel class of haploinsufficient genes which predispose to cancer in a hemizygous (+/-) state but do not show a second mutation in the remaining wild type allele in the tumor. Functional testing of the critical genes by gene transfer and gene disruption strategies is under way and will permit the identification of the putative lung cancer TSG(s), LUCA.To date (September, 2001) we identified tha RASSF1/A gene as multiple TSG involved in many tumors, including lung, breast,prostate, kidney, head&neck (NPC) and others. The HYAL2 gene was identified as a GPI-anchored receptor for the sheep lung cancer retrovirus, JSRV and a sequestration mechanism inactivating HYAL2 product was demonstrated. The Env gene of JSRV was shown to transform human bronchial epithelial cells in vitro and sequester the HYAL2 gene product which alow to study the signal transduction pathways leading to carcinogenesis in this sytem. A new FAS2 gene cDNA polymorphism was shown to be associated with NPC with predictive value in Asian populations. This gene was also identified as a TSG for NPC.Current work is focused on the detection and isolation of the putative human retrovirus that may cause a rapidly rising form of human lung cancer namely bronchioloalveolar adenocarcinoma (BAC).. The 3p12 TSG Cytogenetic deletions and LOH at human 3p12 are a consistent feature of lung cancer specimens and suggest the presence of a tumor suppressor gene(s) (TSG) at this location. Only one gene (DUTT1, Deleted in U Twenty Twenty) was so far cloned from the overlapping region deleted in several lung and breast cancer cell lines (U2020, NCI H2198, HCC38). DUTT1 is the human ortholog of the fly gene ROBO that has homology to NCAM proteins. Extensive analyses of DUTT1 in lung cancer did not reveal any mutations, suggesting another gene(s) at this location could be associated with lung cancer initiation and/or development. We discovered in the overlapping critical region a new small (~230kb), nested homozygous deletion in the SCLC cell line GLC20. This deletion has been PCR-characterized using several polymorphic markers. P1 library screening produced three overlapping clones that cover the whole region and flanks. These clones were used to define by fiber-FISH the location and size of the deletion. Recently several BAC clones covering this region were sequenced by the MIT genome sequencing center providing a genomic tool to discover in silico the resident genes. Several genes represented by EST clusters were detected in the deletion and are being isolated. Subsequent mutation and functional studies will identify the potential 3p12 lung/breast cancer TSG.

VHL TSG（3P25） 为了分析VHL基因的功能及其致癌途径（S），我们获得了基因的整个基因组序列，包括启动子，内含子和侧面，并构建了一组VHL Minigenes（野生型和突变体）和一组无内在的无内在VHL基因驱动的VHL驱动器。然后，我们着手发现由PVHL控制的靶基因。使用差异显示技术使用WT和突变体VHL微型烯稳定转染的UMRC6和786-0细胞发现这些基因。迄今为止（2001年9月）鉴定了六个下调的基因，即Notch2和Dec1，这些基因指定了细胞命运的确定，并且可能具有致癌潜力，两个跨膜型碳纤维性硬脂酶Ca9和ca12，以及两个新的未知基因。 CA9和CA12基因在许多肿瘤类型中因缺氧而过表达，导致功能性PVHL的丧失。CAIX/XII酶可以感觉到细胞内pH值并控制围绕癌细胞的酸度（细胞外pH），从而在癌细胞周围产生脑部的生长，例如在癌细胞中产生脑部的生长，例如，易于挑战。通过单色基因转移，细胞融合和VHL基因转染，携带甲基化VHL内生的肾脏癌细胞中VHL启动子甲基化的甲基化等分析的分析表明，METH+表型在UOK 21中可能在UOK 21中占主导地位。 VHL基因座并证明了在小鼠发育过程中重现了人类VHL甲基化模式，并且与小鼠VHL基因非常相似。该模型将允许研究VHL基因座中的局部甲基化保护机制，以及染色体环境对VHL基因座各种元素的从头甲基化的影响，例如重复序列和VHL CPG启动子。 The future work will be focused (i) on the role of carbonic anhydrases (CAs) in the regulation of tumor pH and its impact on cancer growth, (ii) discovery new specific inhibitors of these enzymes to treat cancer, (iii) deveop and test CAIX/XII cDNA based vaccines to treat cancer, and (iv) the nature of the cis-acting elements in the VHL locus involved in de novo aberrant甲基化。 3P21.3 TSG 我们使用重叠和嵌套的纯合缺失，重叠群建筑，基因组测序，物理和转录本映射，进一步定义了一个〜630-kb的肺癌纯合缺失区域，该区域内具有一个或多个肿瘤抑制基因（S）（TSG）3p21.3染色体上。通过癌细胞，癌细胞系和肺部和乳房的恶性病变中的体细胞遗传学图来鉴定该位置，包括发现几种纯合缺失。分子手动方法和计算预测的组合使我们能够检测，分离，表征和注释一组25个基因，这些基因可能构成了构成该〜630-KB序列中的一组完整的蛋白质编码基因。在〜370-kb的删除重叠区域内发现了其中19个基因的子集。该区域通过嵌套200-kb乳腺癌纯合缺失成两个基因组进一步细分：8个基因位于近端〜120-KB段中，而11个基因位于远端〜250-kb段中。通过计算方法对这19个基因进行了广泛的分析，并通过手动方法对肺癌中表达和突变的丧失进行测试，以鉴定该组内的候选TSG。 Several genes showed loss-of-expression or reduced mRNA levels in non-small cell lung cancer (NSCLC) (CACNA2D2/ (a2d-2), SEMA3B (formerly SEMA(V),) BLU, RASSF1/A (formerly 123F2), and HYAL1) or small cell lung cancer (SCLC) (SEMA3B, BLU, RASSF1/A (formerly 123F2), and透明1）细胞系。我们发现六个基因具有2个或更多的氨基酸序列改变突变，包括：BLU，NPRL2/Gene21，Fus1，hyal1，Fus2和Sema3b。然而，对突变测试的19个基因均未显示出肺癌样品中的频繁（> 10％）突变。这导致我们将该地区的几个基因排除在零星肺癌的经典肿瘤抑制因子中。另一方面，该位置的假定肺癌TSG可能会被肿瘤获得的启动子过度甲基化灭活，或者属于新型的单倍弹性基因，这些基因可容易在半基因亚化（+//-）状态下癌症，但不会在肿瘤中剩下的野生型野生型中的第二突变。通过基因转移和基因破坏策略对关键基因的功能测试正在进行中，将允许鉴定假定的肺癌TSG（S），LUCA，LUCA。至今（2001年9月），我们将THA RASSF1/A基因确定为许多TSG，是许多涉及许多肿瘤，包括许多肿瘤，包括肺，乳房，Prostate，Prostate，Prostate，Prostate，Prostate，Prostate，Prostate，Prostate，Prostate，Prostate，Prostate，Prostate，kidney，Head＆nect＆其他TSG。透明基因被鉴定为绵羊肺癌逆转录病毒，JSRV的GPI锚定受体，并证明了隔离机制灭活透明产物。 JSRV的ENV基因被证明可以在体外转化人支气管上皮细胞，并隔离透明基因产物，该基因产物逐渐研究导致该系统中癌变的信号转导途径。新的FAS2基因cDNA多态性显示与亚洲人群中具有预测价值的NPC相关。该基因也被鉴定为NPC的TSG。电流工作的重点是检测和隔离假定的人逆转录病毒，该逆转录病毒可能导致人类肺癌的快速上升形式，即支支支支支支通电动病毒（BAC）。 3P12 TSG 人类3P12处的细胞遗传学缺失和LOH是肺癌标本的一致特征，建议在此位置存在肿瘤抑制基因（S）（TSG）。到目前为止，只有一个基因（DUTT1，在U二十二十）中被克隆了几个肺癌和乳腺癌细胞系（U2020，NCI H2198，HCC38）的重叠区域。 dutt1是与NCAM蛋白具有同源性的蝇基因机器人的人类直系同源物。 DUTT1在肺癌中的广泛分析没有发现任何突变，这表明该位置的另一个基因可能与肺癌的启动和/或发育有关。我们在重叠的临界区域中发现了一个新的小（〜230kb），嵌套的纯合缺失在SCLC细胞系GLC20中。该删除已使用多个多态标记物进行了PCR特征。 P1库筛选产生了三个覆盖整个区域和侧面的重叠克隆。这些克隆被用来通过纤维化删除的位置和大小来定义。最近，通过MIT基因组测序中心对几个覆盖该区域的BAC克隆进行了测序，该中心提供了一种基因组工具，可以在居民基因中发现。在缺失中检测到以EST簇为代表的几个基因，并正在分离。随后的突变和功能研究将确定潜在的3P12肺/乳腺癌TSG。