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，我们获得了该基因的整个基因组序列，包括启动子、内含子和侧翼，并构建了一组VHL小基因（野生型和突变体）和由VHL启动子驱动的完整无内含子VHL基因。然后我们开始寻找 pVHL 控制的靶基因。采用差异显示技术，使用稳定转染野生型和突变型 VHL 小基因的 UMRC6 和 786-0 细胞来发现这些基因。迄今为止（2001 年 9 月）已鉴定出 6 个下调基因，即 NOTCH2 和 DEC1（它们指定细胞命运决定并可能具有致癌潜力）、两种跨膜型碳酸酐酶 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度表达，导致功能性pVHL丧失。CAIX/XII酶可以感知细胞内pH并控制癌细胞周围环境的酸度（细胞外pH），从而创造有利于肿瘤生长和扩散的微环境。它们还在正常生理学中发挥重要作用，例如眼液、大脑的产生 通过单染色体基因转移、细胞融合和VHL基因转染对携带甲基化VHL内基因的肾癌细胞中VHL启动子的甲基化进行分析表明，在UOK 21细胞中，meth+表型占主导地位，可能是由于VHL位点的顺式作用元件的变化所致。然后，我们创建了表达人类基因组的小鼠转基因模型 VHL 基因座并证明人类 VHL 甲基化模式在小鼠发育过程中重现，并且与小鼠 VHL 基因的甲基化模式非常相似。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 基因座各种元件（例如重复序列和 VHL CpG 启动子）从头甲基化的影响。未来的工作将集中于 (i) 碳酸酐酶 (CA) 在肿瘤 pH 调节中的作用及其对癌症生长的影响，(ii) 发现这些酶的新特异性抑制剂来治疗癌症，(iii) 开发和测试基于 CAIX/XII cDNA 的疫苗来治疗癌症，以及 (iv) 参与从头异常的 VHL 基因座中顺式作用元件的性质 甲基化。 3p21.3 TSG 我们使用重叠和嵌套纯合缺失、重叠群构建、基因组测序、物理和转录图谱来进一步定义染色体 3p21.3 上含有一个或多个肿瘤抑制基因 (TSG) 的约 630 kb 肺癌纯合缺失区域。该位置是通过癌症、癌细胞系以及肺和乳腺癌前病变的体细胞遗传图谱确定的，包括发现了几个纯合缺失。分子手动方法和计算预测的结合使我们能够检测、分离、表征和注释一组 25 个基因，这些基因可能构成了这个约 630 kb 序列中的完整蛋白质编码基因集。在约 370 kb 的删除重叠区域内发现了其中 19 个基因的子集。该区域被嵌套的 200 kb 乳腺癌纯合缺失进一步细分为两个基因组：8 个基因位于近端 ~120-kb 片段，11 个基因位于远端 ~250-kb 片段。通过计算方法对这 19 个基因进行了广泛分析，并通过手动方法测试了肺癌中的表达缺失和突变，以从该组中识别候选 TSG。在非小细胞肺癌 (NSCLC)（CACNA2D2/ (a2d-2)、SEMA3B（以前称为 SEMA(V)、）BLU、RASSF1/A（以前称为 123F2）和 HYAL1）或小细胞肺癌 (SCLC)（SEMA3B、BLU、RASSF1/A（以前称为 123F2）和 HYAL1) 细胞系。我们发现其中 6 个基因具有 2 个或多个氨基酸序列改变突变，包括：BLU、NPRL2/Gene21、FUS1、HYAL1、FUS2 和 SEMA3B。然而，测试突变的 19 个基因中没有一个在肺癌样本中显示出频繁（>10%）的突变率。这导致我们排除了该区域的几个基因作为散发性肺癌的经典肿瘤抑制基因。另一方面，该位置的推定肺癌 TSG 可能因肿瘤获得性启动子高甲基化而失活，或者属于一类新的单倍体不足基因，其在半合子 (+/-) 状态下易患癌症，但在肿瘤中剩余的野生型等位基因中不显示第二个突变。通过基因转移和基因破坏策略对关键基因进行功能测试正在进行中，这将允许鉴定假定的肺癌 TSG（S），LUCA。迄今为止（2001 年 9 月），我们将 RASSF1/A 基因鉴定为涉及许多肿瘤的多个 TSG，包括肺癌、乳腺癌、前列腺癌、肾癌、头颈癌 (NPC) 等。 HYAL2 基因被鉴定为绵羊肺癌逆转录病毒 JSRV 的 GPI 锚定受体，并证明了使 HYAL2 产物失活的隔离机制。 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 HYAL2 基因产物，从而可以研究导致该系统致癌的信号转导途径。一种新的 FAS2 基因 cDNA 多态性被证明与亚洲人群中的鼻咽癌相关，具有预测价值。该基因也被确定为 NPC 的 TSG。目前的工作重点是检测和分离假定的人类逆转录病毒，该病毒可能导致人类肺癌（即细支气管肺泡腺癌（BAC））迅速上升。 3p12 TSG 人类 3p12 处的细胞遗传学缺失和 LOH 是肺癌样本的一致特征，表明该位置存在肿瘤抑制基因 (TSG)。迄今为止，仅从几种肺癌和乳腺癌细胞系（U2020、NCI H2198、HCC38）中删除的重叠区域克隆了一个基因（DUTT1，在U 20 20中删除）。 DUTT1 是果蝇基因 ROBO 的人类直系同源物，与 NCAM 蛋白具有同源性。对肺癌中 DUTT1 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发生和/或发展有关。我们在 SCLC 细胞系 GLC20 中的重叠关键区域中发现了一个新的小（约 230kb）嵌套纯合缺失。该缺失已使用多个多态性标记进行 PCR 表征。 P1 文库筛选产生了三个重叠的克隆，覆盖了整个区域和侧翼。这些克隆用于通过 Fiber-FISH 确定缺失的位置和大小。最近，麻省理工学院基因组测序中心对覆盖该区域的几个 BAC 克隆进行了测序，提供了在计算机中发现常驻基因的基因组工具。 EST 簇代表的几个基因在缺失中被检测到并正在被分离。随后的突变和功能研究将确定潜在的 3p12 肺癌/乳腺癌 TSG。