Cloning tumor suppressor genes (TSG) from human chromosomes 3p and 8p

从人类染色体 3p 和 8p 克隆肿瘤抑制基因 (TSG)

• 批准号: 6433098
• 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/6433098
• 关键词: chromosomes; clone cells; computer assisted sequence analysis; human genetic material tag; human tissue; lung neoplasms; molecular cloning; molecular oncology; neoplasm /cancer genetics; nucleic acid sequence; single strand conformation polymorphism; tumor suppressor genes

The goal of this research is to identify,clone,and characterize TSGs located on chromosomes 3p and 8p that are involved in the origin or development of major human malignancies: carcinomas of the lung, breast,kidney, and prostate.Our accomplishments this year are:(1) the novel pVHL target genes identified by us last year,namely,the carbonic anhydrases, CA9 and CA12 were further analyzed.We showed that the CA9 and CA12 genes are overexpressed in many tumor types due to the loss of VHL or other mechanisms and are involved in the control of the extracellular pH of the miliew surrounding the cells and thus create a microenvironment conducive to tumor growth and spread.Based on these finding Dr. E. Oldfield and a group of surgeons at the NIH Clinical Center initiated a prospective, non-randomized study of the effect of acetazolamide, a strong inhibitor of CAs, in patients with brain hemangioblastomas associated with brain tissue edema and cysts.Future work will focus on the role of carbonic anhydrases and othes genes in the regulation of tumor pH and its potential impact on cancer growth.(2) The 3p21.3 TSG:A subset of 19 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. The 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. (3) 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 cancer TSG.

本研究的目标是鉴定、克隆和表征位于染色体3p和8p上的、参与人类主要恶性肿瘤：肺癌、乳腺癌、肾癌和前列腺癌的起源或发展的TSG。今年我们的成果是：（1）我们去年鉴定的新的pVHL靶基因，即碳酸酐酶CA9和CA12，得到了进一步的进展。 我们分析发现，由于 VHL 缺失或其他机制，CA9 和 CA12 基因在许多肿瘤类型中过度表达，并参与细胞周围环境的细胞外 pH 值的控制，从而创造有利于肿瘤生长和扩散的微环境。基于这些发现，E. Oldfield 博士和 NIH 临床中心的一组外科医生发起了一项前瞻性、非随机研究，研究 乙酰唑胺是一种强效 CAs 抑制剂，用于治疗伴有脑组织水肿和囊肿的脑血管母细胞瘤患者。未来的工作将集中于碳酸酐酶和其他基因在肿瘤 pH 调节中的作用及其对癌症生长的潜在影响。(2) 3p21.3 TSG：在约 370-kb 的删除重叠区域内发现了 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，这将允许识别假定的肺癌 TSG（s），LUCA。 (3) 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，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鉴定潜在的 3p12 肺癌 TSG。