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3 p和8 p上的TSG，这些TSG参与了人类主要恶性肿瘤的起源或发展：肺癌、乳腺癌、肾癌和前列腺癌。我们今年的成就是：（1）我们去年鉴定的新的pVHL靶基因，即碳酸酐酶，我们进一步分析了CA 9和CA 12基因，发现CA 9和CA 12基因在许多肿瘤类型中由于VHL的缺失或其他机制而过表达，并参与了VHL的调控。细胞外pH值的变化，从而创造一个有利于肿瘤生长和扩散的微环境。Oldfield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，探讨乙酰唑胺（一种强的CA抑制剂）对伴有脑组织水肿和囊肿的脑血管母细胞瘤患者的影响，未来的工作将集中在碳酸酐酶和其他基因在调节肿瘤pH值中的作用及其对癌症生长的潜在影响。(2)3p21.3 TSG：在约370-kb的缺失重叠区域内发现了19个基因的子集。 该区域进一步细分为两个基因组的嵌套200 kb的乳腺癌纯合性缺失：8个基因位于近端~120 kb的片段和11个基因位于远端~250 kb的片段。 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的表达缺失和突变进行了测试，以从该组中鉴定候选TSGs。在非小细胞肺癌（NSCLC）（CACNA 2D 2/（a2 d-2）、SEMA 3B（以前称为SEMA（V）、）BLU、RASSF 1/A（以前称为123 F2）和HYAL 1）或小细胞肺癌（SCLC）（SEMA 3B、BLU、RASSF 1/A（以前称为123 F2）和HYAL 1）细胞系中，几种基因表现出表达缺失或mRNA水平降低。 我们发现其中6个基因具有2个或更多个氨基酸序列改变突变，包括：BLU，NPRL 2/Gene 21，FUS 1，HYAL 1，FUS 2和SEMA 3B。 然而，在肺癌样本中，测试突变的19个基因中没有一个显示出频繁（>10%）的突变率。 这使我们排除了该区域中的几个基因作为散发性肺癌的经典肿瘤抑制因子。另一方面，该位置的推定肺癌TSG可能因肿瘤获得性启动子超甲基化而失活，或者属于新的一类单倍不足基因，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，并将允许鉴定推定的肺癌TSG（s），LUCA。（三） 3 p12 TSG：人3 p12的细胞遗传学缺失和洛缺失是肺癌标本的一致特征，表明在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、HCC 38）中缺失的重叠区域克隆了一种基因（DUTT 1，U 2020年中的DUTT）。DUTT 1是与NCAM蛋白具有同源性的果蝇基因ROBO的人类直系同源物。对肺癌中DUTT 1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GLC 20中的重叠关键区域中发现了一个新的小（~ 230 kb）巢式纯合缺失。该缺失已使用几种多态性标记进行PCR表征。P1文库筛选产生覆盖整个区域和侧翼的三个重叠克隆。这些克隆用于通过纤维FISH确定缺失的位置和大小。最近，几个BAC克隆覆盖这一地区的测序由麻省理工学院基因组测序中心提供了一个基因组工具，发现在硅片上的居民基因。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3 p12肺癌TSG。