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

本研究的目的是鉴定、克隆和表征位于染色体3p和8p上的tsg，这些tsg参与了人类主要恶性肿瘤（肺癌、乳腺癌、肾癌和前列腺癌）的起源或发展。我们今年的成果有：(1)我们去年新鉴定的pVHL靶基因，即碳酸酐酶、CA9和CA12进一步分析。我们发现，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，并参与控制细胞周围环境的细胞外pH，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博士和美国国立卫生研究院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（一种强ca抑制剂）对与脑组织水肿和囊肿相关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肿瘤pH调节中的作用及其对肿瘤生长的潜在影响。(2) 3p21.3 TSG：在约370-kb的缺失重叠区发现19个基因亚群。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集：近端~120-kb段有8个基因，远端~250-kb段有11个基因。通过计算方法对这19个基因进行了广泛的分析，并通过人工方法对肺癌中的表达缺失和突变进行了测试，以从该组中确定候选tsg。一些基因在非小细胞肺癌（NSCLC） (CACNA2D2/ (a2d-2)、SEMA3B（以前的SEMA（V)、）BLU、RASSF1/A（以前的123F2）和HYAL1）细胞系或小细胞肺癌（SCLC） （SEMA3B、BLU、RASSF1/A（以前的123F2）和HYAL1）细胞系中表现出表达缺失或mRNA水平降低。我们发现6个基因有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，19个突变基因中没有一个在肺癌样本中显示出频繁的突变率（10%）。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因子。另一方面，该位置的假定肺癌TSG可能因肿瘤获得性启动子超甲基化而失活，或者属于一类新的单倍不足基因，这些基因在半合子（+/-）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。正在通过基因转移和基因破坏策略对关键基因进行功能测试，并将允许确定假定的肺癌TSG(s)， LUCA。(3) 3p12 TSG：人类3p12的细胞遗传学缺失和LOH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, HCC38）中缺失的重叠区域克隆出一个基因（DUTT1, Deleted in U Twenty Twenty）。DUTT1是苍蝇基因ROBO的人类同源物，与NCAM蛋白具有同源性。对DUTT1在肺癌中的广泛分析未发现任何突变，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的重叠关键区域发现了一个新的小的（~230kb）嵌套纯合缺失。这种缺失已经用多态标记进行了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。这些克隆被用来通过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，为在计算机上发现驻留基因提供了基因组工具。在缺失中检测到以EST簇为代表的几个基因，并正在进行分离。随后的突变和功能研究将确定潜在的3p12肺癌TSG。

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.