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.

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我们表明，由于VHL或其他机制的丧失，CA9和CA12基因在许多肿瘤类型中过表达，并且参与了细胞周围的MILIEW的细胞外pH的控制，从而在这些发现的E. Oldfield和NIH的一组临床中启动的临床中心促进了肿瘤的生长，从而产生了一个微环境，从而创造了一个微观环境。与脑组织水肿和囊肿相关的脑血管血管群患者的乙酸唑胺是一种强大的CAS抑制剂。FUTURE工作将集中于碳酸酐酶和OTHES基因在调节肿瘤pH的调节中的作用及其对癌症生长的潜在影响。（2）3p21.3 tsg：3p21.3 tsg：在19个基因中均已被划分。 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，并将允许鉴定推定的肺癌TSG LUCA。 （3）人类3P12处的3P12 TSG：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肺癌TSG。