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: 6289207
• 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/6289207
• 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 found in the deletions overlap of 370-kb were subdivided by a nesting deletion into two gene sets: eight genes lying in the proximal 120-kb segment and 11 genes lying in the distal 250-kb segment. Both gene sets were analyzed extensively by manual and computational methods. Four of the 19 genes showed loss-of-expression or reduced mRNA levels in small cell (SEMA V) or non-small cell (a2d-2) or both (BLU and LUCA1) cancer cell lines. None of the 8 genes showed a frequent (>10%) mutation rate in lung cancer samples leading to conclude that with the exception of the three genes with reduced expression in this set they should be excluded as classical tumor suppressors in sporadic lung cancer. The mutation analysis of the 11 gene set is not yet completed and may reveal a TSG with homozygotic inactivation in tumors. Further mutational analysis in breast tumors and functional testing of the critical genes by gene transfer and gene disruption strategies is ongoing and should permit the identification of the putative TSG(s) among this gene set.(3) The 3p12 TSG:we discovered two new homozygous deletions in SCLC lines (250kb) removing part of the DUTT1 gene. We hypothesized that these deletions may target another cancer gene that may reside in a large intron of DUTT1. To search for this gene we prepared a sequence ready p1phage contig. We also requested the WU Sequencing Center to prepare a 1-mb BAC contig (between markers D3S3681 and D3S1604) that includes the deleted area for sequencing in the near future. (4) The 8p22 TSG:we established the intro-exon structure of the HP gene we cloned last year (GenBank # AFO26219) and are conducting mutation analyses in lung, breast, and prostate cancers. - Carcinogenesis, hereditary cancer, Lung cancer, Oncogenes, Tumor Suppressor, - Human Tissues, Fluids, Cells, etc.

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，研究乙酰唑胺（一种强的CA抑制剂）对脑血管母细胞瘤伴脑组织水肿和囊肿患者的影响，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370-kb的缺失重叠中发现的19个基因的子集通过嵌套缺失被细分为两个基因集：8个基因位于近端120-kb区段，11个基因位于远端250-kb区段。这两个基因集进行了广泛的手动和计算方法分析。19个基因中的4个在小细胞（SEMA V）或非小细胞（a2 d-2）或两者（BLU和LUCA 1）癌细胞系中表现出表达丧失或mRNA水平降低。这8个基因中没有一个在肺癌样品中显示出频繁（>10%）突变率，从而得出结论，除了在该组中具有降低表达的三个基因之外，它们应该被排除作为散发性肺癌中的经典肿瘤抑制因子。11个基因集的突变分析尚未完成，可能揭示肿瘤中具有纯合子失活的TSG。正在进行乳腺肿瘤的进一步突变分析和通过基因转移和基因破坏策略对关键基因进行功能测试，并应允许在该基因集中鉴定推定的TSG。(3)3 p12 TSG：我们在SCLC细胞系（250 kb）中发现了两个新的纯合缺失，去除了部分DUTT 1基因。我们假设这些缺失可能针对另一个可能位于DUTT 1大内含子中的癌症基因。为了寻找这个基因，我们制备了一个序列准备p1噬菌体重叠群。我们还要求WU测序中心准备一个1-mb BAC重叠群（标记D3 S3681和D3 S1604之间），其中包括在不久的将来测序的缺失区域。(4)8 p22 TSG：我们建立了去年克隆的HP基因的内含子-外显子结构（GenBank #AFO 26219），并正在肺癌、乳腺癌和前列腺癌中进行突变分析。- 致癌作用、遗传性癌症、肺癌、致癌基因、肿瘤抑制因子、-人体组织、液体、细胞等。