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 乙酰唑酰胺是一种强效 CA 抑制剂，用于治疗与脑组织水肿和囊肿相关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：对 370-kb 的缺失重叠中发现的 19 个基因的子集进行了细分 通过嵌套删除到两个基因组中：8 个基因位于近端 120 kb 片段中，11 个基因位于远端 250 kb 片段中。通过手动和计算方法对这两个基因组进行了广泛的分析。 19 个基因中有 4 个在小细胞 (SEMA V) 或非小细胞 (a2d-2) 或两者（BLU 和 LUCA1）癌细胞系中表现出表达缺失或 mRNA 水平降低。这 8 个基因中没有一个在肺癌样本中显示出频繁的 (>10%) 突变率，从而得出结论，除了这组中表达减少的三个基因之外，它们应该被排除在散发性肺癌中作为经典肿瘤抑制因子。 11个基因组的突变分析尚未完成，可能揭示肿瘤中纯合失活的TSG。乳腺肿瘤的进一步突变分析以及通过基因转移和基因破坏策略对关键基因进行的功能测试正在进行中，并且应该能够在该基因集中鉴定假定的 TSG。(3) 3p12 TSG：我们在 SCLC 系 (250kb) 中发现了两个新的纯合缺失，去除了 DUTT1 基因的部分。我们假设这些缺失可能针对另一个癌症基因，该基因可能位于 DUTT1 的大内含子中。为了寻找该基因，我们准备了序列就绪的 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 D3S3681 和 D3S1604 之间），其中包括删除的区域，以便在不久的将来进行测序。 (4) 8p22 TSG：我们建立了去年克隆的 HP 基因的内显子结构（GenBank # AFO26219），并正在进行肺癌、乳腺癌和前列腺癌的突变分析。 - 癌变、遗传性癌症、肺癌、癌基因、肿瘤抑制因子、 - 人体组织、体液、细胞等。