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370-kb的缺失重叠中发现了19个基因，通过嵌套缺失将其细分为两个基因集：近端120-kb片段有8个基因，远端250-kb片段有11个基因。通过人工和计算方法对这两个基因集进行了广泛的分析。19个基因中的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突变率（bb0 - 10%），从而得出结论，除了这组基因中表达减少的3个基因外，它们应被排除为散发性肺癌的经典肿瘤抑制基因。11个基因组的突变分析尚未完成，可能在肿瘤中发现纯合子失活的TSG。通过基因转移和基因破坏策略对乳腺肿瘤的进一步突变分析和关键基因的功能测试正在进行中，并将允许在该基因集中识别假定的TSG(s)。(3) 3p12 TSG：我们在SCLC系中发现了两个新的纯合缺失（250kb），删除了部分DUTT1基因。我们假设这些缺失可能针对另一个可能存在于DUTT1大内含子中的癌症基因。为了寻找这个基因，我们准备了一个序列准备好的噬菌体序列。我们还要求吴测序中心准备一个1 mb的BAC序列（在标记D3S3681和D3S1604之间），其中包括被删除的区域，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（GenBank # AFO26219）的内外显子结构，并正在进行肺癌、乳腺癌和前列腺癌的突变分析。-致癌，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。

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.