CLONING TUMOR SUPPRESSOR GENES (TSG) FROM HUMAN CHROMOSOMES 3P AND 8P

本研究的目标是鉴定、克隆和鉴定位于染色体3p和8p上的与人类主要恶性肿瘤：肺癌、乳腺癌、肾癌和前列腺癌的发生或发展有关的TSG。我们今年的成果有：(1)我们去年发现的新的pVHL靶基因，即碳酸氢酶，对CA9和CA12进行了进一步的分析。我们发现，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度表达，并参与控制细胞周围基质的细胞外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NIH临床中心的一组外科医生发起了一项前瞻性的非随机研究，探讨了CA9和CA12的作用。未来的工作将集中在碳酸氢酶等基因在调节肿瘤pH值中的作用及其对肿瘤生长的潜在影响。(2)3p21.3TSG：在370-kb的重叠缺失中发现的19个基因的亚集通过嵌套缺失被细分为两个基因集：8个基因位于120kb的近端，11个基因位于250kb的远端。这两组基因都通过人工和计算方法进行了广泛的分析。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)癌细胞系中表达缺失或降低。这8个基因在肺癌标本中均未显示出频繁的突变率(&gt；10%)，从而得出结论，除了这组基因中表达降低的3个基因外，它们都应该被排除在散发性肺癌中作为典型的抑癌基因。11基因集的突变分析尚未完成，可能揭示肿瘤中存在纯合失活的TSG。进一步的乳腺肿瘤突变分析和通过基因转移和基因中断策略对关键基因的功能测试正在进行中，应该可以在这个基因集中确定可能的TSG(S)。(3)3p12 TSG：我们在小细胞肺癌系(250kb)中发现了两个新的纯合子缺失，移除了部分DUTT1基因。我们推测，这些缺失可能针对另一个癌症基因，该基因可能位于DUTT1的一个大内含子中。为了寻找这个基因，我们准备了一个序列准备好的噬菌体重叠群。我们还要求吴氏测序中心准备一个1mb的BAC重叠群(位于标记D3S3681和D3S1604之间)，其中包括缺失的区域，以便在不久的将来进行测序。(4)8p22TSG：我们建立了去年克隆的Hp基因的外显子内结构(GenBank#AFO26219)，并正在进行肺癌、乳腺癌和前列腺癌的突变分析。-致癌、遗传癌、肺癌、癌基因、肿瘤抑制因子-人体组织、体液、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.