IDENTIFICATION OF 3P RECESSIVE ONCOGENES IN LUNG CANCER

隐性癌基因（肿瘤抑制基因）在肿瘤发生中起主要作用。 人肺癌的发病机制。 3 P是最常见的 肺癌中的染色体区域， 一个或多个新的隐性致癌基因。细胞遗传学和分子 3 p等位基因丢失的证据发生在>80%的小细胞肺癌（SCLC）中， >50%的非小细胞肺癌（NSCLC）。 事实上证据是 提示位于3 p25的几个3 p不同的隐性癌基因， 3p21.3（两个位点）、3p14.2和3 p12 -13。癌前病变研究 表明3 p21等位基因的丢失是最早的改变之一， 在癌前病变中发现（发生在增生阶段） 这表明一个或多个3 p隐性癌基因起着“守门人”的作用 肺癌的分子发病机制。 具体目标是 项目是：（具体目标#1）通过定位克隆分离新的 位于3p21.3的隐性癌基因。 这将使用 独特的试剂，包括完整的粘粒重叠群和cDNA克隆， >25种不同的基因，由Minna博士和他的 合作者覆盖了一系列重叠区域中最短的区域， 在SCLC基因组DNA中发现巢式纯合缺失。 基因将 通过在开放阅读框中寻找突变来鉴定 这些cDNA使用SSCP和DNA测序技术。 具体目标#2 是通过以下方式确定这个位点的功能特征 通过微细胞介导的染色体转移， 将人类染色体3 p的基因导入到携带 3p21.3区域的纯合缺失，然后检测 裸鼠成瘤性抑制，软琼脂集落 细胞培养物中的形成和诱导凋亡（程序化细胞 （死亡）Dr. Killary。 具体目标#3拟用于抑制试验 端粒缩短的发展，以及端粒的丧失。 肺癌中永生细胞生长，引入候选物后 来自3p21.3纯合缺失的cDNA和来自3p21.3纯合缺失的cDNA以及来自3p21.3纯合缺失的cDNA。 使用Shay博士开发的独特检测方法， 实验室 翻译的目标是应用这些信息来开发新的 肿瘤前病变中遗传变化的鉴定方法 用于非常早期的肺癌诊断;用作替代分子 标记;通过生殖系突变寻找遗传易感性， 基因;并有可能开发肿瘤特异性治疗。 研究 翻译涉及细胞遗传学，等位基因丢失， 癌前病变、纯合缺失发现和定位克隆 信息. 所需的步骤是确定候选人3p21.3 隐性癌基因的纯合缺失，决定其开放 肺癌的阅读框序列和表达，筛查 改变一级序列的突变，突变的证明 在肿瘤细胞系、原发性肿瘤和癌前病变中， 突变的存在是否意味着极高的风险 个人和可逆性的病变与化学预防 （替代标记），突变基因的种系遗传试验， 这些是否会导致遗传性癌症易感综合征;以及， 在研究该基因功能的基础上，设计其他诊断试剂， 测试和潜在的治疗方法。 因此，该项目与 项目#2（肺癌的遗传易感性），项目#3 （肺癌的分子早期检测）和项目#4 （肺癌的化学预防）。

Recessive oncogenes (tumor suppressor genes) play a major role in the pathogenesis of human lung cancer. However, 3p is the most frequently involved chromosomal region in lung cancer suggesting the location of one or more new recessive oncogene(s). Cytogenetic and molecular evidence for allele loss of 3p occurs in >80% of small cell (SCLC) and >50% of non-small cell (NSCLC) lung cancers. In fact the evidence is suggestive of several 3p distinct recessive oncogenes located at 3p25, 3p21.3 (two sites), 3p14.2, and 3p12-13. Studies of preneoplasia indicate the loss of 3p21 alleles is one of the earliest alterations found in preneoplastic lesions (occurring at the stage of hyperplasia) suggesting one or more 3p recessive oncogenes functions as "gatekeepers" in the molecular pathogenesis of lung cancer. The specific aims of this project are: (specific aim #1) to isolate by positional cloning a new recessive oncogene residing at 3p21.3. This will be done using the unique reagents including a complete cosmid contig and cDNA clones for >25 different genes, that have been assembled by Dr. Minna and his collaborators covering the shortest region of overlap in a series of nested homozygous deletions found in SCLC genomic DNAs. The gene will be identified by searching for mutations in the open reading frame of these cDNAs using SSCP and DNA sequencing techniques. Specific aim #2 is to determine the functional characteristics of this locus by introducing, through microcell mediated chromosome transfer, a portion of human chromosome 3p into a human lung cancer line bearing a homozygous deletion for the 3p21.3 region and then testing for suppression of tumorigenicity in nude mice, soft agarose colony formation in cell culture, and induction of apoptosis (programmed cell death) by Dr. Killary. Specific aim #3 proposed to test for suppression of telomrase activity, development of telomere shortening, and loss of immortal cell growth in lung cancer, following introduction of candidate cosmids and cDNAs from the 3p21.3 homozygous deletion as well as portions of chromosome 3 using unique assays developed by Dr. Shay's lab. The translational goal is to apply this information to develop new methods for identification of genetic changes in preneoplastic lesions for very early lung cancer diagnosis; use as a surrogate molecular marker; search for genetic predisposition via germline mutations in the gene; and potentially to develop tumor specific therapy. The research being translated involves cytogenetic, allele loss, genetic changes in preneoplasia, homozygous deletion discovery, and positional cloning information. The steps required are identification of candidate 3p21.3 recessive oncogenes in the homozygous deletion, determining their open reading frame sequence and expression in lung cancer, screening for mutations that alter the primary sequence, demonstration of mutations in tumor cell lines, primary tumors, and preneoplastic lesions, testing for whether the presence of the mutation identifies very high risk individuals and the reversibility of the lesions with chemoprevention (surrogate markers), tests for germline inheritance of mutant genes and whether these lead to an inherited cancer predisposition syndrome; and, based on studies of the function of the gene, designing other diagnostic tests and potential therapies. Thus, this project interacts with Project #2 (Genetic Susceptibility to Lung Cancer), Project #3 (Molecular early Detection of Lung Cancer), and Project #4 (Chemoprevention of Lung Cancer).