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Immortalization of SV40-Transformed Human Cells

SV40 转化的人类细胞的永生化

基本信息

批准号：
7274175
负责人：
HARVEY L OZER
金额：
$ 36.76万
依托单位：
UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-12-01 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7274175
关键词：
6q27 Acute Antibodies Apoptotic Biological Assay Candidate Disease Gene Cell Aging Cell Death Cell Line Cell Proliferation Cell fusion Cells Chromosome Transfer Chromosomes, Human, Pair 6 Code Cytogenetics DNA DNA Sequence DNA-Mediated Gene Transfer Databases Deoxycytidine Diploidy Fibroblasts Frequencies Gene Expression Gene Silencing Genes Genomics Goals Growth Haploidy Histone Deacetylation Human Human Cell Line Ligands Localized Longevity Loss of Heterozygosity Mediating Messenger RNA Methylation Modeling Molecular Genetics Mutagenesis Mutation Oncogenes Open Reading Frames Pattern Process Protein Overexpression RNA Interference Receptor Signaling Reporting Reverse Transcriptase Polymerase Chain Reaction Role Series Signal Pathway Simian virus 40 Small Interfering RNA Spheroplasts System Testing Tetanus Helper Peptide Tumor Suppressor Genes Tumor Suppressor Proteins Tumor-Suppressor Gene Inactivation Upper arm Viral Tumor Antigens Yeasts base carcinogenesis cell transformation genome database glycoprotein G immortalized cell neurofibroma notch protein novel response restoration senescence

项目摘要

DESCRIPTION (provided by applicant): Normal human diploid fibroblasts (HF) undergo replicative senescence at the end of their limited lifespan and acute senescence in response to over expression of oncogenes, suggesting senescence is a tumor suppressor mechanism. We have demonstrated that transformation of HF by replication-defective SV40 sequences results in extension of lifespan (which terminates in crisis as apoptotic cell death) and promotes continuous cell proliferation, i.e. immortalization. We have developed an experimental system involving matched sets of SV40-transformed non-immortal HF (SV/HF) and their immortal derivatives to identify cellular genes involved in these processes. We have localized a gene (termed SEN6) with features of a growth suppressor to a region at 6q27, whose inactivation is involved in the immortalization of SV/HF. A series of complementary strategies are proposed to identify SEN6. We will test which of three mechanisms shown to result in inactivation of tumor suppressor genes in humans is (are) responsible; namely, loss of heterozygosity and mutation, haploinsufficiency, and/or gene silencing. Candidate genes for SEN6 identified using the Human Genome Database and the expression pattern of encoded mRNAs and ORF will be tested first by DNA-mediated gene transfer into non-immortal SV/HF for promotion of immortalization by small interfering RNA(s). We will initially assess the role of DLL-1, a ligand for the Notch receptor-signaling pathway, as a candidate based on recent information on the role of the Notch-1 in transformed cells and favorable experimental aspects of DLL-1 (moderate mRNA levels in SV/HF and availability of antibody to DLL-1). We have furthermore identified an SV40-transformed human cell line, which has a single copy of chromosome 6 (mono6) for direct test of the role of haploinsufficiency or as a target for mutagenesis of haploid SEN6. Alternatively, we will exploit YAC and/or BAC transfer to further localize the region encoding SEN6. Toward that goal, we have demonstrated that manipulation of the yeast host can markedly increase DNA transfer to human cells and propose to develop the approach further. Altogether these studies are expected to result in isolation of sequences encoding SEN6 and enhance our understanding of mechanisms of senescence and carcinogenesis as well as transformation/immortalization of SV/HF.

描述（由申请方提供）：正常人二倍体成纤维细胞（HF）在其有限寿命结束时经历复制性衰老，并响应于癌基因的过度表达而发生急性衰老，表明衰老是一种肿瘤抑制机制。我们已经证明，通过复制缺陷型SV 40序列转化HF导致寿命延长（其在凋亡性细胞死亡的危机中终止），并促进连续的细胞增殖，即永生化。我们已经开发了一个实验系统，涉及匹配的SV 40转化的非永生HF（SV/HF）和他们的不朽衍生物，以确定参与这些过程的细胞基因。我们已经定位了一个基因（称为SEN 6）的功能的生长抑制剂的区域在6 q27，其失活参与了SV/HF的永生化。提出了一系列的补充策略来识别SEN 6。我们将测试导致人类肿瘤抑制基因失活的三种机制中的哪一种是负责的;即杂合性和突变的丢失，单倍不足和/或基因沉默。使用人类基因组数据库和编码mRNA和ORF的表达模式鉴定的SEN 6候选基因将首先通过DNA介导的基因转移到非永生SV/HF中以通过小干扰RNA促进永生化来测试。我们将首先评估DLL-1（Notch受体信号传导途径的配体）作为候选物的作用，这是基于Notch-1在转化细胞中的作用的最新信息和DLL-1的有利实验方面（SV/HF中的中等mRNA水平和DLL-1抗体的可用性）。我们还鉴定了SV 40转化的人细胞系，其具有单拷贝的6号染色体（mono 6），用于直接测试单倍不足的作用或作为单倍SEN 6诱变的靶。或者，我们将利用YAC和/或BAC转移来进一步定位编码SEN 6的区域。为了实现这一目标，我们已经证明，操纵酵母宿主可以显着增加DNA转移到人类细胞，并建议进一步开发这种方法。总之，这些研究有望导致分离编码SEN 6的序列，并增强我们对SV/HF的衰老和致癌机制以及转化/永生化的理解。