Immortalization of SV40-Transformed Human Cells

描述(申请人提供)：正常人二倍体成纤维细胞(HF)在其有限的寿命结束时经历复制衰老和急性衰老，以响应癌基因的过度表达，表明衰老是一种肿瘤抑制机制。我们已经证明，通过复制缺陷的SV40序列转化HF会导致寿命的延长(最终导致细胞凋亡)，并促进细胞的持续增殖，即永生。我们已经开发了一套实验系统，涉及SV40转化的非永生HF(SV/HF)及其永生衍生物的配对组，以识别参与这些过程的细胞基因。我们已经将一个具有生长抑制基因特征的基因(称为SEN6)定位在6q27的区域，该基因的失活与SV/HF的永生化有关。提出了一系列补充策略来识别SEN6。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有责任的；即杂合性丢失和突变、单倍体不足和/或基因沉默。利用人类基因组数据库确定的SEN6候选基因及其编码的mRNAs和ORF的表达模式将首先通过DNA介导的基因转移到非永生的SV/HF中以促进小干扰RNA的永生化(S)。我们将根据有关Notch-1在转化细胞中的作用的最新信息以及DLL-1的有利实验方面(SV/HF中适度的mRNA水平和DLL-1抗体的可用性)，初步评估作为候选的Notch受体信号通路配体Dll-1的作用。此外，我们还鉴定了一个SV40转化的人类细胞系，它具有单一的6号染色体(Mon6)副本，用于直接测试单倍体不足的作用或作为单倍体SEN6突变的目标。或者，我们将利用YAC和/或BAC转移来进一步定位编码SEN6的区域。为了实现这一目标，我们已经证明了操纵酵母宿主可以显著增加DNA转移到人类细胞，并建议进一步开发这一方法。总之，这些研究有望分离出编码SEN6的序列，并加深我们对衰老和致癌机制以及SV/HF转化/永生化的理解。

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.