Immortalization of SV40-Transformed Human Cells

描述（由申请人提供）：正常人类二倍体成纤维细胞（HF）在其有限寿命结束时发生复制性衰老，并因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SV40序列转化HF导致寿命延长（在凋亡细胞死亡的危机中终止），并促进细胞连续增殖，即永生。我们开发了一个实验系统，涉及匹配的sv40转化的非不朽HF （SV/HF）及其不朽衍生物，以鉴定参与这些过程的细胞基因。我们已经将一个具有生长抑制特征的基因（称为SEN6）定位在6q27的一个区域，该区域的失活与SV/HF的永生化有关。提出了一系列互补策略来识别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负责任的；即，杂合性缺失和突变、单倍性不足和/或基因沉默。利用人类基因组数据库鉴定出SEN6候选基因，编码mrna和ORF的表达模式将首先通过dna介导的基因转移到非不朽的SV/HF中进行测试，以通过小干扰RNA促进永生。我们将根据Notch受体信号通路的配体DLL-1在转化细胞中的作用的最新信息和DLL-1有利的实验方面（SV/HF中中等mRNA水平和针对DLL-1的抗体的可用性），初步评估其作为候选物的作用。我们进一步鉴定了sv40转化的人类细胞系，该细胞系具有单个6号染色体拷贝（mono6），用于直接测试单倍体功能不全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传输来进一步定位编码SEN6的区域。为了实现这一目标，我们已经证明了对酵母宿主的操纵可以显著增加DNA向人类细胞的转移，并建议进一步发展这种方法。总之，这些研究有望分离编码SEN6的序列，并加深我们对SV/HF的衰老和致癌机制以及转化/永生的理解。

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.