Pardinas J, Pang Z, Houghton J, Palejwala V, Donnelly R J, Hubbard K, Small M B, Ozer H L
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark 07103, USA.
J Cell Physiol. 1997 Jun;171(3):325-35. doi: 10.1002/(SICI)1097-4652(199706)171:3<325::AID-JCP11>3.0.CO;2-9.
Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A lambda cDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can serve as target genes for assessing regulators of gene expression in this process.
正常人类二倍体成纤维细胞（HF）寿命有限，会经历衰老，并且在培养中极少（如果有的话）能自发永生化。由DNA病毒SV40编码的T抗原基因的导入延长了HF的寿命并增加了永生化频率；然而，永生化需要T依赖性和T非依赖性功能。我们之前生成了独立的SV40转化的非永生化（前永生化）HF细胞系，然后从中获得永生化亚系，这是我们多方面方法的一部分，目的是识别负责永生化的功能。在本研究中，我们寻找在永生化过程中差异表达的细胞mRNA。从一个前永生化的SV40转化的HF（HF-C）制备了一个λ cDNA文库。我们用一个经过消减的探针筛选该文库，该探针富集了HF-C中存在且在永生化的AR5细胞中减少的序列。还采用了一种更有限的筛选方法，使用不同策略筛选在AR5中过表达的序列。鉴定出AR5中与信号转导途径相关功能的mRNA水平发生了变化；然而，大多数cDNA编码的是新序列。为了弄清楚哪些改变的mRNA与永生化最相关，我们使用从三对独立的前永生化和永生化（4个细胞系）SV40转化体中制备的RNA进行Northern分析，使用了8个在AR5中表达降低的克隆cDNA。其中三个在其他永生化细胞系中也降低；一个，J4-4（功能未知）在所有测试的永生化细胞系中都降低；第二个，J4-3（可能是PP2C型磷酸酶）在3个匹配组中的2个中降低；第三个，J2-2（功能未知）在2个不相关的永生化细胞系中降低。尽管这些基因的作用尚不清楚，但对它们的进一步分析应该能扩展我们对永生化分子基础的理解。特别是，J4-4和J4-3的表达模式强烈表明它们参与了永生化过程和/或可作为评估该过程中基因表达调节因子的靶基因。
