DIFFERENTIAL GENE-EXPRESSION IN SV40-MEDIATED IMMORTALIZATION OF HUMAN FIBROBLASTS

Normal human diploid fibroblasts (HF) have a limited life span, underg o senescence, and rarely, ii ever, spontaneously immortalize in cultur e. introduction of the gene for T antigen encoded by the DNA virus SV4 0 extends the life span of HF and increases the frequency of immortali zation; however, immortalization requires both T-dependent and T-indep endent 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 func tions responsible for immortalization. In this study we undertook a se arch for cellular mRNAs which are differentially expressed upon immort alization. A lambda cDNA library was prepared from a pre-immortal SV40 -transformed HF (HF-C). We screened the library with a subtracted prob e enriched for sequences present in HF-C and reduced in immortal AR5 c ells. A more limited screen was also employed for sequences overexpres sed in AR5 using a different strategy. Alterations in the level of mRN As in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an ef fort to clarify which of the altered mRNAs are most relevant to immort alization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) S V40-transformants using eight cloned cDNAs which show reduced expressi on in AR5. Three of these were reduced in additional immortal cell lin es as well; one, J4-4 (unknown function) is reduced in all the immorta l cell lines tested; a second, J4-3 (possible PP2C type phosphatase) i s reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown funct ion) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should exte nd our understanding of the molecular bases for immortalization. In pa rticular, the patterns of expression of J4-4 and J4-3 strongly suggest that they are involved in the process of immortalization and/or can s erve as target genes for assessing regulators of gene expression in th is process. (C) 1997 Wiley-Liss, Inc.