Subtractive Hybridization of mRNA from early passage and senescent endothelial cells

Regulation of cellular processes that eventually lead to a state of growth arrest is an important manifestation of in vitro cellular senescence caused and accompanied by variations of the gene expression pattern. Whereas thes e changes at the mRNA level have been studied mainly in fibroblast cultures , we concentrated on endothelial cells that represent an accepted model for Vascular systems and may be involved in the pathogenesis of diseases relat ed to aging. To isolate differentially expressed genes, we created a subtra ctive cDNA library using mRNA from senescent (35 passages) and young (five passages) human umbilical vein endothelial cells (HUVECs). Candidate clones were isolated from the cDNA library, differential expression was confirmed by Northern blot analyses and sequences were compared with a genbank data base. Because many mRNAs were below the detection limit of Northern blot an alysis, we were forced to establish a more sensitive PCR based method (ATAC -PCR) to quantify and confirm altered levels of gene expression. Several mR NAs were found to be upregulated in senescent HUVECs including two componen ts of the extracellular matrix (ECM): plasminogen activator inhibitor and f ibronectin. Elevated expression of both has already been described in senes cent cells. The mRNAs of TGF-beta-inducible gene H3 (beta-IG-H3; ECM protei n), insulin-like growth factor binding protein (IGFBP-3), p53-inducible gen e (PIG3) a protein involved in vesicular transport (SEC13R) and ribosomal p rotein L28 have likewise been shown to be preferentially expressed in senes cent cells. Because studies support the involvement of ECM components, TGF- beta and p53 in tumor suppressing mechanisms, our data supports the hypothe sis that cellular senescence and upregulation of ECM proteins may be associ ated with tumor preventive functions. (C) 2000 Elsevier Science Inc. All ri ghts reserved.