Identification of replicative senescence-associated genes in human umbilical vein endothelial cells by an annealing control primer system

Abstract

Cellular senescence is regulated by specific genes in many organisms. The identification and functional analysis of senescence-associated genes could provide valuable insights into the senescence process. Here, we employed a new and improved differential display reverse transcription-polymerase chain reaction (DDRT-PCR) method that involves annealing control primers (ACPs) to identify genes that are differentially expressed in human umbilical endothelial cells during replicative senescence. Using 120 ACPs, we identified 31 differentially expressed genes (DEGs). Basic local alignment search tool (BLAST) search revealed 29 known genes and two unknown genes. Expression levels of the 29 known genes were confirmed by real-time quantitative RT-RCR and by Western blotting for eight of these genes. CD9 antigen, MHC class I chain-related sequence A (MICA) and cell division cycle 37 homolog (CDC37) were up-regulated, and bone morphogenetic protein 4 (BMP4), dickkopf-1 (DKK1), and transcription factor 7-like 1 (TCF7L1) were down-regulated in old cells. Treatment with recombinant human MICA caused a decrease in cell proliferation and an increase in senescence-associated β-galactosidase staining. Further analysis of differentially expressed genes may provide insights into the molecular basis of replicative senescence and vascular diseases associated with cellular senescence.

Introduction

Replicative senescence is the limited capacity of somatic cells to divide when cultured in vitro and is commonly studied as a model of biological aging (Hayflick and Moorhead, 1961). The phenotype of replicative senescence in human diploid fibroblasts (HDFs) is characterized by irreversible growth arrest in the transition from phase G1 to phase S of the cell cycle (Chen et al., 2000), larger and flattened cell morphology (Wagner et al., 2001a), expression of senescence-associated β-galactosidase (SA-β-gal) (Dimri et al., 1995), short telomeres (Deng et al., 2003, Harley et al., 1990), and altered gene expression (Cristofalo et al., 1998). Senescence occurs in a variety of cell types besides fibroblasts, including glial cells (Huang et al., 2006), keratinocytes (Kang et al., 2005), endothelial cells (Eman et al., 2006, Mueller et al., 1980), and is commonly accompanied by a specific set of changes in cell morphology, gene expression, and function. Using endothelial cells derived from human umbilical vein (HUVECs), in vitro senescence models have been described (Garfinkel et al., 1994, Wagner et al., 2001b) that might contribute to in vivo vascular cell aging and may thereby reveal pathomechanisms relevant to senescence-associated disorders of the human vasculature. Replicative senescence is associated with up- and down-regulation of a variety of genes involved in inflammation, cell cycle regulation, cytoskeleton, etc. (Yoon et al., 2004). Novel genes identified during cellular senescence have been analyzed by serial analysis of gene expression (SAGE) (Untergasser et al., 2002), differential display PCR (DD-PCR) (Linskens et al., 1995), and cDNA microarray technology (Yoon et al., 2004). Recently, an improved method to identify differentially expressed genes (DEGs) was developed that uses annealing control primers (ACPs) (Hwang et al., 2003, Kim et al., 2004).

In this study, to explore novel senescence-associated genes and to investigate their role in cellular senescence, we cultured HUVECs until they reached replicative senescence. We then identified DEGs associated with replicative senescence using ACPs. The expression levels of DEGs were validated by quantitative real-time RT-PCR and Western blot analysis. The possible roles for these genes in replicative senescence are discussed.