[CANCER RESEARCH 62, 6255– 6262, November 1, 2002] Characterization of Epithelial Senescence by Serial Analysis of Gene Expression: Identification of Genes Potentially Involved in Prostate Cancer 1 Gerold Untergasser, Heike B. Koch, Antje Menssen, and Heiko Hermeking 2 Molecular Oncology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18 A, D-82152 Martinsried/Munich, Germany ABSTRACT Evasion of cellular senescence is required for the immortal phenotype of tumor cells. The tumor suppressor genes p16 INK4A , pRb, and p53 have been implicated in the induction of cellular senescence. To identify addi- tional genes and pathways involved in the regulation of senescence in prostate epithelial cells (PrECs), we performed serial analysis of gene expression (SAGE). The gene expression pattern of human PrECs ar- rested because of senescence was compared with the pattern of early passage cells arrested because of confluence. A total of 144,137 SAGE tags representing 25,645 unique mRNA species was collected and analyzed: 157 mRNAs (70 with known function) were up-regulated and 116 (65 with known function) were down-regulated significantly in senescent PrECs (P < 0.05; fold difference > 2.5). The differential regulation of an exem- plary set of genes during senescence was confirmed by quantitative real- time PCR in PrECs derived from three different donors. The results presented here provide the molecular basis of the characteristic changes in morphology and proliferation observed in senescent PrECs. Furthermore, the differentially expressed genes identified in this report will be instru- mental in the further analysis of cellular senescence in PrECs and may lead to the identification of tumor suppressor genes and proto-oncogenes involved in the development of prostate cancer. INTRODUCTION Mammalian somatic cells have a limited proliferative capacity when cultivated in vitro. For instance, human fibroblasts stop dividing after 50 –70 population doublings and enter a terminal arrest state termed replicative senescence (1). Senescent fibroblasts are strongly enlarged and refractory to mitogen stimulation. However, they are metabolically active and survive in culture for several month. A similar limitation of proliferative capacity has been observed for most other cell types (2, 3). Replicative senescence is induced by progres- sive telomere shortening, which occurs during each cell division (4). Telomere erosion presumably generates a DNA damage signal, which leads to activation of p53 and subsequent transcriptional induction of the cdk 3 inhibitor p21 CIP1 . Therefore, prevention of telomere short- ening by ectopic expression of the catalytic subunit of telomerase (hTERT) is sufficient to immortalize primary cells in vitro provided they are cultivated under the appropriate conditions. Induction of a senescence-like phenotype also occurs after aberrant mitogenic signaling and after environmental and genotoxic insults. This form of senescence has been termed cellular senescence as opposed to telomere-associated replicative senescence (5). Similar to apoptosis, cellular senescence is thought to be a mechanism of tumor suppression because it prevents the outgrowth of cells that have acquired mutations in genes rendering them cancerous (6). Consistent with this model, several tumor suppressor genes (e.g., p16) or their products (p53) are activated at the onset of cellular senescence. In addition, mice engineered to display elevated p53 activity show pre- mature aging and a drastically decreased incidence of cancer, sup- porting a role of cellular senescence as a tumor suppressive mecha- nism relevant for the whole organism (7). Recently, it has been shown that mammary epithelial cells have the capacity to spontaneously escape replicative senescence and enter a phase of genomic instability, which may give rise to immortal cells (8). According to calculations by Morris (9), a similar evasion of replicative senescence has to occur for the development of any epi- thelial cancer. Complicating the issue, the presence of senescent fibroblasts pro- motes the proliferation of premalignant and malignant but not normal epithelial cells presumably by generating an altered microenviron- ment (10). Therefore, Krtolica et al. (10) suggested that senescence may promote carcinogenesis in aged organisms while it protects against cancer early in life. Prostatic cancer is the most frequent malignancy in the United States and the second leading cause of cancer deaths in men today (11–14). Among a variety of environmental and genetic factors fa- voring the development of prostatic cancer, aging is the most signif- icant risk factor. It has been estimated that 15–30% of males over the age of 50 and as many as 80% of the males over the age of 80 harbor clinically undetected foci of prostate cancer (15). On the basis of the in vivo expression of pH 6.0 specific -galactosidase, a marker of cellular senescence (16), it has been suggested that the accumulation of senescent prostate epithelial cells within prostatic glands might play a role in the development of prostatic diseases (17). The characterization of senescence in epithelial cells is still in its beginning. However, a detailed characterization of senescence in epithelial cells is necessary to understand how carcinoma circumvent this program. This approach may allow to identify genes involved in the development of prostate cancer, a disease for which relatively few causal genetic events are known. Furthermore, changes in gene ex- pression during senescence of PrECs may provide insights into the aging mechanisms of the prostate. To characterize genome-wide ex- pression during senescence of PrECs, we used SAGE, a quantitative method developed by Velculescu et al. (18). Here, we describe dif- ferentially expressed genes identified by SAGE, which presumably represent components of pathways and mechanisms involved in the induction and maintenance of senescence. Genetic inactivation or deregulation of these genes may lead to immortalization and neoplas- tic transformation of PrECs. MATERIALS AND METHODS Cell Culture. PrECs used for SAGE were derived from a 17-year-old accident victim (Clonetics, San Diego, CA). PrECs were cultivated in PrEC growth medium (Clonetics) on collagen type I vented flasks (BioCoat; BD Falcon, Bedford, MA) according to the supplier’s instructions. PrECs were passaged at 70% confluence by splitting 1:3 using collagenase 1S (Sigma, Deisenhofen, Germany). For qPCR analysis, additional PrEC samples were obtained from two prostate cancer patients (patient 1: 56 years old; patient 2: 63 years old). After Received 4/26/02; accepted 9/5/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supplementary data for this article are available at Cancer Research Online (http:// cancerres.aacrjournals.org). 2 To whom requests for reprints should be addressed, at Max-Planck-Institute of Biochemistry, Molecular Oncology, Am Klopferspitz 18 A, D-82152 Martinsried/Mu- nich, Germany. Phone: 49-(0)-89-8578-2875; Fax: 49-(0)-89-8578-2540; E-mail: herme@ biochem.mpg.de. 3 The abbreviations used are: Cdk, cyclin-dependent kinase; PrEC, prostate epithelial cell; SAGE, serial analysis of gene expression; qPCR, quantitative real-time PCR; ECM, extracellular matrix; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand. 6255 on July 11, 2015. © 2002 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from