Classic mechanisms of tumor response to chemotherapy include apoptosis and mitotic catastrophe. Recent studies have suggested that cellular senescence, a terminal proliferation arrest seen in vitro, may be invoked during the exposure of cancer cells to chemotherapeutic agents. To identify markers associated specifically with the cellular senescence phenotype, we utilized expression data from cDNA microarray experiments identifying transcripts whose expression levels increased as human prostate epithelial cells progressed to senescence. When screened against other growth-inhibitory conditions, including quiescence and apoptosis, many of these transcripts were also upregulated, indicating that similar pathways occur between apoptosis and senescence. A senescent-like phenotype was then induced in several prostate cancer cell lines using 5-aza-2'-deoxycytidine, doxorubicin, or Docetaxel. Treatment with these agents resulted in a significant increase in the induction of senescence-specific genes when compared to nonsenescent conditions. The performance of the panel was improved with fluorescence-activated cell sorting using PKH26 to isolate nonproliferating, viable, drug-treated populations, indicating that a heterogeneous response occurs with chemotherapy. We have defined an RNA-based gene panel that characterizes the senescent phenotype induced in cancer cells by drug treatment. These data also indicate that a panel of genes, rather than one marker, needs to be utilized to identify senescence.

These findings highlight the plasticity of the epigenome in an epithelial organ vulnerable to neoplastic change. They further suggest that chromatin modifications are more susceptible to methyl-deficient diets than DNA methylation at this locus.

The imprinted insulin-like growth factor-2 (IGF2) gene is an auto/paracrine growth factor expressed only from the paternal allele in adult tissues. In tissues susceptible to aging-related cancers, including the prostate, a relaxation of IGF2 imprinting is found, suggesting a permissive role for epigenetic alterations in cancer development. To determine whether IGF2 imprinting is altered in cellular aging and senescence, human prostate epithelial and urothelial cells were passaged serially in culture to senescence. Allelic analyses using an IGF2 polymorphism demonstrated a complete conversion of the IGF2 imprint status from monoallelic to biallelic, in which the development of senescence was associated with a 10-fold increase in IGF2 expression. As a mechanism, a 2-fold decrease in the binding of the enhancer-blocking element CCCTC-binding factor (CTCF) within the intergenic IGF2-H19 region was found to underlie this switch to biallelic IGF2 expression in senescent cells. This decrease in CTCF binding was associated with reduced CTCF expression in senescent cells. No de novo increases in methylation at the IGF2 CTCF binding site were seen. The forced down-regulation of CTCF expression using small interfering RNA in imprinted prostate cell lines resulted in an increase in IGF2 expression and a relaxation of imprinting. Our data suggest a novel mechanism for IGF2 imprinting regulation, that is, the reduction of CTCF expression in the control of IGF2 imprinting. We also demonstrate that altered imprinting patterns contribute to changes in gene expression in aging cells.

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