Supplementary Figures S1-S2 from Epigenetic Down-Regulation of ARF Expression Is a Selection Step in Immortalization of Human Fibroblasts by c-Myc
ARTICLE ABSTRACT
The transcription factor c-Myc is implicated in the pathogenesis of many cancers. Among the multiple functions of c-Myc, activation of hTert and other genes involved in cellular life span contributes to its role as an oncogene. However, the ability of c-Myc to directly immortalize human cells remains controversial. We show here that overexpression of c-Myc reproducibly immortalizes freshly isolated human foreskin fibroblasts. c-Myc–immortalized cells displayed no gross karyotypic abnormalities but consisted of an oligoclonal population, suggesting that additional events cooperated to achieve immortalization. Levels of p53 and p16 were increased, but both p53-dependent DNA damage response and growth arrest in response to p16 overexpression remained intact. A marked decrease in expression of the tumor suppressor ARF occurred in several independently established c-Myc–immortalized cell lines. Methylation-specific PCR showed that the ARF gene was methylated in immortalized but not early-passage c-Myc cells, whereas p16 was unmethylated in both cell populations. Restoration of ARF expression by treatment with a demethylating agent or overexpression by a retroviral vector coincided with inhibition of proliferation and senescence of c-Myc–immortalized cells. Our findings predict that epigenetic events play a significant role in human tumors that express high levels of c-Myc. (Mol Cancer Res 2007;5(11):1181–9)
