Epigenetic Down-Regulation of ARF Expression Is a Selection Step in Immortalization of Human Fibroblasts by c-Myc

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.

Cytoplasmic Poly(A) Binding Proteins Regulate Telomerase Activity and Cell Growth in Human Papillomavirus Type 16 E6-Expressing Keratinocytes

“…Previous studies have shown that hTERT can affect cell growth in culture, both under normal conditions and under stress (6,9,42). As the level of PABPC4 was important in the regulation of hTERT mRNA and telomerase activity in HPV16 E6-expressing HFKs, we wanted to determine whether HPV16 E6-expressing HFKs that overexpressed HA-PABPC4, and therefore had more telomerase activity, would grow better in culture than HPV16 E6/LXSN-expressing cells.…”

Functional genomics identifies therapeutic targets for MYC-driven cancer

“…To broadly identify genes that exhibit a synthetic lethal relation with oncogenic expression of MYC we have used a high-throughput functional genomic approach (16) focused on the druggable genome. As the genetic noise inherent in established cancer cell lines could constitute a potential source of bias, we chose to screen an isogenic pair of primary cells (human foreskin fibroblasts, HFFs), where the only perturbation was overexpression of c-MYC through a retroviral vector (17) and to validate the "hits" in cancer cell contexts. HFFs are unique in that they do not senesce in response to MYC overexpression (17) or introduction of an activated RAS oncogene (18), a property that has been attributed to lack of culture stress.…”

“…As the genetic noise inherent in established cancer cell lines could constitute a potential source of bias, we chose to screen an isogenic pair of primary cells (human foreskin fibroblasts, HFFs), where the only perturbation was overexpression of c-MYC through a retroviral vector (17) and to validate the "hits" in cancer cell contexts. HFFs are unique in that they do not senesce in response to MYC overexpression (17) or introduction of an activated RAS oncogene (18), a property that has been attributed to lack of culture stress. Furthermore, c-MYC overexpression in HFFs recapitulates both the gene expression signatures and cellular phenotypes of MYC-driven cancers (17,19,20).…”

“…HFFs are unique in that they do not senesce in response to MYC overexpression (17) or introduction of an activated RAS oncogene (18), a property that has been attributed to lack of culture stress. Furthermore, c-MYC overexpression in HFFs recapitulates both the gene expression signatures and cellular phenotypes of MYC-driven cancers (17,19,20).…”

Telomerase activation by c-Myc in human mammary epithelial cells requires additional genomic changes

“…A recent study of human foreskin fibroblasts (HFFs) immortalized using the same c-Myc expression vector used in the present work found that c-Myc and telomerase levels did not directly correlate with each other and that immortalization was associated with clonal selection. 18 The authors of this study hypothesized that other events may have occurred to facilitate immortalization of the HFFs. Although karyotypic analysis of the immortalized cells did not reveal any gross chromosomal abnormalities, expression of the tumor suppressor ARF was suppressed by epigenetic means.…”