Overexpression of c-Myc alters G(1)/S arrest following ionizing radiation.

The proto-oncogene c-myc has been implicated as a central switch in the onset or progression of many types of cancer. However, the underlying molecular mechanism(s) responsible for c-Myc-induced tumorigenesis remains elusive. Human c-Myc is a 439-amino-acid protein that when bound to its partner, Max, can interact with DNA and function as a transcription factor (4). Myc-Max heterodimerization, which occurs through the interaction of C-terminal basic helix-loop-helix and leucine zipper motifs, is required for virtually all well-known c-Myc-associated phenotypes: cell proliferation, growth, transformation, and apoptosis (9, 15). In addition to these well-established phenotypes, recent studies have demonstrated that c-Myc expression can cause gross chromosomal aberrations and gene amplifications (16, 35, 36). The onset and progression of tumors depend upon the stepwise accumulation of somatic mutations in proto-oncogenes and tumor suppressor genes. Therefore, destabilizing genomic integrity may accelerate the accumulation of mutations required for the aggressive growth and malignant progression of tumors (6, 29, 34). As such, the c-Myc-induced genomic instability phenotype may provide an informative paradigm for understanding oncogene-induced tumor progression in general. One mechanism crucial to the maintenance of genomic stability is that of the DNA damage-induced checkpoints (12, 24, 44, 56). Cell cycle checkpoints ensure the proper execution of sequential events of the eukaryotic cell cycle; unprepared entry into either S phase or M phase can be fatal. As a cellular surveillance system for DNA damage, the DNA damage-induced checkpoint is a specific class of cell cycle checkpoints that ensure that cells containing damaged DNA do not proceed into S phase or into mitosis (1, 11, 32, 62). Many biochemical components of the G1/S checkpoint have been identified, such as the tumor suppressor p53 and the cyclin-dependent kinase inhibitor (CKI) p21/Cip1 (cyclin-dependent kinase inhibitor protein 1) (22, 61). When stabilized by a DNA damage signal, p53 is homotetramerized and acts as a transcription factor. Importantly, CKI p21/Cip1 links p53 and the DNA damage-induced inhibition of cell cycle progression (11, 13). These roles of p53 and p21/Cip1 in the DNA damage response have been firmly established through recent studies using gene knockout approaches (5, 10, 57, 58). Inhibition of cyclin/Cdk then leads to hypophosphorylation of the tumor suppressor retinoblastoma protein (Rb). The hypophosphorylated form of Rb (pRb) actively sequesters the E2F-1 transcription factor, preventing transactivation of E2F-1 targets. Since E2F-1-mediated transactivation is essential for progression into S phase, Rb-mediated inhibition of E2F-1 activity causes cell cycle arrest at G1/S. The crucial involvement of Rb in the DNA damage-induced G1/S arrest was demonstrated in a study employing Rb−/− nullizygous mouse fibroblasts. Expression of c-Myc is highly regulated throughout the cell cycle (4), and deregulation of c-Myc leads to the alteration of cell cycle regulation, such as a shortened G1 and facilitation of G1/S progression (43). Therefore, we hypothesized that c-Myc overexpression may also override or alter the scheduled cellular response (cell cycle arrest) to DNA damage at the G1/S boundary. In our experiments with nontransformed immortal HMEC lines and normal mortal HMECs, we demonstrated that a transient excess of c-Myc activity was sufficient to alter the G1/S checkpoint. For comparison, two independent controls were also used: an N-terminally truncated c-Myc (c-MycS) (50) and a dominant negative p53 (p53DD) (47). Based on these findings, we suggest a model in which c-Myc can lead to an elimination of a crucial safeguard mechanism for genomic stability in human mammary epithelial cells (HMECs).