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DNA Dynamics and Chromosome Structure

Overexpression of c-Myc Alters G1/S Arrest following Ionizing Radiation

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Pages 1819-1833 | Received 15 Oct 2001, Accepted 15 Nov 2001, Published online: 28 Mar 2023
 

Abstract

Study of the mechanism(s) of genomic instability induced by the c-myc proto-oncogene has the potential to shed new light on its well-known oncogenic activity. However, an underlying mechanism(s) for this phenotype is largely unknown. In the present study, we investigated the effects of c-Myc overexpression on the DNA damage-induced G1/S checkpoint, in order to obtain mechanistic insights into how deregulated c-Myc destabilizes the cellular genome. The DNA damage-induced checkpoints are among the primary safeguard mechanisms for genomic stability, and alterations of cell cycle checkpoints are known to be crucial for certain types of genomic instability, such as gene amplification. The effects of c-Myc overexpression were studied in human mammary epithelial cells (HMEC) as one approach to understanding the c-Myc-induced genomic instability in the context of mammary tumorigenesis. Initially, flow-cytometric analyses were used with two c-Myc-overexpressing, nontransformed immortal lines (184A1N4 and MCF10A) to determine whether c-Myc overexpression leads to alteration of cell cycle arrest following ionizing radiation (IR). Inappropriate entry into S phase was then confirmed with a bromodeoxyuridine incorporation assay measuring de novo DNA synthesis following IR. Direct involvement of c-Myc overexpression in alteration of the G1/S checkpoint was then confirmed by utilizing the MycER construct, a regulatable c-Myc. A transient excess of c-Myc activity, provided by the activated MycER, was similarly able to induce the inappropriate de novo DNA synthesis following IR. Significantly, the transient expression of full-length c-Myc in normal mortal HMECs also facilitated entry into S phase and the inappropriate de novo DNA synthesis following IR. Furthermore, irradiated, c-Myc-infected, normal HMECs developed a sub-G1 population and a >4N population of cells. The c-Myc-induced alteration of the G1/S checkpoint was also compared to the effects of expression of MycS (N-terminally truncated c-Myc) and p53DD (a dominant negative p53) in the HMECs. We observed inappropriate hyperphosphorylation of retinoblastoma protein and then the reappearance of cyclin A, following IR, selectively in full-length c-Myc- and p53DD-overexpressing MCF10A cells. Based on these results, we propose that c-Myc attenuates a safeguard mechanism for genomic stability; this property may contribute to c-Myc-induced genomic instability and to the potent oncogenic activity of c-Myc.

We appreciate the materials and critical advice from Martha Stampfer, Paul Yaswen, Moshe Oren, Trevor Littlewood, Chi Dang, Linda Penn, and Dusty Miller. We also thank Todd Waldman for helpful discussions throughout this study and Geoffrey M. Wahl for sharing experimental information before publication. We gratefully received technical assistance from the microscopic imaging core facilities and the flow cytometry core facilities at the Lombardi Cancer Center, Georgetown University Medical Center. We thank Tim Jorgensen and members of our laboratory for comments on the manuscript.

This study was supported in part by a predoctoral training grant of the DOD breast cancer research program (DAMD17-99-1-9205) to J.-H.S. and NIH grant R01AG1496 to R.B.D.

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