The interplay between MYCN and the DNA damage response: modulation of MYCN expression, its interactions with components of the DNA damage response and cellular responses to N-myc following genotoxic stress

Abstract

The DNA damage response (DDR) forms a signaling cascade rapidly activated upon exposure to genotoxic stress. The DDR safeguards genomic integrity by halting cell cycle progression to allow repair of damaged DNA or by inducing cell death. Myc proteins are key regulators of cell proliferation that transcriptionally control the cell cycle machinery. Amplification of N-myc in neuroblastomas (MNA-NB) is associated with abrogation of the regulatory mechanisms that normally prevent aberrant cell proliferation and the interplay between N-myc and the DDR was here analysed. Initially, an association between N-myc and the cdk inhibitor p21 was investigated in MNA-NB as a possible mechanism by which p21 is functionally suppressed in these cells. Although an N-myc/p21 interaction was not observed, MNA-NB cells appear to express short N-myc isoforms with the potential to associate with p21. Expression of N-myc rendered Rat-1 cells resistant to the cell cycle block imposed by serum starvation but these cells were not able to bypass a G1 arrest imposed by ectopic p21 suggesting N-myc does not abolish p21 activity through regulation at the protein level. Analysis of the N-myc response to DNA damage in MNA-NB cells revealed that N-myc is downregulated in a proteasome-dependent manner in response to UVC or a UV-mimetic carcinogen, 4NQO. This effect was not reproduced with other agents such as IR which like UVC were found to repress cyclin D1 expression likely indicating that alternative DDR signaling pathways differently regulate N-myc. N-myc was found to interact with the DDB2 subunit of the damaged-DNA binding (DDB) complex, a substrate receptor for the DDB1-Cul4ADDB2 E3 ligase. The DDB complex has been implicated in UV-induced protein ubiquitylation suggesting it may play a role in the N-myc response to UVC radiation. These findings highlight the complexities of the DDR and uncover potentially important mechanisms of cell cycle control through regulation of N-myc.