THE CIRCADIAN EXPRESSION OF c-MYC IS MODULATED BY THE HISTONE DEACETYLASE INHIBITOR TRICHOSTATIN A IN SYNCHRONIZED MURINE NEUROBLASTOMA CELLS

Circadian clocks govern the mammalian physiology in a day/night-dependent manner. The circadian oscillator of peripheral organs is composed of the same elements as the central pacemaker at the suprachiasmatic nucleus (SCN). The interaction between the circadian clock and several cell cycle components has been established in recent years, since many key regulators of cell cycle and growth control were proved to be rhythmically expressed. In particular, the proto-oncogene c-Myc has been documented to be under circadian regulation. Given that it is overexpressed in many malignancies, the study of c-Myc mRNA and c-MYC protein regulation by the circadian clock is of great interest. Thus, the aim of this work was to: (a) analyze in detail the circadian oscillations of c-Myc steady-state mRNA levels and to investigate whether c-MYC protein levels display any oscillating pattern, and (b) ascertain whether circadian time is important for reducing c-MYC levels after drug application. For this purpose, we selected trichostatin A (TSA), since it is known that long (≥12 h) treatment durations negatively influence the expression levels of c-Myc and short 2 h treatments up regulate the expression of the central oscillator gene Per1 resulting in the resetting of its rhythm. TSA is a specific inhibitor of histone deacetylases (HDACs), and its application results in increased acetylation levels of histone and non-histone proteins. Our results, using the murine neuroblastoma cell line N2A, show that Per1 and c-Myc steady-state mRNA levels oscillate with the same phase. Moreover, a short 2 h TSA treatment causes a phase-dependent decrease of oscillating c-Myc transcript levels only when applied at the trough of its mRNA rhythm, where a general decrease of c-MYC protein levels is also observed. At the peak of its rhythm, no apparent changes can be observed. These experiments demonstrate for the first time that a significant decrease in c-Myc transcript and protein levels can be achieved after a short TSA treatment applied only at specific circadian times. This is also followed by a reduction in the proliferation rate of the cell population. (Author correspondence: prombona@bio.demokritos.gr)