cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines

Abstract

The phosphorylation of histone H3 is known to play a role in regulation of transcription as well as preparation of chromosomes for mitosis. Various signaling cascades induce H3 phosphorylation, particularly at genes activated by these pathways. In this study we show that signaling can also have the opposite effect. Activators of cAMP signaling induce a rapid and potent loss of H3 phosphorylation. This effect is not mediated through a cAMP metabolite since a membranepermeable form of AMP had no effect on H3 phosphorylation and a phosphodiesterase-resistant cAMP analog efficiently reduced it. cAMP is also the likely regulator of H3 phosphorylation under physiological conditions since only supra-pharmacological doses of cGMP induce the loss of H3 phosphorylation. The loss of phosphorylation is specific for histone H3 since we do not observe drastic losses in total phosphorylation of other histones. In addition, other H3 modifications are unaffected with the exception of lysine 9 methylation, which is elevated. Analysis of cell growth and cell cycle show that cAMP signaling inhibits cell growth and arrests cells at both G1 and G2/M. Similar effects of cAMP signaling on H3 phosphorylation are observed in a variety of mammary adenocarcinoma-derived cell lines. In syngeneic human breast derived cell lines, one diploid and non-transformed, the other derived from a ductal carcinoma, the loss of H3 phosphorylation is significantly more sensitive to cAMP concentration in the transformed cell line.

cAMP signaling regulates histone H3 phosphorylation and mitotic entry through a disruption of G2 progression

“…Based on the fact that changes in the cellular expression profile are required to achieve the G1 block, it is possible that the function of the reversible cAMP-mediated disruption to G2 progression is to allow time for these changes to occur so that the cells efficiently arrest in G1 after mitosis. This is supported by our previous results showing that asynchronously-cycling cells treated with 8-Br-cAMP for 48 hours undergo only one division while untreated cells increase in number by a factor of 5 [37]. …”

“…Our previous study showed that prolonged cAMP signaling results in a block to both G1 and G2 progression and significantly inhibits cell growth [37]. Here we show that the disruption of G2 progression occurs prior to the block to G1 progression.…”

“…We previously reported that cAMP signaling significantly inhibits growth of 1470.2 cells and results in arrest of cell cycle progression in both G1 and G2/M 48 hours after treatment with 8-Br-cAMP [37]. The rapid effects on mitotic entry (Fig.…”

“…We have shown that activation of cAMP signaling by β-adrenergic receptor ligands, forskolin, or 8-Br-cAMP rapidly and specifically downregulates H3 phosphorylation at serines 10 and 28 in a number of mammary-derived cell lines [37]. To further characterize this phenomenon, we continued our studies using 8-Br-cAMP to directly activate cAMP signaling, and avoid the simultaneous activation of other signaling pathways which might be induced by extracellular ligands such as epinephrine.…”

“…3B,C) indicates that there is variability in the rate at which cells recover from the putative checkpoint. The cells remaining in G2/M after prolonged 8-Br-cAMP treatment [37] may be arrested. The ability of these somatic cells to at least partially recover from the cAMP-induced inhibition of G2 progression differentiates them from germ line oocytes undergoing meiosis, in which the cells remain arrested in G2 until they receive a signal to divide [17].…”