cAMP-mediated Inhibition of DNA Replication and S Phase Progression: Involvement of Rb, p21^Cip1, and PCNA

Abstract

cAMP exerts an antiproliferative effect on a number of cell types including lymphocytes. This effect of cAMP is proposed to be mediated by its ability to inhibit G1/S transition. In this report, we provide evidence for a new mechanism whereby cAMP might inhibit cellular proliferation. We show that elevation of intracellular levels of cAMP inhibits DNA replication and arrests the cells in S phase. The cAMP-induced inhibition of DNA synthesis was associated with the increased binding of p21Cip1 to Cdk2-cyclin complexes, inhibition of Cdk2 kinase activity, dephosphorylation of Rb, and dissociation of PCNA from chromatin in S phase cells. The ability of cAMP to inhibit DNA replication and trigger release of PCNA from chromatin required Rb and p21Cip1 proteins, since both processes were only marginally affected by increased levels of cAMP in Rb Ϫ/Ϫ and p21 Cip1Ϫ/Ϫ 3T3 fibroblasts. Importantly, the implications of cAMP-induced inhibition of DNA synthesis in cancer treatment was demonstrated by the ability of cAMP to reduce apoptosis induced by S phase-specific cytotoxic drugs. Taken together, these results demonstrate a novel role for cAMP in regulation of DNA synthesis and support a model in which activation of cAMP-dependent signaling protects cells from the effect of S phase-specific antitumor agents. INTRODUCTIONThe second messenger cyclical AMP (cAMP) plays an important role in regulation of numerous cellular functions. Elevation of intracellular cAMP levels either stimulates or inhibits proliferation of cells depending on the cell type (Pastan et al., 1975;Dumont et al., 1989). Generally, cAMP has an antiproliferative effect on most cells of mesenchymal origin (Blomhoff et al., 1988;Dugan et al., 1999). We have observed that increase in intracellular cAMP leads to accumulation of lymphoid cells in the G1 phase and correlates with rapid dephosphorylation of the retinoblastoma tumor suppressor protein (Rb;Blomhoff et al., 1987Blomhoff et al., , 1988Christoffersen et al., 1994;Naderi and Blomhoff, 1999;Gutzkow et al., 2002). Furthermore, this antiproliferative effect of cAMP in lymphocytes has been shown to be mediated through protein kinase A type I (Skalhegg et al., 1992;Tasken et al., 1994).Rb, is an important inhibitor of cell cycle progression (Wang et al., 1994;Harbour and Dean, 2000). In its hypophosphorylated active form, Rb inhibits transition of cells from G1 into S phase of the cell cycle. In addition, Rb has also been shown to negatively regulate DNA replication and block S phase progression (Chew et al., 1998;Knudsen et al., 1998). The negative effect of Rb on cell cycle progression is countered by its phosphorylation by cyclin-dependent kinases (Cdks). When complexed to their regulatory cyclin subunits, Cdks phosphorylate and thereby inactivate the antiproliferative function of Rb (Mittnacht, 1998). Specifically, inactivation of Rb in G1 is achieved through its sequential phosphorylation by Cdk4/6-cyclin D, Cdk2-cyclin E, and Cdk2-cyclin A complexes (Zarkowska and Mittnacht, 1997;Lundberg and Weinberg...

Chemoresistant KM12C Colon Cancer Cells Are Addicted to Low Cyclic AMP Levels in a Phosphodiesterase 4–Regulated Compartment via Effects on Phosphoinositide 3-Kinase

“…2A-C). Such key changes, commonly associated with negative regulation of progression through the G 1 phase of the cell cycle (17,44), are consistent with the observed accumulation of cells in G 1 (Fig. 2D).…”

“…Interestingly, rolipram can also induce expression of cyclin-dependent kinase (CDK) inhibitors, leading to growth inhibition and differentiation of glioma cells (15), although a high concentration of rolipram was required for these effects. The cAMP-elevating agent forskolin (16), when used at high doses, has been reported to inhibit DNA replication in lymphocytes via PKAmediated effects on p21 CIP1 , leading to dephosphorylation of the retinoblastoma protein (pRb) and disrupted tethering of proliferating cell nuclear antigen to DNA (17). Taken together, these reports tantalizingly suggest that modulating intracellular cAMP, perhaps in a localized manner by targeting particular PDE types, may affect the proliferation of cancer cells.…”

Distinct Action of the Retinoblastoma Pathway on the DNA Replication Machinery Defines Specific Roles for Cyclin-Dependent Kinase Complexes in Prereplication Complex Assembly and S-Phase Progression

“…This was observed with a number of different MCM proteins and did not involve the disruption of the ORC complex. The effect of p16ink4a is not shared with RB, as observed either with activated alleles or through activation/dephosphorylation of endogenous RB (47). p16ink4a function is restricted to G 1 and involves the downregulation of Cdc6 and Cdt1.…”

“…The mechanism through which RB mediates replication control remains a subject of intense study, as both direct and indirect effects of RB on the replication machinery in S phase have been reported (10,26,32,54,66,68). However, the activation of endogenous RB or the introduction of constitutively active alleles of RB attenuates CDK2 activity (through the downregulation of cyclin A) and induces a loss of PCNA activity in S phase (3,47,60). Although these studies connect RB to replication control, the influence of p16ink4a on replication control has not been addressed.…”

cAMP signalling inhibits p53 acetylation and apoptosis via HDAC and SIRT deacetylases

“…This signal transducer is generated by adenylyl cyclases subsequent to stimulation of certain G protein-coupled receptors (GPCRs). Our lab has previously reported that elevation of cAMP in lymphoid cells leads to arrest in the G1 phase of the cell cycle (12)(13)(14), arrest in the S phase and inhibition of apoptosis by anticancer agents (15). Using B cell precursor acute lymphoblastic leukaemia (BCP-ALL) cells as a model system, we also reported that the inhibitory effect of cAMP on apoptosis is p53-dependent (16), and that cAMP antagonizes the disruption of p53-HDM2 interaction by DNA damage (17).…”