Cyclic AMP negatively controls c-myc transcription and G1 cell cycle progression in p210 BCR-ABL transformed cells: inhibitory activity exerted through cyclin D1 and cdk4

Abstract

Raised intracellular cyclic AMP (cAMP) has been demonstrated to exert an antiproliferative effect in myeloid cells. How the antiproliferative activity of cAMP is exerted in p210 BCR-ABL transformed myeloid cells was the subject of this investigation. It was hypothesized that cyclin dependent kinase 4, cdk4, might be a critical target enzyme to affect the related events of c-myc transcription and progression through G1 phase of the cell cycle within cells transformed by p210 BCR-ABL, and further, that cdk4 might be downregulated by cAMP to inhibit proliferation. In order to investigate the regulatory role of cdk4, synchronized cells were studied. In p210 BCR-ABL transformed cells transiting early G1 phase, treatment with a cAMP analogue led to inhibition of cyclin D1 synthesis, and marked reduction of cdk4 kinase activity. Within cells in which cdk4 was inhibited by cAMP, there was augmented interaction of E2F1 with the retinoblastoma protein, pRb in a nuclear matrix-associated cell fraction. As a result of E2F1 sequestration, raised intracellular cAMP was found to inhibit c-myc transcription in p210 BCR-ABL transformed myeloid cells synchronously transiting the early G1 phase of the cell cycle. A target of this transcriptional suppression exerted by cAMP was the E2F site of the c-myc P2 promoter. On the other hand, cyclin D1 content was not reduced by cAMP in these cells when it was applied at a later cell cycle stage at the interface between G1 and S. Corresponding to lack of cyclin D1 inhibition in these later G1-to-S phase cells, cdk4 activity was only modestly suppressed, and c-myc mRNA expression was also inhibited to a lesser degree. These studies show that Rb interaction with E2F1 is regulated by cdk4 and cyclin D1 within p210 BCR-ABL transformed leukemia cells in early G1 phase of the cell cycle. In this context, both cyclin D1 and cdk4 are subject to the level of intracellular cAMP. This interaction between Rb and E2F1, which is subject to the level of cAMP, is critical to transcriptional control of c-myc. Further, pRb regulation of E2F activity affects cellular potential for G1-S phase transition in p210 BCR-ABL transformed myeloid cells, in part, via its effect on c-myc transcription.