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BackgroundB cell precursor acute lymphoblastic leukaemia (BCP-ALL) is the most common paediatric cancer. BCP-ALL blasts typically retain wild type p53, and are therefore assumed to rely on indirect measures to suppress transformation-induced p53 activity. We have recently demonstrated that the second messenger cyclic adenosine monophosphate (cAMP) through activation of protein kinase A (PKA) has the ability to inhibit DNA damage-induced p53 accumulation and thereby promote survival of the leukaemic blasts.Development of BCP-ALL in the bone marrow (BM) is supported by resident BM-derived mesenchymal stromal cells (MSCs). MSCs are known to produce prostaglandin E2 (PGE2) which upon binding to its receptors is able to elicit a cAMP response in target cells. We hypothesized that PGE2 produced by stromal cells in the BM microenvironment could stimulate cAMP production and PKA activation in BCP-ALL cells, thereby suppressing p53 accumulation and promoting survival of the malignant cells.MethodsPrimary BCP-ALL cells isolated from BM aspirates at diagnosis were cocultivated with BM-derived MSCs, and effects on DNA damage-induced p53 accumulation and cell death were monitored by SDS-PAGE/immunoblotting and flow cytometry-based methods, respectively. Effects of intervention of signalling along the PGE2-cAMP-PKA axis were assessed by inhibition of PGE2 production or PKA activity. Statistical significance was tested by Wilcoxon signed-rank test or paired samples t test.ResultsWe demonstrate that BM-derived MSCs produce PGE2 and protect primary BCP-ALL cells from p53 accumulation and apoptotic cell death. The MSC-mediated protection of DNA damage-mediated cell death is reversible upon inhibition of PGE2 synthesis or PKA activity. Furthermore our results indicate differences in the sensitivity to variations in p53 levels between common cytogenetic subgroups of BCP-ALL.ConclusionsOur findings support our hypothesis that BM-derived PGE2, through activation of cAMP-PKA signalling in BCP-ALL blasts, can inhibit the tumour suppressive activity of wild type p53, thereby promoting leukaemogenesis and protecting against therapy-induced leukaemic cell death. These novel findings identify the PGE2-cAMP-PKA signalling pathway as a possible target for pharmacological intervention with potential relevance for treatment of BCP-ALL.

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The differentiation capacity of mesenchymal stem cells has been extensively studied, but little is known on cell cycle-related events in the proliferation and differentiation phases of these cells. Here, we demonstrate that exposure to cAMP-increasing agents inhibits proliferation of adipose stem cells (ASCs). This antiproliferative effect is associated with both reduced cdk2 activity and pRB phosphorylation. Concomitantly, however, the level of cyclin E markedly increases upon cAMP induction, indicating that cyclin E may have cdk2-independent functions in these cells besides its role as a cdk2 activator. Indeed, we found indications of a cdk2-independent role of cyclin E in DNA damage-induced apoptosis. 8-CPT-cAMP sensitizes ASCs to ␥-irradiation-induced apoptosis, an effect abolished by knockdown of cyclin E. Moreover, cAMP induces early activation of ERK, leading to reduced degradation of cyclin E. The cAMP-mediated upregulation of cyclin E was blocked by knockdown of ERK or by an inhibitor of the ERK kinase MEK. We conclude that cAMP inhibits cdk2 activity and pRB phosphorylation, leading to reduced ASC proliferation. Concomitant with this growth inhibition, however, cyclin E levels are increased in a MEK/ERK-dependent manner. Our results suggest that cyclin E plays an important, cdk2-independent role in genotoxic stress-induced apoptosis in mesenchymal stem cells. INTRODUCTIONHuman mesenchymal stem cells (MSCs) have been shown to differentiate not only into mesenchymal lineages (such as osteogenic, chondrogenic, adipogenic, and myogenic), but also into neurogenic, angiogenic, and hepatic lineages (Zuk et al., 2002;Baksh et al., 2004;Boquest et al., 2006a;Schaffler and Buchler, 2007). The pluripotent nature of MSCs makes them potentially ideal candidates for tissue engineering, and they have already demonstrated some efficacy in cell therapy and regenerative medicine Kassem, 2004;Leo and Grande, 2006;Mimeault and Batra, 2006). Adipose tissue contains a supportive stroma that can be easily isolated and provides a rich source of MSCs (Zuk et al., 2002;Boquest et al., 2006a). Thus, adipose tissue represents a valuable source of multilineage stem cells. It is therefore important to understand the biology of adipose stem cells (ASCs).Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a number of different cellular processes, including metabolism, growth, differentiation, and gene regulation (Krebs and Beavo, 1979;McKnight, 1991;Vossler et al., 1997;Daniel et al., 1998;Kim et al., 2005). Most effects of cAMP are mediated through activation of protein kinase A (PKA) or the Epac (exchange protein directly activated by cAMP) family of exchange proteins (Walsh et al., 1968;Bos, 2003). Elevation of intracellular cAMP has both proliferative and antiproliferative effects depending on the cell type. For instance, cAMP stimulates the proliferation of thyroid cells, neurons, and Swiss 3T3 cells, while inhibiting the proliferation of lymphocytes, fibroblasts, and adipocytes (Rozengurt et al., 1981;Blomhoff et ...