Dear Sir,
The ability of the lipidic mediator prostaglandin E 2 (PGE 2 ) to regulate the immune system has been widely explored in the past decade. 1,2 Numerous studies have highlighted the ability of PGE 2 to regulate monocyte macrophages, dendritic cells, as well as T and B lymphocytes. 1,2 The biologic effects of PGE 2 are mediated through interactions with 4 distinct membrane-bound G-protein-coupled EP receptors: EP 1 , EP 2 , EP 3 and EP 4 . EP 1 is coupled to G q/p and ligand binding induces intracellular calcium levels. EP 3 is coupled to G i and inhibits cAMP production. In contrast, EP 2 and EP 4 are coupled to G s and stimulate cAMP production, which leads to gene regulation. 1 Freshly isolated blast cells of acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients increase their cAMP synthesis in response to forskolin, a cAMP-induced agonist. 3 While the presence of functional EP receptors on mature leukocytes is documented, 4 no results are available concerning their presence on immature ones. In view of the potentially important role of PGE 2 in processes of cancer and leukocyte maturation and function, 1,2 we investigated the presence of EP 2 and EP 4 receptors on freshly isolated blast cells from AML and ALL patients by testing the effect of PGE 2 on their cAMP synthesis.
Over a period of 1 year, blood samples recovered on EDTA were obtained from 12 consecutive patients (6 men, 6 women; mean age, 66 years) at diagnosis according to the Helsinki recommendations. Blood samples from patients with more than 85% (range, 85-97%) blast cells as circulating leukocytes were used. Leucocytosis ranged from 11 to 210 G/l. The population (graded according to the French-American-British classification) consisted of 2 AML0, 3 AML1, 2 AML2, 1 AML3, 1 AML4, 1 AML5 and 2 ALL2. Cytogenetic data were available for 11 patients. Six had a normal karyotype. The 5 remaining patients were heterogeneous and comprised one complex karyotype, one with a Philadelphia chromosome, one with a t(15;17), one with an unidentified marker and one with a monosomy 7. Blood mononuclear cells were isolated by separation on a Ficoll gradient and washed 2 times with Hank's balanced salts solution (HBSS). The blast purity (mean, 98%; range, 94-99%) was controlled by flow cytometry analysis (XL II; Coulter, Margency, France). Blast viability (> 95%) was judged by Trypan blue exclusion. Blasts (3 Â 10 6 cells) were stimulated in HBSS with PGE 2 and various EP-specific agonists for 10 min at 378C. In separate sets of experiments, the effect of PGE 2 on cAMP production was investigated in a cell-and timedependent manner. After stimulation, cells were ethanol-extracted and cAMP levels were measured using a commercially available EIA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. The sensitivity of the assay permits detection of 0.1 pmol cAMP. In another set of experiments, EP 2 receptors were studied by reverse transcription-polymerase chain reaction (RT-PCR).
Results indicate that PGE 2 stimulated cAMP production from blast cells of patients with acute leukemia in a cell-dependent (Fig. 1a), time-dependent (Fig. 1b) and dose-dependent manner (Fig. 1c). Stimulation with specific EP agonists showed that PGE 2 enhanced blast cAMP production through EP 2 but not EP 4 receptors (Fig. 1c). The effect of PGE 2 on cAMP production of leuke-mic blasts was a constant finding, although the intensity of stimulation varied from one patient to another (Fig. 1d). In support of data obtained with specific EP agonists, RT-PCR experiments Grant sponsor: the Ligue Contre le Cancer (Comite ´de la Corre `ze).