Genes encoding multiple forms of phospholipase A2 are expressed in immature forms of human leukemic blasts

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate the eicosanoid precursor, arachidonic acid (AA).1, 2 Three distinct families have been documented: low molecular weight soluble forms of PLA2 (sPLA2); Ca2+-dependent high molecular weight PLA2 (cPLA2) and Ca2+-independent high molecular weight PLA2 (iPLA2). The sPLA2 family is implicated in several biological processes such as inflammation and host defense.1, 2 Nine isoenzymes have been identified. Among them sPLA2-IB is the pancreatic one. sPLA2-IIA is constitutively expressed in various organs related to immune response such as bone marrow, spleen and thymus. sPLA2-V is found in several immune cells such as macrophage, mast cells and type II helper T cells. sPLA2-X is expressed in the digestive tract, immune organs and blood leukocytes. The cPLA2 family consisted of four members, cPLA2-IVA, cPLA2-IVB, cPLA2-IVC and cPLA2-IVD; cPLA2-IVA being the central regulator of stimulus-coupled cellular AA release.1, 2 The iPLA2 (PLA2-VI) plays a major role in phospholipids remodeling.

Eicosanoid products of the cyclooxygenase (COX) and lipoxygenase pathways of AA are important mediators of malignant proliferation.3 Deregulated PLA2 activity contributes to the pathogenesis of several malignancies including prostate cancer, ovarian carcinoma and colorectal adenocarcinoma.1, 2 The COX and lipoxygenase pathways of AA have been recently documented on immature leukemic blasts of patients with acute myeloid (AML) and acute lymphoid (ALL) leukemia.4, 5 Thus, freshly isolated AML and ALL blasts express COX-1, produce the AA metabolite prostaglandin E2, express functional EP2 receptors and increase their growth in response to exogenously added prostaglandin E2. The World Health Organization has proposed a classification system that divides AML into several broad groups: AML with genetic abnormalities, AML with multilineage dysplasia, AML related to earlier chemotherapy or radiation and AML not otherwise specified. The latter group contains a subgroup, AML without maturation, which consisted of the weaker immature blast group. In view of the potentially important oncogenic action of PLA2 in leukemic proliferation, quantitative PCR was used to determine the PLA2 mRNAs that were expressed in AML blasts without maturation and ALL blasts.

Blood samples recovered on EDTA were obtained from twenty-one AML patients and ten B-ALL patients at diagnosis according to the Helsinki recommendations. AML patients consisted in a homogeneous group of patients with blasts without signs of maturation and no genetic abnormality. Blood samples with more than 85% blast cells as circulating leukocytes were used. Leukocytosis ranged from 10 to 219 g/l. Immediately after recovery leukemic blasts were isolated by separation on a Ficoll gradient and washed once with Hank's balanced salts solution. The blast purity (>98%) was controlled by flow cytometry analysis. Blast viability (>95%) was judged by trypan blue exclusion. Blast RNA was immediately extracted with Tripure (Roche GmbH, Mannheim, Germany) and was stored at −80 °C until used. As a control group, blood samples were recovered from seven healthy volunteers. Control blood mononuclear cells were recovered and processed exactly as for leukemic blast samples.

In a first set of experiments, we investigated whether mRNAs derived from the four cPLA2 genes were detected in AML and ALL leukemic blasts. As shown in Figure 1 (left part), mRNAs derived from three of the four cloned cPLA2 genes are detected in human leukemic blasts. PLA2-IVD transcripts were not present at detectable levels in AML and ALL leukemic blasts and in blood mononuclear cells from healthy individuals (data not shown). In contrast, PLA2-IVA, PLA2-IVB and PLA2-IVC were detected. Similar amounts of PLA2-IVB and PLA2-IVC transcripts were found in AML and ALL blasts and control cells. Both enzymes show little specificity for the sn-2 fatty acid.1, 2 In contrast, PLA2-IVA transcripts were markedly (P=0.006) reduced in ALL blasts as compared with blood mononuclear cells. PLA2-IVA preferentially hydrolyzes phospholipids containing AA at the sn-2 position. PLA2-IVA is ubiquitously and constitutively expressed in most cells and tissues. One notable exception is mature B and T cells, which do not contain detectable levels of PLA2-IVA.1 Quantitative PCR analysis shows that PLA2-IVA levels were also markedly lowered in immature forms of ALL blasts. The high levels of AA documented in ALL blasts have been earlier related to an elevated Δ6 desaturase activity.6 Low levels of PLA2-IVA in ALL blasts might also explain these elevated AA amounts. PLA2-VI (iPLA2) transcript levels were higher (P=0.003) in AML and ALL leukemic blasts. PLA2-VI was originally reported to mediate phospholipids remodeling and, thus, to act as a housekeeping protein without significant roles in cell growth.1, 2 Several recent studies have shown that PLA2-VI exhibited roles in cell regulation, growth and death. Especially, one mechanism by which PLA2-VI mediates cell growth involves regulation of AA release, p53 and mitogen-activated protein kinase activation.7 A role for PLA2-VI may be suggested in the growth of AML and ALL blasts. The technique employed in this study allowed measurement of the absolute levels of any mRNA. So it was possible to evaluate the levels of each of the isoforms relative to one another. Results indicated the following rank of magnitude for cPLA2 in leukemic blasts: PLA2-VI>PLA2-IVA=PLA2-IVB>PLA2-IVC. Together, these observations might suggest PLA2-VI as a novel and interesting target for drug development for leukemic therapy. However, given the ubiquitous expression of PLA2-VI and its role in glycerophospholipid metabolism, drug strategies targeting PLA2-VI must exhibit selectivity to avoid undesired side effects.

Figure 1
figure1

Quantitative (Q) -PCR analysis of cytosolic phospholipase (PL) A2 and soluble PLA2 transcripts in leukemic blasts.Q-PCR was carried out in duplicate by using TaqMan assay reagents according to the manufacturer's recommendations (Applied Biosystems, Foster City, CA, USA) (product references - PLA2-IB: Hs00386701-m1; PLA2-IIA: Hs00179898-m1; PLA2-IID: Hs00173860-m1; PLA2-IIF: Hs00224482; PLA2-IVA: Hs00233352-m1; PLA2-IVB: Hs00979952-m1; PLA2-IVC: Hs00234345-m1; PLA2-IVD: Hs00603557-m1; PLA2-V: Hs00173472-m1; PLA2-VI: Hs001/85926-m1; PLA2-X: Hs00358567-m1 and PLA2-XII: Hs00830106-s1). Gene expression levels were normalized to 18S RNA (product reference Hs99999901-s1). Amounts of various transcripts were compared with sample with the lowest level of transcripts (a patient who was arbitrarily quoted (1). (•) indicates patients with no detectable transcript. Significance was assessed by using the Kruskal–Wallis test followed by a Mann–Whitney U-test. A Bonferroni adjustment indicated a P<0.016 as significant. ALL, acute lymphoid leukemia; AML, acute myeloid leukemia; C, control; ns, non significant.

In another set of experiments we investigated whether mRNAs derived from the nine sPLA2 genes (i.e PLA2-IB, PLA2-IIA, PLA2-IID, PLA2-IIE, PLA2-IIF, PLA2-III, PLA2-V, PLA2-X and PLA2-XII) were detected in AML and ALL leukemic blasts. PLA2-IIE and PLA2-III were not present at detectable levels in AML and ALL leukemic blasts and control blood mononuclear cells (data not shown). In contrast transcripts for other sPLA2 subtypes could be detected (Figure 1, right part). Results indicated the following rank of magnitude for sPLA2 in leukemic blasts: PLA2-IB>PLA2-XII>PLA2-X>PLA2-IID>PLA2-IIA=PLA2-V>PLA2-IIF. Levels of PLA2-IB and PLA2-X transcripts were higher (P=0.0001) in AML blasts than in control cells. In contrast PLA2-IIA (P=0.0005), PLA2-IID (P=0.0007) and PLA2-V (P=0.0009) were markedly reduced, whereas PLA2-XII and PLA2-IIF ones were unchanged. PLA2-X transcripts were higher (P=0.0006) in ALL blasts than in control cells. In contrast PLA2-IIA transcripts levels were reduced, (P=0.001) whereas PLA2-IB, PLA2-IID, PLA2-IIF, PLA2-V and PLA2-XII ones were unchanged. Such variations of PLA2 subtypes would be of importance for leukemic patients. Thus, coagulation activation was often observed in patients with acute leukemia. PLA2-IIA, PLA2-IID and PLA2-V, which possess potent anticoagulant activity,1 are markedly lowered in AML patients suggesting a putative link between PLA2 activity and coagulation disorders. Bacterial and fungal infections are the major cause of morbidity and mortality in acute leukemic patients. A decreased PLA2-IIA activity which has physiologically significant bactericidal activity,1, 2 and a decreased PLA2-V one that plays a role in innate immunity against fungal invasion1, 2 might be implicated in these infections. Numerous evidences have highlighted that cytosolic PLA2-IVA is the key enzyme for AA release from phospholipids of mammalian cells; AA being the first step for the biosynthesis of eicosanoids. Only those sPLA2 species that have a high specific activity of phospholipids hydrolysis and that can bind well to phosphatidylcholine-rich membranes, the PLA2-V and PLA2-X, have the capacity to release fatty acids when added to mammalian cells. As shown in Figure 1, the increase of PLA2-X transcripts in leukemic blasts is higher than the decrease of their PLA2-V transcripts. Thus, as lipolytic enzyme, PLA2-X might contribute to the generation of lipid mediators. It is interesting that prostaglandin E2, a COX metabolite of AA, was recently reported to stimulate the growth of leukemic blasts through an EP2 receptor-dependent pathway.4

These results show that mRNA from four out of five cytosolic PLA2 (PLA2-IVA, PLA2-IVB, PLA2-IVC and PLA2-VI) and six out of nine sPLA2 (PLA2-IB, PLA2-IIA, PLA2-IID, PLA2-V, PLA2-X and PLA2-XII) are present in leukemic blasts, and that their mRNA transcript levels exhibited important variations as compared with blood mononuclear cells (summarized in Table 1). One of the more notable finding is that leukemic blasts expressed high amounts of PLA2-VI and PLA2-X. This could be extremely significant as these two enzymatic activities play a major role in AA release for the generation of COX- and lipoxygenase-derived lipid mediators. In conclusion, these results indicate that immature forms of leukemic AML and ALL blasts have the potential to express multiple isoforms of cPLA2 and sPLA2, which could be of importance given the potential role of these enzyme activities in inflammation, generation of lipidic mediators, anticoagulant activity and bacterial infection. Investigation of PLA2 transcripts in other AML such as AML with genetic abnormalities and with multilineage dysplasia deserve now to be investigated.

Table 1 Comparison of soluble PLA2 and cytosolic PLA2 transcripts levels in ALL blasts, AML blasts and control blood mononuclear cells

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Acknowledgements

This work was supported in part by a grant from ‘Le Lions Club de la Corrèze, Zone 33 District 103 Sud’.

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Fiancette, R., Vincent, C., Donnard, M. et al. Genes encoding multiple forms of phospholipase A2 are expressed in immature forms of human leukemic blasts. Leukemia 23, 1196–1199 (2009). https://doi.org/10.1038/leu.2009.36

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