meningiomas are the second most common primary intracranial tumor . meningiomas present clinically by causing focal or generalized seizure disorders , focal neurological deficits or neuropsychological decline . a large number of molecular and genetic pathways that are altered in brain tumor cells have been identified . among them , a possible role for the eicosanoid cascade has been suggested in meningiomas . phospholipase a2 ( pla2 ) is the key enzyme involved in eicosanoid generation [ 35 ] . pla2 catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate the eicosanoid precursor arachidonic acid ( aa ) [ 35 ] . four distinct families have been documented : low molecular weight secreted forms of pla2 ( spla2 ) , ca - dependent high molecular weight pla2 ( cpla2 ) , ca - independent high molecular weight pla2 ( ipla2 ) ; and the platelet - activating factor acetylhydrolase ( paf - ah ) . the spla2 family is implicated in several biological processes such as inflammation and host defense [ 3 , 4 ] . nine isoenzymes have been identified in human : pla2-ib , pla2-iia , pla2-iid , pla2-iie pla2-iif , pla2-iii , pla2-v , pla2-x , pla2-xiia , and pla2-xiib . in addition to their function in digestion of dietary phospholipids , host defense against bacteria and aa release from cellular phospholipids for eicosanoid synthesis , two classes of receptors ( m and n ) and several extracellular binding proteins have been identified indicating that spla2 might signal through receptor activation [ 35 ] . in human the cpla2 family consists of four members , pla2-iva , pla2-ivb , pla2-ivc , and pla2-ivd ; pla2-iva being the central regulator of stimulus - coupled cellular aa release [ 35 ] . in human the ipla2 group consists of seven members , ipla2 ( pla2-via-1 ) currently being the best known member and playing major role in phospholipids remodeling and cancer [ 3 , 5 ] . beside its important place for eicosanoid generation , pla2 is also the key enzyme for the generation of the pro - inflammatory lipid mediator paf recently documented in human meningioma . paf - ah activity which hydrolysis paf into the inactive paf precursor , lyso - paf is detected in meningioma . however no results reported whether this enzymatic activity originated from pla2-viia and/or pla2-viib , the plasma paf - ah and the intracellular paf - ah forms , respectively . currently the contribution of pla2 in meningiomas is poorly documented despite the fact that pla2 inhibition decreased the growth of cultured meningioma cells . in view of the potentially important oncogenic action of the various pla2 species , we investigated , at the mrna levels , which of them were expressed in intracranial human meningiomas . the procedure of the present study followed the rules edited by the french national ethics . ethics approval was obtained from the ethics committee of our hospital ( chu dupuytren , limoges , france ) . twenty six patients who underwent surgery for intracranial meningiomas ( from 1998 to 2004 ) were investigated . after undergoing the routine hospital analysis , the excess of sample was kept at 80c until use and this in accordance with the regulations in force in france . no written or oral consent was obtained because it is a study of samples already collected and referred to research prior the french bioethical low ( 2004 ) . normal meninges were not available in our institution in light of our ethic committee law . the low amounts ( 1015  mg ) of available tumors only permitted investigations at the mrna level . there were 16 grade i meningiomas including 8 transitional ( 2 man , 6 women , mean age 60 years ) , 3 meningothelial ( 1 man , 2 women , mean age 60 years ) , and 5 fibrous ( 5 women , mean age 59 years ) . height tumors were grade ii meningiomas : 7 atypical ( 6 men , 1 woman , mean age 58 years ) and 1 chordoid meningiomas ( 1 man , 54 years ) . two tumors were classified as anaplastic grade iii meningiomas ( 2 men , mean age 54 years ) . necrosis was assessed using morphological criteria defined by the who classification of meningiomas :  foci of spontaneous or geographic necrosis  . ( qiagen , courtaboeuf , france ) from 1015  mg of tumor tissue . before rna extraction ( bertin technologies , montigny - le - bretonneux , france ) in 1  ml qiazol lysis reagent and homogenized at 5500  rpm during 2-fold 40  sec in the automated mixer precellys ( bertin technologies ) . then homogenized samples were used for rna purification according to the manufacturer 's protocol . a dnase i digestion step was included for each extraction to avoid rna contamination by genomic dna . rna integrity was checked by capillary electrophoresis on the bioanalyzer 2100 ( agilent technologies , massy , france ) . only rna with an rna integrity number ( r.i.n ) higher than 5.5 was used for reverse transcription . total rna concentration was determined by measuring absorbance at 260  nm with a spectrophotometre nanodrop nd-1000 ( labtech , paris , france ) . total rna was reverse transcribed in single strand cdna using random hexamers and as described in the protocol of the  superscript iii first - strand synthesis system for rt - pcr briefly , 1  g total rna was incubated with 200  u m - mlv reverse transcriptase in the presence of 0.5  mm dntps , 50  ng random hexamers , 5 mm mgcl2 , 10  mm dithiotreitol , and 40  u rnase inhibitor , in a final volume of 20  l 1x rt buffer . reverse transcription was performed as follows in a thermocycler gold 9700 ( applied biosystem ) : denaturation during 5  min at 65c and chilling 1 min on ice , hybridization during 10  min at 25c followed by cdna synthesis during 50  min at 50c ; enzyme was inactivated by a 5  min incubation at 85c and chilling on ice ; finally the destruction of the rna portion of the rna : cdna hybrids was performed by 2  u rnase h during 20  min at 37c . pla2-ib , pla2-iia , pla2-iid , pla2-iie , pla2-iif , pla2-iii , pla2-iva , pla2-ivb , pla2-ivc , pla2-ivd , pla2-v , pla2-vi , pla2-x , pla2-xiia , pla2-xiib , pla2-viia ( plasma paf - ah ) , pla2-viib ( intracellular paf - ah ) , and pla2r transcripts were analyzed using real - time polymerase chain reaction ( pcr ) . pcr was performed in duplicate by using taqman assay reagents ( applied biosystems , foster city , ca ) [ 9 , 10 ] . product references were the following : pla2-ib : hs00386701-m1 ; pla2-iia : hs00179898-m1 ; pla2-iid : hs00173860-m1 pla2-iie : hs00173897-m1 ; pla2-iif : hs00224482-m1 ; pla2-iii : hs00210447-m1 ; 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 ; pla2-xiia : hs00830106-s1 ; pla2-xiib : hs00261432-m1 ; pla2-viia : hs00968593-m1 ; pla2-viib : hs01042135-m1 ; pla2r : hs00234853-m1 . real - time pcr were performed following the recommendations of the manufacturer in a final volume of 20  l with 10  l of 2x universal pcr master mix , 20  ng of cdna in a volume of 9  l ( the amount of cdna is an equivalent based on the initial amount of rna used for the rt reaction and 1  l of a 20x taqman   gene expression specific probe . pcr parameters were the following : 95c for 10  min and forty cycles of 95c/15  sec and 60c/60  sec . amplification products were analyzed on an abi prism 7000 system ( applied biosystems ) [ 9 , 10 ] . gene expression levels were normalized to 18s rna ( product reference : hs99999901-s1 ) according to the manufacturer 's recommendation . amounts of various transcripts were compared to sample with the lowest level of transcripts ( a patient who was arbitrary quoted 1 ) . the relative quantification of gene expression was performed using the comparative ct method ( ct ) ( figure 1(a ) ) . mean ct values were used in the ct calculation by using the  relative quantitation calculation and analysis software for applied biosystems real - time pcr systems  . nonrt controls ( only with rna ) and blank rt controls ( rt without rna ) were run to make sure the amplifications were specifics . significance was assessed by using the kruskal - wallis test followed by a mann - whitney u - test . in a first set of experiments we investigated if mrnas derived from the five intracellular pla2 genes ( four cpla2 and ipla2 ) were detected in meningiomas . as shown in figure 1(b ) , mrnas derived from four of these five cloned pla2 genes are detected . in contrast , pla2-iva , pla2-ivb , pla2-ivc , and pla2-vi were detected in 96% ( 25/26 ) , 100% ( 26/26 ) , 92% ( 24/26 ) , and 100% ( 26/26 ) of tumors , respectively ( figure 2 ) . no difference ( p > .05 , mann whitney u - test ) was found for pla2 transcript amounts in relation to the tumor grade ( figure 1 ) , nor the subtype of meningiomas , the presence of inflammatory infiltrated cells , of an associated edema , mitosis , brain invasion , vascularisation or necrosis ( data not shown ) . the analysis of twenty six patients indicated the following rank of magnitude in human meningiomas : pla2-vi   =   pla2-ivb > pla2-iva > pla2-ivc ( table 1 ) . pla2-ivb and pla2-ivc had little specificity for the sn-2 fatty acid as compared with pla2-iva which preferentially hydrolyses phospholipids containing aa at the sn-2 position [ 35 ] . pla2-vi was originally reported to mediate phospholipid remodeling and , thus , to act as a housekeeping protein without significant role in cell growth [ 3 , 4 ] . however several recent studies have demonstrated 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 mapk activation . of interest , involvements of p53 and mapk kinase have been recently reported in the pathology of human meningiomas [ 12 , 13 ] . a role for pla2-vi together , these observations might suggest pla2-vi as a novel and interesting target for drug development for meningioma 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 .   in a second set of experiments we investigated if 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 , pla2-xiia , and pla2-xiib ) were detected in human meningiomas . pla2-iie , pla2-iif , and pla2-xiib transcripts were not present at detectable levels in tumors while pla2-iid and pla2-x transcripts were detected in only a few number ( 4/26 , 15% ) of them . in contrast , pla2-ib , pla2-iia , pla2-iii , pla2-v and pla2-xiia were detected in 88% ( 23/26 ) , 88% ( 23/26 ) , 77% ( 20/26 ) , 65% ( 17/26 ) , and 96% ( 25/26 ) of tumors , respectively ( figure 3 ) . results indicated the following rank of magnitude for spla2 transcripts in meningiomas : pla2-xiia > pla2-iia > pla2-ib   =   pla2-v   =   pla2-iii   =   pla2-iid > pla2-x . no difference ( p > .05 , mann whitney u - test ) was found for spla2 transcript amounts in relation to the tumor grade ( figure 3 ) , nor the subtype of meningiomas , the presence of inflammatory infiltrated cells , of an associated edema , mitosis , brain invasion , vascularisation or necrosis ( data not shown ) . whether pla2-xiia exhibits a weak aa catalytic activity , a potential role for this enzyme is suggested in membrane fusion or cell division . pla2-xiia was reported to inhibit bone morphogenetic protein ( bmp ) through the loss of activated smad1/4 complexes ; a result of importance since bmp inhibits the tumorigenic potential of human glioblastomas by triggering the smad signaling cascade . apart from its lipolytic and pro - inflammatory activities , pla2-ib acts as receptor ligand to induce cell signaling and subsequent activation of cpla2 , thus indirectly contributing to aa production . pla2-iia elicits a mitogenic response and activates aa metabolism in astrocytoma cells , is critical for neuronal death via reactive oxygen species   and plays a role in cellular senescence . finally , studies have suggested the role of pla2-iia , pla2-iii , and pla2-v as potential prognostic markers in colorectal adenocarcinomas and prostate cancer [ 23 , 24 ] . the data reported in figure 3 suggest that levels of pla2-iia , pla2-iii , and pla2-v transcripts greatly varied between patients ( see the log scale ) . would it be possible that their levels were related to patient outcomes in meningioma tumors ? clearly investigation of a larger number of patients would be of interest to test this hypothesis . in a third set of experiments we investigated paf - ah enzymes that constitute another pla2 subfamily . as shown in figure 4 ( upper panel ) , pla2-viia ( the plasma paf - ah form ) and pla2-viib ( the intracellular paf - ah form ) were present in 100% ( 23/23 ) and 95% ( 22/23 ) meningioma tumors . no difference ( p > .05 , mann whitney u - test ) was found for paf - ah transcript amounts in relation to the tumor grade ( figure 4(a ) ) , nor to other clinical data ( data not shown ) . the present results confirm a previous study reporting paf - ah enzymatic activity in meningioma homogenates . degradation of these bioactive phospholipids by paf - ah may lead to the termination of inflammatory reaction . its presence in human meningioma is consistent with the presence of paf in meningioma tumors [ 6 , 25 ] . finally in a fourth set of experiments we focused our attention on pla2r transcripts in human meningioma . as shown in figure 4 ( lower panel ) , pla2r transcripts were detected in 100% ( 23/23 ) meningioma tumors but without significant link with the tumor grade . the pla2r can act as a ligand for several spla2 thus mediating a variety of biological responses ( such as cell proliferation , cell migration , hormone release , lipid mediator production and cytokine production ) . in turn , pla2r can also play a negative role in spla2 functions by downregulating their exaggerated reactions as pla2r is involved in the degradation / internalization of spla2 . particularly , pla2r deficient mice exhibit resistance to endotoxic shock   and knockdown of the pla2r prevents the onset of replicative senescence and diminishes stress - induced senescence . numerous genes encoding multiples forms of cpla2 , spla2 , and paf - ah are expressed ( at the mrna level ) in human meningiomas where they might act on tumor growth not only by acting on phospholipid remodeling but also by altering the local eicosanoid and/or cytokine networks . it is of evidence that immunhistochemistry would be of importance to confirm the relative expression of the different pla2 forms in human meningioma tumors . the discovery of specific receptors that bind spla2 strongly indicate that these enzymes can exert various biological responses via binding to a receptor , in addition to their enzymatic activity . of interest meningioma tumors expressed pla2r transcripts . further studies are clearly needed to elucidate the contributions of spla2 , cpla2 , ipla2 , and paf - ah in meningioma and to determine their possible relevance in the targeting of new therapeutic interventions .
in view of the important oncogenic action of phospholipase a2(pla2 ) we investigated pla2 transcripts in human meningiomas . 
 real - time pcr was used to investigate pla2 transcripts in 26 human meningioma tumors . 
 results indicated that three ca2 + -dependent high molecular weight pla2 ( pla2-iva , pla2-ivb , pla2-ivc ) , one ca2 + -independent high molecular weight pla2 ( pla2-vi ) and five low molecular weight secreted forms of pla2 ( pla2-ib , pla2-iia , pla2-iii , pla2-v , and pla2-xii ) are expressed with pla2-iva , pla2-ivb , pla2-vi , and pla2-xiia as the major expressed forms . 
 pla2-iie , pla2-iif , pla2-ivd , and pla2-xiib are not detected . 
 plasma ( pla2-viia ) and intracellular ( pla2-viib ) platelet - activating factor acetylhydrolase transcripts are expressed in human meningiomas . 
 however no difference was found for pla2 transcript amounts in relation to the tumor grade , the subtype of meningiomas , the presence of inflammatory infiltrated cells , of an associated edema , mitosis , brain invasion , vascularisation or necrosis . in conclusion 
 numerous genes encoding multiples forms of pla2 are expressed in meningiomas where they might act on the phospholipid remodeling and on the local eicosanoid and/or cytokine networks .