Diverse expression of cytosolic phospholipase A2,

5-lipoxygenase and prostaglandin H synthase 2 in acute pre-B-lymphocytic leukaemia cells

S T I N A F E L T E N M A R K , G~JDMIJNDUR RUNARSSON,* PONTIlS LARSSON, PER-JOHAN JAKOBSSON, MAGNIJS B J ~ R K H O L M * AND HANS-ERIK CLAESSON Department of Medical Biochemistry and Biophysics, Karolinska Znstitutet, Stockholm, and *Division of Medicine, Section of Haernatology and Medical Immunology, Karolinska Hospital, Stockholm, Sweden

Received 20 January 1995; accepted for publication 2 April 1995

Summary. Several lines of evidence suggest that phospho- lipases AZ, leukotrienes and prostaglandins play a role in the proliferation of haemopoietic cells. The expression of genes involved in the biosynthesis of leukotrienes and prosta- glandins was investigated in peripheral B lymphoblasts, isolated from eight patients with acute pre-B-lymphocytic leukaemia (pre B-ALL). RT-PCR analysis demonstrated that four of the investigated pre-B-ALL clones expressed the gene coding for cytosolic phospholipase A2 (cPLA2). but not the gene coding for 5-lipoxygenase. In contrast, the remaining four pre-B-ALL clones expressed 5-lipoxygenase but not cPLA2, suggesting that the transcriptional regulation of these two genes are different and that their cellular functions

are not linked to each other. The capacity of pre B-ALL cells to produce LTB4 and to express the 5-lipoxygenase protein, correlated with the expression of 5-lipoxygenase mRNA. All pre-B-ALL clones expressed genes coding for 5-lipoxygenase activating protein (FLAP), leukotriene A4 hydrolase and prostaglandin (PG)H synthase 1. Seven of the eight pre B- ALL clones expressed PGH synthase 2. In comparison, normal tonsillar B cells did not express CPLA, or PGH synthase 2.

Keywords: arachidonic acid, leukotriene, FLAP (five- lipoxygenase activating protein), leukotriene A4 hydrolase, endothelial cells.

The differentiation of B-lineage cells is blocked at an early stage in acute pre-Rlymphocytic leukaemia (pre-BALL). The disease is characterized by a clonal expansion of immature B cells and suppression of normal haemopoietic stem cells. Multidrug chemotherapy in combination with glucocorticoids is used in the treatment of the disease.

Several reports indicate that phospholipases and products of arachidonic acid have a role in the differentiation and proliferation of certain types of cells (Claesson et al, 1992: Marnett, 1992; Mukherjee et al, 1994: Palmberg et al, 1991). Phospholipases A, (PLA2) (EC 3.1.1.4) are a group of enzymes that are involved in a variety of physiological and pathophysiological processes (Glaser et al, 1993: Hayakawa et al, 1993; Kast et al, 1993). These enzymes can be subdivided into low molecular mass enzymes (10-14 kD) which are cell-associated or extracellular and high mole- cular mass enzymes (85-11OkD) localized in the cytosol

Correspondence: Dr Hans-Erik Claesson. Department of Medical Biochemistry and Biophysics. Doktorsringen 22, Karolinska Institutet, S-17177 Stockholm, Sweden.

0 199 5 Blackwell Science Ltd

(Glaser et al, 1993). The cytosolic, 85 kD, PLA2 (cPLA2) is of particular interest, because several reports indicate that this enzyme is related to the formation of eicosanoids (Lin et al. 1992; Schakwijk et al, 1992). Furthermore. cPLAz seems to have a crucial role in the cytotoxic action of tumour necrosis factor (TNF) and the synthesis of cPLAz is blocked by glucocorticoids (Glaser et al, 1993; Hoeck et al, 1993). The enzyme has been cloned from the human monocytic cell line U937 (Clark et al, 1991; Sharp et al, 1991). Cytosolic PLA, possesses high selectivity for arachidonic acid from the sn-2 position of phospholipids, responds to micromolar levels of calcium, and is active in the reductive milieu (Clark et a / , 1991).

After phospholipase-catalysed liberation of arachidonic acid from the membrane phospholipids, the fatty acid can be further converted to either prostaglandins, thromboxanes or leukotrienes. The relative role of the 85 kD cPLA, for these Merent biosynthetic pathways is not known. Prostaglandin (PG)H synthase (EC 1.14.99.1) (also known as cyclo- oxygenase) catalyses the next step in the conversion of arachidonic acid to prostaglandins and thromboxanes. The

585

586 Stina Feztenmark et a2

enzyme is widely distributed in mammalian tissues. There is, however, no convincing report demonstrating that lympho- cytes express PGH synthase. Recently, it was reported that certain cells express an isoform of PGH synthase, named PGH synthase 2. The gene appears to be a primary response gene that is induced by mitogens and is inhibited by glucocorticoids (Herschman et d, 1993; Hla & Neilson, 199 2; Kujubu et d. 1991: Xie et al, 1991). Both enzymes are inhibited by aspirin and other non-steroidal anti-inflamma- tory drugs (NSAID), but to different extent (Smith, 1992). Interestingly, aspirin has recently been reported to decrease the incidence of colon cancer, but the mechanism of action is not clear (Marnett, 1992).

Biosynthesis of leukotrienes proceeds via 5-hydroperoxy- eicosatetraenoic acid (5-HPETE) and the intermediate leukotriene (LT)A4. These two consecutive reactions are catalysed by 5-lipoxygenase (EC 1.13.11.34) (Samuelsson & Funk, 1989). Leukotriene A4 can be further hydrolysed enzymatically to LTB4, catalysed by the enzyme LTA, hydrolase (EC 3.3.2.6). The cellular synthesis of leuko- trienes is dependent on 5-lipoxygenase activating protein (FLAP) which is an arachidonic acid binding protein (Dixon et al, 1990; Mancini et al, 1993; Miller et al, 1990; Woods et aI, 1993).

Human B lymphocytes express 5-lipoxygenase and produce LTB, to approximately the same extent as granulocytes (Claesson et d. 1993: Jakobsson et al, 1991b, 1992). In contrast, human T lymphocytes apparently lack 5-lipoxygenase activity (Claesson et al, 1993; Jakobsson et al, 1991b, 1992). The mechanism of activation of leukotriene synthesis in intact B lymphocytes is quite different to the activation of leukotriene synthesis in polymorphonuclear leucocytes (PMNL) and monocytes, but the physiological ligand(s) that activates LTB4 synthesis in B cells is not yet known. PMNL and monocytes produce leukotrienes after stimulation with the ionophore A2 3 18 7 or after receptor- mediated activation, in the presence or absence of exogenous arachidonic acid. Intact human tonsillar B lymphocytes and monoclonal B cell lines are dependent, in addition to stimulation with calcium ionophore A2 3 187 and exogenous arachidonic acid, on the presence of a thiol-reactive substance such as diamide (azodicarboxylic acid bis(dimethylamide)), to produce 5-lipoxygenase products (Jakobsson et al, 1992). Leukotriene B4 mediates certain inflammatory and immuno- logical reactions and has been shown to induce the release of a variety of cytokines from monocytes and T cells (Brach et al, 1992; Claesson et al, 1992; Rola-Pleszczynski & Stankova, 1992; Yamaoka & Kolb, 1993). Furthermore LTB, stimu- lates lymphocyte proliferation (Claesson et al, 1992), myelopoiesis (Claesson et al, 1985; Stenke et al, 1993) and expression of the proto-oncogenes c-jos and c-jun (Stankova & Rola-Pleszczynski, 1992).

The present report deals with the gene expression of enzymes involved in the metabolism of arachidonic acid in pre-B-ALL in relation to the stage of B-cell differentiation.

MATERIALS AND METHODS

RPMI 1640 with Hepes buffer 25 mM, L-glutamine and

penicillin were from GIBCO (Paisley, U.K.). Nunclon cell culture bottles were from Nunc A/S (Roskilde. Denmark). Penicillin was from Astra (Sodertalje, Sweden).

HPLC solvents were obtained from Rathburn Chemicals (Walkerburn, U.K.) and synthetic standards of 5-(S)-HETE and LTB4 were from Biomol (Plymouth Meeting, Pa., U.S.A.). The calcium ionophore A23187 was purchased from Calbiochem-Behring (La Jolla, Calif., U.S.A.). Strepto- mycin, diamide and goat anti-rabbit IgG coupled to alkaline phosphatase were from Sigma Chemical Industries (St Louis, Mo.. U.S.A.). PCR primers were obtained from Scandinavian Gene Synthesis AB (Koping, Sweden).

RNasin, Taq DNA polymerase and dNTPs were from SDS Promega (Falkenberg, Sweden) and Moloney murine leukaemia virus (M-MLV) reverse transcriptase was pur- chased from USB (Ohio, Canada). Oligo dT(12-18), restriction enzymes and restriction enzyme buffer (One Phor All Buffer) were from Pharmacia (Uppsala, Sweden). Monoclonal antibodies for flow cytometric analysis were from Dako A/S (Copenhagen, Denmark).

The rabbit anti-human 5-lipoxygenase antibody and the recombinant 5-lipoxygenase were kind gifts from Jilly F. Evans, Merck Frost Centre for Therapeutic Kesearch (Quebec, Canada).

Isolation of cells. The source of human lymphoblasts was previously Ficoll-Isopaque purified and frozen peripheral blood lymphoblasts, collected at diagnosis or relapse, from adult patients (age 16-77) with acute lymphoblastic leukaemia of pre-B phenotype. After thawing, the cells were resuspended in RPMI 1640 (supplemented with Hepes buffer 25 mM, AB serum 5%, L-glutamin 2 mM. streptomycin 0-2mg/ml and penicillin 100 IE/ml) and centrifuged at 400 g for 5 min. The pellet was resuspended in RPMI 1640 (supplemented with 10% AB serum) to a final concentration of 4 x lo7 cells/ml. From this suspension, 25 ml was incubated at 37°C for 45 min in 600 ml Nunclon bottles. The purpose of this incubation was to eliminate contaminat- ing monocytes.

Non-adherent cells were collected and centrifuged at 400 g for 5 min. The pellet was resuspended in PBS and kept on ice for further analysis. Viability was tested by trypan blue dye exclusion. The purity of the cells was estimated by flow cytometric analysis (with FACScan, Becton Dickinson) using antibodies 0 1 4 and CD19. Morphological analysis of the cells was performed by staining with May-Grunwald/Giemsa solution. Platelet contamination was evaluated by morpho- logical examination in Kristensson's solution.

Human umbilical cord vein endothelial cells (HUVEC), human monocytes, BL41-E9 5-A, chronic lymphocytic leukaemia of B-cell type (B-CLL) cells, human tonsillar B cells and washed human platelets, from healthy donors, were isolated as previously described (Claesson et al, 1990, 1988; Hamberg et a!, 1974; Jakobsson et a!, 1991a; Rosen et al, 1986).

Incubation of intact and sonicated cells. The isolated cells (107/sample) were suspended in 1 ml of phosphate-buffered saline (PBS) without calcium. In the intact cell assay, the suspension was prewarmed for 30 s at 3 7°C prior to addition of CaClz, arachidonic acid and ionophore A23187 (final

0 1995 Blackwell Science Ltd, British Journal of Haematolog~ 90: 585-594

Leukotrienes, Prostaglandins and B Lymphocytes 5 8 7 5-LO 5’ ACC ATT GAG CAG ATC GTG GAC ACG C 3’

5’ GCA GTC CTG CTC TGT GTA GAA TGG G 3‘ 5’ GGC CAT CGT CAC CCT CAT CAG CG 3‘ 5’ GCC AGC AAC GGA CAT GAG GAA CAG G 3’

5’ GAA CAA CTG C l T GGA GGA CCA GAG 3’ 5’ TGC AGT CAC GGG ATG CAT GCT TGC 3’ 5’ CCT ACA CCT CCT TCC AGG AGC TC 3’ 5’ AGA CCA GCT TCT TCA GTG TGG CCG 3’

5‘ TTC AAA TGA GAT TGT GGG AAA ATT GCT 3’ 5’ AGA TCA TCT CTG C m GAG TAT C1T 3’

FLAP

LTAl hydrolase:

PGH synthase 1:

PGH synthase 2:

In order to prevent amplification of genomic DNA, the two primers in each set spanned introns.

After amplication, aliquots of the PCR mixture were analysed by electrophoresis in 2% agarose gels. For each sample, 5pl of PCR mixture was mixed with 2p1 loading buffer (0.25% bromophenol blue, 0.25% xylene cyanole FF and 50% glycerol) and loaded to the gel which was then run for 1 h, 65V.

The expected DNA fragments for the different sets of primers were: P-actin (784bp), cPLA2 (554bp), 5- lipoxygenase (488 bp), FLAP (352 bp), LT& hydrolase (624 bp), PGH synthase 1 (285 bp) and PGH synthase 2 (305 bp).

Restriction enzyme analysis of PCH synthase 1, PGH sgnthase 2 and cPLA2. Since the capacity of pre-B-ALL cells to metabolize arachidonic acid to prostaglandins and thromboxanes was not investigated, the identity of the obtained PCR products for PGH synthase 1 and PGH synthase 2 as well as for cPLA2 was confirmed by restriction enzyme cleavage. Aliquots of the amplified PCR products were extracted with one volume of phenol : chloroform 1 : 1 (equilibrated with 0.1 M Tris-HC1, pH 7.6) and centrifuged at 12000g for 15s. The DNA which remained in the chloroform phase after centrifugation was precipitated for 20 min on ice with one volume of ammonium acetate (7.5 M,

pH 7-5) and two volumes of ethanol and was thereafter centrifugated at 15 000 g for 20 min. The supernatants were discarded and the pellets were dried under vacuum and were subsequently dissolved in destilled water. Aliquots of the dissolved cDNA were mixed with restriction enzyme and buffer (Pharmacia, Sweden) in a total volume of lop1 or 4 0 ~ 1 , depending on the restriction enzyme used. The mixtures were incubated at 37°C for 2 h and then heated to 65°C for 20min in order to inactivate the enzyme. To analyse if cleavage was successful, the restriction enzyme mixtures were run in a 2% gelelectrophoresis, 65 V for 1.5 h.

The PGH synthase 1 fragment was cleaved with the restriction enzyme BanII. The reaction mixture contained Bad1 (20 U). restriction enzyme buffer (2 pl) and dissolved DNA in a total volume of 401.11. Cleavage gave rise to two fragments, 1 2 2 and 163 bp, respectively.

The PGH synthase 2 and cPLA2 fragments were both cleaved with the restriction enzyme NcoI. The reaction mixture contained NcoI (30 U), restriction enzyme buffer (2 pl) and dissolved DNA in a total volume of 10 pl. For PGH synthase 2, the cleavage gave rise to two fragments, 144 and 161 bp, respectively, and for cPLAz the sizes of the obtained fragments were 387 and 167 bp, respectively.

The obtained cleavage products for PGH synthase 1, PGH

concentrations 1 mu, 40 ~ L M and 2.5 p ~ , respectively). After incubation at 37°C for 10min. the reaction was terminated by the addition of 1ml methanol. In experiments using diamide ( h a 1 concentration 1 0 0 ~ ~ ) the cells were preincubated in the presence of this drug for 10 min at 20°C.

In the sonicated cell assay, 1 mM EDTA was added to the cell suspension and the cells were sonicated three times for 5 son ice. Subsequently, ATP (final concentration 1 m) was added, and the samples were preincubated for 30 s at 3 7°C before addition of CaCl, and arachidonic acid (final concentrations 2 mM and 40 PM, respectively). The reaction proceeded for 10 min at 37°C and was then terminated by addition of 1 ml methanol.

Analysis of leukotrienes and monohydroxy acids. After incubation. the samples were diluted in PBS (05ml) and PGBl (177.5pmol) was added as an internal standard. Centrifugation was performed in order to get rid of precipitated proteins and cellular debris. The supernatants were extracted by using a disposable octadecyl reverse-phase column (Chromabond, CI8, 200 mg, Machereyl Nagel) and the samples were thereafter subjected to analysis in a reverse-phase HPLC system as described (Jakobsson et al, 1992).

Isolation of total RNA and RT-PCR. Total cellular RNA was isolated as described (Chomczynski & Saachi, 1987). The extracted total RNA was quantitated spectrophotometrically and 2 pg was submitted to reverse transcription in order to produce cDNA. The reverse transcription mixture contained 2 l g of total RNA, 1 x RT buffer ( 5 0 m KCl/lOm Tris- HC1, pH 8.3/2 rn MgCl2/0.O1% gelatin), 5 mM dithiotreithol (DTT), 0.5 m~ dNTPs, 3 7 U RNasin, 200 ng Oligo dT(12-18) and 500 U M-MLV reverse transcriptase in a total volume of 40 p1. The reaction was carried out at 3 7°C for 60 min.

For the PCR reaction, 2 pl of cDNA was mixed with 46 p1 amplification mixture and 2 U Taq DNA polymerase. The amplification mixture was 1 x Taq polymerase buffer (SDS Promega). 0 2 m~ dNTPs, 0-5 m~ each of 5’ and 3‘ primers and 0.5 or 1 . 0 1 ~ MgC12 ( 0 - 5 m ~ for 5-lipoxygenase and FLAP and 1.0 mM for the rest of the primer sets). The reason for the different concentrations of MgClz in the amplification mixture was the finding that different sets of primer pairs varied in their optimal requirement for magnesium ions. Over the mixture, one drop of mineral oil was laid.

Amplification was performed as follows: First cycle, denaturation at 95°C for Smin, annealing at 60°C for 1 min and extension at 72°C for 1.5 min: subsequent cycles, denaturation at 95°C for 1 min, annealing at 60°C for 1 min and extension at 72°C for 1.5 min: last cycle, denaturation at 95°C for 1 min annealing at 60°C for 1 min and 72°C for 10 min.

Twenty-two cycles were carried out for p-actin, 24 cycles for LTA4 hydrolase, 28 cycles for FLAP and 5-lipoxygenase (5-LO) and 34 cycles for cPLA2, PGH synthase 1 and PGH synthase 2.

The following primers were used: p-actin: 5’ GAG GAG CAC CCC GTG CTG CTG A 3’

5’ CTA GAA GCA TTT GCG GTG G 3’ 5’ TTG CAA ACT GCC TCA GCA TCA G 3‘ 5‘ CTC TAG TCC TCC GTT CAA GGA AC 3’

cPLA,:

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588 Stina Feltenmark et a1

synthase 2 and cPLA, were of expected sizes and thereby con6rmed the identity of the transcripts (data not shown).

Western blot analysis of 5-lipoxygenuse. Cells were washed and resuspended in PBS without calcium and magnesium. For each sample, 20 x lo6 cells were centrifuged at 300g for 10 min and the pellet was resuspended in homogeniza- tion buffer (100 pl: PBS without calcium and magnesium, supplemented with 2 m dithiotreithol (DTT). 2 nw ethylene diamide tetraacetate (EDTA), 2 m ethylene glycol tetra- acetate (EGTA), 1 mM phenyl methyl sulfonylfloride (PMSF). leupeptin (10 pg/rnl), bacitracin (100 pg/ml) and 1 mM benzamidine). The suspension was sonicated on ice, using a probe sonicator, three times for 5 s and then centrifuged at 15 0 0 0 g for 10min. The supernatant was mixed with one volume of loading buffer (1 M Tris-HC1, pH 7*5/1% sodium dodecyl sulphate (SDS)/O*l M 2-mercaptoethanol/2 5% gly- cerol/0.02% bromophenol blue) and boiled for 2 min. SDS/ PAGE and Western blotting were performed as described (Medina et ul. 1990). After blotting, the nictrocellulose membrane was incubated overnight at 4°C with rabbit anti- human 5-lipoxygenase antibody, diluted 1 : 400. The second antibody was goat anti-rabbit IgG coupled to alkaline phosphatase (dilution 1 : 1000). The immunoreactive bands were detected with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium as substrates.

RESULTS

Characterization of pre-B-ALL cells The expression of various surface antigens on pre-B-ALL cells are commonly used as markers for the stage of differentiation. Pre-B-ALL cells that do not express the antigens CD10 and CD20/22 are more immature than cells expressing these antigens (Nilsson, 1992). Five of the eight investigated pre-B-ALL clones expressed CD10 (Table I). All clones expressed the &cell antigen CD19. The amount of contaminating monocytes in the cell preparations was estimated by flow cytometry using monoclonal antibodies

Table 1. Characteristics of pre-B-ALL.

raised against the antigen CD14. Only one cell preparation contained >1% of CD14-positive cells (patient H, 1.2%; Table I). Platelet contamination was investigated and always found to be <1%.

Leukotriene synthesis in pre-B-ALL cells Intact pre-B-ALL cells ( lo7 cells/sample) were stimulated with arachidonic acid, calcium ionophore A23 18 7 and diamide. After incubation for l0min at 37°C the reaction was terminated and the products were subsequently analysed by reverse-phase HPLC. In parallel, the capacity of sonicated cells to synthesize these products was investigated. As shown in Fig 1, cells negative for 0 1 0 (patients A and B), representing more immature B-ALL cells, did not possess 5-lipoxygenase activity, because no LTB4 or 5-HETE could be detected. On the other hand, B-ALL cells that expressed the CDl0 antigen (patients E, F, G and H), representing more mature cells, produced both LTB4 and 5- HETE, at amounts similar to those produced by tonsillar B cells, monoclonal B cells, PMNL and monocytes (Claesson et d. 1988. 1993; Jakobsson et al, 1991b. 1992). The most immature of the CD10-positive clones, namely clone E, produced relatively small amounts of these compounds, whereas cells from patient H, which had the most mature pre-B-ALL phenotype, produced the highest amounts of LTB4 and 5-HETE (Fig 1). No 5-lipoxygenase products could be detected in intact cells from either of these six patients stimulated with calcium ionophore A23187 only (data not shown), in agreement with the FACS analysis showing that the cell population was devoid of contaminating PMNL and monocytes.

Detection of mRNAs coding for the enzymes involved in the synthesis of leukotrienes and prostaglandins The expression of genes coding for the enzymes required for production of LTB4 and prostaglandins was investigated in the six patients shown in Fig 1; two additional patients (designated C and D) were included in thii study. Total RNA

Age CR WBC count Blasts CDlO CD19 CD20j22 0 1 4 Patient (yr) Sex (months) ( 109m (XI) (“4 (%I) (“lo) (%I)

A B C D E P G* H

45 16 26 77 43 20 63 72

Male Male Female Male Male Male Male Male

3 4

12 9

18 13 1

n.e.

150 404

30 33 14 60

129 129

95 95 72 89 76 n.d. n.d. 97

0 0 0

70 19 88 78 87

60 76 80 94 91 62 69 87

0 0 0

29 4

30 78 87

0 0 n.d. 0 . 7 0 0 0.4 1.2

Patient data and results from flow cytometric analysis by FACScan. Samples from patients D and F were collected at relapse, the remaining at diagnosis. The antigens CDlO and CD20/22 are maturation markers for B cells. CD19 is a B-cell marker. The antigen CD14 is expressed on monocytes. CR. duration of complete remission: WRC. white blood cell; Blasts, lymphoblasts in peripheral blood as percentage of WRC: n.d, not determined; n.e., not evaluable.

* Patient with chronic myelocytic leukaemia developing a pre-6-ALL. blast crisis.

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Leukotrienes, Prostaglandins and B Lymphocytes 5 89 was isolated and converted to cUNA with M-MLV reverse transcriptase. For comparison, total RNA was also isolated from one patient with chronic lymphocytic leukaemia of B- cell type (B-CLL), human monocytes, human umbilical cord vein endothelial cells (HUVEC), BL41-E95-A cells (a Burkitt lymphoma derived B-cell line), human tonsillar B cells and human platelets. PCR analysis of cPLA, , 5-lipoxygenase, FLAP, LTA4 hydrolase, PGH synthase 1 and PGH synthase 2 was performed. As a quality control, PCR analysis of @actin was performed in all investigated cell populations and the results demonstrated successful isolation of mRNA (data not shown).

The expression of cPLAz was detected in patients A (CD10-), B (CUlO-), C (CD10-) and D (CDlO'), whereas the remaining patients did not express cPLA2 mRNA (Fig 2 ) . As expected, human monocytes also expressed cPLA, mRNA but no signals were observed in HUVEC, B-CLL cells, human tonsillar B-cells or human platelets. In addition, peripheral T lymphocytes from healthy donors did not express mRNA for cPLA2 (data not shown).

Concerning 5-lipoxygenase. the signal pattern was the opposite: patients A (CD10-), B (CD10-), C (CD10-) and D (CDlO'), who all expressed cPLA2, did not express the gene coding for 5-lipoxygenase. On the other hand, patients E, F. G and H (all CD10') expressed 5-lipoxygenase mRNA (Fig 2). These results were in agreement with the capacity of the various pre-B-ALL clones to produce LTB, and 5-HE'IE. as demonstrated in Fig 1. In accordance with earlier observa- tions, human monocytes, B-CLL cells and human tonsillar B-cells expressed 5-lipoxygenase mRNA (Claesson et al, 1993). Neither human platelets nor HUVEC expressed 5- lipoxygenase mRNA.

In contrast to the diverse expression of cPLA2 mKNA and 5-lipoxygenase mRNA in the different pre B-ALL clones, all cell clones expressed the genes coding for FLAP and LTA, hydrolase (Fig 3). Also B-CLL cells, tonsillar B cells and

Fig 1. Leukotriene synthesis in pre-B-ALL. Intact pre-B-ALL cells were incubated with diamide (100 p ~ ) for 10 min at 20°C prior to addition of arachidonic acid ( 4 0 ~ ~ ) and calcium ionophore A23187 ( 2 . 5 ~ ~ ) and thereafter incubated for another period of IOmin at 3 7°C (closed bars). Sonicated pre-B-ALL cells, supple- mented with EDTA (final concentration 1 m ) and ATP (final concentration 1 mM). were incubated with CaCI, and arachidonic acid (final concentrations 2 mM and 4 0 ~ ~ . respectively) for 10min at 3 7°C (open bars). Panel A shows the formation of LTB, and panel B the production of 5-HETE. Patients A and B were negative for C1)10, patients E-H expressed CDIO.

Fig 2. PCR analysis of cPLA2 and 5- lipoxygenase in eight different pre-B-ALL clones, B-CLL cells, human monocytes, HIJVEC. washed human platelets and human tonsillar B cells.

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590 Stina Feltenmark et d

monocytes expressed these genes, whereas platelets expressed neither FLAP nor LTA4 hydrolase. In agreement with earlier reported results, HUVEC expressed LTA4 hydrolase mRNA (Claesson et al. 1990, 1988), but no signal for FLAP was detected.

In the light of the reported effects of prostaglandins/ tromboxanes and the expression of PGH synthase 1 and 2 during cell growth and differentiation, we thought it was of interest to examine the expression of these genes in pre-B- ALL cells. Surprisingly, all clones expressed mRNA coding

Fig 3. PCR analysis of FLAP and LTA4 hydrolase in eight different pre-B-ALL clones, B-CLL cells. human monocytes, HIJVEC, washed human platelets and human tonsillar B cells.

for PGH synthase 1 (Fig 4). Patients with active previously untreated disease or relapse of B-ALL often have low platelet counts. However, since small amounts of contaminating platelets in our cell preparations could be the source of PGH synthase 1 mRNA, we carefully examined our preparations for the presence of platelets. As there were no visible platelets detected in these investigated pre B-ALL cell populations, the platelets were obviously either destroyed during freezing and thawing or eliminated during the purification of the cells. Furthermore, BL41-E9 5-A cells also expressed PGH synthase

Fig 4. PCR analysis of PGH synthase 1 and PGH synthase 2 in eight different pre-B-ALL clones, ECLL cells, human monocytes. HWEC, BL41-E95-A, washed human platelets and human tonsillar B cells.

0 1995 Blackwell Science Ltd. Briffsh Journal of Haenlatology 90: 585-594

Leukotrienes, Prostaglandins and B Lymphocytes 59 1

Fig 5. Western blot analysis of 5-lipoxygenase in two pre-B-ALL clones: patient B (010- ) and patient H (CDlO'). The nitrocellulose membrane was incubated with a polyclonal antiserum against 5-lipoxygenase. Recombinant human 5-lipoxygenase (5-LO, 25 ng) was used as standard.

1, supporting the observation that pre-B-ALL cells in fact expressed PGH synthase 1. In addition, the B-CLL cells used in this study were cultured for 4 d in the presence of growth factors prior to the experiment. Also, these cells expressed PGH synthase 1 mRNA, although to a lower extent than the pre B-ALL cells. Even for human tonsillar B cells, a weak band correlating with PGH synthase 1 was observed. Interestingly, PGH synthase 2 was strongly expressed in six of the pre-B-ALL clones and a weak signal was observed in clone H. Only pre-B-ALL cells from patient F did not express mRNA coding for PGH synthase 2 (Fig 4). As reported earlier, monocytes and HUVEC expressed these two PGH synthase genes (Hla & Neilson, 1992). However, no PGH synthase 2mRNA was detected in the B-CLL cells examined, BL41-E95-A cells, tonsillar B cells or human platelets.

Western blot analysis of 5-lipoxygenuse in pre-B-ALL cells In order to elucidate if the expressed mRNA for 5-lipoxygenase in pre-&ALL cells correlated with the expression of the 5-lipoxygenase protein, one negative clone (patient B) and one positive clone (patient H) for 5-lipoxygenase mRNA were examined. The 15 000 g super- natants of sonicated cells were submitted to SDS/PAGE followed by Western blotting, using a polyclonal anti-human 5-lipoxygenase antiserum. The supernatant of cells originating from patient B (CDlO-) did not reveal any immunoreactive band comigrating with the recombinant 5- lipoxygenase standard, whereas in the sample from patient H (CD10+), a band corresponding to recombinant 5- lipoxygenase was detected (Fig 5). These results correlated well with the capacity of the cells to express 5-lipoxygenase mRNA and to produce LTB4 (Figs 1 and 2).

DISCUSSION

In the present study we have examined the expression of genes involved in arachidonic acid metabolism in blood tumour cells from eight patients with pre-B-ALL. Based on immunophenotyping, the investigated cells represented cells at various stages of differentiation (Table I, Fig 6). Whether the pattern of mRNA expression in the examined pre-B-ALL clones was basically linked to the normal differentiation process, or to abberant enzyme expression related to the malignant process, is not possible to establish from the results of this study. The three most immature clones (patients A, B and C, all CD10-) did not express 5- lipoxygenase mRNA (Fig 2). In addition, pre-B-ALL cells

isolated from one patient (D) with the more mature phenotype (CD10' and CD20/22+) did not express 5- lipoxygenase mRNA either. In contrast, the remaining four CD10' pre B-ALL cell clones (patients E-H) expressed the gene coding for 5-lipoxygenase (Fig 2). The genes coding for FLAP and LTA4 hydrolase were expressed in all clones, independently of their stage of differentiation (Fig 3). The capacity of the investigated pre-B-ALL cells to express the 5- lipoxygenase protein and to metabolize arachidonic acid to 5-lipoxygenase products correlated well with the expression of 5-lipoxygenase mRNA (Figs 1, 2 and 5).

Several reports indicate that the high molecular cPLA2 (8 5 kl)) is the phospholipase that is linked to cellular synthesis of prostaglandins/thromboxanes and leukotrienes (Lin et al. 1992: Schalkwijket al, 1992). Interestingly, cPLA2 mRNA was only expressed in the pre-B-ALL clones that did not express 5-lipoxygenase mRNA (Fig 2). i.e. the three most immature clones and the clone from patient D. The inverse expression of these two genes indicate that cPLA2 (85 kD) is not the key phospholipase related to leukotriene synthesis. However, we cannot exclude the possibility that the cells have a pool of cPLAz proteins, synthesized earlier in the differentiation process. Studies are now in progress in order to investigate this issue. On a molecular and genetic basis, pre-B-ALL constitutes as most other types of neoplasias a very heterogenous group. Biochemical markers that make it possible to subdivide these patients into separate groups could be of importance when evaluating the antitumour effect of different chemotherapeutic agents. Based on the different expression of 5-lipoxygenase and cPLA2 mRNA, the patients investigated could be divided in two separate groups that were not strictly related to the expression of the marker antigens for differentiation of B cells (Fig 6).

Recently, it was reported that a specific 5-lipoxygenase inhibitor (MK-886). at a concentration of 100 nM, inhibited DNA synthesis in a subset of acute myeloid leukaemia cells (Khan et al, 1993). However, MK-886 did not inhibit DNA synthesis in normal bone marrow cells. Possible explana- tions of these results, in the light of the results of this study, could be that leukotrienes have an intrinsic role in cellular differentiation and growth of leukaemia clones expressing 5-lipoxygenase. In addition, it is possible that leukotrienes have an endogenous role in cellular proliferation during a specific differentiation stage or in cells with a certain genetic abberation.

Phospholipases A2 are involved in certain signal- transduction pathways and in a variety of pathophysiological processes (Glaser et al, 1993; Mukhejee et al, 1994). Several

0 1995 Blackwell Science Ltd, British Journal of Huematologg 90: 585-594

592 Stina Feltenmark et a1

Fig 6. Tentative overview of the various stages of differentiation among the pre-B-ALL clones and grouping of the patients according to their different expression of 5-lipoxygenase mRNA and cPI& mRNA. Negative and positive are abbreviated neg. and pos.. respectively.

reports demonstrate that cPLA2 has a crucial role in the cytotoxic action of TNF (Hayakawa et al, 1993; Hoeck et al, 1993). Since certain cell types are very sensitive to the cytotoxic action of TNF, it would be of interest to study the cytolytic effect of TNF on the subset of pre B-ALL cells expressing cPLA2. Glucocorticoids are used in the treatment of pre-B-ALL and in a number of lymphoproliferative disorders. Several reports have shown that glucocorticoids inhibit certain phospholipases A2, but the mechanism of action of these agents is still not clear (Lin et al, 1992; Schalkwijk et al, 1992). The synthesis of cPi;A2 is also blocked by glucocorticoids (Hoeck et al, 1993).

The observation that pre-B-ALL cells expressed mRNA for PGH synthase 1 and 2 was very unexpected. In this study we did not examine if these cells also expressed a functional PGH synthase protein and produced prostaglandins or throm- boxanes. But the finding that pre-B-ALL cells expressed PGH synthase 2, in seven of eight cases, is of interest since the transcription of this gene has been reported to be associated with cell proliferation (Herschman et al, 1993; Hla et al,

1992; Kujubu et al, 1991: Smith. 1992; Xie et al, 1991). Normal tonsillar B lymphocytes did not express PGH synthase 2 (Fig 4). PGH synthase 2 message can be induced by serum and cytokines and is associated with the expression of the v-src oncogene. Furthermore, glucocorticoids at a concentration of 2 nu inhibit the expression of PGH synthase 2 (Xie et al, 1991). Platelets express PGH synthase 1, but not PGH synthase 2 (Fig 4), and inhibition of PGH synthase 1 with NSAID increase the bleeding time due to inhibition of thromboxane synthesis. Treatment of patients suffering from pre-B-ALL with cytostatic drugs leads to thrombocytopenia and severe bleeding problems. Since those NSAU) available today block both PGH synthase 1 and 2, it is not possible to evaluate the potential antitumour effect of these drugs on pre-B-ALL cells. However, more specific PGH synthase 2 inhibitors will hopefully soon be available which can be tested, in combination with chemotherapeutic agents, in pre-B-ALL and other malignant diseases.

In summary, the present report demonstrates an inverse expression of 5-lipoxygenase and cPLAz in pre-B-ALL cells

0 1995 Blackwell Science Ltd, British Journal of Huematology 9 0 585-594

and based on these different gene expressions, the patients could be divided in two subsets. Extended studies will show if response to conventional chemotherapy is linked to the pattern of gene expression. Pre-B-AU cells also expressed PGH synthase 1 and 2. FLAP and LTA4 hydrolase. The effects on proliferation of pre-B-ALL cells of drugs that specifically inhibit cPLA, , 5-lipoxygenase and PGH synthase 2 should be investigated.

ACKNOWLEDGMENTS

The authors are greatly indebted to Ms Helkne Ax:son- Johnson for excellent technical assistance. This work was supported by grants from King Gustaf V's 80-Year Fund, the Funds of Karolinska Institutet. Ulf Widengrens Minnesfond and Cancerfonden (3 5 19).

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