The association of Greig syndrome and mastocytosis reveals the involvement of the hedgehog pathway in advanced mastocytosis.

Mastocytosis is a heterogeneous disease characterized by an abnormal accumulation of mast cells (MCs) in 1 or several organs. Although a somatic KIT D816V mutation is detected in ∼85% of patients, attempts to demonstrate its oncogenic effect alone have repeatedly failed, suggesting that additional pathways are involved in MC transformation. From 3 children presenting with both Greig cephalopolysyndactyly syndrome (GCPS, Mendelian Inheritance in Man [175700]) and congenital mastocytosis, we demonstrated the involvement of the hedgehog (Hh) pathway in mastocytosis. GCPS is an extremely rare syndrome resulting from haploinsufficiency of GLI3, the major repressor of Hh family members. From these familial cases of mastocytosis, we demonstrate that the Hh pathway is barely active in normal primary MCs and is overactive in neoplastic MCs. GLI3 and KIT mutations had a synergistic, tumorigenic effect on the onset of mastocytosis in a GCPS mouse model. Finally, Hh inhibitors suppressed neoplastic MC proliferation in vitro and extend the survival time of mice with aggressive systemic mastocytosis (ASM). This work revealed, for the first time, the involvement of Hh signaling in the pathophysiology of mastocytosis and demonstrated the cooperative effects of the KIT and Hh oncogenic pathways in mice with ASM, leading to the identification of new promising therapeutic targets.

Identification of a novel cDNA, encoding a cytoskeletal associated protein, differentially expressed in diffuse large B cell lymphomas.

Diffuse large B-cell lymphomas (DLBL) constitute an heterogeneous clinico-pathological entity. To characterize molecular events related to histological subtypes, clinical presentation or outcome, we compared the mRNAs expressed in a limited series of DLBL by Differential display-reverse transcription (DDRT) and cloned a differential cDNA, that we called LB1. LB1 open reading frame encodes a 683 amino-acid polypeptide that does not show significant homology upon comparison to protein databases, nor any structural domain relating LB1 to an already known protein family. Immunofluorescence analysis of transfected COS cells showed a cytoplasmic filamentous staining, indicating that LB1 protein is tightly associated with cytoskeletal fibers. Two LB1 transcripts, a major 3.6-3.9 Kb and a minor 2.2 Kb transcripts, were detected among human haematopoietic and non-haematopoietic lines and tissues. LB1 transcripts were abundant in testis, thymus and in tumour derived cell lines, while barely detectable in liver, prostate and kidney. Concerning DLBL, LB1 expression was high in two cases of DLBL, and low or undetectable in four others, confirming the differential expression previously observed in the DDRT experiment. Furthermore, LB1 gene mapped to chromosome 13q14, a region that has been involved as a chromosomal breakpoint in DLBL. The cellular function of LB1 and its relationship with B cell maturation and/or oncogenesis remain to be established.

Use of LiCl in phospholipase C assays masks the impaired functionality of a mutant angiotensin II receptor.

We recently reported that replacement of Tyr302 for Ala in the human angiotensin II type 1 receptor (hAT1) severely impaired its ability to activate phospholipase C (PLC). Another study demonstrated that the same mutation in the rat AT1 receptor only slightly impaired its ability to activate PLC. The most striking difference between the two studies was the use of LiCl in the experimental conditions. Thus, in the present report we evaluated the effect of LiCl on the rate of accumulation of inositol trisphosphate (IP3) in transfected cells stimulated with angiotension II (Ang II). In the presence of LiCl, Ang II caused a significant accumulation of IP3 in COS-7 cells transfected with the hAT1Y302A mutant receptor. In stably expressing CHO cells, stimulation of hAT1Y302A did not induce any IP3 elevation even in the presence of LiCl whereas the hAT1 wild-type receptor increased the production of IP3 exclusively in the presence of LiCl. These results show that LiCl is a convenient tool to enhance the sensitivity of PLC assays. However, in structure-activity relationship studies, it may underestimate or mask the debilitating effect of some mutations.

Short-term desensitization of the angiotensin II receptor of bovine adrenal glomerulosa cells corresponds to a shift from a high to a low affinity state.

Angiotensin II (Ang II) is an important regulator of aldosterone production by bovine adrenal glomerulosa (BAG) cells. Ang II interacts with a specific receptor coupled to a guanyl nucleotide-binding protein (G protein) that controls the activity of phospholipase C. A primary culture of BAG cells was used to study short-term desensitization of the Ang II receptor. After short exposures to Ang II, BAG cells lost some [125I]Ang II binding capacity. This loss was dependent on the duration of the pretreatment and on the concentration of Ang II used. A maximal loss of [125I]Ang II binding of 55 +/- 10% was observed after a pretreatment of 30 min with 30 nM Ang II. The EC50 was 1.3 +/- 0.6 nM (mean +/- SD of three experiments). The desensitization was readily reversible, since most of the binding capacity (higher than 90%) was recovered after a 60-min incubation, at 37 C, in the absence of Ang II. Scatchard studies revealed that the Ang II receptor of BAG cells exists under two affinity states with one dissociation constant of 0.2 nM and another dissociation constant of 1.5 nM. After a 30-min exposure of BAG cells to 10 nM Ang II, an important decrease of high affinity binding sites was observed. The maximal amount of binding sites was similar on control and desensitized cells (around 52,000 receptors per cell). GTP gamma S, a potent activator of G proteins, decreased [125I]Ang II binding to permeabilized BAG cells. This GTP gamma S effect was not observed on permeabilized BAG cells that had previously been desensitized with 10 nM Ang II. These results suggested that, similarly to GTP gamma S, short exposure to 10 nM Ang II caused the uncoupling of Ang II receptor from its G protein. DuP-753 (a selective AT1 angiotensin II type 1 receptor antagonist) markedly unhibited, whereas PD-123319 (a selective AT2 angioten II type 2 receptor antagonist) had no effect on Ang II receptor desensitization, indicating that the AT1 receptor subtype was responsible for the observed phenomenon. Pretreatment of BAG cells with staurosporine (a protein kinase C inhibitor) and R24571 (a calmodulin inhibitor) did not modify Ang II-induced desensitization of AT1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative autoradiographic localization of [125I-Tyr8]bradykinin receptor binding sites in the rat spinal cord: effects of neonatal capsaicin, noradrenergic deafferentation, dorsal rhizotomy and peripheral axotomy.

In vitro receptor autoradiography was used to localize, quantify and characterize [125I-Tyr8]bradykinin binding sites in all major spinal cord segments of normal rats and animals subjected to various chemical treatments and surgical lesions. [125I-Tyr8]bradykinin specific binding sites were predominantly located to superficial laminae of the rat dorsal horn, with the substantia gelatinosa showing the highest density of labelling (values ranging from 3.1 fmol/mg tissue in cervical to 4.5 fmol/mg tissue in lumbar segments). A moderate density (1.8-3.0 fmol/mg tissue) of specific binding was observed in lamina III, whereas in other areas, i.e. laminae I and IV-X, lower amounts of labelling were detected. Within the superficial laminae of the dorsal horn, [125I-Tyr8]bradykinin binding was largely distributed over the neurophil with some perikarya showing concentrations of labelling. In contrast, the ventral horn showed a rather homogeneous distribution of [125I-Tyr8]bradykinin binding over the neuropil, with silver grain alignments surrounding motoneuron perikaryas and proximal processes. Bradykinin, [Tyr8]bradykinin and B2 receptor antagonists (D-Arg[Hyp3,Thi5,D-Tic7,Oic8]bradykinin (Hoe 140), D-Arg[Tyr3,D-Phe7,Leu8]bradykinin, D-Arg[Hyp3, Leu8]bradykinin, D-Arg[Hyp2, Thi5,8,-Phe7]bradykinin D-Arg[Hyp3, D-Phe7, Leu8]bradykinin, Tyr0, D-Arg[Hyp3, D-Phe7, Leu8]bradykinin inhibited [125I-Tyr8]-bradykinin binding with very high subnanomolar affinities, while the B1 receptor agonist (Tyr0,des-Arg10-kallidin) and antagonist ([Leu8]-des-Arg9-bradykinin) did not significantly affect [125I-Tyr8]bradykinin binding at up to micromolar concentrations. Two weeks after unilateral lumbar dorsal rhizotomy (L1-L6) or peripheral lesions of the sciatic nerve, significant decreases ( +/- 50%) in [125I-Tyr8]bradykinin binding sites were found in ipsilateral laminae I-III of lumbar spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional interaction between losartan and central tachykinin NK3 receptors in the conscious rat.

1. The cardiovascular and behavioural effects elicted by the intracerebroventricular (i.c.v.) injection of substance P (SP), neurokinin A (NKA), [MePhe7]neurokinin B ([MePhe7]NKB) or angiotensin II (AII) in the conscious rat were assessed before and 5 min after i.c.v. pretreatment with antagonists selective for angiotensin AT1 (losartan and its active metabolite EXP 3174), angiotensin AT2 (PD 123,319) or tachykinin NK3 (R 486) receptors. 2. I.c.v. administration of 25 pmol AII evoked an increase in mean arterial blood pressure (MAP) and water intake behaviour, accompanied by a transient bradycardia, whereas 25 pmol [MePhe7]NKB caused a transient increase in MAP and heart rate (HR) concurrently with marked wet dog shake behaviour. At the same dose, SP and NKA were more potent than [MePhe7]NKB in increasing MAP and HR, but did not produce water intake or wet dog shake behaviours. 3. Losartan (650 pmol, i.c.v.) reduced significantly the cardiovascular and behavioural responses to AII or [MePhe7]NKB, but not to SP or NKA. While 65 pmol losartan was inactive, 260 pmol inhibited selectively the central effects of AII. Whereas EXP 3174 (6.5 nmol) blocked both AII and [MePhe7]NKB-mediated responses, the dose of 650 pmol blocked only the responses to AII. 4. The central responses to AII and [MePhe7]NKB were not affected by PD 123,319 (650 pmol). On the other hand, the [MePhe7]NKB-induced central effects were significantly reduced by R 486 (650 pmol). The NK3-selective antagonist had no effect against AII. 5. This study provides functional evidence, to support earlier binding data, that losartan (and to some extent its active metabolite EXP 3174) interact with the tachykinin NK3 receptor in rat brain. However,the cardiovascular and behavioural responses induced by central tachykinin agonists (SP, NKA and[MePhe7]NKB) and All are mediated by unrelated mechanisms.

Bovine adrenal glomerulosa cells express such a low level of functional B2 receptors that bradykinin does not significantly increase their aldosterone production.

It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adrenal glomerulosa (BAG) cells. The aim of the present study was to characterize the mechanism of action of BK on these cells. Binding experiments with the radioligand 125I-[Tyr8]BK revealed the presence of a relatively small amount (Bmax = 180 +/- 55 fmol/mg of protein) of high affinity (Kd = 0.65 +/- 0.17 nM) binding sites. BK induced a time- and concentration-dependent increase of [3H]inositol trisphosphate ([3H]IP3) in myo-[3H]inositol-labeled BAG cells. A maximal response was obtained with 10 nM BK and the EC50 value was 1.0 +/- 0.5 nM. 125I-[Tyr8]BK binding and BK-induced IP3 production were inhibited by the selective B2 receptor antagonist Icatibant (1 microM) and unaffected by the selective B1 receptor antagonist [DesArg9, Leu8]BK (1 microM). In fura-2 loaded BAG cells, BK (100 nM) induced a typical biphasic Ca2+ response composed of a rapid and transient increase of intracellular Ca2+ concentration [Ca2+]i which slowly declined to a level that remained above basal level for about 5 min. In the presence of EGTA (2 mM), the rapid and transient calcium increase was unaffected whereas the plateau phase was abolished. Angiotensin II (Ang II, 100 nM) also elicited a typical biphasic response in BAG cells. However the rapid and transient elevation of [Ca2+]i was followed by a sustained plateau phase which remained above the basal level for more than 10 min. Although BAG cells express functional B2 receptors, no secretion of aldosterone was observed after stimulation with 100 nM BK for 120 min. Under the same conditions Ang II increased by about 10-fold the basal level of aldosterone. The lack of effect of BK is probably attributable to its very transient effect on IP3 production. Pretreatment of BAG cells with 100 nM BK for 20 min reduced by 70 +/- 10% their total binding capacity. These results suggest a rapid and very efficient desensitization process. We conclude that BAG cells express functional B2 receptors. The weak production of second messengers and the rapid desensitization process could explain why BK fails to increase aldosterone production in these cells. Since functional B2 receptors are expressed in BAG cells it is likely that under some specific physiological or pathological conditions these receptors may play a significant role in aldosterone secretion. However these conditions remain to be determined.

Stimulation of the angiotensin II type I receptor on bovine adrenal glomerulosa cells activates a temperature-sensitive internalization-recycling pathway.

Angiotensin II (Ang II) is an important regulator of aldosterone production by bovine adrenal glomerulosa cells. On these cells Ang II interacts with the AT1 receptor that is coupled to a G protein controlling the activity of phospholipase C. A primary culture of bovine adrenal glomerulosa cells was used to study the internalization-recycling mechanism of the AT1 receptor after stimulation with Ang II. When cells were pretreated with 10 nM Ang II for 30 min at 37 degrees C and binding studies were performed at 12 degrees C we observed a 48% loss in [125I]Ang II binding. Scatchard analysis revealed that this loss in binding translated into a decreased affinity of the AT1 receptor without any loss in the total amount of binding sites. Under the same conditions an important internalization of [125I]Ang II was invariably observed. These observations suggest that a mechanism was at work to recycle the internalized receptors to the cell surface during the binding studies. Following internalization we indeed observed an externalization of [125I]Ang II. This phenomenon relatively rapid at 37 degrees C was much slower at 12 degrees C and completely inhibited at 4 degrees C. When cells were pretreated with 10 nM Ang II for 30 min at 37 degrees C binding assays at 4 degrees C no longer revealed a loss of binding affinity but rather a 54% reduction in the total amount of binding sites. The maximal binding capacity could be recovered during incubations at 12 degrees C. These results reveal the existence of a dynamic recycling process for the AT1 receptor. In accordance with this interpretation the phenomenon was blocked by monensin, a known inhibitor of receptor recycling. These studies suggest that the stimulation of the AT1 receptor sets in motion an internalization-recycling process that seems to be a fundamental aspect of the AT1 receptor transduction mechanism.

Two NK-3 receptor subtypes: demonstration by biological and binding assays.

The existence of two neurokinin NK-3 receptor subtypes has been suggested on the basis of results obtained in binding assays. In the present study, we have confirmed the two NK-3 receptor subtypes by using data obtained in both biological and binding assays. Experiments have been performed in the rat portal vein and in the guinea-pig ileum treated with NK-1 and NK-2 selective antagonists, namely CP 96345 and SR 48968. Orders of potency of agonists on the rat portal vein are as follows: for neurokinins, NKB > NKA > SP; for tachykinins, KAS > ELE > PHY; and for selective agonist: [MePhe7]NKB >> senktide. On the guinea-pig ileum, the agonist rank orders of potency are: NKB > SP > NKA, ELE > KAS > PHY; and for selective agonist: [MePhe7]NKB = senktide. The apparent affinity of antagonists shows differences in both biological and binding assays. In fact, on the rat portal vein, SR 48968 is almost inactive (pA2 or IC50 approximately 4.8), while R-486 [Trp7, beta Ala8]NKA(4-10) shows a pA2 value of 7.45 and an IC50 of 5.6. An opposite pattern of activity is observed in the guinea-pig ileum, where SR 48968 shows a pA2 of 6.05 and an IC50 of 6.7, while R-486 has a pA2 of 6.1 and an IC50 of < 5.0. These results confirm the existence of two NK-3 sites differing pharmacologically. It is proposed to name NK-3A the receptor of the guinea-pig ileum and NK-3B the receptor of the rat portal vein.

Characterization of NK-1 receptors in guinea pig and rat brain membranes with NK-1 peptides and a non-peptide antagonist.

The major finding of the present investigation is the demonstration of different NK-1 receptors in rat and guinea pig brain membranes with CP 96345 (non-peptide NK-1 antagonist) and R-544 (NK-1 peptide antagonist). We used [3H][Sar9,Met(O2)11]SP, the highly selective ligand for NK-1 receptor to compare NK-1 binding sites in rat and guinea pig brain membranes. Scatchard analysis revealed the existence of a single population of [3H][Sar9,Met(O2)11]SP binding sites in both preparations. The affinity and the maximal number of binding sites were found closely similar in rat (Kd 2 nM, Bmax = 37 fmol/mg protein) and guinea pig brain membranes (Kd = 3 nM, Bmax = 25 fmol/mg of protein). The order of potency of neurokinins to inhibit [3H][Sar9,Met(O2)11]SP binding from rat brain (SP > NKA > NKB) was found different of that observed on guinea pig brain (SP > NKB > NKA). Results obtained with [Sar9,Met(O2)11]SP, [beta Ala8]NKA(4-10) and [MePhe7]NKB suggest that selective agonists cannot discriminate between NK-1 receptors of different species. Using the non-peptide antagonist CP 96345 and the tripeptide R-544, we found that these two NK-1 antagonists discriminate between rat and guinea pig [3H][Sar9,Met(O2)11]SP binding sites.

Non-peptide angiotensin receptor antagonists bind to tachykinin NK3 receptors of rat and guinea pig brain.

[3H]Senktide, a highly selective tachykinin NK3 receptor agonist, was used to study tachykinin NK3 receptors of rat and guinea pig brain. Guinea pig brain membranes had a Kd of 3.9 +/- 0.5 nM and a Bmax of 42 fmol/mg. Dose-displacement experiments with neurokinins and selective tachykinin receptor agonists revealed the following order of potency: [MePhe7]neurokinin B > neurokinin B > substance P > neurokinin A. This order is typical for a tachykinin NK3 receptor. To further characterize the specificity of this receptor, the effects of unrelated compounds such as: bradykinin, angiotensin II, bombesin and their structural analogs were also evaluated on the binding of [3H]senktide. Unexpectedly, the angiotensin AT1 receptor antagonists, DuP 753 (2-n-butyl-4-chloro-5-hydroxymethyl-1-[2'-(1H-tetrazol-5-yl)bip hen yl-4-yl)methyl]imidazole potassium salt), L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'-(1H-tetrazol-5-yl) [1,1'-biphenyl]-4-yl) methyl]-3H-imidazo[4,5-beta]pyridine H2O) and EXP 3174 (2-n-butyl-4-chloro-1-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]i midazole- 5-carboxylic acid), inhibited the binding of [3H]senktide to its receptor in the guinea pig brain membranes with IC50 values of 18 microM, 25 microM and 50 microM, respectively. Similar effects were also observed with rat brain membranes. Angiotensin II, saralasin ([Sar1,Val5,Ala8]angiotensin II, a peptide angiotensin AT1 receptor antagonist) and PD 123,319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl)-4,5, 6,7- tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid, a known non-peptide angiotensin AT2 receptor antagonist) did not inhibit the binding of [3H]senktide to either type of membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of uncoupling agents on AT1 receptor affinity for antagonist analogs of angiotensin II.

AT1 receptor is responsible for most of the physiological effects of Angiotensin II (Ang II). AT1 receptor belongs to the G-protein-coupled receptor (GPCR) family, and it mediates its actions through the coupling of the Gq/11 protein with phospholipase C beta. Classical pharmacology has used the sensitivity of GPCR ligands to uncoupling agents as a criteria to discriminate agonists (which are sensitive) from antagonists (which are insensitive). In this study, the uncoupling agents GTP gamma S and pentosan sulfate (PS) (a low molecular weight polyanion) were used to further characterize the molecular interactions between Ang II analogs and the AT1 receptor. We show that some Ang II antagonists are sensitive to the conformational change of the AT1 receptor induced by uncoupling agents. These results demonstrate that there is no direct relationship between the intrinsic activity of a ligand and its affinity for different conformations of the AT1 receptor and that the sensitivity of GPCR ligands to uncoupling agents can not be used as a criteria to discriminate agonists from antagonists.

The bradykinin B2 receptor couples less efficiently than the angiotensin AT1 receptor to the G protein Gq in transiently transfected COS-7 cells.

The purpose of this study was to compare the efficiency of two different Gq protein-coupled receptors (AT1 receptor for angiotensin II and B2 receptor for bradykinin) to activate phospholipase C (PLC). When the receptors were expressed at a similar level of 0.5 pmol/mg of protein, inositol trisphosphate (IP) accumulation elicited by AT1 receptor was four times higher than that elicited by B2 receptor. Genistein and pertussis toxin did not modify AT1 receptor- or B2 receptor-induced IP accumulation. These results indicate that in COS-7 cells, the two receptors activate PLC beta through G proteins of the Gq family. AT1 or B2 receptors were co-expressed with the alpha subunit of either Gq or G11. Both alpha subunits potentiated to the same extent AT1 receptor-induced IP accumulation. alpha 11 was also as efficient as alpha q to potentiate B2 receptor-induced response. Interestingly, however, the potentiating effect of alpha q and alpha 11 was more important (by 5-fold) on AT1 receptor-mediated response than on B2 receptor-mediated response. These results demonstrate that the extent of activation of PLC beta by different Gq-coupled receptors depends on the level of expression of these receptors and on their coupling efficiency. These are important parameters that determine the relative contribution of specific hormones to different biological processes.

Neurokinin receptors in the guinea pig ileum.

Experiments were performed in the longitudinal muscle strip of the guinea pig ileum to characterize the receptors involved in the contractile response of this preparation to neurokinins. Antagonists for the NK-1 (CP 96345, CP 99994) and NK-2 (SR 48968) receptors, atropine for NK-3 receptors, as well as diphenhydramine (histamine H1 receptor antagonist) and indometacin (cyclooxygenase inhibitor) were used to determine the relative contribution of neurokinin receptors and some endogenous agents to the myotropic effects of substance P (SP) and neurokinin receptor selective agonists. The present findings indicate that the three neurokinin receptor types take part in the contractile activities of SP-related peptides. NK-1 receptors, probably localized in the smooth muscle, are inhibited only by the two CP compounds and not by atropine or the other agents. NK-2 receptors contribute to the contraction by 5-10% and are blocked by SR 48968. NK-3 receptors act indirectly through the release of acetylcholine from the myenteric plexus, since activities of [MePhe7]NKB and senktide are blocked by atropine. Septide behaves as a selective NK-1 receptor agonist and does not show any difference with SP, except for higher sensitivity to CP antagonists. The same is observed with Ac[Arg6,Sar9,Met(O2)11]SP(6-11), another NK-1-selective fragment. Discrepancies between antagonist pA2 values obtained against undeca- and hexapeptide agonists are interpreted as due to a stronger binding affinity of undecapeptide agonists as compared with the hexapeptides. Results of binding assays confirm data from the literature by showing that undecapeptide agonists have higher affinities than hexapeptides, particularly septide,, and such discrepancies (with the biological assays) can also be explained by the reduction or absence of the cationic charge at the N terminal of septide.

Chromatin immunoselection defines a TAL-1 target gene.

Despite the major functions of the basic helix-loop-helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG-GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box-GATA motif and define the first gene regulated by TAL-1.

The MAL gene is expressed in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBL) appears to be a distinct clinicopathologic entity among diffuse large B-cell lymphomas (DLBLs). To find molecular alterations associated with this disease, we compared the mRNAs expressed in 3 PMBLs and 3 peripheral DLBLs by differential display-reverse transcription (DDRT) and identified a mRNA specifically expressed in PMBLs. Sequence analysis showed that this mRNA is encoded by the MAL gene, the expression of which was shown to be restricted to the T-cell lineage during hematopoiesis. MAL gene expression was demonstrated by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) in 8 of 12 PMBLs. However, there was little or no MAL gene expression in 8 peripheral DLBLs. Immunohistochemical analysis evidenced expression of MAL protein in tumoral B cells restricted to the PMBL subtype. Finally, Southern blot studies did not demonstrate rearrangement of the MAL gene. Altogether, our results indicate that MAL expression is recurrent in PMBLs, providing further evidence that PMBL represents a distinct entity among DLBLs. Because MAL protein is located in detergent-insoluble glycolipid-enriched membrane (GEM) domains involved in lymphocyte signal transduction, abnormal expression of MAL protein in the B-lymphoid lineage may have significant implications in PMBL lymphomagenesis.