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Molecular Biology and Genetics |
Division of Molecular Oncology, Department of Pathology, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts 02115 [J. A. M., Y. Y., A. D. B., F. K., N. N., J. I. K., C. R., L. S., G. S. P., J. A. F., J. S.]; Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114 [F. G-C., N. L. H.]; Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 [C. A. G.]; and Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198 [D. W.]
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ABSTRACT |
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INTRODUCTION |
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Another unusual feature of MALT lymphomas is that these neoplasms arise frequently at sites of preexisting inflammation, probably as an outgrowth of a neoplastic clone from among reactive lymphocytes. For example, MALT lymphomas develop in tissues affected by autoimmune processes, as in Hashimoto’s thyroiditis (6
, 7)
and Sjögren’s syndrome (8)
, and they are responsible for the high incidence of lymphoma among patients with these disorders. Furthermore, chronic bacterial infections have been implicated in the development of MALT lymphomas. Gastritis containing the bacterium Helicobacter pylori apparently predisposes to MALT lymphoma of the stomach (9)
, and chronic bacterial infections of the small intestine are believed to have an etiological role in Mediterranean lymphoma or 
heavy chain disease–clinical presentations of lymphomas now regarded as examples of MALT lymphoma (10
, 11)
. Evidence for a direct causal relationship between infection by H. pylori and MALT lymphoma comes from experiments in which cultured lymphoma cells proliferated in response to bacterial antigens only when the antigens were derived from the same strain of H. pylori as was present in the tissues of the particular tumor from which the culture was prepared (12)
. Consistent with these observations is that antibiotic therapy of patients with gastric MALT lymphomas in early phases of the disease has led to complete clinical remissions in the majority of patients treated (5)
.
Insights into the mechanisms underlying the development of MALT lymphoma could help to explain the multiple unusual features observed in this disease, as well as to improved diagnosis and treatment of the tumor. Although antigenic stimuli, such as those resulting from bacterial products or autoantigens, may promote growth of some MALT lymphomas, development of MALT lymphoma very likely requires specific alterations in gene structure and function that drive proliferation of the neoplastic cells. Sites of recombination in recurrent chromosomal rearrangements have often provided clues to the location of genes that have undergone structural or functional changes critical to the malignant phenotype. Several cytogenetic aberrations have been identified within MALT lymphomas, the most common of which appears to be the (11;18)(q21;q21) chromosomal translocation present in about 50% of low-grade MALT tumors (13 , 14) . To search for a gene or genes involved in the pathogenesis of MALT lymphomas, we have analyzed DNA surrounding the 18q21 breakpoint in cells from tumors with this translocation and found that recombination occurs in or around the DNA of a previously undescribed gene having partial homology to genes encoding members of the immunoglobulin gene superfamily and caspase enzymes.
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MATERIALS AND METHODS |
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A cell line, MBCL-1, that was previously established after infection of lymphocytes from a MALT lymphoma of the stomach with EBV5 was also used in these studies. Both the tumor cells and the cell line displayed a karyotype with trisomy 18 but lacked the t(11;18)(q21;q21). This line had been carried continuously in culture for at least 6 months before these studies.
Hybrid cell lines derived from fusions of CHO cells with human cells carrying human chromosome 11 (CHO+11) or human chromosome 18 (CHO+18) as the only human chromosomal constituents (Coriell Cell Repositories, Camden, NJ) were grown in culture according to the conditions recommended by the repository. Total genomic DNA was extracted from CHO, CHO+11, and CHO+18 cell lines using standard procedures (15) and was used as a template for PCR.
YACs, BACs, and ESTs.
YACs were obtained from Centre d’Etude du Polymorphisme Humain megaYAC libraries purchased from Research Genetics (Huntsville, Alabama). YAC clones were expanded in SD media, and the DNA was isolated following standard protocols (16)
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BACs were selected from three-tiered pools of BAC DNA contained within the California Institute of Technology BAC library (Research Genetics) by PCR using primers complementary to DNA in the STSs D18S831, D18S1129, and D18S1026 (17) . Subsequent selections of BACs were performed by PCR amplification using primers based on the nucleotide sequence obtained at the ends of human genomic DNA inserts in previously identified BACS. Reaction conditions for these amplifications were performed as previously described, with 30 cycles of amplification (94°C denaturation, 55°C annealing, and 72°C extension) using minor variations of annealing temperature specific to each primer pair (18) . BAC clones were expanded in Luria-Bertani media containing chloramphenicol (25mg/ml; Ref. 15 ), and the DNA was isolated using the Plasmid Midi-kit (Qiagen, Valencia, CA) following the manufacturer’s instructions.
ESTs were purchased from Genome Systems (St. Louis, MO). Plasmids were expanded in Luria-Bertani media supplemented with ampicillin (100mg/ml), and DNA was purified following standard plasmid preparation protocols (15) .
Fluorescent in Situ Hybridization.
DNA probes for FISH were labeled with digoxigenin- (Boehringer Mannheim, Indianapolis, IN) or biotin-tagged nucleoside triphosphates (Life Technologies, Gaithersburg, MD) using the BioPrime labeling system (Life Technologies). Hybridization was performed on slides of intact nuclei isolated from tissue sections as previously described (19
, 20)
. Slides were treated with 10 mg/ml RNase in 2x SSC for 60 min at 37°C followed by 50 mg/ml pepsin in 0.01 N HCl for 8 min at 37°C. Material on the slides was cross-linked in 10% buffered neutral formalin for 2 min at room temperature and dehydrated in graded alcohols. DNA on the slides was denatured in 70% formamide/2x SSC for 4 min at 72°C and dehydrated in graded alcohols at 4°C. A mixture of probe and COT-1 DNA (Life Technologies) was applied in Hybrisol VI (Oncor, Gaithersburg, MD) and denatured at 80°C for 10 min. Probe was allowed to anneal overnight at 37°C and washed in 0.5x SSC for 5 min at 70°C. Slides were rinsed in PN buffer (0.5 M Na2HPO4 to pH 8.0 with 0.5 M NaH2PO4 and 0.5% NP40 final concentration), and a signal was detected using rhodamine-conjugated antidigoxigenin and/or FITC-conjugated avidin and amplification reagents from Oncor following standard protocols. Results were observed and photographed with a Zeiss Axioskop II Fluorescent microscope (Carl Zeiss, Oberkochen, Germany).
RACE Amplification.
Five-prime RACE was performed using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA). Poly(A)+ RNA isolated from the MBCL-1 cell line was used as a template for first-strand cDNA synthesis carried out with gene-specific primers following the manufacturer’s directions. Nucleotide sequence of the RACE products was determined with standard M13F and M13R primers using the AmpliCycle Sequencing Kit (Perkin-Elmer, Branchburg, NJ) and the GeneAmp PCR 9600 Themocycler (Perkin-Elmer, Norwalk, CT).
IPCR.
Five micrograms of tumor and control DNA were digested to completion with BglII and SpeI restriction enzymes (New England Biolabs, Beverly, MA) according to manufacturer’s instructions. Restriction enzymes were inactivated by standard extraction procedures (15)
and ethanol precipitation. DNA was resuspended in 50 µl of water and quantified by spectrophotometric measurement at a 260 nm wavelength. Digested tumor DNA and control DNA (200 ng in 25 µl final volume) were incubated overnight with 800 units of T4 DNA ligase in 1x ligation buffer (NEB) at 15°C. IPCR (21)
was performed using 8 ng of self-ligated DNA as a template in a total volume of 50 µl under the following reaction conditions: 1x buffer [50 mM Tris-HCl (pH 9.2), 16 mM (NH4)2SO4, 4 mM MgCl2, 0.05% Tween 20]; 200 µM dNTPs; 4 µM of each primer; 2.5U Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ); and 0.05 units of Vent DNA polymerase (NEB). PCR cycling conditions were as follows: 94°C for 2 min; 15 cycles of 94°C for 10 s, 69°C for 210 s; 15 cycles of 94°C for 10 s, 69°C for 210 s plus 10 s per cycle; and 69°C for 8 min. Two rounds of amplification were performed using similar conditions but different pairs of primers nested with respect to one another and 1 µl from the initial round of PCR serving as a template for the second round.
Southern and Northern Blot Hybridization.
Genomic DNA was prepared following standard protocols (15)
with the following modification: frozen tissue was thawed and minced with razor blades in extraction buffer rather than ground frozen with a Waring Blendor. Ten micrograms of genomic DNA were digested with 80–100 units of the indicated restriction enzyme, incubated at 37°C for no less than 6 h, and analyzed by Southern blot as described (15)
. Each digested sample was fractionated on a 0.8% agarose gel in 1x tris acetate EDTA buffer and subjected to electrophoresis for 
25 h at 2.5 V/cm. After depurination, denaturation, and neutralization, DNA was transferred to a Biotrans nylon membrane (ICN, Irvine, CA) in 10x SSC. Blots were hybridized in ExpressHyb Hybridization Solution (Clontech) using probes radiolabeled by PCR with 32P--dCTP (NEN, Boston, MA). Blots were washed two times at room temperature for 20 min (2x SSC, 0.05% SDS) followed by two washes at 50°C for 20 min (0.1x SSC, 0.1% SDS) and exposed to BioMax MS film using a BioMax MS intensifying screen (Eastman Kodak, Rochester, NY) at -80°C.
Total RNA was prepared from the tumor samples and the MBCL-1 cell line using Trizol reagent (Life Technologies) as previously described (15) . Poly(A)+ RNA for Northern blot analysis was selected from the total RNA using the Oligotex mRNA Mini Kit (Qiagen) according to the manufacturer’s instructions. Two micrograms of poly(A)+ RNA were size-fractionated by electrophoresis in a 1% agarose formaldehyde gel run in 3-(N-morpholino) propanesulfonic acid buffer and then transferred to a Biotrans nylon membrane. Both the total RNA tumor Northern blot as well as a Multiple Tissue Northern blot (MTN, Clontech) of poly(A)+ mRNA from various human tissues were hybridized in ExpressHyb Hybridization Solution (Clontech). Blots were washed according to the manufacturer’s instructions for the treatment of the multiple tissue blot.
Determination of Nucleotide Sequence and Computer-assisted Analysis of Derived Sequence.
Sequence data were obtained using the DyeDeoxy Terminator Cycle Sequencing Kit (Perkin-Elmer, Branchburg, NJ) and an ABI DNA Sequencer (Perkin-Elmer, Norwalk, CT). Sequence was analyzed using the National Center for Biotechnology Information BLAST Server and the GCG software package (Genetics Computer Group, Madison, WI). Peptides were analyzed using software available at the ExPASy world wide web server (Swiss Institute of Bioinformatics, Geneva).
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RESULTS |
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5 megabase) intervals. In an initial series of analyses, YAC y766F12 at 99 cM (485 cR), YAC y949C6 at 94 cM, and YAC y943B8 at 83 cM (445 cR) were tested in pairwise hybridizations. In each analysis, two pairs of red/green signals were observed in all nuclei, indicating that the breakpoint does not fall between these YACs. However, when the most centromeric YAC of this initial group, y943B8, was cohybridized to interphase nuclei together with the still more centromeric YAC y830B10 at 79 cM (420 cR), about 40% of the nuclei yielded one paired and one unpaired signal. These observations suggested that the breakpoint falls between y943B8 and y830B10, thereby localizing the breakpoint to the intervening 5 cM. That 60% of the nuclei yielded two paired signals probably reflects the fact that the tissues contain numerous nonlymphoma cells. Hybridization with pairs of successively closer YACs within the contig 18.14 and positioned between y943B8 and y830B10 (Fig. 1A)
established that the breakpoint lies between the DNA of y902A4 and y826H9.
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. The identities of derivative 18 and derivative 11 chromosomes in these analyses were confirmed by rehybridization of the same metaphase with probes specific for the pericentromeric regions of chromosomes 11 and 18 (Fig. 2, B and C)
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Construction of a BAC Contig across the Chromosome 18 Breakpoint.
To identify BACs in the region of the chromosome 18 breakpoint, pools of DNA from a BAC library with 6-fold redundancy (Research Genetics) were screened by PCR using primers from STSs compiled by the Human Genome Project for the region of the YACs described above. D18S831, an STS that lies within DNA of y826H9 and maps adjacent to D18S1129, identified BAC b245Il4; D18S1129 identified BAC b357N13; and D18SlO26, an STS within the DNA of y789F3, identified BAC b54Lll (Fig. 1, A and B)
. To determine the position of the breakpoint with respect to these BACs, labeled DNA from pairs of BACs was used as a probe in two-color FISH on interphase nuclei of case 2. Splitting of the two signals was not observed when b357N13 was tested together with the telomeric flanking BAC b245I14, indicating that the breakpoint does not lie telomeric to b357N13. In contrast, splitting was observed when either of these two BACs was individually tested together with the centromeric flanking BAC b54L11 (Fig. 2D)
, localizing the breakpoint to the interval between b357N13 and b54L11. PCR primers constructed to be complementary to the nucleotide sequence determined for the ends of the BACS were used in PCR amplifications to select additional sets of BACs from the library. Probes produced from DNA of this second generation of BACs (b230N13 and b24P22) centromeric to the breakpoint were tested in two-color FISH with probes produced from second generation telomeric BACs (b32I18 and b79O17). Splitting was again observed, further limiting the breakpoint to a region bounded by b230N13 and b32I18. A third iteration of selection by PCR for BACs containing sequence at the centromeric end of b32I18 insert yielded the BACs b128K1, b246H13, b357H2, and b441H2, with each of the latter three overlapping the centromeric end of b128K1. PCR with primers prepared from the telomeric end of b230N13 identified BAC b205O5, the telomeric end of which was contained within BACs b357H2 and b441H21, indicating that a contiguous overlapping array of specific BACs across the breakpoint could be assembled with BACs selected from the library (Fig. 1B)
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The map deduced for the various BACs in the region of the chromosome 18 breakpoint was further validated by performing PCR amplifications with primers complementary to the ends of the BAC inserts on the DNA of YACs comprising a contig of this region constructed by the Whitehead Genome Project. Additionally, restriction analyses were performed on BAC DNAs, and the results from digests of the individual BACs were compared to define the regions of overlap. The sizes of human DNA inserts in the BACs were calculated based on the total sizes of the restriction fragments produced by digestion of the BAC DNAs.
Identification of a BAC Spanning the Chromosome 18 Breakpoint.
Because b230N13 and b32I18 contain DNA on either side of the breakpoint, it was clear that one or more BACs within the contig between these flanking BACs must span the breakpoint. To determine which of these BACs spans the breakpoint, DNA from each BAC mapping between b230N13 and b32I18 was used as a single color probe on interphase nuclei of the MALT lymphoma designated case 2. Both b128K1 and b246H13 gave only two signals on the majority of interphase nuclei, suggesting that the breakpoint was either not located within the BAC sequence or that the breakpoint was too eccentrically located within the BAC sequence to yield a third signal. BAC b357H2 alone yielded three distinct signals in about 40% of the interphase nuclei. Control FISH with b357H2 to interphase nuclei with normal karyotypes yielded one or two signals in nearly all nuclei examined. To rule out the possibility that the third signal represented spurious cross-hybridization to an irrelevant region of the genome rather than chromosome 18 DNA separated by the translocation, a two-color FISH assay was performed in which b357H2 DNA was labeled with one fluorochrome while the flanking BACs were together labeled with a second fluorochrome. Each of the three signals generated from b357H2 DNA colocalized with signals generated by the flanking BACs, demonstrating the reliability of the three b357H2 signals.
Two-color FISH interphase assays with DNA of b357H2, b32I18, and b205O5, as described above, were repeated on interphase nuclei from cases 1 and 3. In both cases, b357H2 yielded three signals in close proximity to the signal produced by the flanking BACs b32I18 and b20505. These results indicate that in all three of our cases confirmed to have the t(11;18)(q21;q21) by cytogenetic analysis, the chromosome 18 breakpoint lies within DNA of the BAC b357H2.
Localization of a Chromosome 18 Breakpoint in the t(11;18)(q21;q21).
To facilitate ultimately a search for genes in the region of the chromosome 18 breakpoint, DNA of b128K, the smallest of the BACs that mapped to the region, was randomly sheared and subcloned in M13 viral vectors, the inserts of which were then subjected to sequence analysis designed to cover the sequence of the BAC with 6-fold redundancy. This analysis permitted the construction of a 58-kb contig representing the centromeric portion of b128K1, believed to flank the breakpoint region described above. To investigate whether sequence eccentrically located in BAC b128K1 (hence, undetectable by interphase FISH analysis) was rearranged by the breakpoint, probes for Southern blot analysis were generated to test all SpeI and BamHI restriction fragments in the most centromeric 30 kb. Additionally, to extend the analysis in the centromeric direction into a sequence unique to b357H2 (the BAC demonstrated by FISH to contain DNA divided by the translocation), BamHI fragments of 24 kb, 5 kb, and 8 kb were mapped to the portion of b357H2 centromeric to b128K1 using the combined results of restriction digests and Southern blot analysis performed with probes produced from the sequence at the ends of the insert. Nucleotide sequence was obtained from the ends of these fragments, and complementary PCR primers were designed. A probe generated from the centromeric end of the 8-kb BamHI fragment, termed B6, detected a DNA rearrangement in Southern blot analyses (Fig. 3)
with three of five restriction enzyme digests of genomic DNA isolated from case 1. DNA extracted from normal peripheral blood of the patient in case 1 and analyzed in parallel with the tumor DNA showed only unrearranged, germ-line bands in Southern blot autoradiograms. Restriction enzyme digests that demonstrated rearrangements included those with SpeI, BglII, and HindIII. Mapping of restriction sites for these enzymes within B6 further localized the position of recombination in the t(11;18)(q21;q21) to the most centromeric 3 kb of this fragment.
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As a further step toward precise localization of the chromosome 18 breakpoint in case 1, the nucleotide sequence of B6 was determined. DNA fragments containing the fusion of the chromosome 18 nucleotide with the chromosome 11 sequence were then amplified by IPCR using tumor DNA from case 1. "Head-to-head" primers in these reactions were designed to complement the nucleotide sequence at the centromeric end of B6. BglII and SpeI digestion of the IPCR products yielded fragments corresponding in size to those expected from the Southern blot analyses. IPCR of both digests preferentially amplified the smaller rearranged fragments compared to the larger germ-line fragments. IPCR products were ligated into the vector PCR 2.1 (Topo-TA Cloning Kit, Invitrogen, Carlsbad, CA) and subjected to sequence analysis, which produced 39 bp of novel sequence from the BglII template and 190 bp from the SpeI template that diverged from that contained within the B6 of normal DNA (Fig. 4, A and B)
. Oligonucleotide primers designed from these new sequences were used for standard PCR with the following DNA templates: normal human genomic DNA, DNAs from two CHO/human hybrid cell lines containing either chromosome 11 or chromosome 18 as the only human chromosomal constituents (CHO+11 and CHO+18), and the chromosome 18 BAC b357H2. PCR of normal human genomic DNA and CHO+11 DNA yielded identical products of 116 bp, whereas PCR of the other templates showed no amplification products (Fig. 4C)
. Using DNA templates from a panel of chromosome 11q21 YACs, PCR generated the same 116-bp product from YAC y947E2 (data not shown). Additionally, amplification across the translocation breakpoint, using one primer complementary to the putative chromosome 11 sequence and one to the chromosome 18 sequence, yielded a product of predicted size with tumor DNA from case 1 serving as a template but not with normal genomic DNA as a template.
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To further map the unique sequence of the RACE product within the genome, the nonrepetitive portion of the RACE product was generated by PCR, and the resulting fragment was used to probe Southern blots of human genomic DNA and restriction digests of BACs b128K1, b246H13, and b357H2. Similar fragments were detected in the restriction digests of genomic DNA and BAC DNA, indicating that the portion of the RACE product used as a probe consists of a single copy sequence. Analysis of the blots prepared from BAC DNA revealed hybridization to the 24-kb BamHI fragment (B1) of b357H2 centromeric to b128K1. However, there was no hybridization to 5-kb (B10) or 8-kb (B6) BamHI fragments derived from DNA between that of B10 and the end of b128K1. Because B6 contains DNA broken by the t(11;18)(q21;q21) in case 1, recombination in this tumor occurred in the fifteenth intron from the 3' end of a gene, for which we propose the name MALT1.
To determine exon/intron boundaries in the RACE sequence not present in b128K1, cycle sequencing was performed using internal primers from the RACE product and b357H2 DNA as a template. This analysis revealed four exon/intron boundaries demarcated by appropriate consensus splice donor/acceptor sequences. The first exon contains the Alu and LINE iterated sequence elements, with the recombination breakpoint in chromosome 18 of case 1 falling in the third intron 3' of this exon. The entire exon/intron structure deduced for the segment of MALT1 identified thus far is shown in Fig. 1C
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Structural and Expression Analyses of the MALT1 Gene.
The full sequence for MALT1 assembled from genomic sequence, ESTs, and RACE products consists of 3910 bp, including an open reading frame of 2325 bp encoding a polypeptide of 775 amino acids (Fig. 5)
. An in-frame stop codon is located immediately upstream of the putative first ATG, which is found at nucleotide 266 in the sequence. It has homology to members of the immunoglobulin superfamily, with two immunoglobulin-like C2 domains from amino acids 69 to 243 resembling sequence found in both cell adhesion molecules and receptor tyrosine phosphatases. MALT1 is homologous to a conceptually translated open reading frame, F22D3.6, in Caenorhabditis elegans, with 27% identity and 44% similarity between regions extending from amino acid 197 to 569 of MALT1 and from amino acid 92 to 506 of F22D3.6. A ProfileScan of the PROSITE profile database reveals an interleukin-1
-converting enzyme (ICE, or caspase-1) p20 motif from amino acid 292 to 370. These polypeptide sequences share 52% similarity and 27% identity with caspase-1 p20 from amino acid 160 to amino acid 218. However, the alignment terminates abruptly 19 amino acids N-terminal of the active site amino acid (237 in caspase-1 p20). Following submission of this manuscript, much of the sequence of MALT1 was described by Dierlamm et al. (22)
, which they refer to as MLT. However, our sequence differs from that presented by those investigators by including 5' and further 3' untranslated regions, as well as 46 additional N-terminal codons within the open reading frame.
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Northern blot hybridization analysis of polyadenylated RNA from B-cell tumors also indicated the same four MALT1 species, although the relative abundance of the bands varied somewhat among specimens (Fig. 6)
. Several specimens showed an additional faint band corresponding to an RNA of about 7.8 kb. However, a unique band migrating slightly below the 7.8 kb band was detected by MALT1 probes only in the MALT lymphoma from case 1.
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DISCUSSION |
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No matter how diffusely scattered translocation breakpoints may be within DNA about a chromosomal locus, they generally exert their effect in tumors containing them by altering the expression of genes closely linked to the breakpoint. However, in some instances, the distance between a gene and a breakpoint can be relatively great, such as the 1000 kb or more of DNA that may separate translocation breakpoints in chromosome 8 from the MYC gene of some endemic Burkitt’s lymphomas (25) . This fact complicates the determination of the gene directly affected by any translocation that has not been previously characterized at the molecular level. In the present situation, the MALT1 gene covers roughly the middle 65 kb of the BAC b357H2 found by us to span the breakpoint in three examples of the t(11;18)(q21;q21). Although t(11;18)(q21;q21) could act through other genes either contained in b357H2 or in DNA adjacent to the region within this BAC, recombination within MALT1 in at least one case would suggest that this gene plays a role in the pathogenesis of MALT lymphomas. Additionally, FISH using b357H2 as a probe on interphase nuclei from all three tumors with the t(11;18)(q21;q21) yielded signals of roughly equal intensity from the two derivative translocation products, thereby favoring the presence of breakpoints in the mid-portion of the DNA contained in the BAC; that is, the region of DNA covered by MALT1. On the other hand, Northern blot autoradiograms of MALT1 RNA in normal tissues revealed a multiplicity of bands, some of which correspond to rather large RNA species. The size of the gene may therefore be far bigger than the 65 kb we have assigned to it and could constitute a larger target for recombination than we have assumed based on data from ESTs, sequencing of genomic chromosome 18 DNA, and RACE amplification.
Although case 2 was shown by FISH to have the same translocation as case 1, Northern blot analysis of case 2 failed to detect a band similar to the abnormal band seen in case 1. There are several possible explanations for this finding. For instance, an abnormal band may be present at very low intensity in case 2 RNA, either due to variation in the level of transcript among tumors or to a paucity of tumor cells within the tissue sections from which RNA was extracted. It could also be that the breakpoint in the second tumor tested lies within the MALT1 gene but does not affect the RNA sequences recognized by the probes used in this assay. Another possibility is that the breakpoint in the second tumor falls outside the MALT1 gene entirely. Assuming that MALT1 is directly involved in the pathogenesis of MALT lymphoma, the latter explanation would imply that alterations in MALT1 could occur by recombination both within or outside the gene, yet somehow disrupt or alter function in either circumstance. A similar situation has been reported with respect to the MYC gene in translocations of Burkitt’s lymphoma (24) . Finally, the breakpoints within the MALT1 gene could be variable in position and lead to transcripts of size different from the abnormal band produced from RNA of case 1. Some of these transcripts may be unstable or generate bands obscured by the multiple normal bands detected in the Northern blot autoradiograms in normal and tumor tissues. Ultimately, these issues can only be resolved by further structural analysis of the MALT1 gene in MALT lymphomas and by functional studies directed at the ability of this gene in either normal or mutant form to affect the proliferative behavior of lymphocytes in culture and in vivo.
Structural analysis of the coding sequence contained within the MALT1 cDNA identified several regions of homology to known genes. The presence of two immunoglobulin-like C2 domains suggests a transmembrane or extracellular location for the normal MALT1 protein; however, no amino acid sequences compatible with signal peptides or transmembrane regions could be found by computer scans of the cDNA sequence. On the other hand, the protein TITIN is a well-characterized component of striated muscle that also contains immunoglobulin-like C2 domains (25) , so that an intracellular role for MALT1 is a possibility. A second homology of note is the partial caspase motif within MALT1. The exclusion from MALT1 of the critical amino acid in the active caspase protease site (26) would seem to imply that the protein lacks caspase activity but may bind substrate for caspase and conceivably act as a competitive inhibitor of caspase enzymes. According to this line of reasoning, MALT1 would have antiapoptotic function reminiscent of BCL-2, a gene implicated in the malignant transformation of B lymphocytes in follicular lymphoma (27) , another form of indolent B-cell neoplasia. At any rate, an effect of the t(11;18)(q21;q21) on apoptosis is suggested by the recent report of Dierlamm et al. (22) , who describe fusion of MALT-1 (MLT) in this translocation to API2, the product of which is an inhibitor of apoptosis. Investigation of a role for MALT1 in regulating apoptosis or in some other cellular function could possibly gain from genetic studies of the homologous open reading frame F22D3.6 in the Caenorhabditis elegans genome, as well as from analysis of mice carrying transgenes and knock-out mutations for MALT1.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants CA75354, CA72744, and AI013575 from the NIH. 
2 Present address: Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
3 To whom requests for reprints should be addressed, at Division of Molecular Oncology, Department of Pathology, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. Phone: (617) 732-7446; Fax: (617) 732-7449; E-mail: jsklar{at}rics.bwh.harvard.edu
4 The abbreviations used are: MALT, mucosa-associated lymphoid tissue; CHO, Chinese hamster ovary; YAC, yeast artificial chromosome; BAC, bacterial artificial chromosome; STS, sequence-tagged site; EST, expressed sequence tag clone; FISH, fluorescence in situ hybridization; RACE, rapid amplification of cDNA ends; IPCR, inverse PCR; cR, centiRay.
5 L. Soreng, E. Robinson, E. Kieff, and J. Sklar, unpublished data.
Received 5/26/99. Accepted 11/ 1/99.
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E. M. Murga Penas, E. Callet-Bauchu, H. Ye, K. Hinz, N. Albert, C. Copie-Bergman, S. Gazzo, F. Berger, G. Salles, C. Bokemeyer, et al. The translocations t(6;18;11)(q24;q21;q21) and t(11;14;18)(q21;q32;q21) lead to a fusion of the API2 and MALT1 genes and occur in MALT lymphomas Haematologica, March 1, 2007; 92(3): 405 - 409. [Abstract] [Full Text] [PDF]
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 S. M. Cohen, M. Petryk, M. Varma, P. S. Kozuch, E. D. Ames, and M. L. Grossbard Non-Hodgkin's Lymphoma of Mucosa-Associated Lymphoid Tissue Oncologist, November 1, 2006; 11(10): 1100 - 1117. [Abstract] [Full Text] [PDF]
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 B. Streubel, D. Huber, S. Wohrer, A. Chott, and M. Raderer Reverse Transcription-PCR for t(11;18)(q21;q21) Staging and Monitoring in Mucosa-Associated Lymphoid Tissue Lymphoma. Clin. Cancer Res., October 15, 2006; 12(20): 6023 - 6028. [Abstract] [Full Text] [PDF]
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 E. Varfolomeev, S. M. Wayson, V. M. Dixit, W. J. Fairbrother, and D. Vucic The Inhibitor of Apoptosis Protein Fusion c-IAP2{middle dot}MALT1 Stimulates NF-{kappa}B Activation Independently of TRAF1 AND TRAF2 J. Biol. Chem., September 29, 2006; 281(39): 29022 - 29029. [Abstract] [Full Text] [PDF]
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 D. Decaudin, P. de Cremoux, A. Vincent-Salomon, R. Dendale, and L. L.-L. Rouic Ocular adnexal lymphoma: a review of clinicopathologic features and treatment options Blood, September 1, 2006; 108(5): 1451 - 1460. [Abstract] [Full Text] [PDF]
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 M-Q Du and J C Atherton Molecular subtyping of gastric MALT lymphomas: implications for prognosis and management. Gut, June 1, 2006; 55(6): 886 - 893. [Full Text] [PDF]
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T. Samuel, K. Welsh, T. Lober, S. H. Togo, J. M. Zapata, and J. C. Reed Distinct BIR Domains of cIAP1 Mediate Binding to and Ubiquitination of Tumor Necrosis Factor Receptor-associated Factor 2 and Second Mitochondrial Activator of Caspases J. Biol. Chem., January 13, 2006; 281(2): 1080 - 1090. [Abstract] [Full Text] [PDF]
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M. Nakagawa, Y. Hosokawa, M. Yonezumi, K. Izumiyama, R. Suzuki, S. Tsuzuki, M. Asaka, and M. Seto MALT1 contains nuclear export signals and regulates cytoplasmic localization of BCL10 Blood, December 15, 2005; 106(13): 4210 - 4216. [Abstract] [Full Text] [PDF]
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 P. Farinha and R. D. Gascoyne Molecular Pathogenesis of Mucosa-Associated Lymphoid Tissue Lymphoma J. Clin. Oncol., September 10, 2005; 23(26): 6370 - 6378. [Abstract] [Full Text] [PDF]
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L. Ho, R. E. Davis, B. Conne, R. Chappuis, M. Berczy, P. Mhawech, L. M. Staudt, and J. Schwaller MALT1 and the API2-MALT1 fusion act between CD40 and IKK and confer NF-{kappa}B-dependent proliferative advantage and resistance against FAS-induced cell death in B cells Blood, April 1, 2005; 105(7): 2891 - 2899. [Abstract] [Full Text] [PDF]
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 R. Gerl and D. L. Vaux Apoptosis in the development and treatment of cancer Carcinogenesis, February 1, 2005; 26(2): 263 - 270. [Abstract] [Full Text] [PDF]
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 A. Stoffel, M. Chaurushiya, B. Singh, and A. J. Levine Activation of NF-{kappa}B and inhibition of p53-mediated apoptosis by API2/mucosa-associated lymphoid tissue 1 fusions promote oncogenesis PNAS, June 15, 2004; 101(24): 9079 - 9084. [Abstract] [Full Text] [PDF]
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 O. T. Lynch and M. Gadina Ubiquitination for Activation: New Directions in the NF-{kappa}B Roadmap Mol. Interv., June 1, 2004; 4(3): 144 - 146. [Abstract] [Full Text] [PDF]
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Y. Hosokawa, H. Suzuki, Y. Suzuki, R. Takahashi, and M. Seto Antiapoptotic Function of Apoptosis Inhibitor 2-MALT1 Fusion Protein Involved in t(11;18)(q21;q21) Mucosa-Associated Lymphoid Tissue Lymphoma Cancer Res., May 15, 2004; 64(10): 3452 - 3457. [Abstract] [Full Text] [PDF]
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G. Ghannam, A. Takeda, T. Camarata, M. A. Moore, A. Viale, and N. R. Yaseen The Oncogene Nup98-HOXA9 Induces Gene Transcription in Myeloid Cells J. Biol. Chem., January 9, 2004; 279(2): 866 - 875. [Abstract] [Full Text] [PDF]
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 P. C. Lucas, L. M. McAllister-Lucas, and G. Nunez NF-{kappa}B signaling in lymphocytes: a new cast of characters J. Cell Sci., January 1, 2004; 117(1): 31 - 39. [Abstract] [Full Text] [PDF]
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R. W. Tsang, M. K. Gospodarowicz, M. Pintilie, W. Wells, D. C. Hodgson, A. Sun, M. Crump, and B. J. Patterson Localized Mucosa-Associated Lymphoid Tissue Lymphoma Treated With Radiation Therapy Has Excellent Clinical Outcome J. Clin. Oncol., November 15, 2003; 21(22): 4157 - 4164. [Abstract] [Full Text] [PDF]
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L. Franchi, I. Condo, B. Tomassini, C. Nicolo, and R. Testi A caspaselike activity is triggered by LPS and is required for survival of human dendritic cells Blood, October 15, 2003; 102(8): 2910 - 2915. [Abstract] [Full Text] [PDF]
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B. J. P. Huntly, A. Bench, and A. R. Green Double jeopardy from a single translocation: deletions of the derivative chromosome 9 in chronic myeloid leukemia Blood, August 15, 2003; 102(4): 1160 - 1168. [Abstract] [Full Text] [PDF]
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D. Sanchez-Izquierdo, G. Buchonnet, R. Siebert, R. D. Gascoyne, J. Climent, L. Karran, M. Marin, D. Blesa, D. Horsman, A. Rosenwald, et al. MALT1 is deregulated by both chromosomal translocation and amplification in B-cell non-Hodgkin lymphoma Blood, June 1, 2003; 101(11): 4539 - 4546. [Abstract] [Full Text] [PDF]
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B. Streubel, A. Lamprecht, J. Dierlamm, L. Cerroni, M. Stolte, G. Ott, M. Raderer, and A. Chott T(14;18)(q32;q21) involving IGH and MALT1 is a frequent chromosomal aberration in MALT lymphoma Blood, March 15, 2003; 101(6): 2335 - 2339. [Abstract] [Full Text] [PDF]
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 H. Inagaki, J. K. C. Chan, J. W. M. Ng, M. Okabe, T. Yoshino, M. Okamoto, H. Ogawa, H. Matsushita, T. Yokose, Y. Matsuno, et al. Primary Thymic Extranodal Marginal-Zone B-Cell Lymphoma of Mucosa-Associated Lymphoid Tissue Type Exhibits Distinctive Clinicopathological and Molecular Features Am. J. Pathol., April 1, 2002; 160(4): 1435 - 1443. [Abstract] [Full Text] [PDF]
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H. Liu, H. Ye, A. Dogan, R. Ranaldi, R. A. Hamoudi, I. Bearzi, P. G. Isaacson, and M.-Q. Du T(11;18)(q21;q21) is associated with advanced mucosa-associated lymphoid tissue lymphoma that expresses nuclear BCL10 Blood, August 15, 2001; 98(4): 1182 - 1187. [Abstract] [Full Text] [PDF]
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J. I. Koontz, A. L. Soreng, M. Nucci, F. C. Kuo, P. Pauwels, H. van den Berghe, P. D. Cin, J. A. Fletcher, and J. Sklar Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors PNAS, May 22, 2001; 98(11): 6348 - 6353. [Abstract] [Full Text] [PDF]
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 F. Cavalli, P. G. Isaacson, R. D. Gascoyne, and E. Zucca MALT Lymphomas Hematology, January 1, 2001; 2001(1): 241 - 258. [Abstract] [Full Text] [PDF]
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E. Macintyre, D. Willerford, and S. W. Morris Non-Hodgkin's Lymphoma: Molecular Features of B Cell Lymphoma Hematology, January 1, 2000; 2000(1): 180 - 204. [Abstract] [Full Text] [PDF]
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P. C. Lucas, M. Yonezumi, N. Inohara, L. M. McAllister-Lucas, M. E. Abazeed, F. F. Chen, S. Yamaoka, M. Seto, and G. Nunez Bcl10 and MALT1, Independent Targets of Chromosomal Translocation in MALT Lymphoma, Cooperate in a Novel NF-kappa B Signaling Pathway J. Biol. Chem., May 25, 2001; 276(22): 19012 - 19019. [Abstract] [Full Text] [PDF]
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L. M. McAllister-Lucas, N. Inohara, P. C. Lucas, J. Ruland, A. Benito, Q. Li, S. Chen, F. F. Chen, S. Yamaoka, I. M. Verma, et al. Bimp1, a MAGUK Family Member Linking Protein Kinase C Activation to Bcl10-mediated NF-kappa B Induction J. Biol. Chem., August 10, 2001; 276(33): 30589 - 30597. [Abstract] [Full Text] [PDF]
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