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From www.bloodjournal.org by guest on August 3, 2017. For personal use only. Rearrangement of CCNDl (BCLI/PRADI) 3’ Untranslated Region in MantleCell Lymphomas and t(llql3)-Associated Leukemias By Ruth Rimokh, FranGoise Berger, Christian Bastard, Bernard Klein, Martine French, Eric Archimbaud, Jean Pierre Rouault, Benedicte Santa Lucia, Laurent Duret, Michele Vuillaume, Bertrand Coiffier, Paul-Andre Bryon, and Jean Pierre Magaud Rearrangement and overexpression of CCNDl iBCLl/ PRADl),a member of the cyclin G, gene family, are consistent features of t(llql31-bearing B-lymphoidtumors (particularly mantle-cell lymphoma [MCL]). lts deregulationis thought to perturb the G,-S transition of the cell cycle and thereby to contribute to tumor development. As suggested by previously published studies, rearrangement of the 3‘ untranslated region (3’ UTR) of CCNDl may contribute to its activation in some lymphoid tumors. To define further the prevalence ofsuch rearrangements, we report here the result of the molecular study of 34 MCL and six t(llq13)associated leukemias using a set of probes specific to the different parts of the CCNDl transcript. We also sequenced the entire cDNA of the overexpressed CCNDl transcripts in A MAJORITY OF HUMAN hematopoietic malignancies carry nonrandom chromosomal alterations; experimental evidence indicates that genes located at recurring chromosomal breakpoints are directly involved in tumor pathogenesis.’ The t(l1; 14)(q13;q32) translocation and its molecular counterpart, bcl-l rearrangement, are consistent features of the subtype of non-Hodgkin’s lymphoma designated centrocytic lymphoma or the similar intermediate lymphocytic lymphoma:” now referred to as mantle-cell lymphoma (MCL).’ As a result of this translocation, the putative BCLUPRADI proto-oncogene, on chromosome 11, is juxtaposed to an IgH-enhancer sequence located on chromosome 14.9 The BCLUPRADI gene is known under different names (PRADI, cyclin D l , BCLI, DllS287E). We will refer to it as CCNDI, in accordance with the official nomenclature.” Chromosomal breakpoints are widely scattered on chromosome 1lq13, but three hot spots have been individualized: the major translocation cluster (MTC)9lying approximately 110 kb centromeric of the CCNDl gene, and two minor translocation clusters (mTCs), which are less frequently involved: mTCl and mTC2.I’ mTCl is localized approximately 22 kb telomeric of MTC,” andmTC2,”.’’ which has been recently identified, maps to the 5’ flanking region of the CCNDl gene (probe B as described by Williams et all’). CCNDl is a member of the cyclin G, familyI4-l7;its deregulation is thought to perturb the G,-S transition of the cell cycle and thereby to contribute to human tumor development. This hypothesis is in agreement with the finding that CCNDl is overexpressed in the vast majority of the MCL and t( 1lql3)-associated leukemias analyzed so far.ll.18-20 With regard to the mechanisms of activation of CCNDl in lymphoid tumors, one can consider several possibilities. It has beenfirst postulated that activation of CCNDl in t( 11; 14)-carrying tumors is a consequence of its juxtaposition to an immunoglobulin &-acting sequence. According to another hypothesis, CCNDI activation might occur at the posttranscriptional level through rearrangement of its 3’ untranslated region (3’ UTR). In fact, in primary tumors and in Blood, Vol 83, No 12 (June 15). 1994: pp 3689-3696 a t(llql3)-associated leukemia. DNA from four of these 40 patients showed rearrangement of the 3’ UTRof CCNDl coexisting with major translocation cluster (MTC) rearrangement. Southernblot and sequence analyses showed the 3’ AU-rich that, as a result of these rearrangements, region containingsequences involved in mRNA stabilii and in translational control is eliminated. Moreover,the finding that the CCNDl mRNA half-life was greater than 3 hours (normal tissues, 0.5 hours) in three t(llql3)-associated cell linesstresses the importanceofposttranscriptional derangement in the activation of CCNDl.Finally, we did not observe any mutation in the coding frame of the CCNDl cDNA analyzed. 0 1994 by The American Society of Hematology. cell lines with chromosome 1lq13 structural abnormalities, a 1S-kb mRNA can be detected in addition to the normal 4.5kb mRNA. Sequencing of CCNDl cDNA in a few cell lines has shown that the smaller transcript corresponds to a shortened form of the normal 4.5-kb transcript as a result of the use of different polyadenylation signals or of deletions of the 3’ end of the gene.’5,’6,’9 These data suggested that, in some cases, activation of CCNDl might result from the loss of 3‘ regulatory sequences, which are known tobe implicated in mRNA stability and in translational control. Finally, sequence analysis of the CCNDl coding region in primary human tumors has shown that amino acid sustitutions are not required for CCNDl’s oncogenic effects.” The aim of the present study was to determine the incidence and the consequences on the transcription rate of rearrangement of the 3’ UTR of CCNDl in primary human tumors. For this purpose, we first sequenced the coding region and the 3’ UTR ofthe overexpressed CCNDl transcripts in one t(llql3)-associated leukemia. Wenext analyzed breakpoints that map to the CCNDI 3’ UTR in a large series of MCL and t(l lql3)-associated leukemias by using a set Fromthe Luboratoire de CytoginitiqueMoliculaire andthe Laboratoire d’Anatomie Pathologique, Hbpital Edouard Herriot, Lyon, France; Laboratoire deCytoginitique, CRTS, Bois Guillaume, France: and Dipartement d’Himatologie, Centre Hospitalier Lyon Sud, Lyon, France. Submitted August 2, 1993; accepted February 23, 1994. Supported by grants from INSERM (CRE 92 0108,CJF 93-08), CNRS, Hospices Civils de Lyon, Ligue Nationale Contre le Cancer (Comiti du Rhbne, de la Haute-Savoie et de la Sabne et Loire). Address reprint requests to RuthRimokh, PhD, Laboratoire de Cytoginitique Moliculaire, Pavillon E, Hbpital Edouard Herriot, 69437 Lyon Cedex 03, France. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with I 8 U.S.C. section 1734 solely to indicate this fact. 0 1994 by The American Society of Hematology. 0006-4971/94/8312-0028$3.00/0 3689 From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 3690 RIMOKH ET AL of probes specific to the different parts of the CCNDl transcript. The results of MTC, mTC 1, and mTC2 rearrangement analysis of most of the samples presented here have been previously reported.” Finally, when material was available, CCNDl protein level was assessed in these tumors by Westem blot analysis. MATERIALS AND METHODS Tissue samples and cell lines. Thirty-three cases ofMCLwith available frozen material were analyzed (cases no. 1 to 33). Cytogenetic data were not available for any of these MCL cases. Each case was classified by using histologic criteria previously We also analyzed four cases of B-cell chronic lymphocytic leukemia (B-CLL), one case of plasma-cell leukemia carrying a t(11;14)(q13;q32) (cases no. 34 to 38), and one case ofB-CLL carrying a variant t(l1; 19)(q13;q13) translocation (case no. 39). All cases (no. 1 to 39) were included in a previous report.” Cell-suspension and frozen-section immunophenotypic studies were performed as previously reported.26 The following markers were used in this study: IgG, IgM, IgD, IgA, K and X light chains, B1 (CDZO), B4 (CD19), Leu-l4 (CD22), TlOl (CD5), OKT3 (CD3), Leu-9 (CD7), and JS(CDl0). AllMCL cases were monoclonal B cells; 24 of the 30 tested showed CD5 expression and CD10 was positive in only seven of the 31 cases tested. All B-cell leukemias expressed monoclonal immunoglobulin and were positive for markers of the B-cell lineage. The Gob. cell line was derived from the leukemic cells of patient no. 39. Malignant cells continued to proliferate in RPM1 1640 with 20% fetal calf serum only in the presence of recombinant human interleukin-2 (rIL-2,20 to 30 IUlmL; Boehringer Mannheim, Mannheim, Germany). After 3 months of culture, rIL-2 was no longer necessary for the cells to proliferate. It is likely that, as a consequence of its mitogenic effect, rIL-2 favored the occurrence of secondary genetic changes leading to growth factor-independence. Cells from the original culture have now been growing continuously for nearly 1 year. Immunophenotype and karyotype were identical to those seen during the initial examination. Two cell lines carrying a t(11;14)(q13;q32) translocation: Rec-l (referred toas case 40) established from a MCL,”and XG5, a human myeloma cell line”; Ramos, a Burkitt’s lymphoma cell line; reactive lymph nodes, normal tonsils, andhuman fibroblasts were also used in this study. DNA and RNA isolation and analysis. High-molecular weight DNA was extracted from fresh cells or from frozen material following standard procedures. After digestion with appropriate endonucleases as recommended by the suppliers (Boehringer Mannheim), DNA fragments were electrophoresed on 0.8% agarose gels and transferred onto nylon filters. Total cellular RNA was isolated from cultured cell lines or from frozen samples by the acid guanidium thiocyanate-phenol-chloroform method. For Northern blot analysis, poly(A+) or total RNA was size fractionated in formaldehyde-1.2% agarose gels and transferred onto nylon filters. DNA probes and hybridization procedures. A 2.3-kb Sac1 fragment representing the MTC on chromosome 1 lq13 was a gift from Y. Tsujimoto (Philadelphia, PA). Probes “A” and “E” arepolymerase chain reaction (PCR)-amplified DNA fragments encompassing nucleotides 148 to 310 and2,856to 3,091, respectively, on the human PRADl cDNA sequence.14 Probes “B,” “C,” and “D” correspond to CCNDl cDNA subclones (Fig 1). MYC exon 3 probe was a gift from D. Stehelin (Lille, France). Alpha-”P-labeling, prehybridization, hybridization, andwashingwere performed aspreviously described.*’ Preparation and analysis of cDNA library. Poly(A+) RNA extracted from case no. 39 was used to construct an oligo-dT-primed human cDNA library in the lambda gtl 1 vector as recommended by the supplier (Pharmacia, Uppsala, Sweden). Sequencingprocedure and sequence analysis. Overlapping deletions of CCNDl cDNA cloned into Bluescript. SK(-) (Stratagene, La Jolla, CA) were obtained by the unidirectional exonuclease 111 digestion method (Erase-a-Base System; Promega, Madison, WI). Deletion clones were sized on agarose gels, and the inserts ofthe selected clones were sequenced using the double-stranded DNA sequencing technique (dideoxy chain termination procedure) with Sequenase I1 (USB, Cleveland, OH) as described by the manufacturer. Study of CCNDI mRNA stability. Exponentially growing cells (Rec-l, XG5, and Gob, cell lines) were treated with 10 pg/mL of actinomycin D (Sigma Chemical, St Louis, MO). CCNDl mRNA level was then assessed by Northern blot analysis at various times after addition of actinomycin D. The Northern blot was sequentially hybridized to a CCNDl probe andto a MYC exon 3 probeas a control. Western blot analysis. For Western blot analysis, proteins from the pathological samples were solubilized in lysis buffer (1 50 mmol/ L NaCL, 50 mmoVL Tris-HCL, pH 8, 0.5% sodium deoxycholate, 1% Triton X100). Proteins (30 pg) from each sample were then separated by sodium dodecyl sulfate 10% polyacrylamide slab gel electrophoresis and electrophoretically transferred onto nitrocellulose filters. For immunodetection, the filters were incubated with a U750 dilution of a polyclonal rabbit anti-cyclin D antibody (UBI, New York, NY) at room temperature. After washing, fixation of the antibody was shown by using a blotting detection kit for rabbit antibodies (Amersham, Uppsala, Sweden). The polyclonal antibody used in this study was raised against a peptide contained in the Cterminal domain of the protein (residues 208 to 295). RESULTS In a previous study, we showed that inMCL and t(1Iq 13)associated leukemias, the steady-state level of CCNDl transcript is dramatically elevated relative to anyother lymphoid tissues” andthat a 1.5-kbmRNAis predominantly expressed. One case of t( 11; 19)-associated leukemia (case no. 39) expressed, in addition to the 1.5-kb transcript, two aberrant transcripts of 2 and 3 kb, indicating that the CCNDl mRNA may be altered in this tumor (Fig 2 ) . To characterize further the CCNDl transcripts in this lymphoid malignancy, a cDNA library was made from fresh tumoral cells. Screening of this library with probe A allowed us to isolate 12 positive clones. Eleven clones covering less than 1,500 bp may correspond to the 1.5-kb transcript. The longest of them was entirely sequenced; it is almost identical to the region encompassing nucleotides 1 to 1346 on the PRADl cDNAsequence,14 except for two base changes: (1) a G is present at nucleotide 870 instead of an A in the coding sequence; (2) a c is present at nucleotide l100 instead of an A in the 3’ UTR. None of the base changes observed altered the amino acid sequence of the protein. There is no recognizable polyadenylation sequence (AATAAA) anywhere within the sequence of the 1 i clones analyzed, and none of them contains a poly(A) tail. However, the corresponding transcripts are polyadenylated, since poly(A+) selection increased the signal obtained on Northern blot (Fig 2 ) . It is likely that, in this oligo-dT- From www.bloodjournal.org by guest on August 3, 2017. For personal use only. REARRANGEMENT OF CCNDl 3' UNTRANSLATED REGION MTC CEN. + / 3691 5' 3' W I € H I P I I xI - € I l10 kb B BP TEL. CCND1cDNA U U A B p C n O E P SPP L I . 1 . L . 39- cDNA 3 kb & 39- cDNA1.35 kb Fig 1. Comparison of the restriction maps of the 3' end of CCNDl with those of the normal 4.5-kb CCNDl cDNA and of two cDNA clones isolated from case no.39. Black boxes indicate the coding region; dotted line representsthe MER11 sequence; double-headedarrow corresponds to theCCNDT last exon; slided-horizontal lines indicate the position of the probes used in thisstudy. CEN, centromere; TEL. telomere, X, Xbd; E, EcoRI; H, Hindlll;P, P A ; B, BsmHI; Bg, Bgfll; S, Sad. primed library, all the cDNA clones were initiated from the destabilizing signals AUUUApresent in thenormal trannumerous poly(A) stretches present within the 3' end of the script are eliminated. In case no. 39, PCR amplification of CCNDI mRNA. genomic DNAwith primers flanking the CCNDllMERll We also isolated a 3,013-nucleotide long cDNA clone junction (nucleotides 2090 to 2465) yielded a 375-bp fragrepresentative of the 3-kb aberrant transcript. The first 2,275 mentwhose sequence matchedperfectlythat of the 3-kb nucleotides of this clone are identical to nucleotides 2 to aberrant transcript (data not shown), demonstrating that the 2276 of the PRADI cDNA ~equence,'~ with the same base rearrangement occurred atthe genomic leveland was not changes at positions 870 and 1 1 0 0 , and are followed by a the result of a cloning artifact. The fact that the same base sequence (nucleotides 2276 to 3013) unrelated to the changes was observed in the two groups of cDNA suggests CCNDI/PRADI gene (Fig 3). Comparison of this sequence thatthe1.5-kb transcript represents an alternativelyprocessed form of the 3-kb mRNA and that they both originate with those in data bases (EMBL data base) showed that it corresponds to a new member of the MER1 1 sequences from the same allele. On Southern blots containing BamHl and HindIII-cleaved family. In fact, this region has greater than 90% similarity with two members (HUMP45C17, HUMSIGG3) of this famDNA from case no. 39, probes D and E detected two differily of medium reiteration frequency repetitive ~ e q u e n c e s . ~ ~ ~ent * ~rearranged bands in addition to the same germline band (Fig 4). The first explanation of this resultisthat reIt should be notedthat in all the analyzed cDNA clones, arrangement of the 3' end of CCNDI corresponds to an the CCNDl coding frame is retained and that the mRNAinsertion of the MER1 1 sequence at this site. However, as case no. 39 is associated with a variant t(11;19)(q13;q13) translocation, we cannot ruleoutthepossibilitythat the 39 MER1 I sequence originates from chromosome 19 and that the rearranged band detected by probe E corresponds to the A T 4 34 40 der( 19) chromosome. In Fig 4, case no. 39, the intensity of the rearranged bands differs considerably as compared with the residual germline band. This pattern was not observed when using probes A, B, and E (data not shown). This indicates that on the der( 11) chromosome, the 3' CCNDl UTR corresponding to probe D was amplifiedduring the translocation process. This observation and a previously published study" showing that the 3' end of CCNDl was rearranged in one case of lymphoma suggested that, in some tumors, its deregulation might occur at the posttranscriptional level. To test this hypothesis, we haveanalyzed a large series ofMCLand t( 1 Iql3)-associated leukemias using probes specific to the Fig 2. Northern blot analysis of CCNDl expression in MCL or leudifferent parts of the CCNDl transcript (Fig 1). Three of the kemias with rearrangement of the CCNDT 3' UTR. (Left1 10 p g of 40 tumors (cases no. 4, 34, and 40; case no. 39 not included) total RNAfromeach sample was processed for Northern blot analysis showed rearrangement of the 3' end of CCNDI. Southern as described in the Methodsand the filter was hybridized to a CCNDT blot analysis using probes B or C detected a rearranged probe (probe Al. (Right) 5 p g of poly(A'1 RNA (A) and 5 p g of total RNA from case no. 39 were blottedand hybridized to probe A. fragment in BamHI- and Sad-cleaved DNA (Fig 5). None From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 3692 RIMOKH ET A t GCGCAGTAGCAGCGAGCAGCAGAGTCCGCACGCTCCGGCGAGGGGCAGAAGAGCGCGAGGGAGCGCGGGGCAGCAGAAGCGAGAGCCGAGCGCGGACC 98 CAGCCAGGACCCACAGCCCTCCCCAGCTGCCCAGGAAGAGCCCCAGCCATGGAACACCAGCTCCTGTGCTGCGAAGTGGAAACCATCCGCCGCGCGTAC 19 7 17 MetGluHisGlnLeuLeuCysCysGluValGluThrIleArgArgAlaTyr CCCGATGCCAACCTCCTCAACGACCGGGTGCTGCGGGCCATGCTGAAGGCGGAGGAGACCTGCGCGCCCTCGGTGTCCTACTTCAAATGTGTGCAGAAG PrOASpA~aASnLeuLeuAsnAspArgValLeuArgAlaMetLeuLysAlaGluGluThrCysAlaProSerValSerTyrPheLysCysValGlnLys 296 50 GAGGTCCTGCCGTCCATGCGGAAGATCGTCGCCACCTGGATGCTGGAGGTCTGCGAGGAACAGAAGTGCGAGGAGGAGGTCTTCCCGCTGGCCATGAAC 39 5 G~uValLeuProSerMetArgLysIleValAlaThrTrpMetLeuGluValCysGluGluGlnLysCysGluG1uGluValPheProLeuAlaMetAsn 83 TACCTGGACCGCTTCCTGTCGCTGGAGCCCGTGAAAAAGAGCCGCCTGCAGCTGCTGGGGGCCACTTGCATGTTCGTGGCCTCTAAGATGAAGGAGACC 494 116 TyrLwAspArgPheLeuSerLeuGluProValLysLysSerArgLeuGlnLeuLeuGlyAlaThrCysMetPheValAlaSerLysHetLysGluThr ATCCCCCTGACGGCCGAGAAGCTGTGCATCTACACCGACAACTCCATCCGGCCCGAGGAGCTGCTGCAAATGGAGCTGCTCCTGGTGAACMGCTCAAG I1eProLeuThrAlaGluLysLeuCysIleTyrThrAspAsnSerIleArgProGluGluLeuLeuGlnHetGluLeuLeuLwValASnLysLeuLyS 593 149 TGGAACCTGGCCGCAATGACCCCGCACGATTTCATTGAACACTTCCTCTCCAAAATGCCAGAGGCGGAGGAGAACAAACAGATCATCCGCAAACACGCG 692 TrpAsnLeuAlaAlaMetThrProHisAspPheIleGluHisPheLeuSerLysMetProGluAlaGluGluAsnLysGlnIleIleArgLysHiSA~a 182 CAGACCTTCGTTGCCCTCTGTGCCACAGATGTGAAGTTCATTTCCAATCCGCCCTCCATGGTGGCAGCGGGGAGCGTGGTGGCCGCAGTGCAAGGCCTG 79 1 G1nThrPheValAlaLeuCysAlaThrAspValLysPheIleSerAsnProProSerMetValAlaAlaGlySerValValAlaAlaValGlnGlyLeu 215 AACCTGAGGAGCCCCAACAACTTCCTGTCCTACTACCGCCTCACACGCTTCCTCTCCAGAGTGATCAAGTGTGACCC~GACTGCCTCCGGGCCTGCCAG89 0 ASnLwArgSerProAsnAsnPheLeuSerTyrTyrArgLeuThrArgPheLeuSerArgValIleLysCysAspProAspCysLeuArgAlaCySGln 248 GAGCAGATCGAAGCCCTGCTGGAGTCAAGCCTGCGCCAGGCCCAGCAGAACATGGACCCCAAGGCCGCCGAGGAGGAGGAAGAGGAGGAGGAGGAGGTG 989 28 1 GluGlnIleGluAlaLeuLeuGluSerSerLeuArgGlnAlaGlnG1nAsnHetAspProLysAlaAlaGluGluGluGluGluGluGluGluGluVal GACCTGGCTTGCACACCCACCGACGTGCGGGACGTGGACATCTGAGGGCGCCAGGCAGGCGGGCGCCACCGCCACCCGCAGCGAGGGCGGAGCCGGCCC 1088 295 AspLeuAlaCysThrProThrAspValArgAspValAspIle 1187 CAGGTGCTCC~CTGACAGTCCCTCCTCTCCGGAGCATTTTGATACCAGAAGGGAAAGCTTCATTCTCCTTGTTGTTGGTTGTTTTTTCCTTTGCTCTTT CCCCCTTCCATCTCTGACTTAAGCAAAAGAAAAAGATTACCCAAAAACTGTCTTTAAAAGAGAGAGAGAGAAAAAAAAAATAGTATTTGCATMCCCTG 1286 AGCGGTGGGGGAGGAGGGTTGTGCTACAGATGATAGAGGATTTTATACCCCAATAATCAACTCGTTTTTATATTAATGTACTTGTTTCTCTGTTGTAAG 1385 AATAGGCATTMCACAMGGAGGCGTCTCGGGAGAGGATTAGGTTCCATCCTTTACGTGTTTAAAAAAAAGCATAAAAACATTTTAAAAACATAGAAAA 1484 1583 ATTCAGCAMCCATTTTTAAAGTAGAAGAGGGTTTTAGGTAGAAAAACATATTCTTGTGCTTTTCCTGATAAAGCACAGCTGTAGT~~TTCTAGGCA TCTCTGTACTTTGCTTGCTCATATGCATGTAGTCACTTTATAAGTCATTGTATGTTATTATATTCCGTAGGTAGATGTGTAACCTCTTCACCTTATTCA 1682 TGGCTGMGTCACCTCTTGGTTACAGTAGCGTAGCGTGGCCGTGTGCATGTCCTTTGCGCCTGTGACCACCACCCCAACAMCCATCCAGTGACMACC 1781 1880 ATCCAGTGGAGGTTTGTCGGGCACCAGCCAGCGTAGCAGGGTCGGGAAAGGCCACCTGTCCCACTCCTACGATACGCTACTATAAAGAGAAGACGAMT AGTGACATMTATATTCTATTTTTATACTCTTCCTATTTTTGTAGTGACCTGTTTATGAGATGCTGGTTTTCTACCCAACGGCCCTGCAGCCAGCTCAC 1979 GTCCAGGTTCMCCCACAGCTACTTGGTTTGTGTTCTTCTTCATATTCTAAAACCATTCCATTTCCAAGCACTTTCAGTCCAATAGGTGTAGGAAATAG 2078 CGCTGTTTTTGTTGTGTGTGCAGGGAGGGCAGTTTTCTAATGGAATGGTTTGGGAATATCCATGTACTTGTTTGCAAGCAGGACTTTGAGGCMGTGTG 2177 GGCCACTGTGGTGGCAGTGGAGGTGGGGTGTTTGGGAGGCTGCGTGCCAGTCAAGAAGAAAAAGGTTTGCATTCTCACATTGCCAGGATGATAAGTTCA2276 ACTGAGCCTTAAACWIGCAAGTTTTTTATTAAGGGTTTCAAGAGGGGAGGGGGTGTGAGAACATGGAGTAGAGCTCATGCTTCAAAGG~AAAAAACAG 2375 AACAAAGA TCACTTGCTTCTGAGGGAACAGGAGAAAAGGCAACACAGAACTACTGATAAGGGTCCATGTTCAGCGGTGCACGT4 TTATCTTGATAAACA 2474 TTCACCAGGGTGGAGTTTTTCCCCACCGTAGTAAGCCTGAGGGTACTGCAGGAGATCAGGGCG 2573 TCTTGAACAAAAAA TAGGGAAACGTCTGACCACAAA TATCTCAGTCCTTATCTCAACCACGTAAGACAGACATTCCCAGAGCAGCTGTTTATAGACCTCTCCCCAGGAATGCATTCCTTTCCCAGGGTATTAATA 2672 TTAA TATTGCTTGCTAAC;GAAAAGAATTTAGCGATATCCTCCCTACTTGCATGTCCTTTTATAW;CTCTCTGCAAGAAGAAAAATATGGCTTTTTTTGCC 2771 TGACCCTGCAGGCAGTCAGACCTTATGGTTGTCTTCCCTTGTTCCCTAAAATCGCTGTTATTCTGTTTTTTCTCAAGGTGCACTGA TTTCATATTGTTC 2870 AAACACACGTTTTACAATCAATTTGTACAGTTAACACAATTATCACGGTGGTCCTGAGGTGACATACATCCTCAGCTTACGAAGATAAWIGGATTAAGA 2969 3013 GATGAGAGTAAGACAGGCGTAAGAAATTATAAAAGTATTAATTT Fig 3.Sequence of the 3-kb CCNDl aberrant transcript observed in case no. 39.The 295-amino acid coding is shown. The 1 MER1 nucleotide sequence is in italics. *Position of the base changes (nucleotides 870 and 1100)compared with thenormal CCNDl c D N A sequence." The sequence reported here has been deposited in the E M B L data base (accession no. 223022). of the rearranged bands cohybridized tothe immunoglobulin JH probe (data not shown). Probes D and E did not detect anyrearrangedfragment in these cases, althoughtheydetected the same germline bands, as did probes B and C in BamHI- andSad-cleavedDNA.This led us to postulate that, in these cases, the genomic region correspondingto probesD and E was deleted, and that the break occurred between the stop codon and the 5' end of probe D. Interestingly, all of these tumors (case no. 39 included) also demonstrated bcl-l rearrangement with a break in the MTC (Fig 6), each rearrangement being identified on two or more restriction digests. We are indeed unable to specify if the two breaks occurred on one or on both alleles. Northern blot analysis showed that the steady-state level ofthe CCNDl transcripts is dramaticallyelevated in the samples showing complex rearrangements, and that the 1S kb transcript was predominantly represented, the normal 4.5kb mRNA being undetectable (Fig 2 ) . When material was available,Westernblot analysis usingapolyclonalrabbit anti-cyclin D antibody confirmed, at the protein level, that CCNDI is really expressed in thesepathological samples of 35-kD (Fig 7). In fact, a protein with an expected size was detected in cases no. 39 and 40 and in normal human fibroblasts used as positive control. The CCNDI protein was also present in three other cases of MCL (cases no. 9, 19, and 22), but was undetectable in normal lymphoid tissues. To determine if thehighexpression of CCNDI in lymphoidmalignancieswithbreakin the 3' UTRwasa consequence of an increase in stability of its transcript, we assessed CCNDI mRNA half-lifein Rec-landGob. cell lines, which exclusively express short transcripts, and in the XG5 cell line, where both the 1.5-kb and the 4.5-kb transcripts are detected, using the transcriptional inhibitor actinomycin D. It is to be noted that the CCNDI 3' UTR is not rearranged in the XG5 cell line (datanot shown). In normal tissue, the estimated CCNDI mRNA half-life is 0.5 hours.'" From www.bloodjournal.org by guest on August 3, 2017. For personal use only. 3693 REARRANGEMENT OF CCND1 3' UNTRANSLATED REGION c B c 39 H c 39 H B 39 c 39 -23 - 9.4 W C 4 39 3440 - 23 - 9.4 e - 6.7 - 6.7 I. probe D - 9.4 probe E Fig 4. Southern blot analysis of CCNDI3' end rearrangement in case no. 39. DNA from case no. 39 and from normal peripheral blood leukocytes (C) was digested with the indicated enzyme (B, BamHI; H, Hindlll), and processed for Southern blotanalysis as described in the Methods.The filters were hybridized t o probe D andE. The scale is in kilobases. I n XGS. the half-lives of the I .S-and 4.5-kb transcripts are approxitnately 3 hours each: in Rcc-l and Gob.. the short transcripts appeared t o he even more stable. with a halflifegreater than S hours(Fig X). Indeed. n o comparison can he macle with normal B-lymphoid tissues.which do n o t express CCNIII. This showed that increase in mRNA stability could constitute one of the Inechanisms by which CCNIII is deregulated i n MCL and t( I Iql3)-associated leukemias. B c 4c 4 L S c 34 0 c 34 S - 0 -23 W U 4, -9.4 s C 40 C 40 -23 * - '. -94 .4.3 -4.3 probe C .. DISCUSSION The rcsults presented here constitute additional eletnents in favor of the role o f CCNIII i n lymphoid neoplasia antl provide so~neclues with regard to its activation mcchanisms. Cloning and sequence analysis o f CCNDI cDNAs in ;I case of R-CLL showed that the coding franw \vas retained and that the different s i x s of CCNIII mRNA resulted from I n this Ieuketnia. the different 3' untranslatedstructures. CCNIII gene was expressed primarily a s ;I I.5-kb transcript. but two aberrant transcripts o f 2. antl 3 kb were also detected. The absence o f ;I recognizable polyxlcnylation site and of ;I poly(A) tail in the sequence o f I 1 cDNA clones covering less than I .SO0 hp did not allow 11sto specify the nature of I t may correspond. ;IS i t has been theshortesttranscript. previously rcpor~cd.'~.'''t o ;I truncntcd form of the normal 4.5-kh mRNA through the use of different polyaclcn!.latiol~ sign;ds. Sequence analysisshowed that the 3-kb aberrant transcript results from the juxtaposition of a repetitive sequence of the MER1 I family to the CCNDI coding scqucncc. This might first represent a n example of insertional mutagenesis lending to gcncderegulation. ;IS it has been - 23 L z.3 z Fig 6. MTC rearrangement in MCL or leukemias with a break in the 3' end of CCNDI.DNA extracted from the pathological samples and from normal peripheral blood leukocytes (C) was digested with BarnHl endonuclease and processed for Southern blot analysis as described in theMethods. The filter was hybridized to a MTC probe. The scale is in kilobases. -2 probe B probe B Fig 5. Southern blot analysis of rearrangement of the CCNDl 3' end in MCL and tlllql3)-associated leukemias. DNA, extracted from the pathological samples and from normal peripheral blood leukocytes (C), was digested with the indicated enzymes (S,Sad; B, BamHI) and processed for Southern blotanalysis as described in the Methods. The filters were hybridizedt o probes C and B. The scale is in kilobases. - 35 kDa Fig 7. Western blot analysis of the CCNDI protein in MCL and t(llql3)-associated leukemias. Proteins were extracted from pathological samples, normal tonsil IT), reactive lymph node (L), Ramos cell line (R), and normal human fibroblasts (F). lmmunodetectionof the CCNDl protein was performedas described in the Methods. From www.bloodjournal.org by guest on August 3, 2017. For personal use only. RIMOKH ET AL 3694 0 1 2 3 4 5 Hours -3m ccND1: G "2kb -15kb MYC: G CCND1: R MYC: R ~ 1 5 k b 0" -4.5 kb ccND1: x -3kb MYC X Fig 8. Study of CCNDl mRNAstabilii in t(llql3l-bearing cell line: Rec. (RI, Gob. (G), and XG5 (X) cell lines.Total RNA was extracted at the indicated times after addition of actinomycin D and processed for Northern blot analysis as described in the Methods. The filters were sequentially hybridized to a CCNDl probe (probe A) and to a MYC exon 3 probe. The size of CCNDl transcripts is in kilobases. described whenproviral or human repetitive sequence are integrated in the vicinity of certain genes3' However, we have to consider the possibility that this aberrant transcript is the consequence of the t(l I ; 19)(q13;q13), which characterized this leukemia, and that the MER1 I sequence originates from the chromosome 19. The continuation of this workledustoshowthatrearrangement of the 3' end of CCNDl was not a rare event in MCL and in t( 1 Iql3)-bearing leukemias. Using a set of probes specific tothe different parts of CCNDI. such rearrangements were observed in DNA from four of the 40 patients tested. Consistent with previously published works,'5.'" Southern blot and sequence analyses indicated that, as a result of these rearrangements, the greatest part of the CCNDl 3' UTR and. in particular. the AU-rich region containing the mRNA-destabilizing signals AUUUA. is eliminated. It is thus possible that the CCNDI 3' UTR corresponds to a new breakpoint cluster (mTC3). The findingthatthe half-life of CCNDI mRNA is increased in three cell lines carrying a t( I Iq13) translocation stresses the importance of posttranscriptional derangement in activation of CCNDI.The fact that in the XGS cell line the half-lives of the 4.5-kb and 1 .S-kb CCNDI mRNAs are identical allows us to rule out the hypothesis that the AUrichregion contained in the 3' UTR.anddeleted in some tumors, is responsible for the greater stability of the truncated messages. However. it must be pointed out that the half-life of the CCNDl short mRNA is significantly longer in tumors with 3' UTR rearrangement and. thus. that these rearrangements may alter KNA stability. There exist other situations where certain genes implicated in the control of cell proliferation are deregulated throughstabilization of their transcripts. So. the inappropriate expression ofthe M Y C proto-oncogene in Burkitt's lymphomacanresultfrom an increase of the half-life of its transcript.""' However. in this case, the sequences implicated in the control of the mRNA stability are not only localized in the 3' UTR.but also in the S' region encompassing thefirstexonof MYC. It has also been reported that the mRNA stability of different cytokines and cyclins G , is increased in some hematopoietic and solid tumors."'.2J without gross genetic abnormalities ofthe transcripts. From these data. it appears that the AU-rich sequences present in the 3' UTR of certain genes are not always implicated in mRNA stability andthat other mechanisms should exist. Their nature remains to be determined. Comparison of the sequences of therearranged 3' UTRwith those of the short 1 .S-kb transcripts observed in most of the MCLs and of the normal 4.5-kb mRNA should provide data information withregardtotheregionsputativelyinvolved in mRNA stability. AUUUAmotifs in the 3' UTRof RNAscan also help to modulate the efficient translation of these transcripts. In particular. cytokine-derived UA-rich sequence canbe responsible for a translational blockade in ~itro.~'.~' Consistent with this hypothesis is the finding thatin two cases of tumors (cases no. 39 and 40) with deletion of the CCNDI mRNA AU-rich regions, the CCNDI protein is expressed at a high level. Finally, the fact that in most of the MCL andt( 1 lq13)associated leukemias analyzed. CCNDI isprimarily expressed as a I .5-kb transcript".'" prompts us to pinpoint the importance of the translational control of CCNDl expression in the pathogenesis of these tumors. It remained to be determined why a short transcript is also produced in some tumors without detectable rearrangement of the CCNDI 3' UTR. All cases presented here demonstrated rearrangement of both the MTC region and theCCNDI 3' UTR. This observation indicates that, in some cases. activation of CCNDI may be the consequence of two independent genetic events. namely, the juxtaposition of the CCNDI promoting region to IgH enhancer in the t( I 1 ; 14) translocation and the loss of 3' end regulatory sequences. Nucleotide changes leading to amino acid substitutions in From www.bloodjournal.org by guest on August 3, 2017. For personal use only. REARRANGEMENT OF CCNDl 3‘ UNTRANSLATEDREGION the sequence of the CCNDl protein have been described in one cell line.” The present report and the finding that in two primary human tumors the CCNDl coding sequence is normalz1suggest that amino acid changes are not required for CCNDl to develop oncogenic properties in primary human tumors. The results of this study also provide the first evidence that deregulation of CCNDI in MCLs and t(llql3)-associated leukemias leads to the accumulation of abnormally high levels of a normal 35-kD CCNDl protein, which wedid not find expressed in normal lymphoid tissues and in three follicular lymphomas (Fig 7). The normal size of the CCNDI protein in two cases of tumor (cases no. 39 and 40) with a break in the 3’ UTR confirms that the rearrangement did not alter the CCNDl coding frame. ACKNOWLEDGMENT We gratefully acknowledge Jean-Marie Blanchard and Jacques Samarut for helpful discussion, Peter Griggs for reading the manuscript, and B6nidicte Santaluccia for excellent technical assistance. REFERENCES I . Solomon E, Borrow J, Goddard AD: Chromosome aberrations and cancer. Science 254:1153, 1991 2. 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For personal use only. 1994 83: 3689-3696 Rearrangement of CCND1 (BCL1/PRAD1) 3' untranslated region in mantle- cell lymphomas and t(11q13)-associated leukemias R Rimokh, F Berger, C Bastard, B Klein, M French, E Archimbaud, JP Rouault, B Santa Lucia, L Duret and M Vuillaume Updated information and services can be found at: http://www.bloodjournal.org/content/83/12/3689.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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