Expression of ~ 1 3 ~ ~ ' ~ ' Is Restricted To Mature T
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Expression of ~ 1 3 ~ ~ ' ~ ' Is Restricted To Mature T
From www.bloodjournal.org by guest on January 21, 2015. For personal use only. Expression of ~ 1 3 ~ Is ~ 'Restricted ~ ' To Mature T - c e l l Proliferations With t(X; 14) Translocations By Ali Madani, Valerie Choukroun, Jean Soulier, Valere Cacheux, Jean-FranFois Claisse, FranGoise Valensi, Sylvie Daliphard, Bruno Cazin, Vincent Levy, Veronique Leblond, Marie-Therese Daniel, FranGois Sigaux, and Marc-Henri Stern T-cell prolymphocytic leukemia (T-PLL), a rare form of mature T-cell leukemias, and ataxia telangiectasia clonal proliferation, a related condition occurring in patients suffering from ataxia telangiectasia, have been associated t o translocations involving the 14q32.1 or Xq28 regions, where are located the TCLl and MTCP7 putative oncogenes, respectively. The M E P 7 gene is involved in the t(X;14)(q28;qll) translocation associated with these T-cell proliferations. Alternative splicing generates type A and B transcripts that potentially encode two entirely distinct proteins: type A transcripts code for a small mitochondrial protein, ~ 8 ~ ~ and type B transcripts, containing an additional open reading frame, may code for a 107 amino-acid protein, ~ 1 3 ~ ' " . The recently cloned TCLl gene, also involved in translocations and inversions associated with T-cell proliferations, codes for a 14-kD protein that displays significant homology with ~ 1 3 ~ We ~ " have . generated rabbit antisera against this putative ~ 1 3 ~ protein ' ~ ' and screened for expression of p13Mrcp' normal lymphoid tissues and 33 cases of immature and mature lymphoid T-cell proliferations, using a sensitive Western blot assay. We also investigated the MTCPl locus configuration by Southern blot analysis. The ~ 1 3 ~ ' ' protein was detected in the three T-cell proliferations with MTCP7 rearrangements because of t(X; 14) translocations, but neither in normal resting and activated lymphocytes nor "in the . other T-cell leukemias. Our data support the hypothesis that p13Mrcp' and ~ 1 4 " ~form ' a new protein family that plays a key role in the pathogenesis of T-PLL and related conditions. 0 7996 by The American Society of Hematology. T from hemophilic patients with Xq28 deletions including the MTCPI -CELL PROLYMPHOCYTIC leukemia (T-PLL) is a gene." Peripheral blood lymphocytes (PBL) were obtained from rare form of mature T-cell proliferations that has been normal individuals. T and B lymphocytes were separated from a associated with translocations involving the 14q32.1 renormal spleen by 2-aminoethylisothiuronium bromide-treated sheep gion'.* and to a lesser extent the Xq28 A preinvared blood cells rosetting. Leukemic samples were obtained from sive stage of T-PLL, named Ataxia Telangiectasia Clonal our cryopreserved leukemic cell collection, except for some T-PLL Proliferation (ATCP), has been described in the cancer-prone samples which were obtained from different French hematologic genetic disease ataxia telangiecta~ia.~-'' The presence of centers. The cytological diagnosis was reviewed by a member of identical translocations in ATCPs and in T-PLLs supports the French-American-British (FAB) Committee (M-TD). Short-term the view that these translocations play a role in the early cell culture from patient Dol blood sample was also analyzed. A stages of the disease. On this basis, the MTCPl gene was proportion of 230% of clonal cells in the Dol cell culture was estimated by Southern blot technique (data not shown). identified by its involvement in the t(X; 14)(q28;qll) transProduction of antisera againstpl.fMTCP'.The 321 nt open readl ~ c a t i o n . ' ~So . ' ~far, the five t(X; 14) breakpoints cloned ocing frame of the type B MTCPl transcript (orfl07) was amplified curred 5' to or within the MTCPl The MTCPl by the polymerase chain reaction (PCR) using the following primers: gene encodes for several mRNAs generated by alternative 5'-CCGGATCCAAAGGAGCATATCTTCG-3' and 5'-CGGGGAsplicing. The 1.0- to 1.2-kb type A transcripts code for an TCCATGGCAGGAGAGGAT-3'. The PCR product was cloned into unusually small protein of 8 kD ( ~ 8 ~ ~ ' " that ) ' ~ is located the BamHI site of the pGEX2T vector leading to an in frame fusion in the mitochondria.16 Type A transcript^'^-'^ and p8MTCP'15 with the GST gene (pGEX2T-orfl07). The sequence of the construct are highly expressed in T-cell proliferations with t(X; 14). However, we have described another splicing type of MTCPl transcripts (type BI), composed of four additional From the Laboratoire d'Hdmatologie Moldculaire, Hbpital Saint exons, that contains an additional 321 nt open reading frame Louis, Paris; Unite' de Cytogindtique, Hbpital Robert Debrd, Paris: potentially coding for an entirely different protein of 13 Laboratoire d'Himatologie, Amiens; Service d 'Hdmatologie BiolokD (p13MTcp').13 Using Northern blot analysis, this class of gique, Hbpital Necker, Paris: Laboratoire Central d'Hdmatologie, Reims; Service des Maladies du Sang, Lille; Service d'Hdmatologie, mRNAs was found at minute levels in tissues analyzed so H&el-Dieu, Paris: and the Dkpartement d'Hdmatologie, Hbpital far.13 Pitid-Salpe^tridre,Paris, France. The recent cloning of TCLI, the candidate 14q32.1 oncoSubmitted July 12, 1995: accepted October 2, 1995. gene, showed at the protein level an unquestionable homolSupported by l'INSERM, la Fondation contre la Leucdmie, le ogy with p13MTcp'.17.'s Both TCLI and MTCPI gene products Comiti de Paris de la Ligue Nationale Contre le Cancer, la Fondado not share homology with other known sequences and thus tion pour la Recherche Midicale, and la Direction de la Recherche constitute a new gene family. Rearrangements in the vicinity et des Etudes Doctorales. of these genes observed in T-PLLs and related T-cell prolifAddress reprint requests to Marc-Henri Stem, MD, PhD Laboraerations strongly advocate for their roles in the pathogenesis toire d'Hdmatologie Moldculaire, Centre Hayem, Hbpital Saint of T-PLL. Louis, 75475 Paris Cedex IO, France. This homology between ~ 1 4 ~ and ' ~ ' the putative ~ 1 3 ~ ~ " ' The publication costs of this article were defrayed in part by page protein led us to analyze ~ 1 3 ~ ~ ' expression '' in T-PLLs. charge payment. This article must therefore be hereby marked MATERIALS AND METHODS Cells. Cell lines used: cos-7 is a monkey SV40 transformed kidney cell line; RH and 90-109 are lymphoblastoid cell lines derived Blood, Vol 87, No 5 (March 1). 1996: pp 1923-1927 "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact. 0 1996 by The American Society of Hematology. OOO6-497ID6/8705-0047$3.00/0 1923 From www.bloodjournal.org by guest on January 21, 2015. For personal use only. MADANI ET AL 1924 was verified, and Escherichia coli JM109 cells containing the recombinant plasmid were grown in Superbroth medium.'" After a 4-hour induction by IPTG (0.1 mmolk), the GST-p13"rrP' fusion protein was extracted and purified by Glutathione-agarose chromatography as described." New Zealand white female rabbits were immunized with the purified GST-pl 3Mrrp' emulsified with Freund's adjuvant. After three boosts, the antiserum was used without further purification. Obtention of the anti-p8Mrc"' antiserum was previously reported. '' Western blotting. Cell proteins were extracted with the Triple Detergent Lysis Buffer,"' quantified using the BCA kit (Pierce, Rockford, IL), size fractionated on 10% Tris-tricine sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE)," and electrotransferred on nitrocellulose. The membrane was blocked overnight in 10% nonfat dry milk in PBST (phosphate-buffered saline; Tween 20, 0.1%). The antiserum was diluted 1:1,000 in blocking buffer and applied to the membrane. After I hour of incubation, the membrane was washed with PBST and incubated with goat-antirabbit IgGperoxidase conjugate (Boehringer Mannheim, France) for I hour. After three washes in PBST, the membrane was overlayed with the chemiluminescent substrate solution and developed according to the manufacturer's instructions (ECL. Amersham, Arlington Heights, IL). Transient transfections. The orfl07 sequence was cloned in the BamHl site of the eukaryote expression vector pSGS (Stratagene, La Jolla, CA). The recombinant plasmid was transfected into the cos-7 cell line by the calcium-phosphate method.'" Cells were trypsinized after 36 hours and analyzed by Westem blotting after protein extraction. DNA ana/ysi.s. High molecular weight DNA was extracted from leukemic samples, digested by BamHl and Snc I restriction enzymes and the resulting fragments were separated according to size by electrophoresis through a 0.8% agarose gel. After alkaline transfer on N'Hybond membranes (Amersham). samples were hybridized to "P radiolabeled probes." Probes used are schematized see Fig 2A; probes 2.SRB and I.OXH were used on T-PLL DNAs allowing an analysis of approximately SO kb 5' to and within the MTCP l gene: prohe 4.5RB was also used to analyze patient Lec. cDNA cloning and sequencing. A cDNA library from the murine T-cell lymphoma cell line EL4 (Stratagene) was screened with a human MTCP I cDNA probe. Positive clones were in vivo excised according to the protocols of the manufacturer and sequenced by the dideoxynucleotide method (Pharmacia, Uppsala, Sweden). RESULTS Expression analysis of the MTCP 1 prodircts. Considering that the homology between TCLl and MTCPI was restricted to the pl 3MT"" product, we analyzed the expression of p13'7cp' in T-PLLs. Rabbit antibodies against p13MTC"were generated and a sensitive Western blot assay was designed. The specificity of the antibody was controlled in transiently transfected Cos7 cells in which a strong 13-kD signal was detected. corresponding to the predicted molecular weight of the product of B1 transcripts. No 13-kD signal was detected in 90-109 and RH cell lines derived from hemophilic patients with a Xq28 deletion including the MTCPl gene" (Fig I). We then analyzed normal and leukemic T-cell samples. Normal PBLs were found negative for p13M"'P' expression (Fig 1 ) as were phytohemagglutinin (PHA)-activated lymphocytes and normal B lymphocytes (data not shown). A series of acute T-cell lymphoblastic leukemias (9 cases), HTLV-I positive acute T-cell leukemidymphomas (4 cases), Sezary syndromes (4 cases) and large granular lymphocyte proliferations (4 cases) was also found negative for p13MTcp'expression (data not shown). Two previously described T-cell proliferations with known t(X; 14) translocations (Dol and Civ)'j and a series of 10 T-PLLs obtained from different French hematologic centers were then analyzed. Diagnosis was confirmed by reviewing blood or BM smears using FAB criteriaz3 and medical records. A weak but unquestionable signal of 13 kD was found in Civ and Dol, the two patients with a known t(X; 14). Among the 10 other T-PLLs analyzed, only one patient, Lec, expressed the p13Mr'p' protein (Fig I). The expression of the product of type A transcripts of the MTCPl gene, ~ 8 ~ " 'was , also studied in these samples. Contrary to what was observed for ~ 1 3 ~ ' "expression, ' ~8~'"' waq expressed in all patients and in normal PBLs. As previously repOrted,ls higher expression of ~ 8 ~ ' was " found in the proliferations of Dol and Civ with known t(X;14)s. The p8"" protein was also expressed at high levels in patient Lec and, to a lesser extent, in patient Lep (Fig 1). 2 n -18.4 -18.4 -143 -14.3 p*MIcp'_, Fig 1. Expressionof pl3" and ~ 8 in~T-PLLs. " Detergent lysates (30 pg/lane except for Cos-pl3: 3.5 pgllane) were subjected to Tricine SDS-PAGE using a 10% gel. Ponceau S staining was used to verify equal loading and protein integrity. lmmunoblot analyses were performed using an anti-p13MTcp'antiserum ltop panel) and an anti-p8MTcp'antiserum (bottom panel). Samples analyzed are: negative control cell lines with MTCPl deletions IRH and 90-109); transfected COS-7 cells expressing p13MrCP'(Cos p13); normal PBL; an ATCP (Dol), T-PLLs (coded by 8 signals ~ are ~ indicated. " three letters). Molecular weights are indicated in kilodaltons IkD). The p13MrCP'and ~ From www.bloodjournal.org by guest on January 21, 2015. For personal use only. 1925 ~ 1 3 ~ "IN~T-CELL ' PROLIFERATION R A B S R R - B I 1.OXH 14 \ I VI1 II \ Ih b Lec. kb - cation event in patient Lec occurring between the two fragments used as probes. A karyotype obtained from a frozen sample of Lec's leukemic cells confirmed this hypothesis by showing the presence of a t(X: 14). Comparisori of hitrnnrr arid mirrine MTCP 1 arid hirrnan TCL I protein .seyiterices. A murine T-cell lymphoma cell line cDNA library was hybridized to a human MTCPI cDNA probe and one cross-hybridizing type B murine cDNA clone was selected and sequenced. Comparison of murine and human sequences of the open reading frame coding for p13"""" showed a very high level of conservation at the nucleotidic level (92%- of identity, data not shown) and at the deduced amino-acid level (95% of identity) (Fig 3). Comparison of p l 3"'"'c"' sequences with the ~14""' sequence showed a much lower degree of homology (p13'''cp' I' ~14""'. 39% of identity) (Fig 3). S R 4.5Rll GL kb 16, BRRBR - - 2.5RR Lec. XE - 14- 12 GL , DISCUSSION Probe : 1.OXH Based on the results of our Western blot assay, the analysis of the p13""'"' protein in normal and malignant T cells showed a restricted expression in T-cell proliferations with t(X: 14). The p13wrc"' protein was not expressed in resting or activated normal lymphocytes and in other leukemic samples. This protein is homologous to the TCLI gene product. P14rc'.' , which is expressed in T-cell proliferations with I4q32. I trans location^.'^^'^^^' The specific expression of p13""'"' in proliferations with t(X: 14) and its homology with ~14""' also involved in T-PLLs or related T-cell proliferations strongly suggest a role of p13MTc'" in leukemogenesis. The 1 3MTu//p14r<'/./protein family shares no homology with other oncoproteins. and biochemical and cellular analyses in transfected cells and in transgenic animals will be necessary to determine in which oncogenic mechanism they are involved. However, because p 14"'"' expression is restricted in the T-cell lineage to immature thymocytes and activated peripheral lymphocytes, we can hypothesize that the I 3M?"/'/lp 14""' overexpression may interfere with Tcell activation and/or differentiation. It can also be drawn from studies in ataxia telangiectasia patients that TCLI and MTCPl gene rearrangements occur at an early stage of leukemogenesis and are not per se sufficient for the development of malignancy,'."." hypothesis conforted by the demonstration in preleukemic T-cell proliferations of the overexpression of p l 3"""" (this report) or p14"".'4 Chromosomal translocations involving the Xq28 region have been so far rarely reported in malignancy.'5 Taking advantage of our initial characterization of a t(X;14)I3 in 4.5RB BamHI Fig 2. Southern blot analysis of the MTCPl locus. (A) Schematic representation of the MTCPT locus. (HIindicate the MTCPl exons. Restriction enzymes: R, €COR/;B, BamHI; S, Sac I; X, Xho I; H, Hindlll (only relevant Xho I and Hindlll sites are indicated). Fragments used as probes are indicated. (B) Southern blot analysis of patient Lec. Probes and restriction digests are indicated. Molecular weights are indicated in kilobases (kb). Dashes indicate the germline fragments, arrows the rearranged fragments. Southern blot nnalvsis qf the MTCP I locirs. The MTCPl gene was studied by standard Southern blot techniques using two probes and two restriction digests, allowing the analysis of approximately 50 kb 5' to and within MTCPI. T-PLL DNAs were found in germline configuration for the MTCPl locus with the exception of the previously reported Dol and Civ rearrangements and of the Lec rearrangement. A germline 14-kb fragment and an abnormal 16-kb fragment were observed using the I.OXH probe in patient Lec's BamHI digested DNA (Fig 2B). The same blot was then probed using the 4.5RB probe, located 3' to MTCPI exon 1 and hybridizing to the same germline 14 kb BarnHl fragment (see Fig 2A for schematic representation of the MTCPl locus and localization of the probes used). An abnormal 12 kb fragment was found in patient Lec's BarnHI digested DNA. The Southern blot data were thus compatible with a translo- Fig 3. Sequence comparison of human and murine p13M'CP' and human ~ 1 4 " ~ ' Sequence . alignment between human p13''CP' and human ~ 1 4 " ~ is' shown according to Fu et al." Vertical bars indicate identical amino acids. The murine p13M'CP' aminoacid sequence was deduced from the murine type B cDNA sequence (not shown) reported to GenBank under the accession number M U U32332. MAREDVGAPPDELWVBQEGDEYQRTWVAV-VEEETSF-LKARVQQVWPLGD 53 II IIIIIIIIIIIIIIII IIIIIIIIIIII IIIIIII I IIIII IIIIII MAGEDVGAPPDELWVEQEGIYDEYQRTWVAV-VEEETSF-LRARVCQIQWLGD I l l IIII I I I ~ I I II I II 53 EUp14rct' MAECPTLGEAVTDEPDRLWAWEKWYLDEKQEAWLPLTIEIKDRLQLRV'LLRREDVVL~60 Mup13"ncP1 A T K P S E L L T S Q L P L M W Q L Y P ~ R Y M D ~ S ~ W Q I Q B E L ~ ~ Q E ~ P D 107 D .- Eu013-~ EuplQrL1 I III IIIIIIIIIII IIII I IIIIIIII III IIIII IIIIIIII II I IIIII A A R P S E L L T S O L P L M W O L Y P E E R Y M D ~ S ~ W Q I Q E E L ~ ~ O E ~ L L P D D 107 I 1-11 IlilII II I I-- I 11- II IIIII 114 PMTPTQIGPSLLPIMWQLYPDGRYSSDSSFWP.LWEIKIDGVEDMLLELLPDD From www.bloodjournal.org by guest on January 21, 2015. For personal use only. 1926 MADANI ET AL the ataxia telangiectasia clonal T-cell proliferation ‘‘Dol’ ’ selected for its chromosomal aberration,‘ we have screened for MTCPI rearrangement a series of 11 T-PLLs collected without knowledge of the cytogenetic data. This approach allowed us to detect two additional t(X; 14) in the T-PLLs “Civ”” and “Lec” (this report). Considering the way we have recruited T-PLLs in our series, a bias in selecting proliferations with t(X; 14) is unlikely. The rarity of reported t(X; 14) cases in the literature’.’ probably reflects the rarity of the T-cell prolymphocytic type of leukemia. The t(X; 14) translocation, occurring in a significant proportion of T-PLLs and related proliferations, should be considered as a marker of these diseases. Initial hypotheses on the role of MTCPI in leukemogenesis were misled by the fact, so far unprecedented for a mammalian gene, that this gene codes for two differents proteins. Northern blot analyses from us and others’?-’‘ previously detected only significant levels of type A MTCPl transcripts in proliferations with t(X; 14). The MTCPI product coded by these transcripts, the mitochondrial protein p8MTCP’, was found overexpressed in these proliferations. Is The possible relevance in oncogenesis of p13”’C“’ was thus initially underestimated because type B 1 transcripts have been undetected in proliferations with t(X; 14) by Northem blot assay using total RNAs, but nevertheless were coding for low but detectable levels of p13MTCp’. An alternative splicing mechanism contributes to generate the different coding capab es of MTCPl transcripts.I3 The invertly related levels of p13M7CP’and p8M7r“’ proteins in our small series of proliferations with t(X; 14) are compatible with a splicing regulation but could also be related to the localization of the different Xq28 breakpoints. The mechanism by which a high expression of p8M‘Cp’ exists in some T-PLLs without t(X; 14), especially in the T-PLL “Lep,” when compared to normal lymphocytes (Fig 1) has also to be clarified. Data on transcriptional and posttranscriptional regulations of MTCPl are clearly needed to answer these questions. Because the mitochondrial protein p8M““ is overexpressed in T-cell proliferations with t(X; 14) and because its biologic function is still unknown, we cannot rule out its participation in oncogenesis. However, the specific expression of p13M‘cp’ in proliferations with t(X; 14) and the homology of this protein with ~ 1 4 “ ~represent ’ strong evidences for a key role of the p13MTCp‘/p14‘CL’protein family in the pathogenesis of T-PLL and related diseases. Moreover, the biologic importance of p13MTCP’ is reflected by its high degree of conservation in mammals. ACKNOWLEDGMENT We thank H. de ThC and J. HBbert for comments on the manuscript, L. Grollet for oligonucleotide synthesis, A. Metzenberg and D.P. Lillicrap for providing the RH and 90-109 cell lines, B. Boursin for photography, and S. Labaume for help in cellular techniques. We also thank G . Flandrin and H. Dombret for their help and support. REFERENCES 1. 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For personal use only. 1996 87: 1923-1927 Expression of p13MTCP1 is restricted to mature T-cell proliferations with t(X;14) translocations A Madani, V Choukroun, J Soulier, V Cacheux, JF Claisse, F Valensi, S Daliphard, B Cazin, V Levy, V Leblond, MT Daniel, F Sigaux and MH Stern Updated information and services can be found at: http://www.bloodjournal.org/content/87/5/1923.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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