The Class II Tumor-Suppressor Gene RARRES3 Is Expressed in B Cell
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Leukemia (2001) 15, 1521–1526 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu The class II tumor-suppressor gene RARRES3 is expressed in B cell lymphocytic leukemias and down-regulated with disease progression B Casanova1, MT de la Fuente1, M Garcia-Gila1, L Sanz1, A Silva1, JA Garcia-Marco2 and A Garcia-Pardo1 1Departamento de Inmunologı´a, Centro de Investigaciones Biolo´gicas, CSIC, Madrid; and 2Servicio de Hematologı´a, Hospital Universitario Clı´nica Puerta de Hierro, Madrid, Spain The molecular pathogenesis of B cell chronic lymphocytic leu- treatment and poor prognosis9,15 and alterations of the retinoic kemia (B-CLL), the most common form of leukemia, remains acid receptor alpha (RARa) gene at chromosome 17 (6% of unknown. We have used the mRNA differential display tech- 16,17 nique to analyze genes that may be involved in the cases). development/progression of B-CLL. We have identified the To gain some insight on the involvement of these gene alter- tumor suppressor retinoic acid receptor responder 3 ations in the development and progression of B-CLL, we have (RARRES3) as a B-CLL-related gene. RARRES3 maps to chro- used the mRNA differential display technique (DD)18 to ana- mosome band 11q23, a region frequently deleted in lymphopro- lyze gene expression differences in B-CLL and normal B cells. liferative disorders. To assess the potential involvement of Among the differences detected we have identified the reti- RARRES3 in leukemogenesis, we examined 24 cases of B-CLL, 10 of acute lymphocytic leukemia (ALL) and five related cell noic acid receptor responder 3 gene (RARRES3), a recently lines by RT-PCR and sequence analyses. We report a corre- described growth regulatory gene which functions as a class lation between RARRES3 down-regulation and B-CLL pro- II tumor suppressor and mediates some of the growth sup- gression. We also found decreased RARRES3 gene levels in pressive effects of retinoids.19 We have analyzed the ALL cases and in the five cell lines studied. We did not find expression and sequence of this gene in 24 cases of B-CLL mutations in any of the leukemia samples assayed, including and have found a clear correlation between down-regulation those with 11q23 deletion. These results indicate that RARRES3 may play a role in B-CLL progression. Leukemia of RARRES3 expression and B-CLL progression. Furthermore, (2001) 15, 1521–1526. we have extended these analyses to 10 cases of acute lym- Keywords: RARRES3; B-chronic lymphocytic leukemia; mRNA phoblastic leukemia (ALL), another lymphoproliferative dis- differential display; tumor suppressor gene; lymphoproliferative order, and to five Epstein–Barr virus (EBV)-transformed cell disorders lines established from normal and malignant B lymphocytes. Introduction Materials and methods B cell chronic lymphocytic leukemia (B-CLL) is the most com- Patients, cell purification and cell lines mon form of leukemia accounting for 0.8% of all cancers and nearly 30% of adult leukemia.1,2 However, the molecular Peripheral blood samples were obtained with informed con- pathogenesis of B-CLL remains largely unknown and no spe- sent from24 B-CLL and 10 B-cell ALL patients, diagnosed cific gene has been shown to play a major role in this dis- according to established clinical and laboratory criteria (Table ease.1–4 B-CLL progression is usually associated with clonal 1). The B-CLL diagnosis was staged according to Binet and karyotypic alterations described in up to 79% of B-CLL Rai’s classifications.20,21 CD5+ malignant B-lymphocytes were patients.5,6 The most frequent abnormalities are deletions in purified fromthe peripheral blood of B-CLL samplesas pre- chromosome bands 13q12–q14 (30–80% of cases), followed viously described.22 B-lymphocytes from healthy donors were by trisomy 12 (15–35%) and deletions at 11q (20%).5,7–10 11q purified frombuffy coat cells obtained fromthe Centro de deletions correlate with extensive nodal involvement, younger Transfusiones de la Comunidad de Madrid (Madrid, Spain) by age incidence and more aggressive disease progression.5,7,11 Ficoll–Hypaque centrifugation followed by incubation with Some of the reported genetic aberrations in B-CLL affect inter- anti-CD19-coated Dynabeads (Dynal, Oslo, Norway) accord- esting genes, such as LEU2B (located at 13q14.3) which is ing to the manufacturer’s instructions. Purified B cells were frequently deleted in B-CLL,12 although its involvement in the Ͼ95% CD19+ and Ͼ98% viable as determined by flow cyto- disease has not been yet elucidated. Likewise, ATM (ataxia metry and trypan blue dye exclusion, respectively. Although telangiectasia mutated) and RDX (radixin), two potential normal B lymphocytes are not phenotypically identical to B- tumor-suppressor genes, are located at 11q22.3–q23.1, CLL since most of them do not express CD5+ or CD23, they another frequently deleted region. Poor outcome in B-CLL has are closely related cells and generally accepted as control for been associated with absence of ATM protein or loss of the B-CLL cells. ATM gene,11,13 suggesting that mutations of ATM at the germ- The EHEB cell line, established froma patient with B-CLL line could be a risk for development of B-CLL.14 Other fre- by in vitro transformation with EBV, was obtained from the quent abnormalities in B-CLL are deletions of 6q21–q23;9 German Collection of Microorganisms and Cell Cultures deletions/mutations of the P53 tumor suppressor gene at (Braunschweig, Germany). The HUT112, GER112, CO31 and 17p13.1 (16% of cases) which associate with resistance to CO43 cell lines, established from normal B-lymphocytes by in vitro transformation with EBV, were obtained from Dr S Rodrı´guez de Co´rdoba (Centro de Investigaciones Biolo´gicas, Madrid, Spain) and Dr R de Pablo (Hospital Universitario Correspondence: A Garcia-Pardo, Centro de Investigaciones Bio- lo´gicas, CSIC, Vela´zquez 144, 28006 Madrid, Spain; Fax: 34 91 Clı´nica Puerta de Hierro, Madrid, Spain). JM and CEM cells 562 7518 (T cell leukemia) were obtained from Dr C Bernabeu (Centro Received 8 March 2001; accepted 13 June 2001 de Investigaciones Biolo´gicas); RPMI 8866 (B lymphoblastic) RARRES3 expression in lymphoproliferative disorders B Casanova et al 1522 Table 1 Clinical characteristics of patients Patient Sex/Age Morphol Stagea Therapy Karyotype B-CLL patients 1 M/72 Typical C/III No Normal 2 M/NANANANANA 3 M/59 NA B No ND 4 F/71 Atypical C/IV FDR 46,XX, del(11)(q23)[5] 5 F/78 Typical A/I LK 46, XX, del(6)(q15)[10], 46,XX[10] 6 M/49 Atypical C/IV FDR 44–46, XY, −2, der(5)t(5;12) (q35;q12), der(12) t(12;?)(q12;?), del(14)(q12), i(17)(q10),der(18) t(18;21;?)(p11;q11;?), −21, +mar[cp35], 88–115 hiperploidy[10] 7 M/67 Typical C/IV LK 46,XY,del(11)(q23)[12], 46,XY[8] 8 M/77 Typical A/0 No 46,XY[20] 9 F/88 Typical A/1 No No metaphase 10 F/72 Typical A/0 No 46,XX, t(4;13)(q34;q13)[5], t(2;10) (p11;p15), del(11)(q21),− 13,+mar[5], 46,XX[10] 11 M/45 Typical A/0 No 47,XY,+12[13],46,XY,+12,−18[3],46,XY del(13)(q12-q14)[3] 12 F/68 Atypical B/II FDR 45,X,t(X;3)(p22;p23),−7,add(15)(p13),−17, der(19) t(7;19) (p11;p13) ins(19;1)(p13;q11;q44)[16] 13 M/69 Typical C/III FMC 46,XY[20] 14 M/70 Typical A/I HYDREA 46,XY[20] 15 M/67 Typical B/II No 46,XY[20] 16 M/74 NA A/I-II NA NA 17 M/NA NA A/I No NA 18 M/65 Atypical A/0 No 46,XY, del(6)(q16),add(7)(p22)[19], 46, XY, add(7)(p22)[6] 19 F/36 Atypical B/II FDR 46,XX[20] 20 F/73 Atypical A/0 No 46,XX, del(8)(p11)[15], 46,XX[5] 21 M/46 Typical C/IV CHOP 46,XY, del(11)(q13)[20] 22 M/73 Atypical A/0 No 46,XY, del(11)(q23)[16]; 46, XY[4] 23 M/44 Atypical C/IV ESHAP 46,XY, del(11)(q21)[10]; 46,XY, del(11)(q21), der(21)t(11;21) (q12q23;p11)[9]; 47,XY, der(1)del(1)(q10),+der(1)del(1)(p11), del(11) (q21),der(21)t(11;21)(q12q23;p11)[3]; 46,XY[2] 24 M/58 Typical B/II No 46,XY,t(7;11;?)(q36;q12q25;?),del(11)(q22)[15] ALL patients 1 M/16 46,XY[16], 46, XY, t(2;14)(p21;q22)[4] 2 M/20 47, XX, t(9;22)(q34;q11), +der(22) t(q;22)(q34;q11)[16], 46, XX, der(9) t(9;22)(q34;q11)[2], 46, XX, t(9;22)(q34;q11)[2] 3 M/66 35, XY, −3,−4,−5,−7,−9,−13,−14,−15,−16,−17,−19, del(2)(q11)[13], 46,XY, del(11)(q23)[3], 46,XY[4] 4 M/53 46, XY[16], 47, XY, add(3)(q28), +mar[4] 5 F/18 46, XX[20] 6 M/14 46, XY, i(9)(q10)[9] 7 M/12 ND 8 M/20 60, XY, add(1)(q44), +4, +5, +6, +del(6)(q16), +8, ?t(8;14)(q22;q32)?, +11, +12, +14, +15, +i(17)(q10), +18, +21, +2mer[20], 46, XY,[15] 9 M/26 47, XY, +4, t(11;12)(q22;p13)[22] 10 M/NA 49, XY, +X, +der(1) t(1;22)(p11;q11), +8, +8, +13, −21, −22[18] aAccording to Binet et al20 and Rai et al.21 NA, not available; ND, not determined; FDR, fludarabine; FMC, combination of fludarabine, mitoxantrone and chlorambucil; LK, leukeran; CHOP, combination of cyclophosphamide, adriamycin, vincristine and prednisone; ESHAP, combination of etoposide, cisplatin, ara-C and prednisone.