The class II tumor-suppressor gene RARRES3 is ...

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The class II tumor-suppressor gene RARRES3 is expressed in B cell lymphocytic leukemias and down-regulated with disease progression. B Casanova1, MT de ...
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 1 Departamento 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 leukemia (B-CLL), the most common form of leukemia, remains unknown. We have used the mRNA differential display technique to analyze genes that may be involved in the development/progression of B-CLL. We have identified the tumor suppressor retinoic acid receptor responder 3 (RARRES3) as a B-CLL-related gene. RARRES3 maps to chromosome band 11q23, a region frequently deleted in lymphoproliferative disorders. To assess the potential involvement of RARRES3 in leukemogenesis, we examined 24 cases of B-CLL, 10 of acute lymphocytic leukemia (ALL) and five related cell lines by RT-PCR and sequence analyses. We report a correlation between RARRES3 down-regulation and B-CLL progression. We also found decreased RARRES3 gene levels in ALL cases and in the five cell lines studied. We did not find mutations in any of the leukemia samples assayed, including those with 11q23 deletion. These results indicate that RARRES3 may play a role in B-CLL progression. Leukemia (2001) 15, 1521–1526. Keywords: RARRES3; B-chronic lymphocytic leukemia; mRNA differential display; tumor suppressor gene; lymphoproliferative disorders

treatment and poor prognosis9,15 and alterations of the retinoic acid receptor alpha (RARa) gene at chromosome 17 (6% of cases).16,17 To gain some insight on the involvement of these gene alterations in the development and progression of B-CLL, we have used the mRNA differential display technique (DD)18 to analyze gene expression differences in B-CLL and normal B cells. Among the differences detected we have identified the retinoic acid receptor responder 3 gene (RARRES3), a recently described growth regulatory gene which functions as a class II tumor suppressor and mediates some of the growth suppressive effects of retinoids.19 We have analyzed the expression and sequence of this gene in 24 cases of B-CLL and have found a clear correlation between down-regulation of RARRES3 expression and B-CLL progression. Furthermore, we have extended these analyses to 10 cases of acute lymphoblastic leukemia (ALL), another lymphoproliferative disorder, and to five Epstein–Barr virus (EBV)-transformed cell lines established from normal and malignant B lymphocytes.

Introduction

Materials and methods

B cell chronic lymphocytic leukemia (B-CLL) is the most common form of leukemia accounting for 0.8% of all cancers and nearly 30% of adult leukemia.1,2 However, the molecular pathogenesis of B-CLL remains largely unknown and no specific gene has been shown to play a major role in this disease.1–4 B-CLL progression is usually associated with clonal karyotypic alterations described in up to 79% of B-CLL patients.5,6 The most frequent abnormalities are deletions in chromosome bands 13q12–q14 (30–80% of cases), followed by trisomy 12 (15–35%) and deletions at 11q (20%).5,7–10 11q deletions correlate with extensive nodal involvement, younger age incidence and more aggressive disease progression.5,7,11 Some of the reported genetic aberrations in B-CLL affect interesting genes, such as LEU2B (located at 13q14.3) which is frequently deleted in B-CLL,12 although its involvement in the disease has not been yet elucidated. Likewise, ATM (ataxia telangiectasia mutated) and RDX (radixin), two potential tumor-suppressor genes, are located at 11q22.3–q23.1, another frequently deleted region. Poor outcome in B-CLL has been associated with absence of ATM protein or loss of the ATM gene,11,13 suggesting that mutations of ATM at the germline could be a risk for development of B-CLL.14 Other frequent abnormalities in B-CLL are deletions of 6q21–q23;9 deletions/mutations of the P53 tumor suppressor gene at 17p13.1 (16% of cases) which associate with resistance to

Patients, cell purification and cell lines

Correspondence: A Garcia-Pardo, Centro de Investigaciones Biolo´gicas, CSIC, Vela´zquez 144, 28006 Madrid, Spain; Fax: 34 91 562 7518 Received 8 March 2001; accepted 13 June 2001

Peripheral blood samples were obtained with informed consent from 24 B-CLL and 10 B-cell ALL patients, diagnosed according to established clinical and laboratory criteria (Table 1). The B-CLL diagnosis was staged according to Binet and Rai’s classifications.20,21 CD5+ malignant B-lymphocytes were purified from the peripheral blood of B-CLL samples as previously described.22 B-lymphocytes from healthy donors were purified from buffy coat cells obtained from the Centro de Transfusiones de la Comunidad de Madrid (Madrid, Spain) by Ficoll–Hypaque centrifugation followed by incubation with anti-CD19-coated Dynabeads (Dynal, Oslo, Norway) according to the manufacturer’s instructions. Purified B cells were ⬎95% CD19+ and ⬎98% viable as determined by flow cytometry and trypan blue dye exclusion, respectively. Although normal B lymphocytes are not phenotypically identical to BCLL since most of them do not express CD5+ or CD23, they are closely related cells and generally accepted as control for B-CLL cells. The EHEB cell line, established from a patient with B-CLL by in vitro transformation with EBV, was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The HUT112, GER112, CO31 and 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 Clı´nica Puerta de Hierro, Madrid, Spain). JM and CEM cells (T cell leukemia) were obtained from Dr C Bernabeu (Centro de Investigaciones Biolo´gicas); RPMI 8866 (B lymphoblastic)

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Table 1

Clinical characteristics of patients

Patient

Sex/Age

Morphol

Stagea

Therapy

B-CLL patients 1 M/72 2 M/NA 3 M/59 4 F/71 5 F/78 6 M/49

Typical NA NA Atypical Typical Atypical

C/III NA B C/IV A/I C/IV

No NA No FDR LK FDR

7 8 9 10

M/67 M/77 F/88 F/72

Typical Typical Typical Typical

C/IV A/0 A/1 A/0

LK No No No

11 12

M/45 F/68

Typical Atypical

A/0 B/II

No FDR

13 14 15 16 17 18 19 20 21 22 23

M/69 M/70 M/67 M/74 M/NA M/65 F/36 F/73 M/46 M/73 M/44

Typical Typical Typical NA NA Atypical Atypical Atypical Typical Atypical Atypical

C/III A/I B/II A/I-II A/I A/0 B/II A/0 C/IV A/0 C/IV

FMC HYDREA No NA No No FDR No CHOP No ESHAP

24

M/58

Typical

B/II

No

ALL patients 1 M/16 2 M/20 3

M/66

4 5 6 7 8

M/53 F/18 M/14 M/12 M/20

9 10

M/26 M/NA

Karyotype

Normal NA ND 46,XX, del(11)(q23)[5] 46, XX, del(6)(q15)[10], 46,XX[10] 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] 46,XY,del(11)(q23)[12], 46,XY[8] 46,XY[20] No metaphase 46,XX, t(4;13)(q34;q13)[5], t(2;10) (p11;p15), del(11)(q21),− 13,+mar[5], 46,XX[10] 47,XY,+12[13],46,XY,+12,−18[3],46,XY del(13)(q12-q14)[3] 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] 46,XY[20] 46,XY[20] 46,XY[20] NA NA 46,XY, del(6)(q16),add(7)(p22)[19], 46, XY, add(7)(p22)[6] 46,XX[20] 46,XX, del(8)(p11)[15], 46,XX[5] 46,XY, del(11)(q13)[20] 46,XY, del(11)(q23)[16]; 46, XY[4] 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] 46,XY,t(7;11;?)(q36;q12q25;?),del(11)(q22)[15] 46,XY[16], 46, XY, t(2;14)(p21;q22)[4] 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] 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] 46, XY[16], 47, XY, add(3)(q28), +mar[4] 46, XX[20] 46, XY, i(9)(q10)[9] ND 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] 47, XY, +4, t(11;12)(q22;p13)[22] 49, XY, +X, +der(1) t(1;22)(p11;q11), +8, +8, +13, −21, −22[18]

a

According 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.

and RPMI 8226 (myeloma) cells were obtained from Dr F Sa´nchez-Madrid (Hospital de la Princesa, Madrid, Spain).

Chromosome analysis Mononuclear cells from bone marrow and/or peripheral blood for CLL samples and from bone marrow for ALL samples were cultured at 37°C for 3–5 days in the presence of tetradecanoylphorbol-13-acetate (0.05 mg/ml) for CLL samples and 2–3 days without addition of mitogens for ALL samples. At this time standard cytogenetic preparations were made, Gbanded and karyotyped according to the International System for Cytogenetic Nomenclature.23 If available, 20 metaphases were evaluated for each case. Leukemia

mRNA differential display Total RNA was isolated with TRIzol reagent (GIBCO-BRL, Life Technologies, Grand Island, NY, USA) following the manufacturer’s protocol. DNA contamination was removed by treatment with DNAsel (Boehringer Mannheim, Indianapolis, IN, USA) according to the manufacturer’s instructions. mRNA differential display between B-lymphocytes from a normal subject (control cells) and from a B-CLL patient (patient 1 on Table 1) was performed in duplicate as described.18 In brief, total RNA was divided in aliquots of 0.3 ␮g and used to duplicate all subsequent reactions. First-strand cDNA was synthesized using 630 U/␮g RNA of Moloney murine leukemia virus reverse transcriptase (M-MLV) (Amersham Life Science, Cleveland, OH, USA) in three different reverse transcription (RT)

RARRES3 expression in lymphoproliferative disorders B Casanova et al

reactions primed by three downstream one-base anchored oligo-dT primers (oligo-dT11V). Their sequence were 5⬘AAGCT11V-3⬘ (where V can be A, C, or G). 2.0 ␮l of the RT reaction were amplified as described.18 1 ␮Ci of [␣-33P]dATP (2000 Ci/mmol; NEN Life Science Products Inc, Boston, MA, USA) was used per reaction tube. We employed the three oligo-dT11V primers described before in combinations with five upstream arbitrary primers (13 bases long). The arbitrary primers were: AP1 (5⬘-AAGCTTGATTGCC-3⬘), AP2 (5⬘AAGCTTGACTGT-3⬘), AP3 (5⬘-AAGCTTTGGTCAG-3⬘), AP14 (5⬘-AAGCTTCAGCGAA-3⬘) and AP45 (5⬘-AAGCTTACTC CAC-3⬘). The duplicate PCR products were separated on sequencing gels under denaturing conditions. Dried gels were exposed to Kodak X-OMAT AR film (Eastman-Kodak, Rochester, NY, USA) for 24–48 h. Bands that were unique to the control or B-CLL cells and were present in both duplicate reactions were isolated and re-amplified as described.18 Bands successfully amplified were ligated in pGEM-T Easy Vector (Promega Corporation, Madison, WI, USA) and cloned in DH5␣ competent cells.

PCR analysis of RARRES3 expression Total RNA (1 ␮g) was reverse transcribed with 50 mM oligo(dT) primer and 50 U/␮g RNA of M-MLV in a volume of 20 ␮l at 42°C for 45 min. 0.2 ␮l of this reaction were PCR amplified by using the primer RARRES3-1F 5⬘-TCAAGC TTGGAGCACCAGACCTCTC-3⬘ or the primers described by DiSepio et al19 F: 5⬘-TCAAGCTTCCACCATGGCTTCGCCACACCAAGAGCCCA-3⬘ and R: 5⬘-TTGGATCCTGTGGCT GCTTCAGGCGTTGC-3⬘. A control PCR product, corresponding to human glyceraldehyde-3-phosphate dehydrogenase (HGAPDH), was used under the same conditions for normalizing measurement differences (HGAPDH-203F: 5⬘ATGGCACCGTCAAGGCTGAG-3⬘ and HGAPDH-880R: 5⬘AGACCACCTGGTGCCAGTG-3⬘). 1 ␮Ci of [␣-32P]dCTP (3000 Ci/mmol. Amersham International) per reaction tube was used. Aliquots of 4 ␮l were taken from the reaction mixture at cycles 10, 15, 18, 21, 25 and 28 for HGAPDH product, and 21, 25, 28, 30, 35, and 40 for RARRES3 product. The amplified products were separated on a 4% agarose gel, transferred to nylon membranes and radioactivity quantified by Phosphorlmager scanning (Molecular Dynamics, Sunnyvale, CA, USA). For subsequent analyses, we used 18 cycles of amplification for HGAPDH products and 25 cycles for RARRES3 products and proceeded as described above. To quantify the expression of RARRES3 gene we calculated the ratio of the ‘signal of RARRES3 product’ vs ‘signal of HGAPDH product’. Each result was validated by two sets of experiments (PCR and quantification).

Sequence analysis 0.2 ␮l of the RT reaction described above were PCR amplified by using primers RARRES3-1F 5⬘-TCAAGCTTGGAGCACAGACTCCT C-3⬘ and RARRES3-678R 5⬘-TTGGATCCTCC TTCAGTCTTGTTTCAATTAG-3⬘. Amplified products were run on an 2% of agarose gel, purified with the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA) and sequenced with the corresponding forward and reverse primers using the dideoxy terminator reaction chemistry in an automated ABI-Prism 377 sequencing system (Applied Biosystems-Prism-Perkin Elmer, Foster City, CA, USA).

Statistical analysis

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For statistical comparison between groups the Student’s nonpaired t-test was used. Two-tailed statistical significance was determined. A P-value ⬍0.05 was considered significant. Analyses were performed using GraphPad InStat V2.04a software (GraphPad Software, San Diego, CA, USA). Results and discussion To identify genes which may be involved in the development and/or progression of B-CLL, we compared the pattern of gene expression in malignant B lymphocytes from a B-CLL patient (patient 1 in Table 1) and from a normal subject using the mRNA DD method. A 197-bp fragment of cDNA was found to be expressed in normal B cells, but absent in the B-CLL sample (Figure 1a). We initially called this cDNA ‘leukemia related gene-1’ (LRG-1). DNA sequencing of this cDNA and subsequent search for homology in the public GenBank database revealed that LRG-1 showed 99% identity to nucleotides 328–512 of the retinoic acid receptor responder 3 gene (RARRES3, GenBank accession no. AF060228) resulting in highly homologous protein products (Figure 1b). We will therefore refer to LRG-1 as RARRES3. This gene is a wellknown growth regulator whose expression correlates inversely with cell proliferation and is decreased in several cancer cell lines and in some primary tumors such as lymphoma, ureter, kidney, rectal and uterine.19 RARRES3 maps to the 11q23 chromosomal region and thus below the ATM gene.19 Aberrations at 11q regions are recurring abnormalities in various types of neoplasias including lymphoproliferative disorders.24–28 In addition, previous studies have shown that loss of heterozygosity (LOH) at the genomic region 11q22–q23 is one of the most common structural chromosome aberrations in B-CLL,5,7–10 suggesting that a novel tumor suppressor gene may be located in this region. Because RARRES3 is mapped to this hot spot chromosome region and this gene has not been analyzed thus far in hematologic malignancies, we performed expression and mutational analyses to determine whether RARRES3 could play a role in B-CLL pathogenesis. For these studies we used RT-PCR to analyze samples from the 24 B-CLL patients listed in Table 1. The overall incidence of clonal karyotype abnormalities in the B-CLL cases studied was 65% and seven out of 20 patients (35%) had LOH at 11q23 based on chromosome analysis (Table 1). Samples from four normal subjects were used as reference. We also carried out these analyses in four normal EBV-B cell lines and in EHEB cells. As shown in Figure 2a and Table 2, RARRES3 was expressed in normal B-lymphocytes (1.069 ± 0.076, arbitrary units) and this expression was significantly decreased in the normal EBV-lymphocytes and EHEB cell line (Figure 2a, Table 2). These results are in agreement with the role of RARRES3 as a class II tumor suppressor gene, known to be functionally active, but expressed at low levels in cell lines and/or tumors.19 As shown in Table 2, RARRES3 expression values at early stages (A/0–1) of B-CLL did not differ significantly from control levels. At later stages (B and C) however, there was a progressive significant decrease in the expression of this gene. These data indicate that down-regulation of RARRES3 gene expression was clearly related to B-CLL disease progression. Since one of the characteristics of this progression is the transition from a low mitotic activity phase (stage A) to a higher Leukemia

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Figure 2 Expression analysis of RARRES3 gene by RT-PCR. 4 ␮l of each amplified product were run on a 4% agarose gel, transferred to nylon membrane and exposed to PhosPhorlmager scanner to quantify radiactivity. (a) RT-PCR from: P, B-CLL patients; CTR, control subjects; EHEB, B-CLL cell line; HUT112, GER112, CO43, and CO31, EBVcell lines from normal subjects. P6–2 and P8–2, are repeated extractions from patients P6 and P8 obtained at a later stage of the disease. The size of RARRES3 product (primers RARRES3-1F and R) is 550 bp. The size of RARRES3 product at cell line CO31 (amplified with primers F and R) is 526 bp. The size of control product HGAPDH (primers HGAPDH-203F and HGAPDH-880R) is 678 bp. (b) RT-PCR from ALL patients.

Figure 1 (a) Section of differential display gel. Total RNA from BCLL patient 1 (P1) and from a normal subject (CTR) were subjected to differential display (primer combination oligo-dT11G-AP3). P1-R1 and P1-R2, cDNAs amplified from RT reactions 1 and 2 from patient 1. CTR-R1 and CTR-R2, cDNAs amplified from RT reactions 1 and 2 from a normal subject. The position of the band (LRG-1) that was further analyzed is indicated. (b) Multiple sequence alignment showing homology between LRG-1, RARRES3, human H-rev 107 (H-rev), and rat H-rev 107 (R-rev). Consens, consensus sequence. Differences in residues 63 and 118 between LRG-1 and RARRES3 are enclosed in black squares.

proliferation stage reflected by a lymphocyte doubling time of less than 12 months2 and RARRES3 appears to be an inhibitor of cell proliferation,19 the reduced expression of this gene observed in our study could facilitate the increase of CD5+ malignant B-lymphocytes that accompany B-CLL progression. RARRES3 was also significantly decreased in 11q-deleted BCLL samples compared to control values (Figure 2a, Table 2). We have also examined the expression of RARRES3 in 10 B lineage ALL samples. The overall incidence of clonal karyLeukemia

otype abnormalities in the nine cases studied was 88.9% and one patient or 11.1% had deletion at 11q23 (Table 1). As shown in Figure 2b and Table 2, the expression of RARRES3 was also decreased in ALL samples (0.239 ± 0.045) when compared with normal B-cells (1.069 ± 0.076). Therefore down-regulation of this gene could also contribute to the typical lymphocyte high proliferation rate observed in this malignancy.29 Characterization of RARRES3 as a true tumor-suppressor gene would require the identification of loss of heterozygosity or/and inactivating mutations in cancer patients. Thus, we carried out sequence analyses on cDNA samples obtained by RTPCR amplification from EHEB and EBV-transformed cell lines, normal B cells and all B-CLL and ALL samples listed in Table 1. Our analyses revealed three nucleotide differences between the RARRES3 cDNA amplified by us (LRG-1) and the previously reported RARRES3 sequence,19 which resulted in two amino-acid changes (Figure 1b, Table 3). These differences were found in all leukemia samples assayed as well as in T (JM, CEM) and B (RPMI 8866, RPMI 8226) cell lines, suggesting that these nucleotide changes may represent the wild sequence, rather than polymorphism. In this context, we did not find mutations in any of the 36 leukemia samples assayed, including the seven B-CLL and one ALL cases with 11q23 deletion (see Table 1). Failure to detect mutations in these cases might suggest that RARRES3 is not the targeted gene in 11q23 deletions or that the remaining allele, which may account for the observed

RARRES3 expression in lymphoproliferative disorders B Casanova et al

Table 2

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Summary of RARRES3 expression

Study group

Control (n = 4) Control (n = 4) Control (n = 4) Control (n = 4) A/0–1 (n = 10) Control (n = 4) Control (n = 4) Control (n = 4)

vs vs vs vs vs vs vs vs

Study group

Mean ± s.e.m. (relative units)

A/0-I (n = 10) B/II (n = 5) C/III–IV (n = 8) 11q del B-CLL (n = 7) C/III–IV (n = 8) ALL (n = 10) EBV-lymphocytes (n = 4) EBV-lymphocytes + EHEB (n = 5)

1.069 1.069 1.069 1.069 1.226 1.069 1.069 1.069

± ± ± ± ± ± ± ±

0.076 0.076 0.076 0.076 0.208 0.076 0.076 0.076

vs vs vs vs vs vs vs vs

Mean ± s.e.m. (relative units)

P value

± ± ± ± ± ± ± ±

NSa 0.0086 0.0042 0.0245 0.0086 0.0001 0.0336 0.0145

1.226 0.508 0.470 0.619 0.470 0.239 0.525 0.498

0.208 0.124 0.107 0.116 0.107 0.045 0.183 0.144

NS, not significant; P ⬍ 0.05, significant; P ⬍ 0.01, very significant, P ⬍ 0.001, extremely significant.

a

Table 3 Nucleotide and amino acid differences between LRG-1 and RARRES3 sequences

Nucleotide LRG-1/RARRES3 Base pair 217 Base pair 381 Base pair 515

A/G A/G G/A

Amino acid LRG-1/RARRES3 Glu/Gly (position 63) Thr/Ala (position 118) Ala/Ala (position 162)

RARRES3 low expression in 11q23 deleted samples, is inactivated by mechanisms different from mutation or deletion. These alternative mechanisms could affect the expression or inactivate RARRES3 in non 11q23-deleted cases. In this regard, increasing evidence has suggested that silencing the tumor suppressor genes BRCA1, p27Kip1, p53 and p73, and cyclin-dependent kinase inhibitors genes p15 and p16, by promoter methylation, may play a role in the development and/or poor prognosis of some neoplasias.30–34 On the other hand, it cannot be excluded that some alterations (ie mutations, deletions) do not involve the RARRES3 gene coding region itself, but affect its expression by modifying regulatory sequences. Our results do not rule out these alternative mechanisms of tumor suppressor inactivation, and possibly the combination of mono-allelic deletion and/or promoter methylation or mutation of noncoding regulatory elements could take place in the leukemic cases studied. To our knowledge RARRES3 has not been linked so far to the pathogenesis of malignant disease, nor has it been reported in hematologic malignancies. Our study does not suggest a primary role of RARRES3 gene in B-CLL and ALL leukemogenesis and we did not observe the classical tumor suppressor inactivation mechanism consisting in loss of one allele and mutation of the remaining one. However, our results argue in favor of the involvement of RARRES3 function in B-CLL (and possibly ALL) progression. Down-regulation or deletion of RARRES3, a cell proliferation inhibitor,19 may provide a dramatic growth advantage for malignant cells and thus accelerate the leukemic process. Further functional studies will be necessary to determine the exact role of this gene in the development and progression of hematopoietic neoplasms. Acknowledgements This work was supported by grants SAF97-0064-CO3-02 (to AGP) and SAF97-0064-CO3-03 (to AS) from the Comisio´n

Interministerial de Ciencia y Tecnologı´a (CICYT); 08.1/0028/99 (to AGP) and 08.1/012/97 (to AS and AGP) from the Comunidad Auto´noma de Madrid (CAM). B Casanova, MT de la Fuente and L Sanz were supported by fellowships from CAM; M Garcı´a-Gila was supported by a fellowship from CICYT. We thank Drs Felipe Prosper and M Jose´ Terol (Hospital Clinico Universitario, Valencia, Spain) and Dr Ernesto Rolda´n (Hospital Ramo´n y Cajal, Madrid, Spain) for some of the samples and clinical data of B-CLL patients; Dr Santiago Rodrı´guez de Co´rdoba for critical reading of the manuscript and Ms Mercedes Hema´ndez del Cerro for excellent technical assistance.

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