Blood Cells, Molecules, and Diseases 33 (2004) 90–93 www.elsevier.com/locate/ybcmd

Molecular investigation of the stromal cell-derived factor-1 chemokine in lymphoid and patients from Brazil

Gabriela Goncßalves de Oliveira Cavassin,a Fernando Luiz De Lucca,b Nayara Delgado Andre´,a Dimas Tadeu Covas,b,c Maria Helena Pelegrinelli Fungaro,d Ju´lio Ce´sar Voltarelli,b,c and Maria Angelica Ehara Watanabea,*

a Department of Pathological Sciences, Londrina State University, Londrina, PR, Brazil b School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil c Ribeira˜o Preto Blood Center, Ribeira˜o Preto, SP, Brazil d Department of Biology, Londrina State University, Londrina, PR, Brazil Submitted 26 January 2004; revised 21 April 2004 (Communicated by M. Lichtman, M.D., 22 April 2004) Available online 28 May 2004

Abstract

The stromal cell-derived factor-1 (SDF-1) gene contains a common polymorphism, termed SDF1-3VA, in an evolutionarily conserved segment of the 3V-untranslated region (3V-UTR). We compared SDF-1 genotypes in patients diagnosed with lymphoid and . Since the SDF1-3VA variant deletes the MspI restriction site, PCR-restriction fragment length polymorphism (RFLP) analysis was used for identification of genotypes. We identified the heterozygous genotype (3VA/wt) in 38.8% (24/62) of lymphoma patients and in 26.2% (11/42) of lymphoid leukemias. The percentage of 3VA carriers was significantly higher in lymphomas (43.5%) than in lymphoid leukemias (26.2%; P < 0.05). Our study indicates that lymphoma patients from Brazil are more likely to carry the 3VA gene than patients with lymphoid leukemias, suggesting that this polymorphism may be a differential determinant of lymphomas and lymphoid leukemia. D 2004 Elsevier Inc. All rights reserved.

Keywords: SDF-1 chemokine; Lymphoma; Lymphoid leukemia

Introduction conserved cystein residues—CC, CXC, XC and CX3C— with the CC and CXC groups being by far the largest. The development of a large solid tumor requires not only Stromal cell-derived factors 1 alpha and 1 beta (SDF1a engagement of blood vessels, but also infiltrating cells that and SDF1h) are small cytokines belonging to the intercrine comprise a stroma which promotes tumor growth. Ultimately, CXC subfamily originally isolated from a murine marrow tumors spread through the body by metastases, sometimes to stroma cell line. cDNA and genomic clones of human a preferred location depending upon their histological origin SDF1a and SDF1h have been isolated and characterized [1]. Human chemokines are a large family of molecules, and show that cDNAs encode proteins of 89 and 93 amino characterized by structural homologies based on conserved acids, respectively. SDF1a and SDF1h sequences are more cystein residues, as well as binding capacities to particular G than 92% identical to their human counterparts. The ge- protein-coupled receptors [2,3]. Four families of chemokines nomic structure of the SDF1 gene shows that the a and h have been described, based on the relative position of the isoforms arise by alternative splicing of a single gene, and that they are encoded by 3 and 4 exons, respectively [4]. Sequence analysis of the human SDF gene (GenBank accession number L36033) revealed a common variant con- taining a G!A transition in an evolutionarily conserved * Corresponding author. Departamento de Cieˆncias Patologicas, V V ´ segment of the 3 -untranslated region (3 -UTR). This poly- Universidade Estadual de Londrina, Campus Universitario-CEP 86051- h 970, Londrina, PR, Brazil. morphism designated SDF-1-3VA, is present in the SDF-1 E-mail address: [email protected] (M.A.E. Watanabe). transcripts. Because this variant eliminates the MspI restric-

1079-9796/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2004.04.008 G.G. de Oliveira Cavassin et al. / Blood Cells, Molecules, and Diseases 33 (2004) 90–93 91 tion enzyme site, PCR-restriction fragment length polymor- Table 2 phism (RFLP) analysis can be used for rapid identification of SDF1 genotype and 3VA allele frequency on stromal cell-derived factor (SDF1) genotypes genotypes. This variant SDF1 gene allele may play an important regulatory role by increasing production of SDF- SDF-RFLP SDF-RFLP SDF-RFLP 3VA allele wt/wt 3VA/wt 3VA/3VA frequency 1 which bound to SDF chemokine receptor CXCR4 from T cells [5]. Lymphoma 35 24 3 0.242 n =62 (56.5%) (38.8%) (4.8%) The mRNA 3V-untranslated region (3V-UTR) of many Lymphoid leukemia 31 11 0 0.13 genes has been identified as an important regulator of the n =42 (73.8%) (26.2%) (0%) mRNA transcript itself, as well as the translated product [6]. The SDF1 gene variants were identified after PCR amplification of genomic The present report focuses on distribution of the SDF-1 DNA followed by MspI digestion. 3VA/wt and 3VA/3VArepresent in cancer patients attending the Cancer Hospital, Londrina, heterozygous and homozygous genotypes of the mutated form of the Brazil. Genotyping was performed using RFLP analysis SDF-1 gene, respectively. wt/wt represents the wild-type gene. with MspI restriction enzyme mapping. SDF-1 genotyping

Subjects and methods PCR products were subjected to restriction digestion by incubating with MspI (Invitrogen, Life Technologies, USA) Study population for 3 h. at 37jC and then subjected to electrophoresis in 2% agarose gels. SDF-1 wild-type alleles (SDF1-wt) yielded Following approval from the Human Ethics Committee of 100 and 193 base-pair products, while SDF1-3VA alleles the Londrina State University, peripheral blood was collected yielded a 293 base-pair product. from 62 patients with a clinical and histopathological diag- nosis of Hodgkin’s lymphoma (HL-18) or non-Hodgkin’s Statistical analyses lymphoma (NHL-44), and in 42 patients with a clinical and hematological diagnosis of acute lymphoid leukemia (ALL- Molecular data were analyzed statistically by the Chi- 31) or chronic lymphoid leukemia (CLL-11). square test (v2).

Polymerase chain reaction Results Genomic DNAwas isolated from peripheral blood cells by a method described by Miller et al. [7] and 100 ng of DNA In the present study we used PCR-RFLP techniques to were analysed by PCR using primers for SDF 3VUTR-F1 examine SDF-1 genotypes in patients diagnosed with lym- (sense, 5V-CAGTCAACCTGGGCAAAGCC-3V) and SDF phoma or with lymphoid leukemia. Demographic data are 3VUTR-R2 (antisense, 5V-CCTGAGAGTCCTTTTGCGGG- summarized in Table 1. There were no group differences in 3V). Samples were amplified using the kit buffer plus 1.5 age, or gender (Table 1). mmol/l taq polymerase (Invitrogen, Life Technologies, USA). PCR conditions were 5 min denaturation at 94jC, 35 cycles of 1 min at 94jC, 1 min at 60jC and 1 min at 72jC, and 10 min elongation at 72jC in a thermocycler (PCR-Sprint Hybaid). PCR products of 293 base pairs were analysed by electrophoresis in a 2% agarose gel and visualized using UV fluorescence after staining with ethidium bromide.

Table 1 Demographic characteristics of patients in lymphomas and lymphoid leukemias ALL CLL NHL HL (31) (11) (44) (18) Age (years) 6–10 8 0 1 2 11 – 20 12 1 4 2 Fig. 1. SDF1-3VA polymorphism in the lymphoma and lymphoid leukemia 21–30 3 0 5 7 patients. SDF1 genotyping was performed in blood cell DNA by a PCR- >30 8 10 34 7 RFLP technique able to detect G-to-A mutation in the terminal region of the Sex Male 20 8 23 13 SDF-1 3V gene. The rare 3VA homozygotes were mixed with heterozygotes Female 11 3 21 5 in a group referred to as 3VA carriers. Percentage of 3VA carriers in ALL, acute lymphoid leukemia; CLL, chronic lymphocytic leukemia; NHL, lymphomas (43.5%) were significantly higher than in lymphoid leukemias non-; HL, Hodgkin lymphoma. (26.2%; chi-square test). 92 G.G. de Oliveira Cavassin et al. / Blood Cells, Molecules, and Diseases 33 (2004) 90–93

We found that the 3VA/wt heterozygous genotype was in the blood. The mRNA 3V-untranslated region (3V-UTR) of present in 38.8% (24/62) of lymphoma patients, and in many genes has been identified as an important regulator of 26.2% (11/42) of lymphoid leukemia subjects (Table 2). the mRNA transcript, as well as the translated product [6].In There were no statistical differences between acute and most cases, translational control mechanisms result from chronic lymphoid leukemias or between Hodgkin’s and the interaction of RNA-binding proteins with 5V-or3V-UTRs. non-Hodgkin’s lymphomas. This difference for 3VA carriers A multispecies analysis has shown that in most vertebrates, from lymphoma patients was found to be statistically 3V-UTRs are substantially longer than their 5V counterparts, significant by the v2 test ( P < 0.05, Fig. 1). For all samples indicating a significant potential for regulation. In addition, the genotype did not differ from the theoretical distribution the average length of 3V-UTR sequences has increased during given by Hardy–Weinberg equilibrium. evolution, suggesting that their utilization might contribute to organism complexity [15]. Normal B- maturation and proliferation are Discussion regulated by chemokines, and genetic polymorphisms in chemokines and chemokine receptors modify progression Acute lymphoid leukemia, also called lymphoblastic of human immunodeficiency virus-1 infection. In the ac- leukemia (ALL), accounts for about 75–80% of the child- quired immunodeficiency syndrome, the SDF1-3VA chemo- hood leukemias. In this form of the disease, the lymphocyte kine variant was associated with approximate doubling of the cell line is affected, causing overproduction of immature risk in heterozygotes and roughly a fourfold increase in and crowding out other blood cells in the bone homozygotes [16]. marrow and lymphoid organs. This proliferation interferes SDF-1 substantially enhances migration of follicular with normal function of hematopoietic and immune sys- center lymphoma B cells, but not migration of freshly tems. Chronic lymphocytic leukemia (CLL), the most prev- purified B cells. This difference may be alent adult leukemia in the Western world is a neoplastic related to the extended survival of follicular center lympho- disease of advancing age, characterized by a progressive ma versus germinal center B cells. SDF-1 produced in accumulation of functionally incompetent, long-lived small follicular center lymphoma lymph nodes may play a role mature monoclonal B . in the local dissemination of tumor [17]. Non-Hodgkin’s lymphomas comprises many histologi- In our study, the percentage of 3VA gene carriers were cally and perhaps biologically distinct lymphoid malignan- significantly different between the two groups of patients cies, each one with poorly understood but putatively distinct studied: 43.5% for lymphomas and 26.2% for lymphoid etiologies. The majority of lymphoma cases arise in lymph leukemias. The variant SDF-1 gene allele that results from a nodes, but primary extranodal disease accounts for 20–30% 3VUTR substitution may play an important regulatory func- of cases [8,9]. tion by increasing production of SDF-1 by lymph node Hodgkin lymphoma is a malignancy of unknown path- stromal cells. The present work suggests that this polymor- ogenesis. The malignant Hodgkin and Reed/Sternberg cells phism may be responsible for different clinical and biolog- present a heterogeneous and largely uncharacterized pheno- ical behaviour of lymphomas and lymphoid leukemias. type. The hallmark of Hodgkin lymphoma is the presence of large, mononucleated Hodgkin and multinucleated Reed/ Sternberg cells. These cells represent the tumor cells, but Acknowledgments usually comprise less than 1% of the cellular infiltrate in the lymphoma tissue [10]. This study was supported by Conselho Nacional de SDF-1 was initially identified as a bone marrow stromal Desenvolvimento Cientı´fico e Tecnolo´gico-CNPq, Coor- cell-derived factor, and as a pre-B-cell stimulatory factor [11]. denacßa˜o de Aperfeicßoamento de Pessoal de Nı´vel Superior- SDF-1 is also a highly efficient lymphocyte chemoattractant CAPES and Coordenadoria de Po´s-Graduacßa˜o-Londrina [12] and plays an important role in B- and State University-CPG-UEL. homing of hematopoietic stem cells to the bone marrow [13]. The SDF-1 gene variant which has a G!A transition in the 3V-untranslated region may have an important regulatory References function increasing the production of SDF-1 [5].SDF-1 mRNA is abundantly expressed in stromal cells isolated from [1] A.P. Vicari, C. Caux, Chemokines in cancer, Cytokine & Growth the lymph nodes of patients with malignant lymphoma [14]. Factor Rev. 13 (2002) 143–154. In this context, 3VA gene carriers individuals may be good [2] D. Rossi, A. Zlotnik, The biology of chemokines and their receptors, candidates for presenting proliferation of neoplasic cells in Annu. Rev. Immunol. 18 (2000) 217–242. the lymph nodes, because this mutation can be associated to [3] A. Zlotnik, O. Yoshie, Chemokines: a new classification system and their role in immunity, Immunity 12 (2000) 121–127. an increase production of SDF. By contrast, lymphoid leu- [4] M. Shirozu, T. Nakano, J. Inazawa, K. Tashiro, H. Tada, T. 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