Leukemia (2004) 18, 826–831 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00 www.nature.com/leu Activation-induced cytidine deaminase expression in follicular lymphoma: association between AID expression and ongoing mutation in FL MS Hardianti1, E Tatsumi1, M Syampurnawati1, K Furuta1, K Saigo2, Y Nakamachi3, S Kumagai3, H Ohno4,5, S Tanabe6,7, M Uchida6 and N Yasuda8 1International Center for Medical Research (ICMR), Graduate School of Medicine, Kobe University, Kobe, Japan; 2Blood Transfusion Division, Graduate School of Medicine, Kobe University, Japan; 3Department of Laboratory Medicine, Graduate School of Medicine, Kobe University, Japan; 4Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 5Department of Hematology, Nara Social Health Insurance Hospital, Nara, Japan; 6Department of Hematology, Hyogo Prefectural Amagasaki Hospital, Amagasaki, Japan; 7Department of Internal Medicine, Torigata Hospital, Osaka, Japan; and 8Department of General Internal Medicine, Kyoto City Hospital, Kyoto, Japan Activation-induced cytidine deaminase (AID) is required for role in the resistance of some FL cases to a certain therapy.8 The somatic hypermutation (SHM) and class switch recombination mutational analysis of the Ig gene in FLs has revealed some (CSR) of the immunoglobulin (Ig) gene. AID has been reported heterogeneity in SHM characteristic in terms of intraclonal to be specifically expressed in the germinal center (GC). 9,10 Follicular lymphoma (FL) cells are known to be exposed to microheterogeneity and antigen selection. GC reaction, as characterized by a high degree of SHM with Based on the reported heterogeneity of the SHM pattern in FL some heterogeneity in terms of intraclonal microheterogeneity and the possible role of AID involved, AID expression was and antigen selection. The heterogeneity of SHM pattern in FL investigated in the present study. AID mRNA was investigated in intrigued us to investigate the AID expression. AID expression the fresh cells of 15 FL cases and four FL cell lines. AID mRNA was investigated in 19 FL materials consisting of 15 cases of FL fresh cells and four cell lines. In all, 10 fresh cells and three cell was expressed in 10 cases and three cell lines. Furthermore, lines expressed AID, but the others did not. SHM was SHM in IgVH genes was analyzed in 12 FL cases and all cell investigated in 12 fresh cells and four cell lines. The ongoing lines. Independent of the expression or absence of AID mutation was significantly different between AID-positive and transcripts, the IgVH genes in all FL samples were somatically AID-negative FL fresh cells (unpaired Student’s t-test, mutated, indicating that AID can start to be switched off in FL. P ¼ 0.047). Ongoing mutation was not seen in any of the cell The switch off of AID expression in some FL cases may be lines. AID expression was associated with the ongoing muta- tion in FL fresh cells (two-tailed Pearson’s coefficient correla- related to the achievement of high-affinity antibody, as indicated tion, r ¼ 0.899, P ¼ 0.01). The switch off of AID expression may by the cessation of the ongoing mutation in AID-negative FL start in the B-lineage differentiation stage counterpart of FL fresh cells. after optimizing SHM, indicated by the cessation of the ongoing mutation in AID-negative FL fresh cells. Leukemia (2004) 18, 826–831. doi:10.1038/sj.leu.2403323 Materials and methods Published online 26 February 2004 Keywords: FL; AID expression; SHM; ongoing mutation Patients and cell lines Our series consisted of 19 FL materials comprising 15 fresh cells Introduction of FL cases derived from lymph node tissues and four FL cell lines, namely FL18,11 FL 218,12 FL31812 and FL518. The The immunoglobulin (Ig) gene undergoes V(D)J recombination, diagnosis of the 15 cases and the donor patient of FL-518 were class switch recombination (CSR) and somatic hypermutation based on the pathological diagnosis, cell-surface antigen (SHM) to encode a high-affinity antibody. The latter two analysis of lymph-node cell suspension and chromosomal processes mainly occur in the germinal center (GC) of secondary analysis of t(14;18)(q32;q21) (Table 1). lymphoid organ, which is formed by the activated B-cell.1 The isolation of the activation-induced cytidine deaminase (AID) gene, whose product is an enzyme required for SHM and CSR, Reverse transcription polymerase chain reaction was a major achievement for clarifying the molecular mechan- (RT-PCR) for AID ism of the SHM and CSR processes despite the fact that its precise actions remain elusive.2 An initial report showed that 3 cDNA was synthesized using random primer (TaKaRa, Kyoto, AID is specifically expressed in GC. Japan) and M-MLV reverse transcriptase (GibcoBRL, NY, USA) Follicular lymphomas (FLs) are thought to originate from GC B m 4 from 2 g of sample derived RNA extracted by Trizol (Invitro- cells, based on the accumulation of SHM in their Ig genes. The m 5,6 gen, Carlsbad, CA, USA). cDNA (2 l) was then amplified using tumor growth of FLs may be driven by antigen selection, the PCR reaction mixture from Takara Ex-Taq (Takara, Shiga, which involves an active SHM process reflected by the ongoing Japan) in a total volume of 50 ml, following the supplier’s mutations that result in considerable intraclonal microheter- 7 instruction, using a specific AID primers covering the region ogeneity. Ongoing mutation has also been reported to play a from exons 1 to 5, yielding a product of 646 bp.13,14 Briefly, 5 pmol each of an upstream primer (50-GAG GCA AGA AGA Correspondence: E Tatsumi, International Center for Medical Research CAC TCT GG-30) and a downstream primer (50-GTG ACA TTC (ICMR), School of Medicine, Kobe University, Kusunoki-Cho, Chuo- CTG GAA GTT GC-30) were used. After 5 min of incubation at þ Ku, Kobe 650-0017, Japan; Fax: 78 382 5716; 1 E-mail: [email protected] 94 C, 30 cycles of PCR were performed under the following Received 9 June 2003; accepted 7 January 2004; Published online 26 conditions: a denaturation step at 941C for 1 min, an annealing February 2004 step at 561C for 1 min and an extension step at 721C for 2 min AID expression in FL MS Hardianti et al 827 Table 1 Characteristics of FL materials FL material Sex Age (years) CD5 CD10 CD19 CD20 sIg t(14;18)(q32;q21) Case 1 M 52 – + + + Ml + Case 2 M 61 – – + + Gk + Case 3 M 81 – + + + Mk ND Case 4 M 22 – + + + Mk + Case 5 F 38 – – + + Mk ND Case 6 M 53 – – + + Mk + Case 7a F 67 – + ND + Mk ND Case 8 F 48 – + + + Mk + Case 9 F 42 ND – + + Ml + Case 10a F 69 ND + ND + Mk ND Case 11 M 66 – – ND + Ml ND Case 12a M 59 – + ND + Mk + Case 13a M72–+ND+Ml ND Case 14a M57–+ND+Ml ND Case 15 M 61 – + + + Mk ND FL18b M69–+++Gk+ FL218b M73––++Gl + FL318b M67–+++Mk+ FL518b M61–+++Mk+ aExpression of bcl2 protein was detected by immunostaining of the pathological specimen. bAccompanying data represented the donor patient data and CSA of the cell line. M, male; F, female; ND, not determined. (10 min in the last cycle). PCR products (10 ml) were electro- competent cells (TA cloning kit; Invitrogen, Carlsbad, CA, USA). phoresed in a 3% low melting NuSieve GTG Agarose gel (BMA, Following an overnight culture, 15 colonies were picked from a Rockland, ME, USA) in 1 Â TBE and visualized by staining with Luria Bertani (LB) agar plate based on X-Gal screening (Nacalai ethidium bromide. This primer pair can also detect splice Tesque.Inc, Kyoto, Japan). After the insert was checked by PCR, variants consisting of a longer (939 bp) and a shorter (530 bp) five to 10 positive colonies were subcultured overnight in 5 ml product, which were reported to perturb the functionality of of LB medium. Minipreps of plasmid were prepared from the wild-type AID by preventing SHM or interrupting ongoing SHM cultures by alkaline lysis and purified on DNA affinity column in CLL cases.14 by QIAprep Miniprep kit (QIAGENGmbH, Germany). Sequen- Densitometry of AID mRNA levels was carried out using cing was carried out on an ABI Prism 310 sequencer (Applied Scion Image-Release Beta 4.0.2. for Windows. The expression Biosystems, Fostercity, CA, USA) using the dye terminator cycle levels were normalized to the expression level of b-actin sequencing method (BigDye version 3, Applied Biosystems). mRNA15 of each sample. Mutational analysis SHM and ongoing mutation study Final sequences were compared to those of the published A seminested PCR was performed as described previously,16 germline genes with the highest homology in the V-Base (http:// with 500 ng of genomic DNA extracted from lymph node tissues www.mrc-cpe.cam.ac.uk/). The number of somatic mutations for fresh cells and from cell pellets for cell lines, using a within the complementarity determining region (CDR)2 and Genomix kit (Talent SRL, Trieste, Italy). In brief, DNA samples framework (FR)3 in the consensus sequence of each sample were first amplified using the PCR reaction mixture from Ex-Taq were determined. As mutation patterns within CDRs are difficult Takara in a total volume of 50 ml by using 5 pmol of an upstream to interpret, only the R (replacement) to S (silent) mutation ratio primer (FR2A 50-TGG ATC CGC CAG GCT TCN GG-30) and in the FR3 was considered as an indication for antigen 5 pmol of a downstream primer (LJH 50-TGA GGA GAC GGT selection.17 A sequence was regarded as being antigen selected GAC C-30).