Research Article Overexpression of c-Maf Contributes to T-Cell Lymphoma in Both Mice and Human Naoki Morito,1 Keigyou Yoh,2 Yuki Fujioka,1 Takako Nakano,1 Homare Shimohata,1 Yuko Hashimoto,1 Akiko Yamada,1 Atsuko Maeda,1 Fumihiko Matsuno,5 Hiroyuki Hata,5 Atsushi Suzuki,6 Shigehiko Imagawa,3 Hiroaki Mitsuya,5 Hiroyasu Esumi,6 Akio Koyama,2 Masayuki Yamamoto,4 Naoyoshi Mori,7 and Satoru Takahashi1 1Department of Anatomy and Embryology, Biomolecular and Integrated Medical Sciences, 2Pathophysiology of Renal Diseases, Medical Sciences for Control of Pathological Processes, 3Clinical and Experimental Hematology, Major of Advanced Biomedical Applications, 4Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan; 5Department of Internal Medicine II, Kumamoto University School of Medicine, Kumamoto, Japan; 6Cancer Physiology Project, National Cancer Center Research Institute East, Chiba, Japan; and 7Department of Pathology of Biological Response, Nagoya University Graduate School of Medicine, Nagoya, Japan Abstract malignancies. c-Maf is expressed at high levels in multiple c-Maf translocation or overexpression has been observed in myeloma cells carrying the translocation t(14;16) (q32;q23), which human multiple myeloma. Although c-maf might function as results in the fusion of the immunoglobulin heavy chain to the an oncogene in multiple myeloma, a role for this gene in other c-maf gene locus (10–12). Moreover, using gene expression profiling cancers has not been shown. In this study, we have found that by DNA microarray analysis, Hurt et al. (13) detected over- mice transgenic for c-Maf whose expression was direct to the expression of c-Maf in 50% of multiple myeloma cell lines that they T-cell compartment developed T-cell lymphoma. Moreover, have analyzed. However, the t(14;16) (q32;q23) primary transloca- we showed that cyclin D2, integrin B , and ARK5 were up- tion is present in only 5% to 10% cases of multiple myeloma. Thus, 7 the molecular pathogenesis of multiple myeloma characterized by regulated in c-Maf transgenic lymphoma cells. Furthermore, f 60% of human T-cell lymphomas (11 of 18 cases), classified as c-Maf overexpression identifies 50% is not fully understood (14). Although c-maf might function as an oncogene in human multiple angioimmunoblastic T-cell lymphoma, were found to express c-Maf. These results suggest that c-Maf might cause a type of myeloma, there is no evidence to date for the involvement of this T-cell lymphoma in both mice and humans and that ARK5, in gene in the development of other malignancies. In this study, we have generated c-Maf transgenic mice that overexpress c-Maf in addition to cyclin D2 and integrin B7, might be downstream target genes of c-Maf leading to malignant transformation. the T-cell compartment using the human CD2 promoter and locus control region. c-maf transgenic mice developed T-cell lymphoma, (Cancer Res 2006; 66(2): 812-9) providing the first direct evidence that c-maf can function as an oncogene in T cells in vivo. In addition, we analyzed c-Maf Introduction expression in cases of human T-cell lymphoma, showing c-Maf The maf proto-oncogene was originally identified within the overexpression in angioimmunoblastic T-cell lymphoma (AITL) genome of the avian musculoaponeurotic fibrosarcoma virus, AS42 at high frequency. These results indicate that c-Maf can cause a (1). The product of the maf gene and other members of the Maf type of T-cell lymphoma with characteristics similar to multiple family share a conserved basic region and amphipathic helix (bZip) myeloma and suggest that the c-Maf transgenic mouse may be a motif that mediates dimmer formation and DNA binding to the good model to study how the oncogene c-maf contributes to the Maf recognition element (MARE; ref. 2). Large Maf proteins, such pathogenesis of lymphoid malignancies. as c-Maf, MafB, MafA/L-Maf/SMaf, and NRL, contain an acidic domain that mediates transcriptional activation and plays a key Materials and Methods role in cellular differentiation (1–5). Mice. A 1.5-kb full-length cDNA encoding the murine c-Maf protein was c-Maf encodes a Th2-specific transcription factor that activates inserted into a VA CD2 transgene cassette containing the upstream gene the expression of interleukin (IL)-4 and IL-10 in T cells (6). c-Maf regulatory region and locus control region of the human CD2 gene. The VA has also been known to be involved in the regulation of lens vector has been reported to direct expression of an inserted cDNA in fiber cell differentiation (7–9). Recently, it has been determined all T lymphocytes of transgenic mice, with expression being linearly that c-maf functions as an oncogene in multiple myeloma (10–14). proportional to the transgene copy number (15). This c-Maf construct was Multiple myeloma is an incurable neoplasm of terminally injected into BDF1 fertilized eggs to generate transgenic mice. The differentiated B cells characterized by monoclonal expansion of c-Maf transgenic allele was genotyped by PCR using a pair of primers: V V V malignant plasma cells, accounting for f20% of all hematologic sense 5-TGCAGCAGAGACACGTCCTG-3 and antisense 5-TGAGTTCGAA- GATGCAGAGG-3V. Mice were maintained in specific pathogen-free con- ditions in a Laboratory Animal Resource Center. All experiments were done according to the Guide for the Care and Use of Laboratory Animals at the University of Tsukuba. Note: N. Morito and K. Yoh contributed equally to this work. Requests for reprints: Satoru Takahashi, Department of Anatomy and Southern hybridization analysis. High molecular weight DNA was Embryology, Biomolecular and Integrated Medical Sciences, Graduate School of prepared from the tail of each mouse, and 10 Ag of this DNA were Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba digested with NcoI and then subjected to electrophoresis through 0.8% 305-8575, Japan. Phone: 81-298-53-7516; Fax: 81-298-53-6965; E-mail: satoruta@ agarose gels. After electrophoresis, the DNA was transferred to a nylon md.tsukuba.ac.jp. I2006 American Association for Cancer Research. membrane (Zeta-probe; Bio-Rad, Richmond, CA). To generate DNA probes doi:10.1158/0008-5472.CAN-05-2154 for Southern hybridization, we cloned the open reading frame region of Cancer Res 2006; 66: (2). January 15, 2006 812 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. c-Maf and T-Cell Lymphoma Figure 1. Generation of c-Maf-overexpressing mice. A, structures of the mouse c-maf gene locus and the transgenic construct. The c-Maf cDNA was inserted into a vector (VA vector) containing the human CD2 transgene cassette. Southern blotting probe site, restriction sites, and predicted sizes of the endogenous gene and the transgene (NcoI). Arrowheads, positions of genotyping PCR primers (sense and antisense). LCR, locus control region. B, we obtained five transgenic mouse lines (Tg 50, 17, 78, 235, and 312) screened by PCR using tail DNA as templates. C, Southern blot analysis of the endogenous and transgenic c-maf genes in transgenic mice. The 0.3-kb fragment shown in (A) was used as the probe. The 2.8-kb endogenous and 1.1-kb transgenic genes are shown for transgenic lines 50 (Tg 50), 17 (Tg 17), and 78 (Tg 78) mice. The transgene copy numbers for Tg 50 were 6 copies, Tg 17 and 78 were 2 copies, respectively. D, analysis of c-Maf mRNA in thymocytes. c-Maf mRNA was detected in all samples tested. The amount of c-Maf mRNA in samples from Tg 50 mice was several-fold greater than that in samples from wild-type mice through densitometric analysis (Image J). E, immunofluorescence profile analysis of thymocyte subpopulations from c-Maf transgenic mice at 5 weeks of age. Analysis of the total thymocyte population for CD4 and CD8 expression revealed that CD4/CD8 double-negative thymocytes were increased and the thymus contained very few double-positive or single-positive mature thymocytes. Analysis of CD44 and CD25 expression showed that the numbers of CD44+CD25-, CD44+CD25+, and CD44ÀCD25+ thymocytes were increased and that numbers of CD44ÀCD25À thymocytes were decreased in the c-Maf transgenic mice. the c-maf gene. Transgene copy number was determined using Image J intensity and propidium iodide exclusion. The following phycoerythrin (PE), (NIH, Bethesda, MD). To investigate T-cell receptor (TCR) h-chain gene FITC, and peridinin chlorophyll protein (PerCP) labeled monoclonal rearrangement, tail and other DNAs were also examined by Southern antibodies were used: anti-CD4-PE, anti-CD8-FITC, anti-CD25-PerCP, and hybridization as described previously (16). The TCR probes were gel anti-CD44-FITC (BD PharMingen, San Diego, CA). purified and then labeled for hybridization. Hind III-digested DNAs were Histopathologic analysis. Each mouse was bled while under ether hybridized with the Jh2 fragment. anesthesia. At autopsy, organs were fixed with 10% formalin in 0.01 mol/L Flow cytometry analysis. Single-cell suspensions were prepared from phosphate buffer (pH 7.2) and embedded in paraffin. Sections were stained the thymi and splenic tumors of each mouse, which were then blocked with with H&E stain for histopathologic examination by light microscopy. The anti-FcR antibody (2.4G2) for 10 minutes on ice to inhibit the interaction of immunohistochemistry and analysis for CD3, B220, CD4, and CD8 antigens the staining reagents with the cell surface. Multicolor flow cytometric were done using the streptavidin and biotin technique. Anti-CD3 antibody analysis was done using LSR and CellQuest software (Becton Dickinson, was purchased from Serotec (Oxford, United Kingdom). Anti-B220, CD4, Franklin Lakes, NJ) on viable cells as determined by forward light scatter and CD8 antibodies were obtained from BD PharMingen.