Leukemia (1999) 13, 1335–1342 1999 Stockton Press All rights reserved 0887-6924/99 $15.00 http://www.stockton-press.co.uk/leu Selective usage of D-type cyclins in lymphoid malignancies R Suzuki1,2, H Kuroda1, H Komatsu1, Y Hosokawa1, Y Kagami2, M Ogura2, S Nakamura3, Y Kodera4, Y Morishima2, R Ueda5 and M Seto1 1Laboratory of Chemotherapy, 2Department of Hematology and Chemotherapy, and 3Department of Pathology and Clinical Laboratories, Aichi Cancer Center; 4Department of Internal Medicine, Japanese Red Cross Nagoya First Hospital; and 5Second Department of Internal Medicine, Nagoya City University School of Medicine, Nagoya, Japan Three D-type cyclins, cyclin D1, D2 and D3, belong to the G1 some 14,11–13 in much the same way as the c-myc gene is cyclin, which regulates the G1/S transition of the cell cycle, and affected by t(8;14) translocations in Burkitt’s lymphoma14 and feature highly homologous amino acid sequences. The cyclin the BCL-2 gene by t(14;18) translocations in follicular lym- D1 gene was found to be transcriptionally activated in B-lymph- 15 oid malignancies with t(11;14), but available information is lim- phomas. The breakpoints occur at variable distances from ited regarding expression of cyclin D2 and D3 in hematopoietic the cyclin D1 gene, typically up to 120 kb, but the net effect malignancies. We examined the expressions of three D-type appears to be the transcriptional activation of cyclin cyclins to investigate how these homologous genes are differ- D1.12,13,16–18 Several groups including ours have reported entially used. Northern blot hybridization with densitometric cyclin D1 overexpression in B cell malignancies, ie mantle analyses was performed to examine 64 cell lines and 159 cell lymphoma (MCL),18–23 splenic marginal zone lym- patients with various hematopoietic malignancies. Among 24 20,25 lymphoid malignancies, cyclin D1 overexpression was exclus- phoma, hairy cell leukemia, chronic lymphocytic leuke- ively detected in B cell malignancies accompanied by a genetic mia (CLL),20,26 and multiple myeloma/plasma cell leuke- event consisting of 11q13 chromosomal translocation, con- mia.18,27,28 In solid tumors, amplification and overexpression sisting of 13 of 19 (68%) patients with mantle cell lymphoma, of cyclin D1 gene have been demonstrated in a variety of two of 11 (18%) with B-chronic lymphocytic leukemia, and one human cancers including breast carcinomas,29,30 head and of six (17%) with multiple myeloma. The cyclin D2 expression neck squamous cell carcinomas,29,31 and hepatocellular carci- was significantly higher in T cell malignancies than in B cell 32 malignancies (P = 0.003 for cell lines and P Ͻ 0.0001 for patient nomas. Furthermore, mice containing an MMTV-cyclin D1 samples, respectively). In the T cell malignancies, cyclin D2 transgene were reported to develop breast tumors, thereby overexpression was predominantly recognized in those with confirming in vivo that cyclin D1 is indeed an oncogene.33 mature phenotype. Furthermore, cyclin D2 expression was An increased cyclin D2 expression has been reported in B- upregulated by phytohemagglutinin (PHA) stimulation of nor- CLL cases34 and human T-lymphotropic virus type I (HTLV-I) mal T-lymphocytes, suggesting that this simply represents the 35 proliferation status of mature T cells. Although cyclin D3 was infected T cell lines. Although no translocational activation ubiquitously expressed, its expression was reduced in lymph- of the cyclin D2 gene has been found in human cancers, oid malignancies with cyclin D1 or D2 overexpression. In cyclin D2 amplification was noted in colorectal cancer,36 B myeloid leukemias, although three D-type type cyclins were dif- cell lymphoma37 and B-CLL.38 Cyclin D3 gene has not yet ferentially expressed, no preference for particular D-type been identified as a proto-oncogene. cyclins was found. This selective usage of D-type cyclins in The three D-type cyclins are highly homologous in amino lymphoid malignancies suggests an existence of a regulatory acid sequences in the cyclin box (homology 73–83%), com- mechanism among three D-type cyclins. 10 Keywords: cyclin D; overexpression; acute leukemia; malignant paring with other types of cyclin A, B, C, and E (21–36%). lymphoma; adult T cell leukemia In addition, these three D-type cyclins possess similar function to regulate the cell cycle of G1/S phase transition.39,40 In spite of the similarities in sequence and function, their chromo- Introduction somal localizations are different: cyclin D1 is located at 11q13, cyclin D2 at 12p13, and cyclin D3 at 6p21, indicating Cyclins were first identified as proteins whose levels fluctuate that they may possess different biological roles. The dramatically during the cell cycle,1 but later they came to be expression of these D-type cyclins has not yet been examined recognized as regulatory subunits for serine/threonine kinases systematically in hematopoietic malignancies, and it thus of the cdc2 or cyclin-dependent kinase family.2 Cyclin D1, remains unclear how these three similar genes are used in the first identified as a PRAD1 gene product in parathyroid aden- hematopoietic system. We therefore investigated their correla- omas,3–5 was cloned by the rescue of a budding yeast lacking tive expression in various hematopoietic malignancies to clar- G1 cyclins.6,7 Separately, three murine cyclin-like genes, ify the difference and the biological significance in employ- CYL1, CYL2 and CYL3, were isolated as colony-stimulating ment of these three D-type cyclins. factor 1 responsive genes, and CYL1 was proved to be the murine cyclin D1 gene.8 Human cyclin D2 and D3 were identified as homologues of the murine CYL2 and CYL3 Materials and methods genes.9,10 In B cell malignancies with 11q13 translocation, BCL-1 Cell lines locus on chromosome 11, just upstream of cyclin D1 gene, fuses to the immunoglobulin heavy chain gene on chromo- The cell lines used in this study are listed in Table 1. All these cell lines were grown in suspension at between 0.2 and 1.0 × 106 cells/ml in RPMI 1640 medium containing 10% fetal Correspondence: R Suzuki, Laboratory of Chemotherapy, Aichi Can- cer Center, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan; calf serum. Cultures for FLAM-76 required an additional Fax: 81 52 763 5233 4 ng/ml of interleukin-6, for IPAT-1, KPNT-1 and WHN-2, Received 19 March 1999; accepted 14 May 1999 70 U/ml of interleukin-2, and those for SKNO-1 and OIH-1, D-type cyclins in hematopoietic malignancies R Suzuki et al 1336 Table 1 Cell lines used in this study Cell line Cell line B cell lines T cell lines Precursor B cell leukemia NALM-6 Precursor T cell leukemia HPB-ALL NALM-18 MOLT-3 KOCL-33 MOLT-4 KOCL-44 Jurkat KOCL-45 CCRF-CEM KOCL-48 T cell lymphoma Karpas299 KOCL-51 SUDHL1 KOCL-50 AST-1 RS 4;11 Hut-78 KOPB-26 Hut102 NALL-1 ATL HTLV-I(+) ATN-1 KOPN-1 IPAT-1 B cell lymphoma SUDHL4 KPNT-1 SUDHL6 ATL HTLV-I(−) ATL-5T SUDHL9 WHN-2 SUDHL10 Myeloid leukemia cell lines RC-K8 Karpas422 Myeloblastic leukemia HL60 ACOL-1 Kasumi-1 Burkitt’s lymphoma Raji SKNO-1 Daudi NKM-1 Manca HYT-1 NCU-L3 SKK-1 Mantle cell lymphoma SP-49 OIH-1 SP-53 NC-02 Myeloma with t(11;14) NOP-2 Monocytic leukemia ME-1R KMS-12 NOMO-1 FLAM76 THP-1 Myeloma NOP-1 IMS-M1 AMO1 U937 NOL3 Erythroid leukemia HEL K562 Breast cancer ZR75-1 Megakaryoblastic leukemia CMK MEG-01 ATL, adult T cell leukemia; HTLV-I, human T-lymphotropic leukemia virus type-I. 10 ng/ml of granulocyte–macrophage colony-stimulating Peripheral blood lymphocytes factor. Peripheral blood lymphocytes (PBL) were obtained from nor- mal volunteers under informed consent, and were separated Patients by Ficoll–Hypaque gradient centrifugation. Anti-CD4, anti- CD8 and anti-CD19 monoclonal antibodies combined with A total of 159 patients with various hematopoietic malig- immunomagnetic beads (MBL, Nagoya, Japan) were used for fractionation of T cells and B cells, according to the manufac- nancies diagnosed at Aichi Cancer Center Hospital, Nagoya, turer’s protocol. The PBLs obtained from healthy individuals Japan was examined. These patients consisted of nine cases of precursor B-lymphoblastic leukemia, 20 of diffuse large B were cultured in RPMI 1640 with 10% fetal calf serum in the presence of 30 ␮g/ml PHA-P (Difco Laboratories, Detroit, MI, cell lymphoma (DLBL), 21 of follicular center cell lymphoma ° (FCL), 19 of MCL, 11 of B-CLL, six of myeloma, seven of pre- USA) in a 5% CO2 incubator at 37 C for 1 to 48 h. cursor T-lymphoblastic leukemia/lymphoma (T-ALL/LBL), four of peripheral T cell lymphoma (PTCL), 21 of adult T cell leuke- mia (ATL) and 31 of acute myeloid leukemia (AML). The clini- Northern blot analysis cal records of all these cases were reviewed. The diagnosis of the lymphoid malignancies was based on REAL classi- Total RNA was extracted with the guanidinium isothiocyanate fication,41 and of AMLs on French–American–British classi- and cesium chloride method. Five micrograms of total RNA fication.42,43 For patients with leukemia, bone marrow or per- was applied to each lane and Northern blot analyses were ipheral blood samples containing more than 80% of leukemic performed using Hybond-N plus filter (Amersham-Japan, cells were selected for this study. For patients with lymphoma, Tokyo) by capillary blotting method according to the manu- biopsied specimens containing more than 50% of lymphoma facturer’s instructions. Probes were radiolabeled using a ran- cells determined by flow cytometric analyses at diagnosis dom primer DNA labeling kit (Nippon gene, Tokyo) with ␣- were selected, and those with less tumor cells were excluded 32P-dCTP.