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 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, D1, D2 and D3, belong to the G1 some 14,11–13 in much the same way as the c-myc is cyclin, which regulates the G1/S transition of the , 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 and D3 in hematopoietic the 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 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 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 ( 73–83%), com- mechanism among three D-type cyclins. 10 Keywords: ; overexpression; acute leukemia; malignant paring with other types of , 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/ 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 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 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 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. Sheared human placenta DNA was included in the from this study. hybridization solution at a concentration of 100 ␮g/ml to D-type cyclins in hematopoietic malignancies R Suzuki et al 1337 eliminate cross-hybridization with ribosomal RNA. Washing Cyclin D3 expression was detected in most of the cell lines conditions were done to a final stringency of 0.1 × SSC, 0.1% examined: 27 of 31 B cell lines, 14 of 15 T cell lines and 15 SDS, 65°C. Total RNA from cell line ZR75-1 for cyclin D1, of 17 myeloid cell lines. However, its level was reduced in Hut102 for cyclin D2 and Raji or CMK cell lines for cyclin lymphoid malignancies overexpressing cyclin D1 or D2 D3 were applied to each gel as a standard for Northern analy- (Figure 2a, b and c). Thus, cyclin D3 overexpression was sis (Figure 1). The probes used for cyclin D1 mRNA expression noted in 22 of 31 B cell lines, 10 of 15 T cell lines and 10 of was a 0.9 kb PstI/HindIII fragment of the cDNA clone ␭P1-4, 17 myeloid cell lines. which contained a part of the open reading frame (ORF), a kind gift from Dr Andrew Arnold.4 Human cyclin D2 cDNA probe was a 0.85 kb total ORF fragment, a generous gift from D-type cyclin expression in patients’ tumors Dr Gordon Peters.44 The cyclin D3 probe was a 2.0 kb cDNA clone ␭A7, generously provided by Dr Andrew Arnold.45 The The results are summarized in Table 2. Cyclin D1 expression loading and integrity of the RNA on each gel were checked was observed in 16 patients with B cell malignancies, but by 28S ribosomal RNA level visualized by ethidium bromide none with T cell malignancies. These cyclin D1 overexpress- staining. A part of the examination was performed by repro- ing malignancies included 13 of 19 MCLs, two of 11 B-CLLs, bing the filters for the detection of different cyclins after con- one of six myelomas. In the myeloid malignancies, the cyclin firming the reproducibility of the reprobing. For comparison, D1 overexpression was also infrequently seen in two of 31 films exposed for 24-h were used for densitometric analysis. AMLs. Cyclin D2 was expressed in 24 patients with B cell, 24 with T cell, and 21 with myeloid . It was identified as Densitometric analysis overexpression in four of nine precursor-B ALLs, one of 20 DLBLs, one of 21 FCLs, two of 11 B-CLLs, one of six mye- Densitometric scanning of autoradiographic signals was per- lomas, three of seven T-ALL/LBLs, three of four PTCLs and 15 formed by using an Image Analyzer V10 (Toyobo, Osaka, of 21 ATLs, with a significantly higher incidence in T cell than Japan). The cyclin D1 signal of ZR75-1 was arbitrarily defined in B cell neoplasms (P Ͻ 0.0001). as 100% and the expressions of each of the sample were cal- Cyclin D3 was expressed in the majority of the cases exam- culated and then corrected according to the corresponding ined. However, its expression was reduced in lymphoid 28S ribosomal RNA levels on ethidium bromide staining. The malignancies with cyclin D1 or D2 overexpression, while it standard intensity for cyclin D2 (Hut 102 cell line) was calcu- was not always decreased in AML cases, supporting the obser- lated as 170 relative percent, and that for cyclin D3 (Raji or vation in the cell lines. Namely, overexpression of cyclin D3 CMK) as 100%. The expression of all other cell lines and was observed in all of nine pre-B ALLs, 12 of 20 DLBLs, 13 patient samples was evaluated by comparing with these stan- of 21 FCLs, one of 19 MCLs, six of 11 B-CLLs, three of six dards. The values from patient samples were re-corrected myelomas, three of seven T-ALL/LBLs, one of four PTCLs and according to the tumor cell ratio determined by phenotypic two of 21 ATLs, with significantly higher incidence in B cell analyses. Expression levels of less than 5 relative percent were than in T cell neoplasms (P = 0.0013). defined as no expression. Because cyclin D1 is silent in nor- mal lymphocytes, detectable expression of cyclin D1 was regarded as overexpression. Overexpression of cyclin D2 and Relationship between cyclin D1 overexpressing D3 was defined as an expression higher than that of PBL for B-lineage cases and 11q13 status lymphoid malignancies, and that of bone marrow mono- nuclear cells for myeloid leukemias. Statistical analyses for In 13 cases of MCL with cyclin D1 overexpression, cyto- comparison of the two groups were performed with ␹2 tests. genetic data was available in five cases. Although one of the five cases showed normal karyotype (46,XX), the remaining four cases demonstrated cytogenetic abnormalities including Results 11q13 translocation; three with t(11;14)(q13;q32) and one with t(11;22)(q13;q11). Another five cases were proved to D-type cyclin expression in cell lines have 11q13 translocation by Southern blotting, and three cases (including the one case with normal karyotype) showed In B cell lines, cyclin D1 overexpression was observed in five germline configuration. In six cases of MCL without cyclin D1 cell lines derived from MCL and myeloma carrying 11q13 mRNA expression, one case showed abnormal karyotype with translocations (Figure 2a). No T cell lines expressed cyclin D1 45,XY,−20,del(11)(q23). Using Southern blotting analyses, no except for one cell line, AST-1, derived from a PTCL patient genetic rearrangement was found in five cases examined (Figure 2b). Cyclin D1 was also expressed in seven of 17 including the one karyotyped case. myeloid cell lines without 11q13 translocations (Figure 2c). In two cases of B-CLL with cyclin D1 overexpression, one Cyclin D2 expression was observed in five of 31 B cell showed complexed karyotypes including an 11q13 abnor- lines, eight of 15 T cell lines and 15 of 17 myeloid cell lines mality, and the other, a normal karyotype. One case of mye- (Figure 2a, b and c). Among these, one B cell lymphoma cell loma with cyclin D1 overexpression showed a karyotype with line, three myeloma cell lines, three T cell lymphoma cell 46,XY,t(11;14)(q13;q32). lines, five ATL cell lines and 13 myeloid leukemia cell lines were regarded as overexpressing cyclin D2. The incidence of cyclin D2 overexpression was significantly higher in T cell D-type cyclin expression in normal lymphocytes lines than in B cell lines (P = 0.003). Its expression level had a predominant tendency in T cell lines of mature phenotype Cyclin D1 expression was not recognized in either the stimu- and was the same for HTLV-1 positive and negative T cell lated or unstimulated lymphocytes derived from healthy vol- lines. unteers (Figure 3). Although both cyclin D2 and D3 were D-type cyclins in hematopoietic malignancies R Suzuki et al 1338

Figure 1 Northern blot analysis of various hematopoietic cell lines with D-type cyclins. 5 ␮g of total RNA was applied to each lane. ZR 75- 1, Hut102 and CMK were used as positive controls of cyclins D1, D2 and D3, respectively. Only one D-type cyclin was expressed in SKNO- 1 (cyclin D2), THP1 and U937 (cyclin D3), whereas all three were expressed in HL60 and ME-1R cell lines.

expressed in T cells and B cells, cyclin D2 expression was than genetic translocation might be responsible for this higher in T cells, while cyclin D3 in B cells. The expression unusual expression of cyclin D1 in myeloid leukemia. The level of cyclin D2 was increased more than 10 times in 2 to incidence of cyclin D1 expression in cell lines derived from 6 h by PHA-P stimulation, while that of cyclin D3 was once AML and myeloma was conspicuously higher than in patient decreased in 1 to 2 h and then increased in 6 to 12 h. samples. It seems likely that cyclin D1 expression enhances growth advantage and is responsible for the subsequent estab- lishment of cell lines, as has been reported in Discussion /MTS1/INK4A alteration in various tumors.47 In the present study, cyclin D1 expression was observed in Cyclin D2 overexpression has been reported in ATL cell the majority of MCLs, as well as in a subset of B-CLLs and lines, and was attributed to HTLV-I infection and Tax1 gene myelomas, but not in the other B cell or T cell malignancies. involvement.35 In our study, however, this overexpression was All these cyclin D1 positive cases were restricted in the neo- observed in the majority of mature T cell malignancies includ- plasms having possibilities to possess 11q13 translocations. ing those without HTLV-I association. These include the ATL- This exclusive detection of cyclin D1 overexpression in these 5T and WHN-2 cell lines, established from HTLV-I negative diseases is considered to reflect a genetic alteration of cyclin ATL.48,49 Moreover, in the present study, cyclin D2 expression D1 gene in lymphoid malignancies, which is in line with the level was demonstrated to be increased by PHA stimulation literature.46 Indeed, nine of the 13 cases of MCL with cyclin in normal T cells from healthy volunteers not infected with D1 overexpression showed cytogenetic and/or genetic abnor- HTLV-I. These data suggested that the cyclin D2 over- mality of 11q13 region. Because cytogenetic analysis is not expression in T cell lineage represented the proliferation of always informative due to a low proliferative rate in lym- mature T cells, and was not necessarily caused by HTLV-I phoma cells and Southern blotting analysis is not always cap- infection nor Tax1 gene involvement. Indeed, in ATL patient able of detecting all of the 11q13 translocation due to the samples, cyclin D2 expression levels were lower than those wide range of the breakpoint region, the negative result from in ATL cell lines, which might reflect their respective acti- cytogenetic or Southern blotting analysis does not necessarily vation status or cytokine levels in the culture medium. exclude the presence of 11q13 translocation. In MCL, six Cyclin D2 expression has also been reported in B-CLL and cases (32%) did not express cyclin D1, and the results were lymphoplasmacytic lymphoma cases.34 Werner et al38 in line with those from immunohistochemical, karyotypic and described that 29 of 34 B-CLL cases expressed cyclin D2, genetic analyses. These cyclin D1-negative MCL cases ful- although cyclin D3 expression was not detected in their 10 filled the morphological criteria of MCL, and might constitute cases examined. In our present study, although the number is a distinct entity from cyclin D1-positive typical MCL, as has relatively small, expression of cyclin D2 and D3 was observed been proposed by our group.23 in five and 10 out of 11 cases, respectively, which was some- Cyclin D1 expression was also observed in seven myeloid what different from the data of Werner et al. Because B-CLL leukemia cell lines and two patient samples derived from is rather rare in Japan compared with Western countries,50 the AML, although the expression levels were lower than those in incidence of cyclin D2 expression in B-CLL might reflect epid- B cell malignancies bearing 11q13 translocations. All of these emiological differences as mentioned in the low incidence myeloid samples did not have 11q13 translocations by kary- and frequencies of BCL-2 rearrangement in Japanese follicular otypic analyses, thus an as yet unknown mechanism other lymphoma.51,52 These discrepancies do not deny the possi- D-type cyclins in hematopoietic malignancies R Suzuki et al 1339

Figure 2 The expression level of D-type cyclins in cell lines. Northern blot films were scanned with densitometer and the expressions of each sample were calculated and were corrected by the 28S ribosomal RNA levels on ethidium bromide staining. The cyclin D1 signal of ZR75-1 cell line was arbitrarily defined as 100%, and the cyclin D2 expression of Hut102 (170%) and cyclin D3 expression of Raji or CMK (100%) were used as standard for each cyclin. The levels less than 5 relative percent were defined as no expression. (a) D-type cyclin expression in B cell lines. Cyclin D1 was exclusively expressed in cell lines with t(11;14). Cyclin D2 overexpression was detected in myeloma cell lines without 11q13 translocations and one B cell lymphoma line, ACOL-1. In the remaining cell lines, cyclin D3 dominantly expressed. (b) D-type cyclin expression in T cell lines. Cyclin D1 was expressed in AST-1 cell line at a relatively low level. In T cell lymphomas and ATL cell lines, cyclin D2 was overexpressed, and cyclin D3 was decreased in its expression except for anaplastic large cell lymphoma line, Karpas 299 and SUDHL1. Cyclin D3 was strongly expressed in T-ALL cell lines as recognized in precursor-B ALL cell lines. (c) D-type cyclin expression in myeloid leukemia cell lines. Three D-type cyclins were expressed independently with no apparent inclinations. bility that cyclin D2 acts as a candidate oncogene in B-CLL, ietic malignancies analyzed in the present study. Our data but further investigations are needed to determine the signifi- confirmed that cyclin D1 is silent in lymphoid malignancies cance of cyclin D2 expression in B-CLL. without 11q13 translocations, and demonstrated that cyclin Reports on murine CYL-1, CYL-2 and CYL-3 suggest that D3 rather than cyclin D2 was ubiquitously expressed. Cyclin the expression of cyclin D2 is ubiquitous in growth factor- D3 expression was generally high in B cell malignancies and dependent hematopoietic cells, whereas that of cyclin D1 and even in normal B cells, but was reduced in MCL and multiple cyclin D3 is restricted to macrophages and T cells, respect- myeloma in which cyclin D1 or D2 was overexpressed. This ively.8 This is clearly not the case with the human hematopo- agrees with the recent report of Ott et al,53 describing low or D-type cyclins in hematopoietic malignancies R Suzuki et al 1340 Table 2 D-type cyclin expression in patient samples

Cyclin D1 Cyclin D2 Cyclin D3 overexpression (%) expression overexpression expression overexpression (%) (%) (%) (%)

B cell malignancies (n = 86) 19 28 10 77 51

precursor B-ALL (n = 9) 0 44 44 100 100 DLBL (n = 20) 0 35 5 90 60 FCL (n = 21) 0 19 5 100 62 MCL (n = 19) 68 0 0 16 5 B-CLL (n = 11) 18 45 18 91 55 MM (n = 6) 17 67 17 83 50

T cell malignancies (n = 32) 0 75 66 81 19

T-ALL/LBL (n = 7) 0 43 43 86 43 PTCL (n = 4) 0 75 75 50 25 ATL (n = 21) 0 86 71 86 10

AML (n = 31) 6 68 58 87 58

M0 (n = 2) 0 0 0 0 0 M1 (n = 5) 20 80 80 100 60 M2 (n = 5) 20 80 60 80 40 M3 (n = 5) 0 100 100 100 80 M4 (n = 5) 0 80 60 80 60 M5 (n = 5) 0 20 20 100 60 M7 (n = 4) 0 75 50 100 75

ALL, acute lymphoblastic leukemia; DLBL, diffuse large B cell lymphoma; FCL, follicle center lymphoma; MCL, mantle cell lymphoma; CLL, chronic lymphocytic leukemia; MM, multiple myeloma; LBL, lymphoblastic lymphoma; PTCL, peripheral T cell lymphoma, unspecified; ATL, adult T cell leukemia; AML, acute myeloid leukemia.

Figure 3 D-type cyclin expression in normal lymphocytes. Cyclin D1 was not detected in any of the samples. The expression of cyclin D2 was more intense in T cells than in B cells, and was upregulated more than 10 times by PHA in 2 to 6 h. Cyclin D3 expression was more intense in B cells than in T cells. The expression of cyclin D3 was once downregulated in 1 to 2 h after PHA stimulation and then upregulated in 12 to 24 h after PHA stimulation. D-type cyclins in hematopoietic malignancies R Suzuki et al 1341 missing expression of cyclin D3 in MCL. In the normal and 7 Lew DJ, Dulic V, Reed SJ. Isolation of three novel human cyclins neoplastic cells of T cell lineage, cyclin D3 and D2 were pref- by rescue of G1 cyclin (Cln) function in yeast. Cell 1991; 66: erentially expressed in their immature and mature stage, 1197–1206. 8 Matsushime H, Roussel MF, Ashmun RA, Sherr CJ. Colony-stimul- respectively. It is similarly suggested in the mature T cells that ating factor 1 regulates novel cyclins during the of the cyclin D2 is expressed through the cell cycle of G1/S tran- cell cycle. Cell 1991; 65: 701–713. sition, and cyclin D3 is consecutively downregulated. In PHA- 9 Inaba T, Matsushime H, Valentine M, Roussel MF, Sherr CJ, Look stimulated normal PBLs, cyclin D3 expression decreased in AT. Genomic organization, chromosomal localization, and inde- the first 1 to 2 h and subsequently upregulated in 12 to 24 h. pendent expression of human cyclin D genes. Genomics 1992; Because PBLs are a heterogeneous population including T 13: 565–574. 10 Xiong Y, Menninger J, Beach D, Ward DC. Molecular cloning and cells and B cells, it is possible that cyclin D2 upregulation in chromosomal mapping of CCND genes encoding human D-type PHA-stimulated PBLs reflects a direct activation of T cells, and cyclins. Genomics 1992; 13: 575–584. that later upregulation of cyclin D3 arises from B cells stimu- 11 Tsujimoto Y, Yunis J, Onorato-Showe L, Erikson J, Nowell PC, lated by certain factors derived from PHA-activated T cells.54 Croce CM. Molecular cloning of the chromosomal breakpoint of Three D-type cyclins are differentially and selectively B-cell lymphomas and leukemias with the t(11;14) chromosome expressed in lymphoid malignancies, suggesting their different translocation. Science 1984; 224: 1403–1406. role in the cell cycle control and the oncogenic activities. 12 Rosenberg CL, Wong E, Petty EM, Bale AE, Tsujimoto Y, Harris NL, Arnold A. PRAD1, a candidate BCL1 oncogene: mapping and Regarding myeloid leukemias, no tendencies in the expression in centrocytic lymphoma. Proc Natl Acad Sci USA expression of three D-type cyclins were recognized in contrast 1991; 88: 9638–9642. to lymphoid malignancies. All three D-type cyclins are 13 Withers DA, Harvey RC, Faust JB, Melnyk O, Carey K, Meeker expressed with a variable intensity in myeloid cell lines, HL- TC. Characterization of a candidate bcl-1 gene. Mol Cell Biol 60, NKM-1, ME-1R, IMS-M1 and Meg-01. On the other hand, 1991; 11: 4846–4853. none of the three cyclin Ds was expressed in three AML (two 14 Croce CM, Nowell PC. Molecular basis of human B cell neoplasia. Blood 1985; 65: 1–7. M0 and one M4) case, which was not observed in any of the 15 Seto M, Jaeger U, Hockett RD, Greninger W, Bennett S, Goldman lymphoid malignancies. It is suggested that certain other mol- P, Korsmeyer SJ. Alternative promoters and exons, somatic ecules might substitute the function of D-type cyclins in these mutation and deregulation of the Bcl-2-Ig fusion gene in lym- cases. Also, further investigation is needed to identify how phoma. EMBO J 1988; 7: 123–131. these three D-type cyclins are differentially employed in 16 Rabbitts PH, Douglas J, Fisher P, Nacheva E, Karpas A, Catovsky myeloid malignancies. D, Melo JV, Baer R, Stinson MA, Rabbitts TH. Chromosome abnor- In conclusion, selective usage of three D-type cyclins in malities at 11q13 in B cell tumours. Oncogene 1988; 3: 99–103. 17 Williams ME, Meekers TC, Swerdlow SH. Rearrangement of the lymphoid malignancies suggests the existence of an underly- chromosome 11 bcl-1 locus in centrocytic lymphoma: analysis ing regulatory mechanism and a functional difference of these with multiple breakpoint probes. Blood 1991; 78: 493–498. three D-type cyclins. The fact that no preference for particular 18 Seto M, Yamamoto K, Iida S, Akao Y, Utsumi K, Kubonishi I, D-type cyclins was found in myeloid leukemia suggests that Miyoshi I, Ohtsuki T, Yawata Y, Namba M, Motokura T, Arnold such a regulatory mechanism may operate predominantly in A, Takahashi T, Ueda R. Gene rearrangement and overexpression lymphoid lineage. of PRAD1 in lymphoid malignancy with t(11;14)(q13;q32) translo- cation. Oncogene 1992; 7: 1401–1406. 19 Komatsu H, Iida S, Yamamoto K, Mikuni C, Nitta M, Takahashi T, Ueda R, Seto M. A variant chromosome translocation at 11q13 Acknowledgements identifying PRAD1/cyclin D1 as the BCL-1 gene. Blood 1994; 84: 1226–1231. The authors are grateful to H Onishi, M Sugiyama, and H 20 Bosch F, Jares P, Campo E, Lopez-Guillermo A, Piris MA, Villamor N, Tassies D, Jaffe ES, Montserrat E, Rozman C, Cardesa A. PRAD- Suzuki for excellent technical assistance. 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