British Journal ofHuernatology, 1995, 91, 1025-1030

RAPID PAPER

Increased expression of the PRAD-I ICCNDI gene in hairy cell leukaemia

FRANCESC B 0 S C H . l E L ~ A S c A M P 0 , 2 ’ 3 PEDRO JARBS,2’3 STEFANIA PITTALUGA,4 JOSEP M U N O Z , 2 IRACEMA N A Y A C H , 2

MIGUEL ANGEL P I R I S , ’ CHRIS DEWOLF-PEETERS,4 ELAINE s. JAFFE,6 CIRIL ROZMAN,’ EMILIO MONTSERRAT’ AND

ANTONIO C A R D E S A ~ ‘Postgraduate School of Haematology ‘Farreras Valenti’, Hospital Clinic, University of Barcelona, Barcelona, Spain; ’Department of Anatomic Pathology, Hospital Clinic, University of Barcelona, Barcelona, Spain; Department of Basic Medical Sciences, University of Lleida, Lleida, Spain; 4Department of Pathology, University of Leuven,

Leuven, Belgium; 5Department of Anatomic Pathology, Hospital ‘Virgen de la Salud’, Toledo, Spain; and ‘Hematopathology Section, Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.

3

Received 16 August 1995: accepted for publication 19 September 1995

Summary. The PRAD-l/CCNDl gene encodes Cyclin D1, a cyclin involved in cell cycle regulation at the GI-S transition. Over-expression of this gene is a highly specific molecular marker of mantle cell lymphomas (MCLs), but it may also be up-regulated in some chronic lymphoprolifera- tive disorders, mainly chronic lymphocytic leukaemia.

We have examined PRAD-l/CCNDl gene expression by Northern blot and Western blot analysis in a series of 18 hairy cell leukaemias (HCLs), nine other splenic malignant lymphoproliferative disorders, and three normal/reactive spleens. Over-expression of the mRNA PRAD-l/CCNDl gene was observed in 16/18 HCLs, including one case of hairy cell leukaemia variant, whereas this molecular alteration was not found in other cases examined. mRNA levels varied from

case to case, but they were lower than those observed in MCLs. At the protein level, Western blotting analysis showed Cyclin D1 protein expression in the 11 HCLs analysed. No bcl-1 rearrangements were seen with the MTC, p94PS and PRAD-1 (A-PI-4) probes used, and no PRAD-l/CCNDl gene amplification was detected in any case. These findings indicate that PRAD-l/CCNDl is over-expressed at mRNA and protein levels in a high number of HCLs. However, the levels of expression are much lower than in MCLs, and this expression is not associated with bcl-1 rearrangements or PRAD-l/CCNDl gene amplification.

Keywords: hairy cell leukaemia, t( 11:14), PRADl/CCNDl, Cyclin D1, oncogenes.

Hairy-cell leukaemia (HCL) is a rare monoclonal prolifera- tion of mature activated B lymphocytes that is clinically characterized by pancytopenia, splenomegaly, and an increased risk of opportunistic infections (Bouroncle et al, 1958). Peripheral blood and bone marrow (BM) examina- tion often demonstrate hairy cells, and BM morphology is, in almost all cases, diagnostic. The hairy cells have a characteristic immunophenotype. with expression of CDllc and CD25 in addition to monoclonal pan B-cell markers (CD19, CD20 and CD22), and lack of CD5 expression (Robbins et al, 1993).

Chromosomal abnormalities studies in HCL are limited by the rarity of the disease and the difficulty in obtaining cell material and metaphase cells. The frequency of clonal

Correspondence: Dr Elias Campo. Department of Anatomic Pathol- ogy, Hospital Chic, ViUarroel 170, 08036-Barcelona, Spain.

Q 199 5 Blackwell Science Ltd

abnormalities detected varies from 25% to 67% (Juliusson & Garton, 1993; Golomb et al, 1978; Ueshma et al. 1983: Brito-Babapulle et al, 1986: Haglund et al, 1994: Kluin- Nelemans et al, 1994). Although no specific chromosome alterations have been described in HCL, recent reports have drawn attention to some abnormalities, mainly those involving chromosome 5 (Haglund et al, 1994: Kluin- Nelemans. 1994). However, there are few studies of molecular genetic alterations in HCL. Some genes have been implicated in HCL tumorigenesis on the basis of the karyotypic findings (i.e. RAS and RASA genes) (Haglund et al, 1994). and an increased expression of the src proto- oncogene without genetic abnormalities was found (Lynch et al, 1993). PRAD-I/CCNDl which encodes Cyclin D 1 is a gene

initially identified on chromosome 1 l q l 3 in parathyroid adenomas (Motokura et al, 1991; Rosenberg et d, 1991b).

1025

1026 Rapid Paper This gene product seems to play an important role in the cell cycle control at the GI-S transition, probably by interacting with the retinoblastoma gene protein (Jiang et al, 1993; Dowdy et al, 1993). Moreover, transfection studies have demonstrated that PRAD-ZICCNDI may function as a cooperating oncogene in the malignant transformation of cells (Hinds e t al, 1994). The PRAD-I/CCNDI gene expression is up-regulated in cell lines or lymphoproliferative disorders with the t( 11;14) translocation (Rosenberg et al, 1991 b; Seto et al, 1992) and it is considered the bcl-1-related gene. In addition to translocations, other mechanisms may induce PRAD-I ICCNDI gene over-expression in neoplasms, as over-expression also may occur with gene amplification in some solid tumours, particularly in breast and squamous cell carcinomas of head and neck (Lammie et al, 1991; Schuuring et al, 1992; Tares et al, 1994). In occasional tumour cell lines and human neoplasms over-expression of this gene is not associated with gross genetic abnormalities, indicating that other mechanism may control its expression (Faust & Meeker, 1992).

Recently, we have demonstrated that PRAD-I/CCNDI over-expression in lymphoproliferative disorders is a highly specific and sensitive molecular marker of mantle cell lymphomas (MCL) (Bosch et al, 1994). This gene was also up-regulated in a minor subset of B-chronic lymphocytic leukaemias and in the two HCL cases analysed (Bosch et al, 1994). The frequency of PRAD-IICCNDI gene over-expres- sion in HCL and the possible mechanism leading to this upregulation are unknown.

The aims of this study were to deline the frequency of PRAD-I ICCNDI gene over-expression in a series of 18 HCL, to investigate the mechanism leading to the over-expression of this gene, and to elucidate the role that the PRAD-I / CCNDl gene over-expression may play in the development of this chronic lymphoproliferative disorder.

MATERIALS AND METHODS

Tissue samples. A total of 30 spleens (from 18 patients with HCL, six with splenic marginal zone lymphomas (SMZL), one with follicular lymphoma (FL), two with chronic lympho- cytic leukaemias (CLL), and three with non-malignant conditions) and two blood samples from patients with HCL were included in the study on the basis of the availability of snap-frozen samples for molecular studies. Two of the HCL and the six SMZL were included in a previous study (Bosch et al, 1994). These cases were obtained from the files of the Departments of Pathology of the Hospital Clinic, University of Barcelona, Barcelona, Hematopathology Section, Labora- tory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Md., U.S.A., and Department of Pathology, Leuven University, Leuven, Belgium.

RNA extraction and Northern blotting analysis. Total cellular RNA was isolated from frozen tissues by guanidine isothiocyanate extraction and cesium chloride gradient centrifugation. 8pg of total RNA from each sample were electrophoresed on a denaturating 1.2% agarose formalde- hyde gel and transferred to Hybond-N membranes (Amersham, U.K.). As a positive control, 5 pg of total RNA

from a squamous cell carcinoma of the larynx previously known to have PRAD-1 ICCNDI gene amplification and mRNA over-expression, and 8 pg of RNA from a MCL that also over-expressed this gene were included in all the gels. The membranes were prehybridized, hybridized with the [a- 32P]dCTP labelled PRAD-1 cDNA probe, washed, and the filters autoradiographed for 24-72 h as previously described (Bosch et al. 1994). Longer exposures (2-3 weeks) were also taken to observe possible baseline signals.

Hybridization signals of different radioautographic expo- sures within the linear response range were quantified using a UVP-GDS5000 video densitometer (UVP Inc., San Gabriel, Calif.). The hybridization signals of each case were normal- ized to the respective 28s RNA band for loading differences and to the signal of the same control case (MCL) present in all the blots to standardize expression levels. Expression is presented as arbitrary units related to the expression of the MCL control case.

DNA extraction and Southern blotting analysis. DNA extraction was performed from additional frozen material available in 13 cases. High molecular weight DNA was obtained by the standard proteinase K/RNAse treatment and phenol-chloroform extraction as described previously (Bosch et al, 1994). DNA from each case (lObg) was digested with EcoRI, HindIII, BarnHI and SstI restriction enzymes (BRL, Gaithersburg, Md.). The digested DNA was separated on 0.8% agarose gels, and transferred to nylon membranes (Hybond-N, Amersham, Amersham. U.K.) for Southern blotting. The membranes were prehybridized, hybridized, and washed accordingly previously described methods (Bosch et al, 1994). The filters were then autoradiographed using intensifying screens at - 70°C.

Table 1. PRADl/CCM?l gene expression in HCL.

Densitometric Protein analysis (AU) expression bcl-1

Case HCLjMCL (Western blot) rearrangement

1025 0.1 1032 0.2 1284 0.24 1340 0.34 4893 0 2 6 4989 0.1 5276 0.1 5480 <0.1 5568 0 1 8 6220 0.12 6425 0.1 204 1.05 220 0.2

1583 0.21 1820 0.1 2412 c O . 1 417 0.2

5958 0.2

+ + + + + + ND + ND + + ND ND + + ND ND ND

G G G G G G G ND ND G G ND G G G G G G

Abbreviations: AU, arbitrary units of mRNA cyclin U1 over- expression: HCL, hairy cell leukaemia: G, germline: ND, not done.

c) 1995 Blackwell Science Ltd, British JoumI of Haematolog~ 91: 1025-1030

Rapid Paper 102 7 Probes. Probes were radiolabelled using a random primer

DNA labelling kit (Promega Corp., Madison, Wis.) with [a- 32P]dCTP. The PRAD-I/CCNDI probe used was 1.4 kb EcoRI fragment (A-P1-4) of the pPL-8 partial cDNA clone of PRAD-IICCNDI gene (kindly provided by Dr A. Arnold, Massachussets General Hospital, Boston, Mass.) (Motokura et ul, 1991).

Southern blots were hybridized with two bcl-1 transloca- tion breakpoint probes including a 2-3 kb Sac1 fragment for the MTC (Tsujimoto et aI, 1984) (kindly provided by Dr Y. Tsujimoto, Winstar Institute, Philadelphia, Pa.) and a 460 bp SmaI-BarnHI DNA fragment from p94PS (Meeker et al, 1991) (kindly provided by Dr T. C. Meeker, IJniversity of California, San Francisco, Calif.).

Western blot. Tissue lysates were obtained from 10 frozen sections of 30pm. The cryostat sections were incubated in 300 p1 of ice-cold lysis buffer (50 m~ Tris-C1. pH 8, 150 m~ NaCI, 0.02% sodium aide, 0.1% SDS, 1% NP-40 and 0.5% sodium deoxycholate) containing 150 pg/ml phenylmethyl sulphonylfluoride (PMSP) and aprotinin 50 pg/ml for 30 min at 4°C. The cell debris was sedimented by centrifugation at 14000 rpm at 4°C for 20min. The clarified supernatants were collected, and the protein content of the lysate was

determined by the Bradford protein assay (Bio-Rad, Her- cules, Calif.). 80 pg of total cellular protein were run per lane on a 12% SDS-PAGE and electroblotted to a nitrocellulose membrane (Amersham, Amersham, U.K.). Membrane was blocked by overnight incubation in 5% dry milk and 0*1%1 tween-20 at 4°C. The blocked membrane was then incubated with the DCS-1 anti-Cyclin D1 monoclonal antibody (Novocastra, Newcastle, U.K.) at final dilution 1 : 50 for 1 h 30 min. The membrane was then washed three times with PBS and 0.25% gelatine for 15min each and exposed to sheep antimouse conjugated to horseradish peroxidase (Amersham, U.K.) at a I : 1000 dilution for 1 h 30 min. After washing, bound antibody was detected with chemiluminescence detection procedures according to the manufacturer recommendations (Amersham, Amersham. U.K.).

RESULTS

The results of Northern blot analysis performed in the 18 HCL studied are summarized in Table I. PRAD-I/CCNDI expression was clearly observed in 16/18 cases examined (Fig 1). The levels of PRAD-IICCNDI gene expression varied

Fig 1. (A) Northern blot analysis of H a s , other splenic lymphomas, and normal spleens. All the HCLs show Cyclii D1 expression. Two transcripts of normal size are seen in most of the cases. In case 6220 the signal of the 4.5 kb message is very low. No signal is seen in normal spleen and other splenic lymphomas. Total RNA from a squamous cell carcinomas of the larynx (control) and a mantle cell lymphoma (MCL) were used as positive controls. (B) The ethidium bromide-stained gel of all cases showing loading of RNA in each lane.

@) 1995 Blackwell Science Ltd, British Journal of Haernatology 91: 1025-1030

102 8 Rapid Paper

Fig 2. Western blot analysis of Cyclin D1 protein in HCLs. All the HCLs and the mantle cell lymphoma (MCL) used as positive control show protein expression whereas the signal in samples from a follicular lymphoma (FL) and the two normal spleens (NS) is negative.

from case to case. The mean of expression was 0.23 arbitrary units (AU) (range 0.1-1.05 AU). A low signal was observed in two cases (AU < 0.1). No correlation was observed between the levels of PRAD-I ICCNDI mRNA over-expres- sion and the clinical or pathologic characteristics of the patients. One case (no. 1820) was a hairy cell variant (Catovsky & Foa, 1990) and the PRADIICCNDI level was similar to other typical H a s . No PRAD-I/CCNDI over- expression was observed in any of the spleens from the other (six SMZL, two CLL, one FL) lymphoproliierative disorders or normal spleens analysed. In some of these cases, however, a very low signal was also detected after longer exposures (1-2 weeks).

The PRAD-IICCNDI signal in the majority of the over- expressed cases showed two transcripts of 4.5 and 1.5 kb, with the intensity of the smaller transcript being higher than the 4-5 kb message, as previously described in MCLs (Seto et al, 1992: Bosch et al, 1994). However, three cases showed almost no signal of the 4.5 kb message. No transcripts of anomalous size were detected in any of the cases.

DNA was available for Southern blot analysis in 1 3 cases. No bcl-I rearrangements with the MTC, p94PS and PRADl (a-P1-4) probes used were observed. Similarly, no PRAD-I/ CCNDl gene amplification was detected in any of the cases.

In order to determine whether the PRAD-I/CCNDI mRNA expression was also associated with an increase in protein levels, protein extracts were obtained from additional tissue available in 11 HCL. Samples from two mantle cell lymphomas, two follicular lymphomas, and two normal spleens were also included as positive and negative controls. Western blotting analysis showed Cyclin D1 protein expression in the 11 HCLs and MCLs but it was negative in the non-Hodgkin's lymphomas and normal spleens (Fig 2).

DISCUSSION

Chromosomal translocations and deregulation of the trans- posed genes are important mechanisms in the pathogenesis of several non-Hodgkin's lymphomas. The t( 11: 14)(q13:q32) translocation and bcl-1 rearrangement have been shown to be selectively associated with mantle cell lymphomas and less frequently with other B-cell lympho- proliferative disorders. Several studies have consistently

identified PRAD-IICCNDI as the gene activated by this translocation (Motokura et al, 1991: Withers et al, 1991; Komatsu et al, 1994). We and others have recently shown that PRAD-I ICCNDI gene over-expression at mRNA (Rosenberg et al. 1991b; Bosch et al, 1994: Rimokh et al, 1993) and protein levels (Yang et al, 1994) is a highly specific characteristic of mantle cell lymphomas. Only some aggressive CLLs bearing the t(ll:14) and two HCLs were found to express high levels of PRAD-I/CCNDI in addition to MCLs (Bosch et ul, 1994: Yang et al, 1994).

In the present study we have shown that PRAD-I/CCNDl is almost constantly expressed in H a s . However, the levels of mRNA expression are 2-10-fold lower than those observed in the 19 MCL examined in our previous study (Bosch et al, 1994). We have also found by Western blot analysis that Cyclin D 1 is expressed at the protein level in all HCL examined. Recently, Zukerberg et a1 (199 5) have observed protein expression in tumour cells in one of 15 HCL examined by immunohistochemistry using an anti- PRADl polyclonal antibody. This study was performed on formalin-fixed paraffin-embedded material. The low expres- sion of PRAD-I/CCNDI in HCLs in comparison to MCLs may explain the low number of positive cases detected by immunohistochemistry.

PRAD-I /CNNDI over-expression in mantle cell lympho- mas and other lymphoproliferative disorders is associated with translocations involving the 1 lq13 region (Rosenberg et al, 1991b: Bosch et al, 1994: Rimokh et al, 1993). Over- expression may also occur with gene amplification in some solid tumours, particularly in breast and head and neck carcinomas (Lammie et al, 1991: Schuuring et al, 1992: Jares et aI, 1994). We have not detected the presence of bcl-1 rearrangements or amplifications in any of the HCLs examined with three different probes, indicating that those are not the mechanisms implicated in PRAD-7 ICCNDI expression in this disorder. Bcl-1 rearrangements may occur in a 110 kb region centromeric to PRAD-IICCNDI gene (Rosenberg et ul. 1991a, b: Withers et al, 1991). Previous studies have detected bcl-1 rearrangements in up to 70% of the MCLs using different probes spanning this breakpoint region (Williams et al, 1992). Although we cannot completely rule out the possibility of rearrangements beyond the regions examined in our HCLs, translocations do

0 1995 Blackwell Science Ltd, British Journal of Haemutology 91: 1025-1030

Rapid Paper 1029 not seem to be the main mechanism implicated in PRAD-II CCNDZ expression in this disorder. The low levels of mRNA expression in HCLs compared to those observed in M C h might be related to different mechanisms in gene activation. In addition, some MCLs and cells lines carrying l lq13 translocations have PRAD-I/CCNDI mRNA messages of anomalous sizes (Seto et ul, 1992; Withers et al, 1991; Rimokh et al, 1993). These anomalous transcripts are related to rearrangements in the 3’ region of the gene (Seto et al. 1992; Komatsu et ul, 1994). No anomalous transcripts were seen in any of the HCLs in our study, supporting the idea that translocations do not seem to occur in this disorder.

The biological significance of PRAD-I ICCNDZ expression in HCL is unclear. Among haemopoietic cells, macrophages are the only cells that normally express PRAD-I ICCNDI and use this pathway in their cell-cycle progression (Withers et al, 1991; Inaba et al, 1992; Matsushime et al, 1991, 1994). Several studies have shown some phenotypic and functional similarities between hairy cells and cells of monocyte/ macrophage lineage (Knowles, 1992). Although the B-cell lineage of HCL is now well established (Korsmeyer et ul, 1983), PRAD-IICCNDI expression could also be a func- tional characteristic shared by these cells.

Cyclin D1 participates in the cell-cycle control at Gl/S transition and is required for G 1 progression in €3-cell lines with bcl-1 rearrangements (Lukas et al, 1994). Cyclin D1 over-expressing cells have abnormal proliferative character- istics with shortened G 1 phase and less dependence on growth factors (Jiang et al, 1993; Quelle et al, 1993). Conversely, microinjection of anti-Cyclin D1 antibodies or antisense vectors arrest the cells in G 1 (Quelle et al, 1993; Baldin et al, 1993). The role of Cyclin D1 in HCIs is not clear. In spite of the Cyclin D1 expression, the proliferative activity of this disorder is very low. Similarly, Cyclin D1 has also been found in some resting cells, such as murine hepatocytes, with no significant variation in its levels throughout cell- cycle progression, indicating that expression of this cyclin is not always associated with high proliferative activity (Loyer et al, 1994). The biological significance of Cyclin D1 in these circumstances is not known.

In conclusion, we have shown that the PRADZ /CCNDI gene is over-expressed at the mRNA and protein levels in the majority of HCLs. However, the levels of expression are much lower than seen in MCLs. This expression is not associated with bcl-1 rearrangements or PRAD-I/CCNDI gene ampli- fication. The biological significance of PRAD-2 ICCNDI gene expression in HCL warrants further investigation.

ACKNOWLEDGMENTS

The authors thank Dr A. Arnold (Massachussets General Hospital, Boston, Mass.), Y. Tsujimoto (Winstar Institute, Philadelphia, Pa.), and T. C. Meeker (University of California, San Francisco, Calif.) for the gift of the probes.

This work was supported by grant SAF 1195/93 from CICYT, Ministerio de Educaction y Cien&a, and grant FIJC 94/P-CR from International Foundation ‘Josep Carreras’. Francesc Bosch is the recipient of the Young Investigators in Haematology Award 1995 from the Catalan Society of

Haematology. Pedro Jares is a fellow supported by the Spanish Ministerio de Educaci6n y Ciencia. Josep Muiioz holds Research Fellowships from the Hospital Clinic of Barcelona. Iracema Nayach is a fellow supported by a grant from CIRIT, Generalitat de Catalunya.

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0 1995 Blackwell Science Ltd. British Journal ofHaematology 91: 1025-1030