Protein Expression Analysis of Chromosome 12 Candidate Genes in Chronic Lymphocytic Leukemia (CLL)

Leukemia (2005) 19, 1211–1215 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu Protein expression analysis of chromosome 12 candidate genes in chronic lymphocytic leukemia (CLL)

D Winkler1, C Schneider1, A Kro¨ber1, L Pasqualucci2, P Lichter3,HDo¨hner1 and S Stilgenbauer1

1Universita¨t Ulm, Innere Medizin III, Ulm, Germany; 2Institute for Cancer Genetics, Columbia University, New York, USA; and 3Deutsches Krebsforschungszentrum, Heidelberg, Germany

The pathogenic role of trisomy 12 in chronic lymphocytic cells from healthy donors and lymphoma cell lines (JVM-2, leukemia (CLL) remains unresolved, but recently an upregu- EHEB and JURKAT). lated RNA expression level has been observed for chromosome 12 candidate genes. In the current study, the protein expression of chromosome 12 candidate genes was characterized by comparing CLL cases with (n ¼ 58) or without (n ¼ 16) trisomy Materials and methods 12, CD19 þ -B-cells and cell lines (JVM-2, EHEB, JURKAT). Immunoblotting was performed to quantify the levels of AID, Sample characterization APAF-1, ARF3, CCND2, CDK2, CKD4, GLI, MDM-2, p27, Smac/ DIABLO and STAT6 (signal transducer and activator of CD19 þ -B-cells were isolated from buffy coats obtained transcription 6). The cell lines showed distinct expression from healthy donors using anti-CD19 magnetic microbeads patterns for CCND2, MDM-2, p27, Smac/DIABLO and STAT6, and displayed higher levels of CDK2 and CDK4 than the CLL (Miltenyibiotech, Bergisch Gladbach, Germany) according to cases. JURKAT and the CLL cases expressed uniformly high the manufacturer’s instructions. B-cell purity was 497% as levels of p27, but low levels of CCND2. AID expression in the assessed by FACS analysis. CLL cases was weak with slight variations regardless of the Tumor samples were prepared by Ficoll density-gradient subgroup affiliation. The expression of the investigated pro- centrifugation from the blood of patients with CLL and teins was independent of the trisomy 12 status as well as of the molecular cytogenetic analysis as well as VH sequencing were VH mutation status. The comparison of CD19 þ -B-cells with 2,11 CLL revealed higher protein levels in CLL for CDK4, p27, Smac/ performed as described previously. CLL cases showing either DIABLO and STAT6. Further studies including protein expres- a trisomy 12 (n ¼ 58) or a normal karyotype (n ¼ 16) were sion experiments in genetic high-risk subgroups of CLL have to selected. In addition to trisomy 12, two cases had a 17p elucidate whether these proteins qualify as candidates for deletion, one an 11q deletion and 16 cases a 13q deletion. For targeted CLL therapies. the trisomy 12 group, the tumor purity of the sample was defined Leukemia (2005) 19, 1211–1215. doi:10.1038/sj.leu.2403778 by the percentage of cells with trisomy 12 as detected by FISH. Published online 19 May 2005 Keywords: B-CLL; cell lines; trisomy 12 The criteria for the selection of cases with a normal karyotype by FISH were high lymphocyte counts in combination with a high percentage of CD5 þ and CD19 þ cells as measured by FACS. According to these criteria, the median content of clonal cells Introduction was 80% (range: 70–88%) in the trisomy 12 cohort and 88% (range: 44–98%) in the group with a normal karyotype. An Chronic lymphocytic leukemia (CLL) is the most common unmutated VH status was observed in 32 of 45 cases in the leukemia in the Western world and is characterized by typical trisomy 12 group and in 11 of 16 cases in the group with a genomic aberrations, trisomy 12 being among the most frequent normal karyotype. ones.1,2 In several studies, the presence of trisomy 12 has been Clinical data were available for 32 patients. Treatment-free shown to be associated with a more aggressive clinical survival (TFS) after a median follow-up of 59.7 months was 58.3 course.1,3–5 The role of genes potentially involved in the months in the trisomy 12 group (n ¼ 17) vs 60.0 months in cases pathogenesis of CLL with trisomy 12 is not established. While with a normal karyotype (n ¼ 15; P-value 0.57). Patients with the critical region of most aberrations in CLL could be narrowed unmutated VH status (n ¼ 20) had a significantly shorter TFS down to a set of candidate genes, this has been difficult in the with 31.1 months compared to patients with mutated VH genes case of trisomy 12. Earlier studies indicated that the bands (n ¼ 12; TFS of 86.4 months; P-value 0.012). 12q13–q15 comprise the minimally gained region.3,6–8 Recent RNA expression analyses showed an overexpression of genes located to chromosome 12 in CLL cases with trisomy 12 as Cell lines compared to cases without this aberration.9,10 The aim of the The human lymphoma cell lines EHEB (Epstein–Barr virus (EBV)- current study was to characterize the protein expression patterns transformed CLL), JVM-2 (EBV-transformed B-PLL/leukemic of chromosome 12 candidate genes with oncogenic potential or mantle cell lymphoma) and JURKAT (non-EBV-transformed functional relevance in apoptosis, cell cycle, signal transduction T-ALL) were purchased from DSMZ (Braunschweig, Germany) or somatic hypermutation (for details see Table 1) when and cultured in RPMI 1640 (Seromed, Berlin, Germany) comparing CLL cases with or without trisomy 12, CD19 þ -B- supplemented with 10% fetal calf serum (Serva, Heidelberg, Germany). To allow for a comparison of different blots, lysates Correspondence: Dr S Stilgenbauer, Department of Internal Medicine of the cell line EHEB were loaded on all gels as an internal III, Universita¨t Ulm, Innere Medizin III, Robert-Koch-Str. 8, Ulm standard. To avoid expression differences of cell cycle proteins 89081, Germany; Fax þ 49 731 500 24405; E-mail: [email protected] in the cell lines, all cells used for the blots shown in Figure 1 Received 17 November 2004; accepted 24 March 2005; Published were harvested at a single point of time. To gain more online 19 May 2005 information about expression differences in dependence Protein Expression in CLL with trisomy 12 D Winkler et al 1212 Table 1 Investigated proteins with molecular weight and potential functional relevance for the pathogenesis of CLL in the order of their chromosomal localization

Protein Chromosomal Molecular Functional relevance in CLL localization weight (kDa)

AID Activation-induced cytidine deaminase 12p13 24 Somatic hypermutation/CSR recombination CCND2 Cyclin-D2 12p13 35 Cell cycle control p27 Cyclin-dependent kinase inhibitor 1B 12p13.1–p21 27 Cell cycle control (KIP1) STAT6 Signal transducer and activator of 12q13 100 Signal transduction and activation of transcription 6 transcription ARF3 ADP-ribosylation factor 3 12q13 20 Member of the RAS superfamily CDK2 Cyclin-dependent kinase 2 12q13 33 Cell cycle control GLI Glioma-associated oncogene homolog 12q13.2–q13.3 124 Oncogene CDK4 Cyclin-dependent kinase 4 12q14 34 Cell cycle control MDM-2 Mouse double minute 2 homolog 12q14.3–q15 90/76/57 Oncogene APAF-1 Apoptotic protease activating factor-1 12q23 130 Key regulator of mitochondrial apoptosis pathway Smac/DIABLO Second mitochondria-derived activator of 12q24.31 22 Promotion of apoptosis caspase The protein selection was based on the gene locus and involvement in apoptosis, cell cycle, signal transduction, somatic hypermutation or tumorigenic potential. Emphasis was put on bands 12q13–q15.

a J

J R b

CLL (trisomy 12) V

CLL (normal karyotype) V

- M

nd u nd m - B

2 p90 u u m u m u p76 MDM-2 STAT6 p57 Actin Actin

U CLL CLL J

U c JV R d CLL (normal karyotype) E

(normal karyotype) (trisomy 12) H

B m m m u m nd u u 2 m m u u u u u p27 CCND2 Actin Actin

CLL (trisomy 12) E

e V

R f

V CLL (trisomy 12) M

B T

2 nd nd u m m u m u u nd u nd m CDK4 Smac Actin Actin

g J CLL (trisomy 12)

CLL (normal karyotype) h H

T 2 m u nd m nd u u m m m u AID ARF3 Actin Actin

Figure 1 Representative immunoblots showing chromsome 12 candidate gene expression in CLL samples and cell lines (JVM-2, EHEB, JURKAT). Karyotype and VH mutation status (u ¼ unmutated VH;m¼ mutated VH;nd¼ not done) are highlighted. Each lane was loaded with 20 mg of total protein. (a) MDM-2 shows a speciﬁc pattern for each of the cell lines and restriction of expression to p90 and p57 in CLL samples. (b) Lower expression of STAT6 in JURKAT as compared to CLL samples, JVM-2 and EHEB. (c) Higher expression of p27 in CLL samples as well as in JURKAT when compared to JVM-2 and EHEB. No difference of p27 expression was detected between the CLL subgroups with or without trisomy 12 and mutated or umutated VH. (d) Restriction of CCND2 expression to JVM-2 and EHEB. (e) Lower expression of CDK2 (not shown) and CDK4 in CLL irrespective of the karyotype when compared to the cell lines. (f) Smac/DIABLO shows a double band in JVM-2 and a uniformly high expression in the genetic CLL subgroups. (g) No differences in ARF3 expression neither when comparing CLL cases with cell lines nor among the cell lines or CLL cases. Similar results were seen for APAF-1 and GLI (not shown). (h) Low but detectable expression and subtle differences in expression levels of AID, which were independent of the VH mutation status in the CLL samples.

of the time point of harvest, the JVM cells already used Immunoblot analyses for the experiments shown in Figure 1 but freshly harvested EHEB cells were used for the blots shown in A total of 1–10 Â 107 cells were lysed in 0.5–1 ml lysis buffer Figure 2. (10 mM Tris base; 5 mM EDTA; 130 mM NaCl; 1% Triton X-100;

Leukemia Protein Expression in CLL with trisomy 12 D Winkler et al 1213 (normal karyotype; (Cell Signaling Technology Inc., Beverly, USA) (see Table 1). uae VH) mutated nuae VH) unmutated

CD19+-B-cells Actin blots were performed to control for protein content. tioy12; (trisomy CLL CLL JVM-2 EHEB Results and discussion

a Specific expression patterns of the investigated proteins were STAT6 identified when comparing CLL cases and the cell lines. The Actin most striking differences among the cell lines were found for CCND2, CDK4, MDM-2, p27, Smac/DIABLO and STAT6 b p27 (Figure 1). The analysis of MDM-2 (Figure 1a) revealed a highly specific pattern for each cell line clearly differing from CLL cases Actin with a lower expression of the p57 band in JURKAT as compared to JVM-2 and EHEB. EHEB had the highest expression c CCND2 of the p57 band of the three cell lines, which all showed at least three MDM-2 bands (p90, p76, p57). The expression of STAT6 Actin (Figure 1b) was low in JURKAT, while EHEB and JVM-2 as well as the CLL samples showed strong signals. The expression of p27 d CDK4 (Figure 1c) was higher in JURKAT as compared to EHEB and ARF3 JVM-2, but similar when comparing JURKAT and the CLL samples. In contrast, CCND2 (Figure 1d) was not expressed by AID JURKAT and the CLL samples, but clearly detectable in EHEB and JVM-2, possibly influenced by EBV transformation of EHEB Actin and JVM-2.12 Interestingly, the expression levels of p27 and e CCND2 varied in EHEB but not in JVM-2 between the two series CDK2 of experiments shown in Figures 1 and 2. As we used the same JVM-2 samples but two different EHEB samples for the Actin experiments shown in the two figures, this finding clearly f demonstrates that the expression of cell cycle proteins in Smac continuous cell lines can vary over time and may be influenced Actin by the time point of their harvest. CDK4 expression was lower in CLL samples when compared to the three cell lines (Figure 1e). g Smac/DIABLO (Figure 1f) displayed a double band in JURKAT APAF-1 and JVM-2, possibly representing different splice variants.13 The Actin expression of ARF-3 (Figures 1g and 2d), APAF-1 (Figure 2g) and GLI was as strong in the patient samples as in the cell lines and Figure 2 Immunoblots showing chromosome 12 candidate gene no difference in expression was seen neither among the CLL expression in CD19 þ -B-cells (lane 3). To allow comparison with the cases nor among the cell lines. Overall, the protein expression results shown in Figure 1, two CLL samples as well as JVM-2 and EHEB were included. Each lane was loaded with 20 mg of total protein. pattern of the two B-lineage cell lines EHEB and JVM-2 seems to (a) Lack of STAT6 expression in CD19 þ -B-cells. (b) No p27 differ from that of CLL cases, especially with regard to cell cycle expression in CD19 þ -B-cells. Detection of a higher p27 expression proteins. Notably, neither cell line, although reportedly estab- in the EHEB cell sample used for the experiment series shown in lished from CLL patients, exhibits typical CLL characteristics, for Figure 2 when compared with the sample used for the experiment example, absence of known cytogenetic abnormalities of CLL, series shown in Figure 1. (c) No difference in expression of CCND2 including trisomy 12.14 In fact, JVM-2 carries the t(11;14) between CLL samples and CD19 þ -B-cells. Lower expression of 15 CCND2 in the EHEB cell sample than in the first experiment series. (d associated with mantle cell lymphoma. Therefore, both cell and e) CD19 þ -B-cells and CLL samples with the same expression lines may serve as positive controls or internal expression levels of ARF3, AID and CDK2. In contrast, the expression level of standards, but may be of limited usefulness as model systems for CDK4 is slightly higher in CLL than in CD19 þ -B-cells, but still CLL. Especially, the variation of cell cycle protein expression significantly lower than in JVM-2 and EHEB. (f) Lower expression of shown in EHEB in this study renders subject to caveat data on Smac/DIABLO in CD19 þ -B-cells when compared to CLL cases. A cell cycle progression in continuous cell lines. double band is observed in JVM-2. (g) Similar expression level of 16 APAF-1 in CLL, CD19 þ -B-cells and cell lines. Several variants of MDM-2 protein have been described, and the proteins p57, p59, p67 and p90 were overexpressed in different combinations in 64% of CLL cases when compared to normal volunteers.17 MDM-2 protein expression did not pH 7.4) containing protease inhibitors (Roche, Basel, Switzer- distinguish the genetic CLL subgroups in the present analysis, land) and the lysate was cleared by centrifugation. NuPage Gel but in all CLL samples coexpression of p90 and p57 was system (Invitrogen, Karlsruhe, Germany) was used for electro- observed (Figure 1a). If this phenomenon is restricted to CLL, phoresis. After protein transfer to Immobilon-P polyvinylidene cases with trisomy 12 and cases with a normal karyotype difluoride membranes (Millipore, Bedford, USA), immunoblot- remains to be defined. ting was performed using antibodies detecting ARF3, APAF-1, STAT6 has been shown to be frequently activated via CCND2, CDK2, Smac/DIABLO, STAT6 (signal transducer and phosphorylation and translocated to the nucleus in Hodgkin’s activator of transcription 6) (BD Biosciences, Palo Alto, USA), lymphoma.18 In contrast, no constitutive nuclear translocation CDK4, MDM-2 (Oncogene, San Diego, USA), GLI, p27 has been found in CLL cells.19 STAT6 was strongly expressed in (Santa Cruz Biotechnology, Heidelberg, Germany) and AID all CLL cases as well as in EHEB and JVM-2 without significant

Leukemia Protein Expression in CLL with trisomy 12 D Winkler et al 1214 differences between the CLL subgroups (Figure 1b), whereas no cannot be explained by a differential expression of the expression was detected in CD19 þ -B-cells (Figure 2a). investigated proteins. A protein that antagonizes the proliferative effect of CDK– Interestingly, recent microarray and quantitative PCR stu- cyclin complexes, p27, was highly expressed in all CLL cases dies9,10 revealed an upregulation of the RNA levels of genes comparable with the expression level in JURKAT cells located on chromosome 12 (such as p27 and CDK4) in CLL (Figure 1c). A strong p27 protein expression in CLL has also cases with trisomy 12. In the CLL subgroup with trisomy 12, 17 been reported in other studies20,21 and high levels of p27 have of the top 25 deregulated genes were localized on chromosome been shown to be associated with a poor prognosis in CLL.21 12 and all were upregulated, indicating a gene dosage effect as The current study confirms a strong expression of p27 in CLL potential pathomechanism.9 However, this upregulation of (Figure 1c), but all genetic subgroups investigated showed genes located on chromosome 12 on the RNA level was not similar levels of p27. In addition, no expression of p27 was observed in the current study on the protein level despite a high found in CD19 þ -B-cells (Figure 2b) pointing to a subgroup- tumor load of the samples investigated. This may be due to independent involvement of p27 in the pathogenesis of CLL. The additional post-transcriptional mechanisms of deregulation or overexpression of CCND2 mRNA has been described in CLL as due to a lower sensitivity of immunoblotting to detect subtle compared to normal resting B–lymphocytes,22 and an upregula- expression differences. The upregulations observed on the RNA tion of the CCND2 protein in CLL cells has been reported upon level were 1.36-fold for CDK4 and 1.32-fold for p27,10 stimulation with CpG oligonucleotides and interleukin-2.20 In differences that may well be beyond the limits of quantitation contrast, the current study revealed generally low expression of immunoblotting. levels of CCND2 as well as of CDK2 and CDK4 in CLL The analysis of CD19 þ -B-cells from healthy donors in irrespective of the trisomy 12 status or the VH status when comparison to CLL cases revealed higher protein expression compared to EHEB and JVM (Figure 1d and e). Remarkably, the levels in CLL for CDK4, p27, Smac/DIABLO and STAT6. expression of CDK4 in CLL was still higher than in CD19 þ -B- Whether these proteins qualify as candidates for targeted cells, where no expression of CDK4 was detected (Figure 2d). therapies of CLL has to be further evaluated, especially with No expression differences between CD19 þ -B-cells and CLL regard to genetic high-risk subgroups of CLL (patients with 17p were seen for CCND2 and CDK2 (Figure 2c and e). These results deletion/p53 mutation and 11q deletion, respectively). are concordant with a model in which cell cycle stimulation plays a minor role as compared to the inhibition of apoptosis in the accumulation of CLL cells in the peripheral blood.23 Acknowledgements It has been observed that AID mRNA is preferentially expressed in CLL cases with unmutated VH genes.24 AID is an This work was supported by the Landesforschungsschwerpunkt essential component of the canonical somatic hypermutation Baden-Wu¨rttemberg and the DFG (STI 296/1-1). The AID process in healthy B cells and its expression in CLL is potentially antibody (Cell Signaling Technology Inc., Beverly, USA) was relevant to the disease. Interestingly, a subsequent study showed kindly provided by Dr R Dalla-Favera. AID mRNA to be expressed only in a very small fraction of the CLL clone, this fraction varying from patient to patient and from 25 time to time. Accordingly, the present analysis of AID protein References expression revealed very low but detectable levels in EHEB as well as in the CLL subgroups with slight differences in 1 Juliusson G, Oscier DG, Fitchett M, Ross FM, Stockdill G, Mackie expression levels among the patient samples, which were MJ et al. Prognostic subgroups in B-cell chronic lymphocytic independent of the VH mutation status and the karyotype leukemia defined by specific chromosomal abnormalities. N Engl J (Figure 1h). Moreover, the expression level of AID in CD19 þ -B- Med 1990; 323: 720–724. cells was very similar to that of CLL cases (Figure 2d). Thus, AID 2Do¨hner H, Stilgenbauer S, Benner A, Leupolt E, Kro¨ber A, Bullinger protein expression detected by immunoblotting does not seem L et al. Genomic aberrations and survival in chronic lymphocytic to be a surrogate marker for the VH mutation status in CLL. leukemia. N Engl J Med 2000; 343: 1910–1916. 3 Oscier DG, Stevens J, Hamblin TJ, Pickering RM, Lambert R, Smac/DIABLO was easily detectable in CLL samples with an Fitchett M. Correlation of chromosome abnormalities with expression level as high as in EHEB regardless of the CLL laboratory features and clinical course in B-cell chronic lympho- subgroup affiliation defined by the karyotype and the VH cytic leukaemia. Br J Haematol 1990; 76: 352–358. mutation status (Figure 1f). Despite the presence of XIAP in CLL 4 Matutes E, Oscier D, Garcia-Marco J, Ellis J, Copplestone A, cells and its upregulation in conditions where apoptosis is Gillingham R et al. Trisomy 12 defines a group of CLL with atypical prevented, no caspase-inhibiting effect of IAP has been morphology: correlation between cytogenetic, clinical and labora- 26 tory features in 544 patients. Br J Haematol 1996; 92: 382–388. detected. This phenomenon might be partly explained by 5 Knauf WU, Knuutila S, Zeigmeister B, Thiel E. 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