On: 15 November 2007
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Leukemia and Lymphoma
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Protein expression and cellular localization in two
prognostic subgroups of diffuse large B-cell lymphoma:
Higher expression of ZAP70 and PKC-β II in the
non-germinal center group and poor survival in patients
deficient in nuclear PTEN
Marie Fridberga; Anna Servina; Lola Anagnostakib; Johan Linderothc; Mattias Berglundd; Ola Söderberge; Gunilla Enbladd; Anders Rosénf; Tomas Mustelin g; Mats Jerkemanc; Jenny L. Perssonb; Anette Gjörloff Wingrena
aDepartment of Tumor Biology, Lund University, Malmö University Hospital, Malmö, Sweden
bDepartment of Pathology, Lund University, Malmö University Hospital, Malmö, Sweden
cDepartment of Oncology, Lund University, Lund, Sweden
dDepartment of Oncology, Radiology and Clinical Immunology, Section of Oncology, Uppsala University, Uppsala, Sweden
eDepartment of Genetics and Pathology, Uppsala University, Uppsala, Sweden
fDepartment of Biomedicine and Surgery, Division of Cell Biology, Linköping University, Linköping, Sweden gLaboratory of Signal Transduction, The Burnham Institute, La Jolla, CA, USA
First Published on: 09 October 2007
To cite this Article: Fridberg, Marie, Servin, Anna, Anagnostaki, Lola, Linderoth, Johan, Berglund, Mattias, Söderberg, Ola, Enblad, Gunilla, Rosén, Anders, Mustelin, Tomas, Jerkeman, Mats, Persson, Jenny L. and Wingren, Anette Gjörloff (2007) 'Protein expression and cellular localization in two prognostic subgroups of diffuse large B-cell lymphoma: Higher expression of ZAP70 and PKC-β II in the non-germinal center group and poor survival in patients deficient in nuclear PTEN', Leukemia and Lymphoma, 48:11, 2221 - 2232
To link to this article: DOI: 10.1080/10428190701636443 URL:http://dx.doi.org/10.1080/10428190701636443
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ORIGINAL ARTICLE: RESEARCH
Protein expression and cellular localization in two prognostic subgroups
of diffuse large B-cell lymphoma: Higher expression of ZAP70 and
PKC-b II in the non-germinal center group and poor survival in patients
deficient in nuclear PTEN
MARIE FRIDBERG
1, ANNA SERVIN
1, LOLA ANAGNOSTAKI
2, JOHAN LINDEROTH
3,
MATTIAS BERGLUND
4, OLA SO
¨ DERBERG
5, GUNILLA ENBLAD
4, ANDERS ROSE
´ N
6,
TOMAS MUSTELIN
7, MATS JERKEMAN
3, JENNY L. PERSSON
2, &
ANETTE GJO
¨ RLOFF WINGREN
11
Department of Tumor Biology, Lund University, Malmo¨ University Hospital, Malmo¨, Sweden,2Department of Pathology, Lund University, Malmo¨ University Hospital, Malmo¨, Sweden,3Department of Oncology, Lund University, Lund, Sweden,
4
Department of Oncology, Radiology and Clinical Immunology, Section of Oncology, Uppsala University, Uppsala, Sweden,
5
Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden,6Department of Biomedicine and Surgery, Division of Cell Biology, Linko¨ping University, Linko¨ping, Sweden, and7Laboratory of Signal Transduction, The Burnham Institute, La Jolla, CA, USA
(Received 15 May 2007; revised 17 July 2007; accepted 15 August 2007)
Abstract
Patients diagnosed with diffuse large B-cell lymphoma (DLBCL) show varying responses to conventional therapy, and this might be contributed to the differentiation stage of the tumor B-cells. The aim of the current study was to evaluate a panel of kinases (ZAP70, PKC-b I and II and phosphorylated PKB/Akt) and phosphatases (PTEN, SHP1 and SHP2) known to be frequently deregulated in lymphoid malignancies. De novo DLBCL cases were divided into two subgroups, the germinal center (GC) group (14/28) and the non-germinal center (non-GC) or activated B-cell (ABC) group (14/28). ZAP70 and PKC-b II were expressed in a significantly higher percentage of tumor cells in the clinically more aggressive non-GC group compared with the prognostically favourable GC group. Also, the subcellular localization of PKC-b I and II differed in DLBCL cells, with the PKC-b I isoform being expressed in both the cytoplasm and nucleus, while PKC-b II was found exclusively in the cytoplasm. Loss of nuclear PTEN correlated with poor survival in cases from both subgroups. In addition, five cell lines of DLBCL origin were analyzed for protein expression and for mRNA levels of PTEN and SHP1. For the first time, we show that ZAP70 is expressed in a higher percentage of tumor cells in the aggressive non-GC subgroup of DLBCL and that PKC-b I and II are differently distributed in the two prognostic subgroups of de novo DLBCL.
Keywords: B cell, lymphoma, germinal center, DLBCL, ZAP70, PKC-b
Introduction
Diffuse large B-cell lymphomas (DLBCL) represent the most common type of non-Hodgkin lymphomas in Western countries and are characterized by heterogeneous clinical, immunophenotypic and ge-netic features [1 – 6]. Although DLBCL is one of the most chemotherapy-responsive human malignancies,
many newly diagnosed patients will not be cured with conventional anthracycline-based chemotherapy [1]. The variability in response to therapy suggests underlying heterogeneity in a disease that is largely treated with a homogeneous approach.
Using cDNA microarrays, two subgroups of DLBCL have recently been identified, which differ in their ability to be cured by the multi-agent
Correspondence: Anette Gjo¨ rloff Wingren, Department of Biomedical Laboratory Science, Health and Society, Malmo¨ University, Malmo¨ Sweden. Tel:þ46-40-391105. Fax: þ46-40-391222. E-mail: anette.gjorloff-wingren@hs.mah.se
ISSN 1042-8194 print/ISSN 1029-2403 online Ó 2007 Informa UK Ltd. DOI: 10.1080/10428190701636443
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chemotherapy that is used at present [7 – 9]. The germinal center (GC) subgroup expresses genes characteristic of normal GC B-cells and has been associated with a better outcome, whereas the activated B-cell like (ABC) group expresses genes characteristic of activated blood B-cells and has been associated with a poor outcome. Moreover, a type 3 (a group of unclassified cases) group was also identified, with similar outcome as the ABC like-group [8]. The ABC like-group and the type 3 like-group together, are commonly termed the non-germinal center (non-GC) group. Bcl6, CD10 and IRF4 (MUM1) have been shown to be differently ex-pressed in the GC and non-GC subgroups using gene expression arrays as well as immunohistochem-istry [7 – 10]. As shown by others, these three markers might successfully subdivide GC and non-GC DLBCL [11]. Both CD10 and Bcl6 are considered as GC markers while IRF4 has been found to be expressed in plasma cells and a subset of cells in the apical light zone of the GC [12 – 14].
In a study by Bai et al., bcl6 and CD10 expression was positively correlated with the apoptotic index and the proliferation-associated protein Ki67 [15]. The authors proposed several lines of evidence indicating a positive correlation of CD10 expression with proliferation and apoptosis in both normal and malignant B-cells. Indeed, GC-cells are character-ized by high proliferation and have the propensity to undergo apoptosis and upregulate CD10 expression on apoptotic induction, whereas CD10 is absent on other subsets of mature B-cells that are not char-acterized by high apoptosis [16 – 19]. The protein tyrosine kinase (PTK) ZAP70 is a key mediator of T-cell receptor (TCR) signalling and was until recently believed to be restricted to B-cell chronic lymphocytic leukaemia (B-CLL), where its expres-sion is an important negative prognostic factor. ZAP70 has now been reported to be expressed in normal and malignant B-cells of various differentia-tion stages but its funcdifferentia-tion in B-cell receptor (BCR) signalling is so far not well understood [20,21].
The protein kinase C-b (PKC-b) gene encodes two mRNAs that originate from alternative splicing of the C-terminal exons [22]. Little is known about the potentially different biological roles of the two isoforms of PKC-b, I and II, but in a recent report they were shown to exhibit different membrane translocation patterns in response to angiotensin II type IA receptor agonist activation [23]. The PKC-b gene is over-expressed in refractory rather than in curable DLBCL [24] and the PKC-b activity en-hances B cell proliferation and survival. PKC-b II has further been implicated to be involved in poor response to chemotherapy and survival in DLBCL [25,26]. B-cells that are deficient of the tumour
suppressor PTEN are hyperproliferative in response to mitogenic stimuli [27] and exhibit a lower thresh-old for activation through the BCR. Loss of PTEN function is one of many different genetic alterations used by tumors to activate PI3K/Akt pathway and confers selective growth and survival advantage [28]. In a recent paper, nuclear PTEN was proposed to exert tumor suppressor functions by maintaining chromosomal integrity [29]. The protein tyrosine phosphatase (PTP) SHP1 can also be a tumour suppressor in hematopoietic cells where it antago-nizes growth-promoting and potentially oncogenic effects of tyrosine kinases [30].
In this study, two prognostic subgroups of DLBCL were divided by the use of immunohistochemistry in a GC and a non-GC group, based on the expression of CD10, Bcl6 and IRF4 [12,31]. We provide novel data showing that de novo DLBCL cases express ZAP70 and PKC-b II in a significantly higher amount in the prognostically unfavourable non-GC group, compared to the GC group. No differences in PTEN, SHP1, SHP2, PKC-b I or phosphorylated Akt expression were found between the groups, but interestingly, the localization pattern of the two PKC-b isoforms differed. Further, we show that deficiency of nuclear PTEN expression correlates with poor survival in de novo DLBCL cases. 5 DLBCL cell lines, with different origin, were also analyzed for their expression of CD10, Bcl6, IRF4, ZAP70, PKC-b I and II, phosphorylated Akt, PTEN, SHP1 and SHP2. Studies of the expression of the most commonly mutated tumor suppressor and proliferation genes is of outmost importance to clarify the molecular background of DLBCL and may provide new insight for future treatment of this disease.
Materials and methods
Tissue array and immunohistochemistry
A total of 28 pieces of paraffin embedded de novo DLBCL tissues from 8 men and 20 women with a median age of 70 and diagnosed between 2001 and 2006, were selected by a pathologist from the Department of Pathology, University Hospital in Malmo¨, Sweden (Ethical approvement No. LU 210/ 2006). Representative areas in all paraffin blocks were chosen and tissue arrays were constructed as described earlier [32]. In brief, punches of 0.6 mm were taken from each block and mounted manually in a recipient block. Four micrometer sections were dried, deparaffinized, rehydrated and treated in a microwave for 10 min with target retrieval solution pH 9.9. This was followed by incubation in an automated immunohistochemical staining machine
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(Techmate 500, Dako, Copenhagen, Denmark) with primary antibodies: mouse anti-human CD10 and mouse anti-human ZAP70 (Serotec, Oxford, UK), mouse anti-human Bcl6 (Chemicon/Millipore, Bedford, MA), mouse anti-human MUM1/IRF4 (Abcam, Cambridge, UK), rabbit anti-human p-Akt 1/2/3, rabbit human SHP1, rabbit anti-human SHP2, mouse anti-anti-human PKC-b I and rabbit anti-human PKC-b II (Santa Cruz Biotech-nology, Santa Cruz), and rabbit monoclonal anti-human PTEN (Cell Signaling, Danvers, MA). Dako real envision detection system peroxidase/DAB (Dako, Copenhagen, Denmark) was used for in-cubation with secondary antibodies and visualisation. Slides were counterstained with haematoxylin. Analyses were performed independently by one pathologist and two researchers. There was a 90% agreement between the observers, and in cases of variance, the pathologist determined the outcome. To classify patients into the GC- or non-GC group, we used the three marker-model previously described by Hans et al. and Berglund et al. [12,31]. In brief, cases were considered GC-positive if CD10 alone or both CD10 and Bcl6 were positive. If both CD10 and Bcl6 were negative, the cases were considered GC-negative. If CD10 was negative and Bcl6 positive, IRF4 determined the outcome: Positivity for IRF4 designated the patient material to the non GC-group while negative IRF4 staining ment the GC group. The staining intensity for ZAP70, PKC-b I and II, p-Akt, PTEN, SHP1 and SHP2 was graded from 0 to 3. 0¼ no staining, 1 ¼ weak staining, 2¼ moderate staining and 3 ¼ strong staining. For measuring the fraction of positive cells, we used a 25% cut off to classify patients as positive or negative for all markers. Further, proportion of tumor cells less than 25% positive were scored as 1; greater than 25%, but less than 50% positive were scored as 2; greater than 50%, but less than 100% positive were scored as 3. Cell lines were fixed in paraformalde-hyde and dehydrated before being paraffin em-bedded and prepared as described for the patient material. The cell lines were also placed in the GC/ non-GC group by use of the three marker model. Pearson Chi-Square tests and Mann Whitney tests were performed and Statistical calculations analyzed, with SPSS for Windows 12.0.1 (2002, SPSS Inc.).
Western blot
106 106cells were lysed with 50ml lysis buffer (9 mM Hepes, 0.08 mM EDTA, 0.08 mM EGTA, 9 mM KCL, 5%NP40, complete protease inhibitor (Roche, Mannheim, Germany)). Routinely 15ml protein lysates/lane were run on 10% SDS gels. Gels were blotted on PVDF membranes
(Immobilion-P, Millipore, Bedford) using 45 V for 1.5 h or 35 V for 2 h, followed by blocking in 5% milk in PBS in 0.05% Tween20 (TW20). Routinely, primary Abs in 5% milk (in PBSþ 0.05% TW20) were incubated for 2 h at RT or overnight at 48C. Following washing (PBS in 0.05% TW20 PBS) and incubation with HRP-conjugated secondary Ab for 45 min at RT, membranes were developed with the ECL chemiluminsecent method (Amersham Bios-ciences, UK) and Fluor-S-Max detector. Mem-branes were routinely stripped of primary Ab and reprobed with actin as a protein-loading control. The Abs used for Western blot were rabbit anti-actin (I-19) and rabbit anti-SHP1 (C-(I-19) from Santa Cruz Biotechnology. Rabbit anti-IgG-HRP used as sec-ondary Ab was from Dako (Glostrup, Denmark).
Cells and culturing conditions
The following B cell lines have been described elsewhere [33]. In brief, FLEB 14 – 4 (pro-B) is established by EBV transformation of B cells from an ALL child, Nalm-6 (pre-B) is EBV-negative, con-taining intracellularm-chains, Rael and Daudi (small mature B) are derived from Burkitt lymphoma (BL) patients and express surface IgG and IgM, respec-tively, U-698 (B-blast) is derived from a non-Hodgkin lymphoma and LP-1 is a plasmacytoma representing plasma cells near terminal differentia-tion. The cells were cultured in RPMI 1640 medium (Invitrogen, San Diego, CA) supplemented with 10% fetal calf serum and garamycin 0.1 g/ml.
The EBV-negative DLBCL cell lines U-2932, U-2904, U-2940, U-2946 and ULA were from Department of Oncology, Genetics and Pathology, Uppsala, Sweden. The DLBCL cell lines used in this study represent (i) de novo DLBCL: U-2946 and ULA [34], (ii) transformed DLBCL from Hodgkin lymphoma: U-2932 [35] and U-2940 [36] and (iii) transformed DLBCL from follicular lymphoma: U-2904 [37]. Amini et al. previously showed that U-2932 carries high-level amplifications of the chromosomal bands 18q21 (BCL2-loci) and 3q27 (BCL6-loci) and overexpression of these genes. Also, p53 is overexpressed in U-2932 due to a point mutation [35]. U-2940 display microinstability and multiple chromosomal rearrangements described in both Hodgkin and non-Hodgkin lymphoma, and some of these aberrations are shared by U-2932 [36]. For U-2904, Sambade reported translocation of t(8;14) typically seen in Burkitt lymphoma where c-myc is overexpressed, and of t(14;18) associated with follicular lymphoma and Bcl2 overexpression [37]. The de novo DLBCL cell line U-2946 is unpublished. The cells were cultured in RPMI 1640 medium (Invitrogen, San Diego, CA) supplemented
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with 10% fetal calf serum and garamycin 0.1 g/ml, except from ULA which was cultured in equal amounts of IMDMþ glutaMAX and Opti-MEM I (Invitrogen, San Diego, CA), 10% fetal calf serum and garamycin 0.1 g/ml.
PCR
Total RNA from the cell lines (106 106cells) were isolated with TRIzol (Invitrogen, San Diego, CA) and cDNA from 2mg of RNA was prepared using First Strand cDNA Synthesis Kit according to manufacturers instructions (Amersham Biosciences, Amersham, UK). The PCR was performed in a total volume of 50ml reaction mix, consisting of 5 ml Taq buffer which was added to 4 ml MgCl2 (Fermentas,
Burlington, Canada), 2ml of 10 6 dNTP, 2 ml Taq polymerase (Finnzymes, Espoo, Finland), 34 ml of RNAse free water, 1ml cDNA and 1 ml of forward and reverse primer, respectively. For SHP1 and PTEN, the following PCR program was used: Denaturation at 958C for 1 min followed by 30 cycles of 958C for 1 min, annealing of primers for 1 min at 578C and elongation for 1.5 min at 728C. Final extension was done for 7 min at 728C. For GAPDH the following PCR program was used: Denaturation at 948C for 2 min followed by 45 cycles of 948C for 30 s, annealing of primers for 1 min at 558C and elongation for 1 min at 728C. Final extension was done for 10 min at 728C.
PCR primers
Oligonucleotide primers were as follows: PTEN, 50 -GAA ACT ATT CCA ATG TTC AGT GGC-30 and 50-CTG ATC TTC ATC AAA AGG TTC ATT CTC-30 (expected size 587 bp), SHP1, 50-A GTT GAA TTC ATG TGC GAG GGT GGA CGC TAC ACA-30and 50-A GTT GAA TTC GGT ACT GGT AAT GCC AGA TCT CCC-30 (expected size 735 bp), GAPDH, 50-CGA CCA CTT TGT CAA GCT CA-30 and 50-AGG GGT CTA CAT GGC AAC TG-30 (expected size 228 bp).
PTP assay
In order to measure the activity of SHP1, 106 106cell lysates were incubated with anti-SHP1-antibody (rabbit anti-human SHP1) for 2 h in 48C, and immunoprecipitated with protein G-beads (Amersham Biosciences, Amersham, UK) for 1 h, in 48C. A PTP-activity assay was then per-formed, where 20ml beads solution were incubated with 20ml buffer (100 mM Bis-Tris pH 6.0, 150 mM NaCl, DTT 5 mM) and 60ml pNPP-solution (phosphorylated substrate), for 45 min in
378C. As a negative control and background, one sample was immunoprecipitated with mouse anti-human HA-antibody (Roche, Basel, Switzerland). After 45 min, OD was measured at 405 nm. The OD correlates to the degree of free phosphate-groups, and this indicates how active the PTP is in the analyzed cells.
Results
Division into the subgroups and clinical data
The 28 patient samples were divided into a GC- and a non-GC group based on the expression of GC markers CD10, Bcl6 and post-GC marker IRF4 as analysed by immunohistochemical analysis and as described in ‘‘Material and methods’’. A 25% cut off to classify cases as positive or negative for all markers was used. Fourteen patients (4 men, 10 women) were classified as belonging to the GC group and 14 patients (4 men, 10 women) constituted the non-GC group (Table IA). Although comprised of a rather small group of DLBCL cases, the sub-group division correlated significantly with Ann Arbor stage classi-fication [38] as analyzed by Pearson Chi-Square test. Indeed, we found that the non-GC group were characterized by a more advanced stage disease (stage III – IV) compared with the GC group (p-value¼ 0.022) (Table IB). However, no signifi-cant differences in elevated S-lactate dehydrogenase (S-LDH) (p-value¼ 0.450), high International prog-nostic index, IPI, (p-value¼ 0.256), high Ki67 levels (p-value¼ 0.135) or survival (p-value ¼ 0.445) could be found between the two subgroups, when analyzed by Pearson Chi-Square test.
The cases of the non-GC group displayed significantly higher number of ZAP70 and PKC-b II positive cells compared to the GC group
First, the DLBCL cases were stained and analyzed for expression of ZAP70, PKC-b I, PKC-b II and
Table IA. Classification of the de novo DLBCL cases into the GC/non GC-groups.
Antibody GC group (N) Non-GC group (N)
CD10 11/14 0/14
Bcl6 10/14 6/14
IRF4 4/14 11/14
Cases were designated to the GC-group if they were positive for CD10 or CD10 and Bcl6. If they were negative for CD10 and Bcl6, they were considered to belong to the non-GC group. If CD10 was negative and Bcl6 positive, the expression of IRF4 determined the outcome. Cases were placed in the GC group if IRF4 was negative and in the non GC-group if IRF4 was positive.
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phosphorylated PKB/Akt (p-Akt) (Table II). A Mann Whitney test was performed showing that the non-GC group displayed a significantly higher ZAP70 expression in terms of percent positively stained tumor cells, compared with cases in the GC-group (p-value¼ 0.039). Also, in the non-GC group, 51 – 100% of PKC-b II positive tumor cells were found in 11 out of 14 cases, compared with in only 2 out of 14 cases in the GC group (Table II). This difference in percentage positively stained PKC-b II tumor cells resulted in a statistical significant difference between the GC and non-GC groups (p-value¼ 0.001). PKC-b I and p-Akt were expressed in a high percentage of tumor cells in both subgroups (p-value¼ 0.246 and p-value¼ 0.769, respectively).
Different subcellular localization pattern of the PKC-b I and II isoforms in DLBCL cases
Interestingly, the subcellular localization differed between the two PKC-b isoforms, with PKC-b I being expressed in both cytosol and nucleus, whereas PKC-b II was found exclusively in the cytoplasm [Figures 1B and 1C]. Moreover, PTEN, SHP2, Zap70 and p-Akt were detected in both cytoplasm and nucleus, while SHP1 was detected only in the cytoplasm [Figures 1(A) – 1(G)].
Trends towards a stronger PTEN staining intensity in the non-GC group
In the non-GC group, PTEN was found to be expressed in 13 out of 14 cases, as displayed
by 26 – 50% and 51 – 100% positively stained tumor cells (Table III). We detected nuclear PTEN in 7 cases in the GC group and in 8 cases in the non-GC group (data not shown for PKC-bI, p-Akt or SHP2). No significant differences were found between the groups in terms of percent positively stained cells for PTEN (p-value¼ 0.246), SHP1 (p-value¼ 0.164) or SHP2 (p-value ¼ 0.982) when analyzed by a Mann Whitney test, but a trend towards a stronger PTEN staining intensity (graded 0 – 3) in the non-GC group was found (p-value¼ 0.094) (Table III).
Absence of nuclear PTEN correlates with poor survival The de novo DLBCL cases analyzed in this study were related to survival and a higher percentage of cells lacking the expression of nuclear PTEN staining was found to correlate with poor survival (p-value¼ 0.059) (Table IV). No such correlation was found between any of the other proteins analyzed. The statistical analyses were performed by Mann Whitney tests.
Table IB. Clinical data of de novo DLBCL cases. GC group (%) Non-GC group (%)
Stage I – II 64 21 Stage III – IV 36 79 p-Value 0.022 Normal S-LDH 57 43 Elevated S-LDH 43 57 p-Value 0.450 Low IPI 64 43 High IPI 36 57 p-Value 0.256 Low Ki67 57 27 High Ki67 43 64 p-Value 0.135 Survival 64 50 p-Value 0.445
The cases divided into the GC or non-GC group were scored for clinical characteristics. Stage I – II and III – IV are according to Ann Arbor stage classification, S-LDH; S-lactate dehydrogenase, IPI; International Prognostic Index, Ki67; proliferation marker (low Ki67¼ expression in 570% of cells, high Ki67 ¼ expression in470% of cells); Survival as before 2006-12-31.
Table II. The expression of protein tyrosine kinases ZAP70, PKCb I, PKCb II and p-Akt in the GC- and non-GC group of de novo DLBCL cases. GC group (N) % non-GC group (N) % ZAP70 0 – 25% 5 36 1 7 26 – 50% 4 28 2 14 51 – 100% 5 36 11 79 Total 14 14 p-Value 0.039 PKCb II 0 – 25% 5 39 0 0 26 – 50% 6 46 3 21 51 – 100% 2 15 11 79 Total 13 14 p-Value 0.001 PKCb I 0 – 25% 1 7 1 7 26 – 50% 0 0 4 28 51 – 100% 13 93 9 65 Total 14 14 p-Value 0.246 p-Akt 0 – 25% 0 0 0 0 26 – 50% 2 14 1 7 51 – 100% 12 86 13 93 Total 14 14 p-Value 0.769
DLBCL tissue was stained by immunohistochemistry and the number of positive cells in percent is indicated for each marker: 0 – 25%, 26 – 50% and 51 – 100%. For statistical analysis, a Mann Whitney test was performed.
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Figure 1. Immunohistochemical stainings of de novo DLBCL tissue. A. ZAP70, B. PKC-b I, C. PKC-b II, D. p-Akt, E. PTEN, F. SHP1, G. SHP2. All tissues A-G were graded ‘‘3’’, for strong staining intensity. Magnification 406. (Please see colour online)
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ZAP70, PKC-b I and II, p-Akt, PTEN, SHP1 and SHP2 expression in 5 DLBCL cell lines of GC origin We further analyzed 5 DLBCL cell lines for protein expression. First, CD10, Bcl6 and IRF4 were analyzed and all DLBCL cell lines were placed in the GC group according to the three marker-model [31]. U-2932 was positive for all three markers, U-2904 and U-2946 were negative for Bcl6, U-2940 was negative for both CD10 and IRF4 and ULA was negative for IRF4 (Table V). Next, we analyzed the cell lines for the staining intensity of ZAP70, PKC-b I and II, p-Akt, PTEN, SHP1 and SHP2 by immuno-histochemistry (Table V). U-2904 and U-2946, were strongly positive for ZAP70 when analyzed with immunohistochemistry and Western blot (data not shown), whereas U-2932, U-2940 and ULA were negative. PKCb II was negative in U-2940 and ULA and rather weakly expressed in the other three cell lines. PKCb I was strongly expressed in U-2932, U-2946 and ULA and weaker expressed in U-2904
and U-2940. The cytoplasmic and nuclear PKC-b I and cytoplasmic PKC-b II was also seen in all DLBCL cell lines. p-Akt was detected in all cell lines, with weak expression. PTEN expression was strong in all DLBCL cell lines. A strong SHP1 expression was detected in U-2940, U-2946 and ULA, whereas a weak expression and in fewer cells was seen in U-2932 and U-2904 (Table V). SHP2 expression was strong in the two de novo DLBCL cell lines U-2946 and ULA, and weaker in the other three cell lines. The levels of PTEN protein expression as measured by immunohistochemical stainings corre-lated well with the mRNA levels in the majority of the cell lines tested (Figure 2). A correlation between SHP1 protein and mRNA expression in the cell lines was also found, with an even expression level in all DLBCL cell lines (Figure 2). The exception was U-2904, which showed a SHP1 mRNA level equal to the other cell lines, but displayed a very low protein expression of the PTP.
Table III. Trend towards a stronger staining intensity of PTEN in the non-GC group of de novo DLBCL cases, but no significant differences between groups regarding percent positive stained cells for PTEN, SHP1 and SHP2.
GC group (N) % Non-GC group (N) % PTEN (intensity) 0 5 36 1 7 1 3 21 2 14 2 4 29 8 57 3 2 14 3 21 Total 14 14 p-Value 0.094 PTEN 0 – 25% 5 36 1 7 26 – 50% 1 7 2 14 51 – 100% 8 57 11 78 Total 14 14 p-Value 0.246 SHP1 0 – 25% 2 14 0 0 26 – 50% 5 36 3 21 51 – 100% 7 50 11 78 Total 14 14 p-Value 0.164 SHP2 0 – 25% 1 7 0 0 26 – 50% 0 0 1 7 51 – 100% 13 93 13 93 Total 14 14 p-Value 0.982
DLBCL tissue was stained by immunohistochemistry and the staining intensity is indicated using a 4-grade system ranging from 0¼ no staining, 1 ¼ weak staining, 2 ¼ moderate staining, 3¼ strong staining. The number of positive cells in percent is indicated for each marker: 0 – 25%, 26 – 50% and 51 – 100%. For statistical analysis, a Mann Whitney test was performed.
Table IV. Correlation of protein expression and survival in de novo DLBCL. Mors* (N) % Survival (N) % Nuclear PTEN 0 – 25% 9 75 6 37 26 – 50% 3 25 7 44 51 – 100% 0 0 3 19 Total 12 16 p-Value 0.059 Cytoplasmic PTEN 0 – 25% 3 25 3 19 26 – 50% 3 25 0 0 51 – 100% 6 50 13 81 Total 12 16 p-Value 0.241 PKCb II 0 – 25% 2 17 3 20 26 – 50% 4 33 5 33 51 – 100% 6 50 7 47 Total 12 15 p-Value 0.905 SHP1 0 – 25% 2 17 0 0 26 – 50% 3 25 5 31 51 – 100% 7 58 11 69 Total 12 16 p-Value 0.507 ZAP70 0 – 25% 3 25 3 19 26 – 50% 3 25 3 19 51 – 100% 6 50 10 62 Total 12 16 p-Value 0.599
DLBCL tissue was stained by immunohistochemistry and the number of positive cells in percent is indicated for each marker: 0 – 25%, 26 – 50% and 51 – 100%. For statistical analysis, a Mann Whitney test was performed.
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PTEN and SHP1 mRNA was detected in all DLBCL cell lines
To investigate if the mRNA levels of SHP1 and PTEN were similar to protein levels, DLBCL cell
lines was analyzed by isolating total RNA and preparing cDNA. PTEN and SHP1 mRNA could be detected in all the DLBCL cell lines, as analyzed by PCR [Figure 2(A)]. To compare the mRNA levels between the cell lines, the intensity of the PTEN and SHP1 bands was measured by a volume analysis report. To compensate for any unequal loading and to measure the total RNA content, the GAPDH intensity for each cell line was measured and standardized. Subsequently, the PTEN and SHP1 intensity for the cell lines could be calculated and related to each other, by dividing each PTEN and SHP1-value with their corresponding GAPDH-value, respectively [Figure 2(B)].
Biological activity of SHP1 in the cell lines
To confirm biological acticity of the SHP1 protein in the DLBCL cell lines, we performed an in vitro PTP activity assay by immunoprecipitating SHP1 from the same number of cells from each of the DLBCL cell lines and thereafter assaying against the pNPP substrate. The SHP1 activity in U-2940 was about two times higher as compared to the activity in the other DLBCL cell lines tested (Figure 3). U-2904 displyed the weakest SHP1 activity, which is con-sistent to our observation in immunohistochemical analysis.
Table V. Germinal center marker expression and staining intensity of ZAP70, PKCb I and II, p-Akt, PTEN, SHP1 and SHP2 in 5 DLBCL cell lines.
U-2932 U-2904 U-2940 U-2946 ULA Positive (þ) /negative (7) CD10 þ þ 7 þ þ Bcl6 þ 7 þ 7 þ IRF4 þ þ 7 þ 7 GC/non-GC GC GC GC GC GC Staining intensity ZAP70 7 þþþ 7 þþþ 7 PKCb II þþ þ 7 þþ 7 PKCb I þþþ þþ þþ þþþ þþþ p-Akt þþ þþ þ þ þþ PTEN þþ þþþ þþ þþþ þþþ SHP1 þþ þ þþþ þþþ þþþ SHP2 þþ þ þ þþþ þþþ
The 5 DLBCL cell lines were stained for the indicated markers and analyzed for percent stained cells (CD10, Bcl6 and IRF4) and for staining intensity (the PTKs and PTPs). With a 25% cut off, the cell lines were considered positive or negative for the three GC-markers. For staining intensity,7¼no staining, þ ¼ weak staining, þþ ¼ moderate staining and þþþ ¼ strong staining.
Figure 2. SHP1 and PTEN mRNA expression in the 5 DLBCL cell lines. A. PCR was performed on cDNA prepared from the cell lines (1. U-2932, 2. U-2904, 3. U-2940, 4. U-2946, 5. ULA) as described in Material and methods. GAPDH indicates total cDNA level and loading. B. The intensity of the PTEN and SHP1 bands was measured for each cell line and described in a volume analysis report. The PTEN and SHP1 value was divided with the value of the GAPDH-intensity for each cell line, to compensate for any unequal loading. The quotients from one representative experiment for PTEN (left) and SHP1 (right) respectively, are pictured.
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SHP1 expression is strong in the early B cell differentiation, but declines with further maturation SHP1 expression was also investigated at various B cell differentiation stages in cell lines (Figure 4). By Western blot analysis, we show that SHP1 was strongly expressed in the pro-B FLEB 14-4 and the pre-B Nalm-6 cell lines, weakly expressed in the three mature Daudi, Rael and U-698 B cell lines, but not detectable in the plasma cell line LP-1. The biological SHP1 activity as measured by the in vitro PTP activity assay was in line with Western blot results (data not shown).
Discussion
Tyrosine phosphorylation is a rapidly reversible process catalysed by PTKs and reversed by PTPs.
Abnormalities in tyrosine phosphorylation play a role in the pathogenesis of numerous inherited or acquired human diseases, from cancer to immune deficiencies. Mutations of PTKs and loss of PTPs have been reported in numerous experimental and clinical cancers [39 – 41]. At least 30 different PTPs have been reported to be affected and loss can occur by genetic or epigenetic mechanisms. Here, we have used immunohistochemistry to analyze the protein expression of ZAP70, PKC-b I and II, p-Akt, PTEN, SHP1 and SHP2, critical for the BCR signaling capacity, in 28 cases of de novo DLBCL and in 5 cell lines of DLBCL origin. Further, we have analyzed the cell lines for mRNA expression of SHP1 and PTEN, and for biological activity of SHP1.
The DLBCL cases in this study were divided into two groups, a GC and a non-GC group, based on their expression of CD10, Bcl6 and IRF4 [12,31]. Only de novo DLBCL cases were included in the study, since other DLBCL cases with earlier history of a low-grade malignant lymphoma can be more similar to the ancestral lymphoma type at the molecular level [31,42]. Fourteen cases were classi-fied as belonging to the GC group (CD10 alone or both CD10 and Bcl6 were positive) and 14 cases to the clinically more aggressive non-GC group. Since the patients were diagnosed rather recently (2001 – 2006), a follow up of 5-year survival was not possible to perform. However, cases of the non-GC group were found to display a more advanced stage disease, stage III-IV, compared to the GC group (p¼ 0.022) and the survival was higher in the GC group. Also, clinical characteristics such as elevated S-LDH and high Ki67 stains were detected in the prognostically unfavourable non-GC group. Between the two groups, there were significant differences in the number of cells positively stained for ZAP70 and PKC-b II. Indeed, the two proteins were expressed in a higher number of cells in the non-GC group, compared to the GC group. This is particularly interesting, since ZAP70 is associated with poor prognosis in B-CLL and was recently found to be expressed in ABCs [20]. In T-cells, ZAP70 is recruited to the TCR upon activation, and from here the protein functions in the activation of down stream targets for proliferation and survival, such as the phosphatidyl inositol-3 kinase. In a recent report, ZAP70 was proposed to function differently in BCR signalling, compared to TCR mediated signaling [43]. It seems to mediate inhibition of events such as BCR internalization that would otherwise terminate the signalling response [43]. In two previous studies, ZAP70 was reported to be absent in DLBCL tissue analyzed by immunohistochemistry [44,45]. Our findings indicate a possible role for ZAP70 in DLBCL development, and it’s importance as a
Figure 3. Biological activity of SHP1 in the DLBCL cell lines. The SHP1 activity assay procedure is described in Material and methods. In short, SHP1 was immunoprecipitated from equal amounts of cells from each cell line and incubated with a phosphorylated substrate, pNPP, before OD was measured. OD correlates to the activity in the cell lines. Mean values from two experiments are shown.
Figure 4. Expression of SHP1 in B cell lines representing different maturation stages. 106 106cells from B cell lines FLEB 14 – 4 (pro-B), Nalm-6 (pre-B), Daudi, Rael (small B), U-698 (B-blast) and LP-1 (plasma B) were lysed, run on SDS-gel and transferred to PVDF-membranes. The membranes were incubated over-night with antibodies to human SHP1. The membrane was stripped of antibody and reprobed with antibodies to human actin.
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possible prognostic marker in DLBCL needs to be further explored. PKC-b I and II showed not only a different distribution between the GC and the non-GC group, but also a different subcellular localiza-tion pattern. PKC-b I expression was similar in both groups and was localized to both nucleus and the cytoplasm. Interestingly, PKC-b II was absent in the nucleus, and instead detected in the cytoplasm, with a significant over-expression in the non-GC group which comprises patients with a more advanced disease. This is in line with other studies showing that membrane PKC-b II expression predicts for inferior survival in DLBCL [25,26]. Espinosa et al. reported PKC-b II expression in the membrane, cytoplasm and nucleus in DLBCL cells [26]. We can only detect PKC-b II expression in the cytoplasm and the reason for the difference in PKC-b II localization between our studies can only be specu-lated. Different antibodies were used for immuno-histochemical staining, but in both studies the antibody recognized the C-terminal part of PKC-b II. To our knowledge, our study is the first to show the different distribution of PKC-b I and II in the GC and the non-GC groups. The phosphorylation and activation of PKB/Akt is negatively regulated by PTEN. When analyzed here, no correlation in either expression or staining intensity of p-Akt and PTEN was found and Akt was found to be phosphorylated in a high percentage of cells in both subgroups. However, we report a trend towards a stronger staining intensity of PTEN (cytoplasmic and nuclear) in the non-GC group of DLBCL cases. Moreover, here we show a correlation between the absence of nuclear PTEN expression and inferior survival in de novo DLBCL cases. PTEN has a well-defined role in plasma membrane signaling but is also found in the nucleus in a number of different normal and tumor cell types [46,47]. Recent studies have suggested that phosphatase-independent functions of PTEN mod-ulate the activity and stability of p53 within the nucleus [48,49]. Shen et al. showed recently that PTEN-deficient mouse embryonic fibroblasts con-tained an increased number of chromosomal frag-ments, breakage of chromatids and other chromosomal instabilities, suggesting that one of PTENs nuclear tumor suppressor functions might be to guard the genome integrity [29].
In this study of 28 de novo DLBCL cases, we could not detect any significant differences (p-value¼ 0.164) between SHP1-positive DLBCL cells in the two prognostic subgroups, but the cases in the non-GC group contained a higher percentage of SHP1 positive cells. Previous immunohistochem-ical stainings of lymphoid tissues have shown that SHP1 is downregulated in B cells forming GCs, while it is expressed in earlier and later stages of B
cell differentiation [50]. We also confirm these results by showing that B cell lines of the early stages in the B cell differentiation express higher levels of SHP1, compared to mature B cells and plasma cells. SHP2, which was expressed in both cytoplasm and nucleus in DLBCL tumor cells, was detected in equal amounts in both subgroups.
5 DLBCL cell lines with varying clinical back-ground were analyzed in our study. The cytoplasmic PTEN expression was strong in all DLBCL cell lines with an expression in 90 – 95% of the cells analyzed. Sambade et al. conclude that chromosomal aberra-tions in combination with strong CD19 expression may point to a deregulation of the CD19-PI3K pathway in U-2940 [36]. A strong SHP1 expression in a high percentage of cells was also detected in U-2940, U-2946 and ULA, whereas a weaker expres-sion in a fewer number of cells were found in U-2932 and U-2904. The fact that many of the DLBCL cases in our study expressed SHP1 despite previous reports of SHP1 downregulation in many lymphomas [30], led us to further investigate this PTP. The SHP1 expression correlated with biological activity in the DLBCL cell lines. Indeed, the most pronounced SHP1 activity was measured in U-2940 cells and the lowest activity in U-2904, which also displyed the highest and lowest protein expression, respectively.
In conclusion, here we show for the first time a significantly higher expression of ZAP70 in the clinically more aggressive non-GC group, compared to the GC group of de novo DLBCL cases. Further, we report different cellular localization of PKC-b I and II in DLBCL tissue, and over-expression of PKC-b II, but not of PKC-b I, in the non-GC group of DLBCL. We also provide novel information about the tumor suppressors PTEN and SHP1, and report that the absence of nuclear PTEN correlates with poor survival in DLBCL. Given that this disease is cured in only 40% of the cases, analyses of molecules required for cancer cell proliferation or survival are of outmost importance.
Acknowledgement
The authors thank Elise Nilsson for invaluable technical assistance. This work was supported by grants from the American Cancer Society for technology transfer, the Alfred O¨ sterlund Founda-tion, the Greta and Johan Kock FoundaFounda-tion, the Magnus Bergvall Foundation, the Medical Faculty of Lund, the Kungliga Fysiografiska Sa¨llskapet, Svenska La¨karesa¨llskapet, the Siv-Inger and Per-Erik Anders-son Foundation, the Berta Kamprad Foundation, the Cancer Foundation at Malmo¨ University Hospital, Swedish Cancer Association (A.R.) and by grants from the National Institute of Health (T.M.).
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