bcl-2 overexpression leads to increases in suppressor of ... · the rare hematologic disorder,...

Bcl-2 Overexpression Leads to Increases in Suppressorof Cytokine Signaling-3 Expression in B Cellsand De novo Follicular Lymphoma

Gary J. Vanasse,1 Robert K. Winn,4 Sofya Rodov,1 Arthur W. Zieske,2 John T. Li,4 Joan C. Tupper,3

Jingjing Tang,5 Elaine W. Raines,5 Mette A. Peters,6 Ka Yee Yeung,6 and John M. Harlan3

Departments of 1Internal Medicine and 2Laboratory Medicine, Yale University School of Medicine, New Haven,Connecticut and Departments of 3Internal Medicine, 4Surgery, and 5Pathology, School of Medicine,and 6Center for Expression Arrays, University of Washington, Seattle, Washington

AbstractThe t(14;18)(q32;q21), resulting in deregulated

expression of B-cell-leukemia/lymphoma-2 (Bcl-2),

represents the genetic hallmark in human follicular

lymphomas. Substantial evidence supports the

hypothesis that the t(14;18) and Bcl-2 overexpression

are necessary but not solely responsible for neoplastic

transformation and require cooperating genetic

derangements for neoplastic transformation to occur. To

investigate genes that cooperate with Bcl-2 to influence

cellular signaling pathways important for neoplastic

transformation, we used oligonucleotide microarrays to

determine differential gene expression patterns in

CD19+ B cells isolated from EM-Bcl-2 transgenic mice

and wild-type littermate control mice. Fifty-seven genes

were induced and 94 genes were repressed by >_ 2-fold in

EM-Bcl-2 transgenic mice (P < 0.05). The suppressor of

cytokine signaling-3 (SOCS3) gene was found to be

overexpressed 5-fold in B cells from EM-Bcl-2 transgenic

mice. Overexpression of Bcl-2 in both mouse embryo

fibroblast-1 and hematopoietic cell lines resulted

in induction of SOCS3 protein, suggesting a

Bcl-2-associated mechanism underlying SOCS3

induction. Immunohistochemistry with SOCS3 antisera

on tissue from a cohort of patients with de novo

follicular lymphoma revealed marked overexpression

of SOCS3 protein that, within the follicular center

cell region, was limited to neoplastic follicular

lymphoma cells and colocalized with Bcl-2 expression

in 9 of 12 de novo follicular lymphoma cases examined.

In contrast, SOCS3 protein expression was not

detected in the follicular center cell region of benign

hyperplastic tonsil tissue. These data suggest that Bcl-2

overexpression leads to the induction of activated signal

transducer and activator of transcription 3 (STAT3)

and to the induction of SOCS3, which may contribute

to the pathogenesis of follicular lymphoma.

(Mol Cancer Res 2004;2(11):620–31)

IntroductionFollicular lymphomas comprise approximately one third of

all cases of non–Hodgkin’s lymphoma in humans. Follicular

lymphomas are initially clinically indolent and chemosensitive

but have a natural history marked by multiple relapses,

becoming progressively chemoresistant and ultimately remain-

ing incurable. Twenty-five percent to 60% of follicular

lymphomas also transform into more aggressive subtypes of

non–Hodgkin’s lymphoma (1-3). Eighty-five percent of

follicular lymphomas harbor t(14;18)(q32;q21), resulting in

juxtaposition of the B-cell-leukemia/lymphoma-2 (Bcl-2)

proto-oncogene with the immunoglobulin heavy chain (IgH)

locus, typically upstream of one of the JH segments (4-8).

Deregulated expression of Bcl-2 prolongs survival of B and T

lymphocytes via abrogation of the majority of apoptotic

pathways (8-10). Substantial evidence supports the hypothesis

that t(14;18) and Bcl-2 overexpression are necessary but not

solely responsible for the genesis of follicular lymphomas. EA-Bcl-2 transgenic mice uniformly develop polyclonal B-cell

hyperplasia, but only 5% to 15% eventually progress to

aggressive monoclonal B-cell lymphomas following a pro-

tracted latency period and often in conjunction with cooperating

cytogenetic lesions (10-13). Further evidence supporting the

notion that t(14;18) is not causative for the development of

follicular lymphoma is that B cells harboring t(14;18) have

been detected by PCR screening of peripheral blood and

hyperplastic lymphoid tissue from healthy individuals (14-16),

and this phenomenon seems to increase with age (17). Finally,

the rare hematologic disorder, persistent polyclonal B-cell

lymphocytosis, is characterized by chronic stable polyclonal

lymphocytosis that, despite the presence of Bcl-2 rearrange-

ments into the IgH locus, fails to overexpress Bcl-2 (18). These

data suggest that, although t(14;18) is sufficient to initiate an

oncogenic pathway, Bcl-2 alone is a relatively weak oncogene

and requires additional cooperating genetic lesions for neoplas-

tic transformation to occur. Although molecular analysis of

human follicular lymphoma has revealed numerous cytogenetic

Received 7/6/04; revised 9/20/04; accepted 10/6/04.Grant support: NIH grants CA78254 (G.J. Vanasse) and 5U24DK058813-02(K.Y. Yeung), American Society of Hematology fellow scholar grant (G.J.Vanasse), and NIH research grant CA-16359 from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Notes: G.J. Vanasse is a past American Society of Hematology fellow scholar andmember of the Yale Cancer Center.Requests for reprints: Gary J. Vanasse, Section of Hematology, Department ofInternal Medicine, Yale University School of Medicine, 333 Cedar Street, WWW-403, Box 208021, New Haven, CT 06520. Phone: 203-737-2340; Fax: 203-785-7232. E-mail: [email protected] D 2004 American Association for Cancer Research.

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alterations potentially important for propagation of a neoplastic

clone (19), the significance of these secondary chromosomal

abnormalities in influencing clinical course and pathogenesis of

follicular lymphoma remains to be determined.

The application of oligonucleotide and cDNA microarray

technology to the study of non–Hodgkin’s lymphoma has

provided insights into gene expression patterns that differentiate

malignant B cells from their normal counterparts, has defined

prognostic subgroups, and has identified potential therapeutic

targets (20-22). Gene profiling studies on follicular lymphoma

B cells have revealed a genetic signature similar to germinal

center B cells; have identified differentially expressed genes

involved in cellular pathways important for cell cycle reg-

ulation, cell adhesion, cellular signaling, and B-cell develop-

ment; and have shown that transformation of follicular

lymphoma into diffuse large B-cell lymphomas requires distinct

genetic alterations (20, 23-27). However, much of the gene

expression analyses have been generated on follicular lympho-

ma B cells obtained from patients heavily treated for relapsed

disease, on t(14;18)+ cell lines rather than on primary cells, or

on RNA isolated from whole tissue biopsies rather than from

purified follicular lymphoma cells. Therefore, these studies may

be compromised in their ability to distinguish early, primary

genetic events important for the genesis of follicular lymphoma

from the multitude of secondary genetic changes associated

with disease progression, therapeutic intervention, or the cel-

lular microenvironment.

The use of microarray technology to analyze gene

expression profiles in animal models of proto-oncogene

deregulation may facilitate the identification of those primary

genetic events important for tumorigenesis in humans. We

hypothesized that gene expression profiling of primary,

polyclonal B cells overexpressing Bcl-2 could serve as a tem-

plate for the identification of candidate genes, the deregulation

of which affects pathways important for the biology of Bcl-2-

associated lymphomas. Using oligonucleotide microarrays to

analyze nonmalignant B cells from EA-Bcl-2 transgenic mice,

we aimed to identify differentially expressed genes that also

exhibited correlative deregulated expression in human follicular

lymphoma. In the present study, we show that CD19+ B cells

isolated from EA-Bcl-2 transgenic mice overexpress the

suppressor of cytokine signaling-3 (SOCS3) gene when com-

pared with littermate control (LMC) mice. SOCS3 induction is

mediated by overexpression of Bcl-2 in a manner independent

of the site of transgene insertion. We also provide evidence

showing overexpression of SOCS3 protein in a cohort of

patients diagnosed with de novo follicular lymphoma. Taken

together, these studies suggest that the Bcl-2-associated

induction of SOCS3 represents an early genetic event

influencing cellular pathways important for the pathogenesis

of follicular lymphomas in humans.

ResultsPurification of B Cells from El-Bcl-2 Transgenic and Wild-type Mice

To characterize Bcl-2-mediated cellular signaling pathways

important for the development of follicular lymphoma in

humans, we used differential gene expression in Bcl-2-

overexpressing polyclonal B cells as a template to identify

genes also deregulated in de novo follicular lymphoma. We

purified primary B cells from EA-Bcl-2 transgenic mice and

wild-type LMC mice by negative selection and did oligonu-

cleotide microarray analyses to formulate a differential gene

expression profile. Phenotypes of the mice were similar to that

reported previously, with EA-Bcl-2 transgenic mice exhibiting

B-cell hyperplasia as described (28). At the time of analysis, all

mice were healthy and without evidence of tumor formation.

Single cell suspensions of splenocytes were prepared from

spleens harvested from six 24-week-old EA-Bcl-2 transgenic

and five age-matched wild-type LMC mice (C57BL/6 strain).

Immunomagnetic bead depletion was used to isolate naive B

cells of primarily B2 subtype and devoid of B1 subtype, T cells,

monocytes, and natural killer cells. Negative selection of B cells

was done to avoid B-cell activation and its resultant gene

profile, which may confuse interpretation of the microarray

analysis. B cells were phenotyped and analyzed by flow

cytometry, revealing a >97% CD19+ pure population without

detectable CD4+, CD8+, or CD56+ (data not shown).

Oligonucleotide Microarray AnalysisTo identify Bcl-2-mediated differential gene expression, we

compared the CD19+ B-cell gene expression between trans-

gene-positive and LMC mice by oligonucleotide microarray

analysis. Target transcripts (15 Ag) from individual mice from

each cohort were hybridized to Affymetrix murine U74v2 A, B,

and C chipsets (Affymetrix, Santa Clara, CA) consisting of

>36,000 genes and expressed sequence tags. Individual array

results obtained from the six EA-Bcl-2 transgenic and the five

LMC mice were summarized as one experimental array and one

control array, respectively, and ratios built and analyzed using

Rosetta Resolver version 3.1 software (Rosetta Biosoftware,

Seattle, WA) were used to identify genes exhibiting z2-fold

differential expression (P < 0.05). Comparison of the two

composite arrays revealed that a total of 151 genes were

differentially expressed according to our parameters, with

57 genes induced and 94 genes repressed (Fig. 1; Table 1). Of

this group, 103 represented known genes, whereas 48 were

cDNAwithout known function or homology based on Genbank

database references. Analysis revealed differential expression of

both known and novel genes associated with cellular pathways

important in apoptosis, B-cell growth and differentiation, cell

cycle, intracellular signaling, inflammatory response mediators,

immunity, and DNA damage repair. Genes associated with

antiapoptotic pathways (HSP1a and HSP1b) as well as me-

diators of intracellular signaling (SOCS3 and MAP3K11) were

induced. Conversely, reduced expression was noted in the

proapoptotic gene Bid; the c-myc and c-myb proto-oncogenes;

cyclin D2 , an important regulator of progression through G1

phase of the cell cycle; and several genes associated with in-

nate immunity (Pgrp , CR2 , and TRAF1). To validate the array

results, we measured mRNA levels by real-time quantitative

reverse transcription-PCR (RT-PCR) using SYBR Green I.

Differential gene expression was confirmed in 18 of 20 genes

tested (Table 2). The fold change for several genes tested by

real-time quantitative RT-PCR was proven greater than that

reported on the microarray, suggesting that microarray analysis

Bcl-2-Associated SOCS3 Induction in Follicular Lymphoma

Mol Cancer Res 2004;2(11). November 2004

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may underestimate the amplitude of differential gene expres-

sion. Interestingly, the SOCS3 gene was found to be induced

2-fold on the microarray, and this was highly statistically

significant (P = 0.003). To confirm SOCS3 induction, real-time

quantitative RT-PCR and RNase protection assays were done

on RNA samples from B cells from array mice. Both methods

revealed 5-fold induction of SOCS3 mRNA in B cells from

EA-Bcl-2 transgenic mice, and this was consistent across

samples (Table 2). An accumulating body of evidence indicates

SOCS3 to be an important negative regulator of inflammatory

and immune responses. However, Bcl-2-associated transcrip-

tional deregulation of SOCS3 has not been reported previously.

Therefore, SOCS3 seemed an interesting candidate gene

warranting further investigation. The complete differential gene

expression analysis has been deposited in the Gene Expression

Omnibus at http://www.ncbi.nlm.nih.gov/geo/.

SOCS3 Protein Is Overexpressed in Distinct Strains ofEl-Bcl-2 Transgenic Mice

To determine whether SOCS3 protein levels were induced in

EA-Bcl-2 transgenic mice relative to LMC mice, we did

Western blot analysis on whole cell lysates prepared from four

matched pairs of transgenic and control mice. To control for the

possibility that SOCS3 induction was due to the site of

transgene insertion and not mediated by Bcl-2, we also

prepared whole cell lysates from CD19+ B cells isolated as

described from a distinct strain of EA-Bcl-2 transgenic mice

(29), which as a result of transgene insertion leads to Bcl-2

overexpression in both B and T cells. Otherwise, both of the

EA-Bcl-2 transgenic strains exhibit phenotypically similar

B-cell hyperplasia as well as a similar incidence of lymphoma

development (29). Probing with SOCS3 antisera revealed a

marked increase in SOCS3 protein in both strains of EA-Bcl-2transgenic mice relative to their respective LMC mice (Fig. 2).

These results indicate that the induction of SOCS3 occurs

independently of the site of transgene insertion, thereby

decreasing the likelihood that SOCS3 induction is the result

of insertional mutagenesis. In contrast, probing with antisera

recognizing the SOCS family members CIS , SOCS1 , and

SOCS5 failed to reveal detectable protein expression of these

other SOCS family members in B cells from either strain of

EA-Bcl-2 transgenic mice (data not shown).

Overexpression of Bcl-2 Leads to Increased SOCS3Expression

We then wanted to determine whether SOCS3 induction was

due to overexpression of Bcl-2 or merely a response to antigen-

driven polyclonal B-cell hyperplasia common in EA-Bcl-2transgenic mice. A retroviral construct containing a human Bcl-

2 cDNA and an IRES-enhanced green fluorescent protein

(EGFP) was then overexpressed in bothmouse embryo fibroblast

(MEF-1) and monocyte/macrophage hematopoietic (JAWSII)

cell lines. Western analysis for Bcl-2 revealed undetectable

endogenous expression of Bcl-2 in MEF-1 and JAWSII cells

(Fig. 3A). Whole protein lysates from each cell line were

prepared as described and measured for SOCS3 protein levels.

When probed with SOCS3 antisera, MEF-1 cells overexpressing

the Bcl-2:EGFP construct exhibited marked overexpression of

SOCS3 protein compared with MEF-1 cells expressing EGFP

alone, where SOCS3 levels were proven undetectable (Fig. 3B).

FIGURE 1. Oligonucleotide microarray analysis of murine CD19+ B cells overexpressing Bcl-2 . Affymetrix murine U74v2 A, B, and C chipsets were usedto study CD19+ B cells obtained from EA-Bcl-2 transgenic and transgene-negative LMC mice. Normalized intensity data from individual arrays obtained fromsix EA-Bcl-2 transgenic and five LMC mice were summarized as one combined transgenic intensity experiment and one combined control intensityexperiment, respectively, and analyzed using Rosetta Resolver version 3.1 software to identify genes exhibiting z2-fold differential expression (P < 0.05).The combined LMC intensity experiment was used as the baseline. Red crosses, induced expression; green crosses, reduced expression.

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Table 1. Differential Gene Expression Analysis of Murine CD19+ B Cells Overexpressing Bcl-2

Accession (Unigene) Gene/Protein Name Fold Change P

Oncogene and tumor suppressor proteinsAA839840 Putative RNA polymerase II elongation factor �2.00 0.02M12848 c-myb proto-oncogene (c-myb ) �2.00 0.04L00039 c-myc proto-oncogene (c-myc ) �2.04 0.01Z31359 Neoplastic progression 2 gene (Npn2 ) �2.02 0.03

Intracellular signaling mediators and stress response proteinsM12571 Heat shock protein 70.3 (HSP1a) 11.12 <0.001AF109906 Heat shock protein 70.1 (HSP1b) 7.30 0.007U24703 Reelin 6.03 0.003AI842663 Osmotic stress protein 94 (OSP94) 3.95 0.02X78667 Ryanodine receptor 2 (Ryr2) 3.08 0.007AI846606 Hypothetical protein similar to MAP3K11 2.44 0.03AV084051 IL-1 receptor antagonist 2.26 0.03AV374868 SOCS3 2.05 0.003M90388 Protein tyrosine phosphatase 70zpep �2.00 0.02AI153935 Phosphatidylinositol 3-kinase, regulatory subunit, p150 �2.00 0.01AA175606 Putative InB~ protein �2.00 0.04Y09632 Rabkinesin-6 �2.14 0.03AW122494 Ras-GRF2 �2.15 0.008AI594690 Choline/ethanolaminephosphotransferase 1 �2.34 0.04AV087622 Annexin 4 �3.01 0.03U96635 Nedd4 �3.25 0.01AW050293 Putative elongation factor Tu �3.97 <0.001L35302 Tumor necrosis factor receptor – associated factor 1 (TRAF1 ) �4.13 0.03

Apoptosis and cell cycle regulation proteinsAB021861 Apoptosis signal-regulating kinase 2 2.76 <0.001L31532 Bcl-2a exon 2 2.00 0.03U75506 BID BH3-only domain protein �2.00 0.01L31532 Bcl-2h �2.02 0.01AI447296 Ectodysplasin A receptor – associated death domain (EDAR) �2.07 0.01AI605650 DNase g precursor �2.37 0.02AA119627 Protein similar to M-phase phosphoprotein 9 �2.37 0.04AI152882 Transglutaminase 2 �5.92 <0.001M83749 Cyclin D2 �5.92 0.04

Transcription factors and DNA binding proteinsAV349362 Myelin transcription factor 1 (Myt 1) 2.59 0.003AI841913 Sclerostin-like protein 2.59 0.03AI527205 Coup transcription factor 2 2.41 0.03AW050036 Brain abundant membrane signal protein (Basp1) homologue 2.06 0.02U08185 B-lymphocyte – induced maturation protein 1 (BLIMP1) 2.00 0.01AA162644 Putative transcription regulator NT fin 12 �2.20 0.02AF077861 Id2 gene �2.20 0.03AI415206 IFN-induced Mx protein �2.24 0.009AI957146 Putative MASL1 gene �2.28 0.01AI594455 Trichorhinophalangeal 1 (TRPS1) �2.50 0.01AA960657 Putative INF-g-induced protein IFI16 �2.97 0.04AI019193 T-cell transcription factor 7 (Tcf 7) �4.71 <0.001

Receptors and cell surface proteinsAI647643 Signal recognition particle 54-kDa protein (Srp54) 3.29 0.004AF010254 C1 inhibitor 3.20 0.02AF107847 Golgi protein 55 isoform 2.77 0.02AI608001 Src H3 domain bp 4 2.67 0.009AA510989 Protein similar to IL-6 receptor a 2.53 0.04AI426271 Paired Ig-like type 2 receptor a 2.52 0.04AJ132336 Chemomokine receptor 9 (CCR9) 2.37 0.03AW124738 Lanthionine synthetase C– like protein (Lancl-1) 2.23 0.02AI849185 Muscleblind-like protein 2.19 0.01M29281 Complement receptor 2 (CR2 ) �2.00 <0.001M63695 Cd1d1 �2.00 0.003AV340322 IFN-g-induced Mg11 protein homologue �2.00 0.008M18194 Fibronectin �2.05 0.004AI747561 Mucolipin 3 �2.15 0.01AI851899 Transmembrane protein 25 (Tmem25) �2.20 0.03AF076482 Peptidoglycan recognition protein precursor (Pgrp ) �2.23 0.04U29678 Chemokine receptor 1 (CCR1) �2.47 0.04U05265 Glycoprotein 49B (gp49B) �2.98 0.04M65027 Glycoprotein 49A (gp49A) �3.37 0.01L08115 CD9 �3.84 0.004AI853884 Chemokine binding protein 2 �5.17 0.007

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Table 1. Differential Gene Expression Analysis of Murine CD19+ B Cells Overexpressing Bcl-2 (Cont’d)


AF000236 RDC1 orphan chemokine receptor �5.89 0.008AA822679 Hematopoietic cell signal transducer �5.97 0.01

Miscellaneous proteins and cDNAAV253089 cDNA 10.57 0.01V00793 IgG1-C region 10.16 0.002X67210 Rearranged IgG2b H-chain 4.11 0.01AI451032 IgG1 H chain 4 3.90 0.04AA416072 cDNA 3.73 0.03D14625 IgG3 H chain 8 3.71 0.02D78344 IgG 3.41 0.03AW047643 cDNA 3.21 0.009J00475 IgH DFL16.1 3.11 0.002Ai850363 Muscle glycogen phosphorylase (Pygm) 2.92 0.04AV038316 cDNA 2.79 0.03AF002719 Secretory leukoprotease inhibitor (SLPi) 2.58 0.007AW061234 cDNA 2.54 0.03AI786089 Kininogen precursor 2.46 0.04AA517032 cDNA 2.45 0.02AA789553 Alstrom syndrome 1 (Alms1) protein homologue 2.39 0.02M90766 Ig J chain 2.35 0.006AI853664 cDNA 2.31 0.006AV080003 IgH J558 family 2.25 0.03AV258047 cDNA 2.23 0.03J03482 Histone H1 gene 2.21 0.02AV281523 cDNA 2.20 0.01AV210037 cDNA 2.17 0.03AF000913 WS2a43 mutated IgH 2.12 0.01AI314284 hypothetical protein 2.00 0.02AV259552 cDNA 2.00 0.03AU045276 cDNA 2.00 0.04AV320218 cDNA 2.00 0.03M34597 Ig germ line E chain Vx-J2-C2 2.00 0.003AV217136 cDNA 2.00 0.01AV297816 cDNA 2.00 0.009AV174430 cDNA 2.00 0.04AV207625 Gene similar to protein phosphatase-2 inhibitor 2.00 0.02AV225591 Protein similar to mouse glutathione peroxidase �2.00 0.03AV310830 cDNA �2.00 0.03AI448839 cDNA �2.00 0.01M16819 Mouse tumor necrosis factor-h �2.00 0.02AI462391 Hypothetical protein �2.07 0.03AV235558 cDNA �2.09 0.008AV012076 cDNA �2.10 0.02AI152709 cDNA �2.10 0.04AW045191 cDNA �2.17 0.006AV101344 DNA ligase-3 �2.17 0.02AI662280 cDNA �2.17 0.03M60474 Myristoylated alanine-rich protein kinase C substrate �2.18 0.009AV128327 cDNA �2.20 0.008M19436 Myosin light chain �2.20 0.03AF072697 Shyc �2.23 0.04AI842144 cDNA �2.25 0.009X51941 Methylmalonyl CoA mutase �2.25 0.04AI627038 cDNA �2.32 0.03AI481498 Procollagen, type V, a1 �2.34 0.009AI182009 cDNA �2.35 <0.001AV212587 cDNA �2.35 0.03AV368065 Hypothetical protein �2.39 <0.001AV229080 cDNA �2.41 0.03AV332560 cDNA �2.48 0.04AA671194 cDNA �2.51 0.01X12905 Properdin factor �2.53 0.03AI853854 ATP binding cassette, subfamily C protein �2.55 0.006AI530075 cDNA �2.55 0.03AI841689 Chemokine-like factor superfamily-3 �2.56 0.03AW229312 cDNA �2.67 0.02AW124025 Putative helicase-like protein non –Hodgkin’s lymphoma �2.72 0.03AW214234 cDNA �2.74 0.01AV365271 Nedd4 �2.77 <0.001AV271750 cDNA �2.81 0.02AA712022 cDNA �2.82 0.002AI835567 Tubulin, g2 chain �2.86 <0.001

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JAWSII cells overexpressing the Bcl-2: EGFP construct

also revealed overexpression of SOCS3 protein compared with

cells expressing EGFP alone (Fig. 3B). In addition, to assess

whether Bcl-2-associated induction of SOCS3 is linked to

activation of signal transducer and activator of transcription

(STAT) 3, we measured phospho-STAT3 levels relative to

STAT3 in Bcl-2-overexpressing cells. When probed with

phospho-STAT3 antisera, both MEF-1 and JAWSII cells over-

expressing the Bcl-2:EGFP construct exhibited overexpression

of phospho-STAT3 protein compared with their respective EGFP

control cells, which failed to express detectable levels of

phospho-STAT3 protein (Fig. 3C). Western blots were then

stripped and the same blots were reprobed with STAT3 antisera,

revealing no difference in the levels of STAT3 protein between

Bcl-2-overexpressing and control cells (Fig. 3C). Probing with

antisera recognizing the SOCS family members CIS , SOCS1 ,

and SOCS5 failed to reveal detectable protein expression of these

specific family members in either MEF-1 or JAWSII cells

overexpressing Bcl-2:EGFP (data not shown). These data

indicate that the induction of SOCS3 is associated with

overexpression of Bcl-2 and not simply a physiologic response

to increased cell turnover. Furthermore, overexpression of Bcl-2

mediates SOCS3 induction via cellular pathways linked to

activation of STAT3.

SOCS3 Is Expressed at High Levels in a Cohort ofPatients with De novo Follicular Lymphoma

To determine whether Bcl-2-associated induction of SOCS3

may occur in human follicular lymphoma, we measured SOCS3

protein levels by immunohistochemistry in paraffin-embedded

biopsies from 12 patients diagnosed with de novo follicular

lymphoma prior to the initiation of therapy. Follicular lym-

phoma tissue specimens were diagnosed as either histologic

grades I or II, and all harbored t(14;18) with concomitant

marked overexpression of Bcl-2 in the follicular center cell

region (Fig. 4). Immunostaining with two distinct antibodies to

SOCS3 revealed marked overexpression of SOCS3 protein that,

within the follicular center cell region, was limited to neoplastic

follicular lymphoma cells and colocalized with Bcl-2 in 9 of 12

de novo follicular lymphoma cases examined (Fig. 4; Table 3).

The antibodies stained mainly the nucleus of positive cells, with

slight cytoplasmic staining also noted in some cases. In con-

trast, SOCS3 protein was not detected by immunostaining in

germinal center follicular B cells from benign hyperplastic

tonsil tissue (Fig. 4). SOCS3 staining was also noted in normal

as well as neoplastic follicular lymphoma cells outside the fol-

licular center cell region.

DiscussionAlthough t(14;18) represents an early initiating genetic event

required for the development of follicular lymphoma, it is clear

that cooperating genetic errors are required to deregulate

cellular pathways for neoplastic transformation to occur. In our

search for genes that cooperate with Bcl-2 to mediate neoplastic

Table 1. Differential Gene Expression Analysis of Murine CD19+ B Cells Overexpressing Bcl-2 (Cont’d)


AV092014 cDNA �2.87 0.04AW228014 Hypothetical protein �2.93 0.001M20878 TCR h chain, VDJ region �3.06 0.04M21050 Lysozyme M �3.16 0.001AI429433 cDNA �3.18 <0.001X70057 Serine protease gene �3.67 0.04AI159157 cDNA �4.20 0.02AA289585 cDNA �4.60 0.008AI450988 cDNA �4.64 0.02U34277 Platelet-activating factor acetylhydrolase �4.87 0.03AV260742 cDNA �4.91 0.04X51547 Lysozyme P precursor �5.81 0.004AI844675 cDNA �5.92 0.01AI450144 cDNA �6.95 0.002AV312050 cDNA �8.70 0.02X15313 Myeloperoxidase �26.73 <0.001

NOTE: Comparison of composite arrays generated on CD19+ B cells obtained from both EA-Bcl-2 transgenic and transgene-negative LMC mice revealed 151 genesexhibiting z2-fold differential expression (P < 0.05), with 57 genes induced and 94 genes repressed. Genes are denoted according to their Genbank accession nos. Genes aregrouped according to their function as reported.

Table 2. Selected Differential Gene Expression in EA-Bcl-2Transgenic Mice as Determined by Microarray Analysis,RT-PCR, and RNA Protection Assay

Gene Microarray P RT-PCR RNA Protection Assay

SOCS3 2.05 0.003 5 5HSP1a 11.12 <0.001 ND NDIgG1-C 10.16 0.002 10 NDIgG2b H chain 4.11 0.01 10 NDCCR9 2.37 0.03 7 NDBcl-2 a exon 2 2.00 0.03 2 NDBlimp 1 2.00 0.01 5 NDc-myb �2.00 0.04 �4 NDCR2 �2.00 <0.001 �4 NDBID �2.00 0.01 �2 NDc-myc �2.04 0.01 �3 NDNEDD4 �3.25 0.01 �4 NDCyclin D2 �5.92 0.04 �8 ND

NOTE: Microarray, RT-PCR, and RNA protection assay data reflect the foldchange for the average of EA-Bcl-2 transgenic animals examined calculatedrelative to the signal observed for the average of LMC control samples. ND, notdetermined.



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transformation in follicular lymphoma, our analysis revealed

that overexpression of Bcl-2 was associated with induction of

the SOCS3 gene in both murine CD19+ B cells and human

follicular lymphoma tissue specimens. To our knowledge, this

is the first description of an association between Bcl-2 and

SOCS3 as well as the first report of the induction of SOCS3

RNA and protein in purified populations of murine B cells and

human de novo follicular lymphoma cells. Our data suggest

that SOCS3 transcript levels in normal B cells are barely

detectable, with transcriptional induction noted only in con-

junction with overexpression of Bcl-2. The finding that SOCS3

expression colocalized with that of Bcl-2 and was noted pri-

marily throughout neoplastic germinal center follicles indicates

that overexpression of SOCS3 originated from malignant follic-

ular lymphoma B cells and not from associated normal cells.

Previous studies examining gene expression in follicular

lymphoma have reported discordant results regarding SOCS3

induction, a finding potentially due to multiple factors. First,

studies have used different microarray platforms as well as

varied statistical methodologies for determining differential

gene expression, thus resulting in difficulty with database

comparisons. Second, differences exist between studies

concerning the cell type examined. Our arrays were done on

primary murine CD19+ B cells overexpressing Bcl-2. Bohen

et al. (25) noted SOCS3 overexpression in follicular lymphoma

tissue from a group of patients noted to be nonresponders to

rituximab. However, in this study, interpretation of differential

gene expression may be complicated by the fact that analysis

was done on a mixed population of cells rather than purely

isolated neoplastic B cells. In comparison with studies

examining human follicular lymphoma cells (23, 26, 27) or

cultured t(14;18)+ cell lines (24), which have not revealed

SOCS3 induction, SOCS3 gene induction in murine B cells

examined in our study may reflect differences between murine

and human B-cell biology or differences inherent to the

developmental stage of the B cell examined. Finally, selection

biases resulting from heterogeneity in patient populations and

tumor biology as well as of Bcl-2 expression levels may

contribute to variable levels of SOCS3 expression.

The SOCS3 gene is a member of a family of cytokine

suppressors that inhibit cytokine-mediated signaling via classic

negative feedback loop inhibition (30-34). Transcripts

FIGURE 2. SOCS3 protein is induced in distinct strains of EA-Bcl-2 transgenic versus LMC mice. Western blot for SOCS3 on whole protein extracts(30 Ag/lane) from CD19+ B cells isolated from individual mice from two distinct strains of EA-Bcl-2 transgenic mice and their respective transgene-negativeLMC mice. Top, Western blot for SOCS3 in EA-Bcl-2 transgenic strain 1 (lanes a , c , d , and f ), LMC strain 1 (lanes b and e ), EA-Bcl-2 transgenic strain 2(lanes g and i), and LMC strain 2 (lanes h and j ). Bottom, Western blot for actin (lanes a-j ) to confirm equivalent protein loading.

FIGURE 3. Overexpression of Bcl-2 inducesSOCS3 protein levels in MEF-1 and JAWSIIcells via activation of STAT3 . Both MEF-1 andJAWSII cells were transduced with a retroviralconstruct harboring either a fusion Bcl-2:EGFPor EGFP alone, and whole protein lysates wereisolated at 48 hours. Western blot for Bcl-2 ,SOCS3 , or phospho-STAT3 was then doneon whole proteinlysates (30 Ag/lane). A. Top,Western blot for Bcl-2 in MEF-1 cells harboringBcl-2:EGFP (lane a) and EGFP control (lane b)and JAWSII cells harboring Bcl-2:EGFP (lane c )and EGFP control (lane d ); bottom, Westernblot for actin (lanes a -d) to confirm equivalentprotein loading. B. Top, Western blot forSOCS3 in MEF-1 cells containing Bcl-2:EGFP(lane a) and EGFP control (lane b ) and JAWSIIcells containing EGFP control (lane c ) and Bcl-2:EGFP (lane d ); bottom, Western blot for actin(lanes a -d) to confirm equivalent protein load-ing. C. Top, Western blot for phospho-STAT3 inboth MEF-1 cells (lane a) and JAWSII cells(lane c ) containing Bcl-2:EGFP and in respec-tive EGFP control cells (lanes b and d); bottom,Western blot for STAT3 (lanes a-d ).

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encoding various SOCS family members are typically present at

very low or undetectable levels but are rapidly up-regulated in

response to a wide range of cytokines and hormones (33). The

expression of SOCS3 is tightly controlled via transcriptional

regulation primarily mediated through STAT family proteins

STAT3 and STAT5 (35-38). SOCS3-mediated feedback

inhibition is primarily regulated via signaling through Janus-

activated kinase and STAT family proteins and has been

extensively reviewed elsewhere (33, 39-43). SOCS3 expres-

sion has been primarily noted in murine T cells and has been

shown to regulate both T-cell development and activation via

numerous mechanisms (44-47). The recent generation of

mice lacking functional SOCS3 in hepatocytes, macrophages,

and neutrophils reveals SOCS3 to be an essential regulator of

interleukin-6 (IL-6) signaling via mediation of gp130-related

cellular signaling pathways (48-50) as well as a negative

regulator of granulocyte colony-stimulating factor signaling

(51). In addition to its role as a suppressor of IL-6-mediated

signaling, SOCS3 may also have qualitative and quantitative

influence over cellular responses to IL-6 (33). Specificity of

SOCS3 activity may thus be nonredundant and dependent on

specific cytokine receptor interactions, thus possibly revealing

a central role for SOCS3 in directing gp130-related cytokines

toward either proinflammatory or anti-inflammatory cellular

responses.

Although SOCS3 would seem to negatively regulate

inflammatory responses (33), its role in tumorigenesis and the

underlying mechanisms that regulate its expression in B cells

remain to be defined. Several studies have examined SOCS3

expression in a diverse group of tumors of hematopoietic cell

origin. Chronic myelogenous leukemia cell lines as well as

leukemic cells from patients with chronic myelogenous

leukemia blast crisis were noted to constitutively express

SOCS3, resulting in attenuation of IFN-a signaling and re-

sistance to its antiproliferative effects (52). Similarly, SOCS3

overexpression in cancer cells derived from patients with

cutaneous T-cell lymphoma was found dependent on aberrant

expression of STAT3, representing a pathway that also results

FIGURE 4. Immunostaining reveals overexpression of SOCS3 inde novo follicular lymphoma. All staining was done on paraffinbiopsies by Vectastain ABC detection. Representative case ofde novo follicular lymphoma (no. 3 in Table 3) showing (A and B)Bcl-2 -positive staining of neoplastic follicular lymphoma cells withinthe follicular center cell region. Concomitant SOCS3-positive stainingis seen within the follicular center cell region limited to neoplasticfollicular lymphoma cells using (C and D) anti-SOCS3 antibody 1(Zymed) and (E) anti-SOCS3 antibody 2 (Santa Cruz Biotechnology).F and G. Representative case of benign hyperplastic tonsil germinalcenter B cells showing negative staining for SOCS3. Originalmagnification, �10 (A, C, and F) and �50 (B, D, E, and G).



627



in decreased responsiveness of cutaneous T-cell lymphoma

cells to IFN-a (53). In addition, acute myeloid leukemia cells

bearing IL-6-mediated constitutive STAT3 phosphorylation

were found to also constitutively overexpress SOCS1 and

SOCS3 (54). In contrast to our study, oligonucleotide micro-

array analysis of IL-6-dependent multiple myeloma cells

revealed STAT3-mediated induction of SOCS3 via Bcl-2-

independent cellular pathways (55). It is possible that Bcl-2-

associated induction of SOCS3 is restricted to an earlier stage of

B-cell development rather than in late-stage plasma cells or

memory B cells. Collectively, these studies indicate that IL-6-

dependent, STAT3-mediated pathways serve as important regu-

lators of SOCS3 expression levels in diverse hematopoietic

tumors.

The cellular pathways required for Bcl-2-mediated induction

of SOCS3, as well as the degree to which SOCS3 induction

influences the biology of de novo follicular lymphoma in

humans, remain to be elucidated. Given that Bcl-2 is not a

transcription factor, Bcl-2 overexpression likely induces

SOCS3 indirectly by modulating pathways that deregulate

factors necessary for transcriptional up-regulation of SOCS3. It

remains to be determined whether Bcl-2 and SOCS3 function

in cooperation to cause an oncogenic hit important for neoplas-

tic transformation of B cells or whether SOCS3 may be

suppressing propagation of malignant B cells harboring Bcl-2

overexpression. It is well known that forced expression of

oncogenes in concert with Bcl-2 overexpression cooperate to

deregulate pathways that speed the pace of tumorigenesis as

well as influence morphology of the neoplastic clone.

Transgenic animals with combined overexpression of c-myc

and Bcl-2 develop lymphomas of a more primitive histology

and with markedly decreased latency compared with transgenic

animals solely overexpressing Bcl-2 (13), most likely reflecting

combined deregulation of apoptotic and cell cycle pathways. It

remains to be determined whether SOCS3 might also cooperate

with Bcl-2 in affecting neoplastic transformation of B cells. The

finding that SOCS3 is overexpressed in our cohort of follicular

lymphoma and is also overexpressed in other hematopoietic

tumors suggests that SOCS3 deregulation activates cellular

pathways important for tumorigenesis and does not serve as

a tumor suppressor gene. On the other hand, similar to its

emerging role in immunity as a negative regulator of cellular

signaling that mediate inflammatory responses, SOCS3 over-

expression in de novo follicular lymphoma may serve a reg-

ulatory role to suppress the proliferative capacity of the

neoplastic clone and select for a more indolent lymphoma

phenotype. In this scenario, the loss of SOCS3 induction may

then predispose to a more aggressive phenotype such as seen

in transformed follicular lymphoma. Determining whether

SOCS3 induction is present in intermediate and high-grade

non–Hodgkin’s lymphoma subtypes and whether it carries

prognostic significance will help discern whether SOCS3

overexpression influences neoplastic transformation or acts

as a tumor suppressor.

Taken together, our study suggests that the induction of

SOCS3 in B cells is an early genetic event mediated by

overexpression of Bcl-2 and that SOCS3 may cooperate with

Bcl-2 in deregulating cellular pathways important for the

pathogenesis of de novo follicular lymphoma in humans.

Examination of the cellular pathways by which Bcl-2 over-

expression leads to the induction of SOCS3 and other

downstream effectors affected by deregulated expression of

SOCS3 should provide important insight into the genesis of

follicular lymphoma in humans as well as identify potential

novel signaling intermediaries that lend to the development of

novel targeted therapies.

Materials and MethodsB-Cell Isolation

Single cell suspensions were prepared from individual

spleens from two distinct strains of male and female 24-week-

old EA-Bcl-2 transgenic mice (28, 29) and 24-week-old

transgene-negative LMC mice (C57BL/6 strain). Naive un-

touched B cells were isolated from murine spleen cells by

negative selection using an immunomagnetic bead B-Cell

Isolation Kit (Miltenyi Biotec, Inc., Auburn, CA) according to

the manufacturer’s instructions. Following immunomagnetic

bead isolation, a small aliquot of cells was phenotyped by flow

cytometry to assess B-cell purity.

Flow Cytometry and AntibodiesImmunophenotyping was done on cell suspensions using a

FITC-conjugated monoclonal antibody directed against murine

CD19 (BD Biosciences/PharMingen, San Diego, CA). An

irrelevant isotype-matched control antibody was used in all

experiments. Analysis was done within 1 hour using a dual-

laser FACSCalibur instrument (Becton-Dickinson, Franklin

Lakes, NJ).

RNA IsolationTotal RNA was prepared from B cells isolated as above

using the RNeasy Midi Kit (Qiagen, Valencia, CA) according to

the manufacturer’s instructions. RNA quality was examined

by the RNA 6000 LabChip Kit on the 2100 bioanalyzer

(Agilent Technologies, Palo Alto, CA). Quantity and absor-

bance at 260/280 nm of total and cRNA were assessed by

UV spectrophotometer.

Table 3. SOCS3 Immunostaining Patterns of Neoplastic BCells in Paraffin-Embedded Biopsies from Cases ofFollicular Lymphoma

Follicular Lymphoma Case Anti-SOCS3 Antibody 1 Anti-SOCS3 Antibody 2

1 + +2 + +3 + +4 � �5 + +6 +/� +/�7 + +8 � �9 � �10 + +11 +/� +/�12 + +

NOTE: Immunostaining using two distinct SOCS3 antisera: anti-SOCS3 antibody1 (Zymed) and anti-SOCS3 antibody 2 (Santa Cruz Biotechnology). �, all tumorcells negative; +/�, staining in >50% of the tumor cells; +, staining in >90% ofthe tumor cells.

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Gene Expression Analysis by DNA OligonucleotideArrays

Double-stranded cDNA was synthesized from total RNA,

amplified as cRNA, labeled with biotin, and hybridized to

Affymetrix murine U74v2 A, B, and C Array chipsets, which

were washed and scanned at the University of Washington’s

Center for Expression Arrays according to procedures devel-

oped by the manufacturer. Image processing was done using

Affymetrix MAS-5 software. The quality of hybridization and

overall chip performance was determined by visual inspection

of the raw scanned data and the MAS-5 generated report file.

The raw data were loaded into the Rosetta Resolver Gene

Expression Data Analysis System (56, 57) via standard

methods. Using the Resolver system, the normalized intensity

data from all control experiments and from all transgenic

experiments were summarized as one combined control

intensity experiment and one combined transgenic experiment,

respectively. These combined experiments take the spread and

distribution of the individual experiments into consideration,

hence facilitating our analysis without losing information.

Resolver uses an error-weighed approach to compute expres-

sion log ratios for each probe set based on the spread of the

replicate measurements. The software then computes a con-

fidence level, called P, for each probe set based on this error

estimate. Background correction in the Resolver system is done

on individual perfect match and mismatch probes. Resolver

adopts an error model (56) and a background correction

strategy in estimating the probe set intensity levels. Their error

model is derived from extensive like-versus-like experiments,

and their background correction approach uses local back-

ground estimates for probe sets in different regions of the chip.

In effect, the error model minimizes false-positives, particularly

at low expression values.

Real-time Quantitative PCR AnalysisReal-time quantitative RT-PCR analysis was done using a

LightCycler (Roche Diagnostics, Basel, Switzerland). Reverse

transcription was done by using SuperScript II (Invitrogen,

Carlsbad, CA). PCR primers were designed with MacVector

software (Accelrys, San Diego, CA). The nucleotide sequences

of the primer pairs are available on request. PCR reactions were

optimized using the FastStart DNA Master SYBR Green I Kit

(Roche Diagnostics) after verifying that no amplification was

noted in the no-template controls. To ensure that any DNA

contamination was removed by DNase I treatment of total

RNA, real-time quantitative RT-PCR was done on non-reverse-

transcribed RNA. No amplification was observed in these

conditions for differentially expressed genes examined. The

size of the PCR product for each gene was verified by gel

electrophoresis. Signals for genes from each RNA sample were

normalized to that sample’s signal for glyceraldehyde-3-

phosphate dehydrogenase. The fold change for experimental

samples was calculated relative to the signal observed for

control samples.

RNase Protection Assay32P-labeled riboprobes were incubated with total RNA

(10 Ag) and then subjected to RNase digestion using a

RiboQuant kit (BD Biosciences/PharMingen) according to the

manufacturer’s instructions. Following electrophoresis on a 5%

polyacrylamide gel containing urea (8 mol/L), radiolabeled

bands from experimental and control sample lanes were

quantitated using PhosphorImager and normalized to the values

for glyceraldehyde-3-phosphate dehydrogenase and L32 in the

same samples.

Cell CultureMEF-1 cell lines (American Type Culture Collection,

Manassas, VA) were cultured in DMEM with glucose (4.5 g/L)

enriched with 10% heat-inactivated fetal bovine serum (Hyclone

Laboratories, Logan, UT), L-glutamine (2 mmol/L), nonessential

amino acids mixture (100�), and sodium pyruvate (1 mmol/L)

in the presence of penicillin (100 units/mL) and streptomycin

(100 Ag/mL), all purchased from BioWhittaker, Inc. (Walkers-

ville, MD). Mouse bone marrow cells (JAWSII; American Type

Culture Collection) were cultured in a-MEM with ribonucleo-

sides, deoxyribonucleosides enriched with 20% heat-inactivated

fetal bovine serum (Hyclone Laboratories), L-glutamine

(4 mmol/L), and sodium pyruvate (1 mmol/L) in the presence

of granulocyte macrophage colony-stimulating factor (5 ng/mL;

R&D Systems, Inc., Minneapolis, MN).

Construction of Expression VectorsA cDNA encoding the intron-less open reading frame of the

717-bp human Bcl-2a (pORF-hBcl-2; InvivoGen, San Diego,

CA) was cloned into shuttle plasmid SL1180 (Amersham

Pharmacia Biotech, Piscataway, NJ) using the NcoI and NheI

(New England Biolabs, Inc., Beverly, MA) restriction enzyme

sites. The pORF-hBcl-2 was subsequently cloned into the

EcoRI and XhoI (New England Biolabs) sites on the multiple

cloning region of the bicistronic retroviral expression plasmid,

pBMN-IRES-EGFP (kindly provided by Dr. Garry Nolan,

Stanford University, Palo Alto, CA). High-titer, second-

generation helper free retrovirus was produced by calcium

phosphate–mediated transfection of the Phoenix ecotropic

packaging cell line (American Type Culture Collection) with

either 24 Ag of the hBcl-2 expression plasmid or pBMN-IRES-

EGFP control plasmid. Recombinant retroviral supernatant

was collected 48 hours after transfection and filtered through

a Millex-HV 0.45 Am filter (Millipore Corp., Bedford, MA).

For transduction, cell culture medium from f70% confluent

MEF-1 cells or JAWSII cells in six-well plates (Corning Inc.,

Corning, NY) were replaced with 2.5 mL of retrovirus

supernatant and centrifuged for 2 hours (1,430 � g at 32jC)and then incubated for 10 hours (5% CO2, 37jC). On com-

pletion of the incubation period, retroviral supernatant was

replaced by appropriate normal growth medium for each cell

type. Cells were sorted for stable retrovirus transfection based

on EGFP expression using a FACSVantage SE cell sorter

(Becton-Dickinson).

Western Blot AnalysisCell lysis and preparation of whole protein lysates were

done as described (58). Proteins were separated by SDS-PAGE

and blotted onto nitrocellulose membrane. Filters were probed

with primary antibodies recognizing either SOCS3 or SOCS1



629



(Zymed, South San Francisco, CA), CIS (Novus Biologicals,

Littleton, CO), SOCS5 (Imgenex, San Diego, CA), STAT3 or

phospho-STAT3 (Upstate, Charlottesville, VA), or Bcl-2 (BD

Biosciences/PharMingen) followed by horseradish peroxidase–

conjugated anti-rabbit IgG (BD Biosciences/PharMingen) and

detected using an enhanced chemiluminescence kit (Amersham

Pharmacia Biotech).

Tissue SpecimensParaffin-embedded biopsies of newly diagnosed, de novo

follicular lymphoma and benign hyperplastic tonsil were

obtained from the Critical Technologies Shared Resource of

the Yale Cancer Center according to approved Human

Investigation Committee protocols. In each case, the diagnosis

had been made based on conventional histologic and

immunohistologic examination according to the criteria of the

WHO classification (59).

ImmunohistochemistrySlides containing 4-Am tissue sections were subjected to a

conventional antigen retrieval protocol for 2 minutes using a

pressure cooker and prepared as described (60). Slides were

then incubated overnight at 4jC with one of two distinct

antibodies to SOCS3 [a rabbit polyclonal antibody to SOCS3

(Zymed) and a goat polyclonal antibody to SOCS3 (Santa Cruz

Biotechnology, Santa Cruz, CA)] or Bcl-2 (BD Biosciences/

PharMingen) followed by detection the next day using a

Vectastain ABC detection kit (Vector Laboratories, Burlingame,

CA) according to the manufacturer’s instructions. Sections were

stained in parallel without primary antibody to provide a

negative control for each reaction. Two authors of this study

(G.J.V. and A.W.Z.) independently evaluated the immunostain-

ing results.

AcknowledgmentsWe thank Noel Blake for assistance with flow cytometry; Vicki Morgan-Stephensen, Annie Minard, and Kristine Eiting for technical assistance; Drs.David Rimm and Robert Camp (Yale Department of Pathology) for providingtissue specimens and helpful advice; and Dr. Nancy Berliner for critical review ofthe article.

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2004;2:620-631. Mol Cancer Res Gary J. Vanasse, Robert K. Winn, Sofya Rodov, et al. Center.Hematology fellow scholar and member of the Yale Cancer

G.J. Vanasse is a past American Society ofNotes:research grant CA-16359 from the National Cancer Institute.Hematology fellow scholar grant (G.J. Vanasse), and NIHand 5U24DK058813-02 (K.Y. Yeung), American Society of

NIH grants CA78254 (G.J. Vanasse)11Follicular LymphomaDe novoCytokine Signaling-3 Expression in B Cells and

Bcl-2 Overexpression Leads to Increases in Suppressor of

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