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Molecular Cancer Research 2:620-631 (2004)
© 2004 American Association for Cancer Research

Cancer Genes and Genomics

Bcl-2 Overexpression Leads to Increases in Suppressor of Cytokine Signaling-3 Expression in B Cells and De novo Follicular Lymphoma1

Gary J. Vanasse1, Robert K. Winn4, Sofya Rodov1, Arthur W. Zieske2, John T. Li4, Joan C. Tupper3, Jingjing Tang5, Elaine W. Raines5, Mette A. Peters6, Ka Yee Yeung6 and John M. Harlan3

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

Requests for reprints: Gary J. Vanasse, Section of Hematology, Department of Internal 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: gary.vanasse{at}yale.edu


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The 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 Eµ-Bcl-2 transgenic mice and wild-type littermate control mice. Fifty-seven genes were induced and 94 genes were repressed by ≥2-fold in Eµ-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 Eµ-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.

Key Words: Bcl-2 • SOCS3 gene • follicular lymphoma • microarray • B cells


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Follicular 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 remaining 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. Eµ-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 protracted 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 rearrangements 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 neoplastic transformation to occur. Although molecular analysis of human follicular lymphoma has revealed numerous cytogenetic 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 regulation, cell adhesion, cellular signaling, and B-cell development; 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 lymphoma 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 cellular 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 template 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 Eµ-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 Eµ-Bcl-2 transgenic mice overexpress the suppressor of cytokine signaling-3 (SOCS3) gene when compared 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.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Purification of B Cells from Eµ-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 Eµ-Bcl-2 transgenic mice and wild-type LMC mice by negative selection and did oligonucleotide microarray analyses to formulate a differential gene expression profile. Phenotypes of the mice were similar to that reported previously, with Eµ-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 Eµ-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 Analysis
To identify Bcl-2-mediated differential gene expression, we compared the CD19+ B-cell gene expression between transgene-positive and LMC mice by oligonucleotide microarray analysis. Target transcripts (15 µg) 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 Eµ-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 ≥2-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 cDNA without 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 mediators 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 innate 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 may underestimate the amplitude of differential gene expression. 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 Eµ-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 transcriptional 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/.



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  		FIGURE 1. Oligonucleotide microarray analysis of murine CD19+ B cells overexpressing Bcl-2. Affymetrix murine U74v2 A, B, and C chipsets were used to study CD19+ B cells obtained from Eµ-Bcl-2 transgenic and transgene-negative LMC mice. Normalized intensity data from individual arrays obtained from six Eµ-Bcl-2 transgenic and five LMC mice were summarized as one combined transgenic intensity experiment and one combined control intensity experiment, respectively, and analyzed using Rosetta Resolver version 3.1 software to identify genes exhibiting ≥2-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

 

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

 
SOCS3 Protein Is Overexpressed in Distinct Strains of Eµ-Bcl-2 Transgenic Mice
To determine whether SOCS3 protein levels were induced in Eµ-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 Eµ-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 Eµ-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 Eµ-Bcl-2 transgenic 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 Eµ-Bcl-2 transgenic mice (data not shown).



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  		FIGURE 2. SOCS3 protein is induced in distinct strains of Eµ-Bcl-2 transgenic versus LMC mice. Western blot for SOCS3 on whole protein extracts (30 µg/lane) from CD19+ B cells isolated from individual mice from two distinct strains of Eµ-Bcl-2 transgenic mice and their respective transgene-negative LMC mice. Top, Western blot for SOCS3 in Eµ-Bcl-2 transgenic strain 1 (lanes a, c, d, and f), LMC strain 1 (lanes b and e), Eµ-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.

 
Overexpression of Bcl-2 Leads to Increased SOCS3 Expression
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 Eµ-Bcl-2 transgenic mice. A retroviral construct containing a human Bcl-2 cDNA and an IRES-enhanced green fluorescent protein (EGFP) was then overexpressed in both mouse 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). 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 overexpressing 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.



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  		FIGURE 3. Overexpression of Bcl-2 induces SOCS3 protein levels in MEF-1 and JAWSII cells via activation of STAT3. Both MEF-1 and JAWSII cells were transduced with a retroviral construct harboring either a fusion Bcl-2:EGFP or EGFP alone, and whole protein lysates were isolated at 48 hours. Western blot for Bcl-2, SOCS3, or phospho-STAT3 was then done on whole proteinlysates (30 µg/lane). A. Top, Western blot for Bcl-2 in MEF-1 cells harboring Bcl-2:EGFP (lane a) and EGFP control (lane b) and JAWSII cells harboring Bcl-2:EGFP (lane c) and EGFP control (lane d); bottom, Western blot for actin (lanes a-d) to confirm equivalent protein loading. B. Top, Western blot for SOCS3 in MEF-1 cells containing Bcl-2:EGFP (lane a) and EGFP control (lane b) and JAWSII cells containing EGFP control (lane c) and Bcl-2:EGFP (lane d); bottom, Western blot for actin (lanes a-d) to confirm equivalent protein loading. C. Top, Western blot for phospho-STAT3 in both MEF-1 cells (lane a) and JAWSII cells (lane c) containing Bcl-2:EGFP and in respective EGFP control cells (lanes b and d); bottom, Western blot for STAT3 (lanes a-d).

 
SOCS3 Is Expressed at High Levels in a Cohort of Patients 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 lymphoma 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 contrast, 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 follicular center cell region.



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  		FIGURE 4. Immunostaining reveals overexpression of SOCS3 in de novo follicular lymphoma. All staining was done on paraffin biopsies by Vectastain ABC detection. Representative case of de novo follicular lymphoma (no. 3 in Table 3
Table 3. SOCS3 Immunostaining Patterns of Neoplastic B Cells in Paraffin-Embedded Biopsies from Cases of Follicular 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 antibody 1 (Zymed) and anti-SOCS3 antibody 2 (Santa Cruz Biotechnology). –, all tumor cells negative; +/–, staining in >50% of the tumor cells; +, staining in >90% of the tumor cells.

) showing (A and B) Bcl-2-positive staining of neoplastic follicular lymphoma cells within the follicular center cell region. Concomitant SOCS3-positive staining is seen within the follicular center cell region limited to neoplastic follicular 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 germinal center B cells showing negative staining for SOCS3. Original magnification, x10 (A, C, and F) and x50 (B, D, E, and G).

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
Although 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 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 conjunction with overexpression of Bcl-2. The finding that SOCS3 expression colocalized with that of Bcl-2 and was noted primarily throughout neoplastic germinal center follicles indicates that overexpression of SOCS3 originated from malignant follicular 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 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 expression 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-{alpha} signaling and resistance 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 in decreased responsiveness of cutaneous T-cell lymphoma cells to IFN-{alpha} (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 microarray 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 regulators 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 neoplastic 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 overexpression in de novo follicular lymphoma may serve a regulatory 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 overexpression 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 Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
B-Cell Isolation
Single cell suspensions were prepared from individual spleens from two distinct strains of male and female 24-week-old Eµ-Bcl-2 transgenic mice (28, 29) and 24-week-old transgene-negative LMC mice (C57BL/6 strain). Naive untouched 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 Antibodies
Immunophenotyping 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 Isolation
Total 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 absorbance at 260/280 nm of total and cRNA were assessed by UV spectrophotometer.

Gene Expression Analysis by DNA Oligonucleotide Arrays
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 developed 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 expression log ratios for each probe set based on the spread of the replicate measurements. The software then computes a confidence 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 background 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 Analysis
Real-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 Assay
32P-labeled riboprobes were incubated with total RNA (10 µg) 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 Culture
MEF-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 (100x), and sodium pyruvate (1 mmol/L) in the presence of penicillin (100 units/mL) and streptomycin (100 µg/mL), all purchased from BioWhittaker, Inc. (Walkersville, MD). Mouse bone marrow cells (JAWSII; American Type Culture Collection) were cultured in {alpha}-MEM with ribonucleosides, 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 Vectors
A cDNA encoding the intron-less open reading frame of the 717-bp human Bcl-2{alpha} (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 µg 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 µm filter (Millipore Corp., Bedford, MA). For transduction, cell culture medium from ~70% 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 x g at 32°C) and then incubated for 10 hours (5% CO2, 37°C). On completion 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 Analysis
Cell 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 (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 Specimens
Paraffin-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).

Immunohistochemistry
Slides containing 4-µm 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 4°C 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 immunostaining results.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We 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 providing tissue specimens and helpful advice; and Dr. Nancy Berliner for critical review of the article.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 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.

Notes: G.J. Vanasse is a past American Society of Hematology fellow scholar and member of the Yale Cancer Center. Back

Received July 6, 2004; revised September 20, 2004; accepted October 6, 2004.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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