Original Article

Leukemia (2006) 20, 715–723. doi:10.1038/sj.leu.2404099; published online 2 February 2006

Decoupling of normal CD40/interleukin-4 immunoglobulin heavy chain switch signal leads to genomic instability in SGH-MM5 and RPMI 8226 multiple myeloma cell lines

W Y K Hwang1,2,6, C A Gullo1,3,6, J Shen1,3, C K Poh1,2, S C Tham1,3, G Cow1, M Au1,4, E W E Chan5 and G Teoh1,2,5

  1. 1Multiple Myeloma Research Laboratory (MMRL), Singapore Health Services (SingHealth) Research Facilities, Singapore, Singapore
  2. 2Department of Haematology, Singapore General Hospital (SGH), Singapore, Singapore
  3. 3Department of Clinical Research (DCR), SGH, Singapore, Singapore
  4. 4Division of Research, SGH, Singapore, Singapore
  5. 5Faculty of Medicine, National University of Singapore (NUS), Singapore, Singapore

Correspondence: Dr G Teoh, MMRL, SingHealth Research Facilities, 7 Hospital Drive, Block A #02–05, Singapore 169611, Republic of Singapore. E-mail: ghetkh@sgh.com.sg

6These authors have contributed equally and share first authorship in this paper.

Received 1 August 2005; Revised 7 November 2005; Accepted 23 November 2005; Published online 2 February 2006.

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Abstract

The processes mediating genomic instability and clonal evolution are obscure in multiple myeloma (MM). Acquisition of new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32) in MM, often heralds transformation to more aggressive disease. Since the combined effects of CD40 plus interleukin-4 (IL-4) mediate IgH isotype class switch recombination (CSR), and this process involves DNA double strand break repair (DSBR), we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormal DNA DSBR and lead to genomic instability and clonal evolution. In this study, we show that MM cell lines that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger maximal growth of tumor cells. We further demonstrate that CD40 triggering induced both DNA DSBs as well as newly acquired karyotypic abnormalities in MM cell lines. Importantly, these observations were accompanied by induction of activation induced cytidine deaminase expression, but not gross apoptosis. These data support the role of abnormal CD40 signal transduction in mediating genomic instability, suggesting a role for the CD40 pathway and intermediates in myelomagenesis and clonal evolution in vivo.

Keywords:

DNA double stand breaks, DNA repair, DNA protein kinase, Ku proteins, activation-induced cytidine deaminase

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Introduction

Maturation and transformation of normal B cells into immunoglobulin (Ig) secreting plasma cells involve the recombination of variable, diversity and joining (V(D)J) Ig gene segments; somatic hypermutation (SH); as well as isotype class switch recombination (CSR) of the Ig heavy chain (IgH) switch region. The process of IgH isotype CSR first involves cleavage of switch region DNA, which results in the creation of DNA double-stranded breaks (DSBs).1 This is followed by ligation of cleaved DNA by the error-prone process of nonhomologous end joining (NHEJ) of DNA. Triggering of normal B cells via a combination of CD40 plus interleukin-4 (IL-4) consistently induces maximal Ig isotype class switching, that is, from IgM through IgE.2 These signals (i.e. CD40 plus IL-4) also induce the expression of cell cycle regulatory proteins that facilitate both G1 cell cycle exit;3 as well as rescue from apoptosis4 in normal B cells.

Multiple myeloma (MM) is a malignancy of post-switched, terminally differentiated B cells, that is, plasma cells. The premalignant precursor cell in MM is believed to be a preswitched B cell, that is, the postgerminal center plasmablast.5 We hypothesize that malignant transformation of preswitched B cells to postswitched MM cells would involve, at least in part, the process of IgH isotype CSR. Whether CD40 and/or IL-4 mediated intracellular signal transduction contributes to this myelomageneic process is presently unknown. However, current available information suggests that both normal B cells as well as MM cells are able to respond, and to respond differently, when triggered via CD40 and/or IL-4. For example, CD40 induces vascular endothelial growth factor (VEGF) secretion and MM cell migration, suggesting a role for CD40 in regulating MM homing and angiogenesis.6 This effect of CD40 triggering appears to be primarily mediated via activation of PI3K/AKT/NF-kappaB signaling.7 Moreover, IL-6 secretion and proliferation of MM cells was triggered by coculture with CD40 ligand (CD40L) transfected NIH3T3 cells;8 and inhibited by incubation with anti-CD40 blocking antibodies.9 Furthermore, clonotypic B cells from MM patients expressing different isotype classes analyzed using allele-specific oligonucleotide reverse transcription polymerase chain reaction (ASO-RT-PCR) were detected both before and after culturing with CD40L and IL-4.10 In aggregate, these data suggest that although MM cells are postswitched cells, they continue to respond to CD40 and/or IL-4 in the context of IgH isotype CSR, possibly in an abnormal fashion. Accordingly, we hypothesize that MM cells could undergo abnormal DNA DSBR by NHEJ when triggered by signals that normally induced IgH isotype CSR signal. We further hypothesize that this process could result in genomic instability and contribute to clonal evolution.

In this study, we demonstrate that MM cell lines require different conditions versus normal B cells for optimal CD40 and/or IL-4 activation. We confirm that growth and DNA synthesis of MM cell lines is regulated, at least in part, by the CD40 pathway. Moreover, we demonstrate that IL-4 signal transduction is decoupled from CD40 in MM cells, and that CD40 alone is sufficient to trigger growth pathways. Accordingly, CD40 triggering induced DNA DSBs and karyotypic changes in MM cell lines, suggesting that CD40 could mediate genomic instability in MM cell lines. Importantly, these biological sequelae were accompanied by induction of activation-induced cytidine deaminase (AID) expression but not apoptosis; further suggesting involvement of the CD40-dependent DSBR pathway, rather than part of a more generalized catabolic process.

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Materials and methods

Cell lines and culture

RPMI 8226 human MM (CCL-155) and CESS Epstein–Barr virus (EBV)-immortalized human B lymphocytic (TIB-190) cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD). EBV-negative human normal B splenocyte cell line11 was a kind gift of Professor Kenneth Anderson (Dana-Farber Cancer Institute, Boston, MA). The EBV-negative SGH-MM5 human MM cell line (CD10+ CD19- CD20- CD38+ CD40+ CD45+ CD56+ CD138+) was developed in our laboratory from a patient with MM using a modified Dexter-type long-term tissue culture system, which was previously described.10 Informed consent and approval by the Singapore General Hospital Institutional Review Board was obtained for the use of patient material. All cell lines were cultured in complete media consisting of 90% RPMI 1640 with L-glutamine media, 10% fetal bovine serum (FBS), 25 IU/ml penicillin, 25 mug/ml streptomycin, and additional 5 mM L-glutamine. Cell cultures were maintained at 37°C with 5% CO2 in a humidified atmosphere.

CD40 and/or IL-4 triggering

Cell lines in log phase growth (initiating viable cell density=5.0 times 105 cells/ml). were triggered using sCD40L (0.0, 0.1, 1.0, 10.0 or 20.0 ng/ml, Peprotech, Rocky Hill, NJ) and/or IL-4 (0.0, 0.1, 1.0, 10.0 or 20.0 ng/ml, R&D Systems, Minneapolis, MN) in complete culture media for up to 5 days. Viable cell density was determined using standard Trypan blue exclusion assays; and DNA synthesis was determined using tritiated thymidine (3H-TdR, Perkin Elmer Life Sciences, Boston, MA) incorporation assays. For the latter, cell lines were first incubated with 0.25 muCi/well of 3H-TdR for 3 h. Next, cell lines were harvested using a cell harvester (Tomtec Mach III Auto, Hamden, CT) and counted on a beta plate reader (Wallac 1450 MicroBeta TriLux, Turku, Finland). Experiments were performed in triplicate and analyzed using the Mann–Whitney test (SPSS for Windows, version 10.0).

Comet assay

The comet assay was performed at neutral pH (nondenaturing conditions) to determine the presence of DNA DSBs, as previously described.12 Briefly, nontriggered as well as CD40 and/or IL-4 triggered SGH-MM5 or RPMI 8226 MM cells, or normal B splenocytes, or CESS EBV-immortalized normal B cells (1.4 times 106 cells/ml) were first embedded in 0.75% low melting point (LMP) agarose in a sandwiched manner on glass microscope slides. Next, cells were lysed and DNA unwound using neutral pH (pH 7.4) 1 times Tris borate buffer (TBE) for 30 min, followed by electrophoresis at 0.52 V/cm for up to 2 h. The lengths of the comet tails were detected using SYBR Green II Gel staining (Qiagen Gmb, Hilden, Germany) and fluorescence microscopy (Leica DC500, Leica Camera AG, Frankfurt, Germany). Images were analyzed using the Leica Q Fluro (V12.0) software (Leica Camera, AG). The relative ratio of the lengths of the comet tails was determined by normalizing the cell nuclear diameter to an arbitrarily assigned representative nontriggered SGH-MM5 MM cell, which was cultured in media alone. Experiments were performed in triplicate and analyzed using the Mann–Whitney test (SPSS for Windows, version 10.0).

Chromosome G-banding

Conventional chromosomal G-banding was performed for karyotype analysis, as previously described.13 Briefly, tumor cells were first cultured in hypotonic colcemid solution for 45 min, followed by 24 h in RPMI 1640 medium. Next, 20 metaphases were analyzed and chromosomal abnormalities classified according to the International System for Human Cytogenetic Nomenclature (ISCN), 1995.14

Western immunoblotting

Whole cell extracts (WCE) were obtained from cell lines (3.0 times 106 cells/sample). using EBC1 lysis buffer, which contains 50 mM tris pH 8.0, 150 mM NaCl, 0.1% NP-40, 0.5 mug/ml phenylmethylsulfonyl fluoride (PMSF), 50 mM NaF, 1 mM NaVO4, and one Complete® protease inhibitor tablet (Roche Diagnostics GmbH, Mannheim, Germany) in every 50 ml of lysis buffer. Proteins were quantified using Bradford's method (BioRad, Hercules, CA), and resolved (20 mug/sample) in a 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) gel. Next, proteins were transferred onto a polyvinylidine difluoride (PVDF) membrane (Schleicher & Schuell, Keene, NH), and blocked for 2 h using tris-buffered saline (TBS) – 20 mM tris pH 7.6 and 150 mM NaCl, which contains 1.0% Tween-20 (Sigma-Aldrich, St Louis, MO). Membranes were probed using goat antiactivation-induced cytidine deaminase (AID) monoclonal antibody (mAb) 1:200 dilution (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h, then washed thrice with TBS containing 0.2% Tween-20 (TBST), and then reprobed using horse raddish peroxidase (hrp)-conjugated donkey anti-goat IgG mAb 1:15 000 dilution (Santa Cruz Biotechnologies) for 2 h. Next, membranes were washed six times with TBST, and chemiluminescene detection was preformed using ChemiGlow reagents and filmless imaging on the FluoChem Imager (both from Alpha Innotech, San Leandro, CA). Spot densitometry was performed using AlphaEaseFC software (Alpha Innotech).

Annexin-V/propidium iodide (PI) dual staining

Annexin-V/PI dual staining (BD Pharmingen, San Diego, CA) was used to assay apoptosis/necrosis of cell lines. Briefly, cell lines (1.0 times 105 cells/sample) were first washed, pelleted and resuspended in 100 mul of binding buffer. Next, cell lines were stained using fluorescein isothiocynate (FITC)-labelled annexin-V (5.0 mul/sample) and PI (2.0 mul/sample), for 15 min at room temperature in the dark, according to the protocol provided by the manufacturer. Another 400 mul of binding buffer was then added, followed by indirect immunofluorescence flow cytometric analysis on the Cytomics FC500® cytometer (Beckman Coulter, Miami, FL). CD40-triggered cell lines were analyzed 2 and 4 h after culture in sCD40L. Ultraviolet light (UV)-irradiated (120 mJ/cm2, UV Stratalinker 1800, Stratagene, La Jolla, CA) cell lines were used as positive controls for the induction of apoptosis/necrosis. Analysis of UV-irradiated cell lines was performed 2 h after UV-irradiation. Non-UV-irradiated and non-CD40-triggered cell lines were used as negative controls. When tumor cells are analyzed shortly after introduction of a proapoptotic signal, cells in early apoptosis will demonstrate Annexin-V positivity (quadrant 4, Q4); whereas cells in the intermediate stages of apoptosis will demonstrate both Annexin-V and PI positivity (Q2). In contrast, necrotic cells or cells in the late stages of apoptosis will stain only for PI (Q1).

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Results

Effect of sCD40L and/or IL-4 on proliferation of SGH-MM5 and RPMI 8226 MM cell lines

The combinatorial effects of sCD40L and/or IL-4 on the growth of SGH-MM5 or RPMI 8226 MM cell lines were first compared to normal B splenocytes or CESS EBV-immortalized normal B cells. As indicated in Table 1, optimal triggering of both SGH-MM5 or RPMI 8226 MM cell lines required 1.0 ng/ml of sCD40L for 4 h and/or 1.0 ng/ml of IL-4 for 4 h. Optimal triggering conditions are defined by the amount of cytokine(s) and time needed to maximally achieve proliferation of the cell lines, in which excess time or cytokine(s) leads to an overgrowth and inhibition of proliferation as measured by cell number and thymidine incorporation. By contrast, optimal triggering conditions for both normal B splenocytes or the CESS cell line were substantially longer, that is, 1.0 ng/ml of sCD40L for 3 days and/or 1.0 ng/ml of IL-4 for 3 days. Analysis (Mann–Whitney test) of growth curves of MM cell lines triggered using optimal conditions for sCD40L and/or IL-4 demonstrate that significant (P<0.01 for both cell lines) proliferation of SGH-MM5 (Figure 1a) and RPMI 8226 (Figure 1b) MM cell lines was induced using sCD40L alone. The use of IL-4 alone had no effect on MM cell line proliferation; and in fact abrogated the growth of CD40 triggered MM cell lines. In contrast, neither sCD40L alone or IL-4 alone induced growth of both normal B splenocytes (Figure 1c) or the CESS cell line (Figure 1d); that is, sCD40L and IL-4 were required to significantly (P<0.01 for both cell lines) induce cell proliferation. These data suggest that sCD40L was an independent growth factor regulating the growth of MM cell lines, whereas IL-4 was not. Moreover, the data also suggests that IL-4 could potentially be a negative regulator of CD40 triggered MM cell growth.

Figure 1.
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Effect of sCD40L and/or IL-4 on proliferation of SGH-MM5 and RPMI 8226 MM cell lines. SGH-MM5 (a) or RPMI 8226 (b) MM cell lines, or normal B splenocytes (c), or CESS EBV-immortalized normal B cells (d) (initiating cell density=0.5 times 106 cells), were cultured in complete media alone; or in complete media containing sCD40L (0.0, 0.1, 1.0, 10.0 or 20.0 ng/ml) and/or IL-4 (0.0, 0.1, 1.0, 10.0 or 20.0 ng/ml) for up to 5 days. Viable cell density was determined using Trypan blue exclusion assay and growth curves were obtained for each of the 25 possible sCD40L and/or IL-4 triggering combinations. Only data for optimized sCD40L and/or IL-4 triggering conditions (Table 1) is shown. Experiments were performed in triplicate (plusminuss.e.m.), analyzed using the Mann–Whitney test and displayed as box plots.

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Effect of sCD40L and/or IL-4 on DNA synthesis in SGH-MM5 and RPMI 8226 MM cell lines

In order to confirm the uncoupling of IL-4 triggering from CD40 triggering in SGH-MM5 and RPMI 8226 MM cell lines, we compared DNA synthesis in MM cell lines with normal B cell lines under optimal conditions of CD40 and/or IL-4 triggering (Table 1). We demonstrate that DNA synthesis was increased in CD40 triggered SGH-MM5 (Figure 2a) and RPMI 8226 (Figure 2b) MM cell lines (P<0.01 for both cell lines), but not in nontriggered MM cell lines, or MM cell lines triggered using IL-4 alone, or sCD40L plus IL-4. In contrast, DNA synthesis in normal B splenocytes (Figure 2c) or the CESS cell line (Figure 2d) was increased only when triggered using both sCD40L and IL-4 (P<0.01 for both cell lines). Triggering of normal B cells using IL-4 alone or sCD40L alone had no effect on DNA synthesis. These data confirm that the IL-4 signaling pathway is indeed uncoupled from CD40 in MM cell lines, and that IL-4 could in fact be a negative regulator of CD40 triggered MM cell growth. These data further suggest that abnormal uncoupling of CD40 and IL-4 signal transduction in MM cell lines could impact on other biological sequelae or signaling pathways downstream of CD40, including Ig isotype CSR and DNA repair.

Figure 2.
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Effect of sCD40L and/or IL-4 on DNA synthesis in SGH-MM5 and RPMI 8226 MM cell lines. SGH-MM5 (a) or RPMI 8226 (b) MM cell lines, or normal B splenocytes (c), or the CESS cell line (d) (initiating cell density=0.5 times 106 cells), were triggered using optimal triggering conditions for sCD40L and/or IL-4 for each cell line (see Table 1). DNA synthesis was assessed using standard 3H-TdR (0.25 muCi/well for 3 h) incorporation assays. Triplicate experiments were performed (plusminuss.e.m.), analyzed using the Mann–Whitney test and displayed as box plots.

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Generation of DNA DSBs in CD40 triggered SGH-MM5 and RPMI 8226 MM cell lines

Since the most effective signals mediating Ig isotype CSR by NHEJ in normal B cells are CD40 and IL-4, and this process involves the generation of DNA DSBs, we next investigated whether CD40 triggering alone (i.e., uncoupled from IL-4) could induce DNA DSBs in MM cell lines. We performed standard nondenaturing comet assays at neutral pH to assay for DNA DSBs and observed that there was significant (P<0.01 for both cell lines) induction of DNA DSBs in both SGH-MM5 (Figure 3a and e) as well as RPMI 8226 (Figure 3b and f) MM cell lines following CD40 triggering. In contrast, triggering of MM cell lines via IL-4 alone or using the combination of IL-4 and sCD40L was not associated with any significant (P>0.05) induction of DNA DSBs. Moreover, although CD40 plus IL-4-triggered SGH-MM5 MM cells appeared to demonstrate some degree of induction of DNA DSB (Figure 3a), this was not statistically significant (P>0.05) from nontriggered and CD40-triggered tumor cells. This was due to the wide range in the lengths of the comet tails measured in this assay. Further statistical analysis confirmed that comet tail lengths between CD40-triggered and CD40 plus IL-4-triggered SGH-MM5 MM were significantly (P<0.05) different (Figure 3e). Triggering of normal B splenocytes (Figure 3c and g) or the CESS cell line (Figure 3d and h) using sCD40L alone or IL-4 alone also failed to induce DNA DSBs. However, the combination of sCD40L and IL-4 was, as expected, associated with significant (P<0.05 for both cell lines) induction of DNA DSBs in these normal B cell lines, and served as positive controls. These data firstly confirm that the CD40 signaling pathway is uncoupled from IL-4 in MM cell lines. Secondly, they demonstrate that CD40 activation of MM cell lines, in the absence of IL-4, could result in a variety of downstream biological consequences (i.e., cell proliferation and DNA damage) that could enhance the survival and growth of tumor cells. Since DNA DSBs could potentially lead to novel karyotypic abnormalities and genomic instability, these data further suggest that CD40 activation in MM cell lines could regulate clonal evolution in MM.

Figure 3.
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Generation of DNA DSBs in CD40 triggered SGH-MM5 and RPMI 8226 MM cell lines. SGH-MM5 (a, e) or RPMI 8226 (b, f) multiple myeloma (MM) cell lines, or normal B splenocytes (c, g), or CESS EBV-immortalized normal B cells (d, h) were triggered using optimal triggering conditions for soluble CD40 ligand (sCD40L) and/or interleukin-4 (IL-4) for each cell line for up to 5 days (Table 1). Comet assays were performed at neutral pH to assess the generation of DNA double-strand breaks (DSB). Fluorescence microscopic images (original magnification times 40) are presented for non-triggered cell lines (ad, column 1), or cell lines triggered using sCD40L alone (ad, column 2), or both sCD40L and IL-4 (ad, column 3). Normalization was performed for the diameter of the cell nucleus. Experiments were performed in triplicate, analyzed using the Mann–Whitney test and displayed as box plots. The asterix (*) indicates an additional specific statistical analysis performed using the Mann–Whitney test to better determine statistical differences between CD40-triggered and CD40 plus IL-4 -triggered SGH-MM5 MM cell lines.

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Generation of new karyotypic abnormalities in the CD40 triggered SGH-MM5 MM cell line

In order to show that CD40 triggering induced new karyotypic abnormalities, we performed conventional chromosomal G-banding on the CD40 triggered SGH-MM5 MM cell line (Table 2). As can be seen in Figure 4, after 1 day of CD40 activation of the SGH-MM5 MM cell line, we observed the translocation of additional genetic material onto the short arm of chromosome 4, that is, add(4)(p12). Moreover, after 8 days of CD40 activation of the SGH-MM5 MM cell line, we observed the following new karyotypic changes in addition to add(4)(p12); that is, isochromosome 13q or i(13)(q10); additional genetic material on the short arm of chromosome 14 or add(14)(p13); deletion of both the long and short arms of chromosome 19 or der(19)del(19)(p13.3)del(19)(p13.3); and trisomy chromosome 22 or +22 (Figure 4). While the gradual development of new chromosomal abnormalities may occur in tumor cell lines in long-term culture, the development of a relative abundance of new abnormalities over the course of 8 days following CD40 triggering is unusual and not seen in non-triggered cultures of these cells (data not shown). These data suggest that CD40 triggering could indeed induce new karyotypic abnormalities in MM cell lines. Moreover, since all these cumulative chromosomal aberrations are mechanistically derived from DNA DSBs, it is tempting to speculate that CD40-induced DNA DSBs could potentially mediate genomic instability in MM cell lines.

Figure 4.
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Generation of new karyotypic abnormalities in the CD40 triggered SGH-MM5 MM cell line. Karyotype of the SGH-MM5 MM cell line was studied after 1 and 8 days of CD40 triggering. Numerous novel chromosomal abnormalities were progressively induced in CD40 triggered tumor cells, which were not seen in cultures performed without triggering over the same time period.

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Upregulation of activation induced cytidine deaminase (AID) expression in CD40 triggered SGH-MM5 MM cell line

To further elucidate the mechanisms involved in CD40 induced DNA damage, we studied the expression of AID in the SGH-MM5 MM cell line following CD40 triggering. AID is a 198-residue RNA-editing enzyme that facilitates DNA DSB repair (DSBR), which occurs during IgH isotype CSR.15 As can be seen in Figure 5, AID expression progressively increased after 2 and 6 h of CD40 triggering in the SGH-MM5 cell line; whereas there was no increase in tubulin (control) expression. When MM cells and normal B lymphocytes were subjected to the combinations of sCD40L alone, sCD40L plus IL-4 or IL-4 alone, maximal induction of AID expression occurred with sCD40L alone in MM cells. By contrast, maximal AID exprerssion was observed with IL-4 triggering for normal B splenocytes (see Table 3). Specifically, CD40 appears to be the stronger mediator of AID expression in MM cells, whereas AID expression in normal B cells is primarily regulated by IL-4.

Figure 5.
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Upregulation of AID expression in CD40 triggered SGH-MM5 MM cell line. The SGH-MM5 MM cell line was triggered using sCD40L (1.0 ng/ml) for 2 h or 6 h, and the AID expression was analyzed using Western immunoblotting (first panel). Membranes were stripped and reprobed for tubulin expression to confirm equal protein loading (second panel). Relative AID expression was determined using image densitometry and presented graphically, in the bar chart showing percentage increase in AID from baseline (normalized to tubulin) (third panel).

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CD40 triggering of SGH-MM5 and RPMI 8226 MM cell line is not associated with apoptosis

Since CD40 triggering in a variety of cells may inhibit cell growth or induce apoptosis; and CD40-triggered MM cells may undergo both p53-dependent as well as Fas-independent apoptosis, we next investigated whether DNA DSBs induced by CD40 triggering could be part of a more generalized degenerative process (i.e., DNA fragmentation during apoptosis), rather than a more physiological event (i.e., IgH isotype CSR). As can be seen in Figure 6, CD40 triggering of SGH-MM5 (from 2.7 to 2.1% at 2 h, and 3.5% at 4 h) and RPMI 8226 (from 4.0 to 2.9% at 2 h, and 5.9% at 4 h) MM cell lines is not associated with any significant apoptosis during the period in which AID expression is induced. In fact, at 2 h, the percentage of cells expressing Annexin-V appears to decrease, rather than increase. In contrast, CD40 activated normal B splenocytes (from 5.1 to 8.8% at 2 h, and 7.6% at 4 h) show a modest but consistent increase in the percentage of apoptotic cells at both time points. Moreover, control UV-irradiated cell lines all show definite apoptosis – 45.7% early apoptosis and 35.7% established apoptosis in SGH-MM5; 21.5% early apoptosis and 46.1% established apoptosis for RPMI 8226; and 25.4% early apoptosis and 61.9% established apoptosis for normal B splenocytes. These data suggest that CD40-induced DNA DSBs are not related to generalized DNA damage associated with DNA fragmentation and apoptosis. In contrary, these data suggest that CD40-induced DNA DSBs could indeed be related, at least in part, to physiological processes induced by CD40, for example, IgH isotype CSR.

Figure 6.
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CD40 triggering of SGH-MM5 and RPMI 8226 MM cell line is not associated with apoptosis. SGH-MM5 (top panel) or RPMI 8226 (middle panel) MM cell lines, or normal B splenocytes (bottom panel) were stimulated using sCD40L (1.0 ng/ml) for 2 h or 4 h and analyzed for apoptosis using dual Annexin-V/PI staining and indirect immunofluorescence flow cytometric analysis. Ultraviolet light irradiated cell lines were used as positive controls, and non-UV-irradiated, non-CD40-triggered cell lines were used as negative controls. Experiments were performed in triplicate.

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Discussion

The interaction resulting from an IL-4 stimulus, and CD40 engagement by CD40L or a microbial activator such as lipopolysaccharide (LPS) is critical to normal B-cell maturation and function. CD40 and IL-4 synergism plays a key role in regulation of V(D)J recombination in developing B lineage cells, SH of the Ig hypervariable gene segments in follicular B cells, and IgH isotype CSR in postgerminal center plasmablasts; as well as protection against apoptosis16 and maintenance of B-cell division, survival and differentiation. The combinatorial interaction between CD40 and IL-4 signals may be related to the increase in IL-4-dependent CD40 expression;17, 18 and/or enhanced CD40L expression on B cells (which in turn triggers a 'reverse' signal influencing the function of other B cells);19 and/or enhancement of NF-kappaB and STAT-6 mediated gene expression of cytokine, cell cycle regulatory and inflammatory response genes.20 Although MM cells are terminally-differentiated plasma cells that have undergone V(D)J recombination, SH and IgH isotype CSR, prior studies have suggested that MM cells might still respond to these signals, especially CD40. In fact, response of MM cells to CD40 might also be pro-oncogenic depending on the status of p53.21 Whether IL-4 also produces a pro-oncogenic effect is less clear. We demonstrate in the present study that CD40 alone is sufficient to induce MM cell growth, and that addition of IL-4 to cell cultures might in fact abrogate the pro-proliferative effects of CD40. Thus, IL-4 signal transduction appears to be decoupled from CD40 in MM cells, although it is presently not clear whether MM cells possess an alternative IL-4 signal transduction pathway or permit CD40 signal transduction via novel molecules. The ability of CD40 to physically associate with Ku proteins,22 suggests that novel signal transduction molecules could potentially mediate yet unknown biological sequelae.

Multiple myeloma is frequently characterized by karyotypic abnormalities involving the IgH gene locus.23 Previous studies have suggested that the early myelomageneic event may involve germinal center B cells, where illegitimate recombination of the IgH switch region at 14q32 and non-Ig chromosomal partners (e.g. cyclin D1, FGFR3 and MMSET) have been observed.24 Moreover, errors introduced into the IgH locus due to defects in the NHEJ system have been shown to induce abnormal gene translocations and tumor formation in mice.25 It has been suggested that when this primary translocation results in juxtaposition of an oncogene near the Ig enhancer and subsequent dysregulation of the oncogene, genomic instability results and further global and random chromosomal abnormalities, not necessarily involving the IgH switch region, occur. As the CD40/IL-4 signal is the principal trigger in effecting IgH isotype CSR in normal plasma cells,26 we postulate that the normal mechanisms of IgH CSR are dysfunctional in postswitched in MM cells due to CD40/IL-4 signal dysregulation, and that this results in abnormal NHEJ and consequent genomic instability and tumor progression in response to CD40 triggering.

A key enzyme that was studied during CD40 triggering of MM cells was AID;27, 28, 29 which is involved in activating IgH isotype CSR in a number of ways. Firstly, indirectly by editing mRNAs encoding for nucleases or proteins involved in IgH isotype CSR, or facilitating breaks at single-stranded regions when germline transcription reveals open chromatin.15 And secondly, by directly creating DNA lesions at IgH S regions before actual CSR occurs.30 Constitutive expression of AID protein occurs in normal and transformed germinal center B cells;31 and is absent in MM, B-precursor lymphoblastic leukaemia and mantle cell lymphoma.32 Moreover, this unique enzyme that specifically regulates IgH isotype CSR,33 is upregulated principally by IL-4 and to a lesser extent CD40L in normal B cells.34 In this present study, we demonstrate that unlike normal B cells that respond primarily to IL-4, triggering of MM cells using CD40L alone resulted in significant upregulation of AID. Importantly, we excluded the effects of CD40 and/or IL-4 in SH of the variable (VH) region of the IgH gene by specifically using MM cell lines that contained deletions in the VH complementarity determining region 3 (CDR3); that is, SGH-MM5 (data not shown) and RPMI 8226.35 Hence, induction of AID expression would suggest genetic events occurring as part of IgH isotype CSR rather than as part of SH. In aggregate, our data is consistent with the fact that MM arising from AID-expressing germinal center B cells acquire critical and characteristic primary translocations involving the IgH locus (11;14, 6;14, 4;14 and 14;16), prior to infiltration of the blood and bone marrow.36 Accordingly, we propose that the induction of nonapoptotic genomic instability by CD40 in MM cells suggests that the investigation into CD40 pathway intermediates could yield important clues to tumorigenesis and karyotypic evolution in MM.

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Acknowledgements

This work was supported by research grants from the National Medical Research Council (NMRC), Singapore (grant #s NMRC/0489/2000, NMRC/0758/2003 and NMRC/0734/2003); SingHealth (grant #s SU007/2001, SU008/2001, SU009/2001 and SU089/2003); and the Department of Clinical Research, SGH. We gratefully acknowledge the invaluable contributions of Ms Janice Chow and Ms Lim Lay Feng (Research Coordinators, MMRL) in proofreading this manuscript, and Ms Stephanie Fook Chong (Statistician, DCR) for statistical analyses.