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[Cancer Research 66, 3996-4000, April 15, 2006]
© 2006 American Association for Cancer Research

Priority Reports

Immunoglobulin Heavy Chain Locus Events and Expression of Activation-Induced Cytidine Deaminase in Epithelial Breast Cancer Cell Lines

Gavin Babbage1,2, Christian H. Ottensmeier1,2, Jeremy Blaydes2, Freda K. Stevenson1,2 and Surinder S. Sahota1,2

1 Genetic Vaccine Group and 2 Cancer Sciences Division, School of Medicine, University of Southampton, Southampton, United Kingdom

Requests for reprints: Surinder S. Sahota, Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, School of Medicine, University of Southampton, Tremona Road, Southampton SO16 6YD, United Kingdom. Phone: 44-2380-798768; Fax: 44-2380-701385; E-mail: s.s.sahota{at}soton.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
When cells transform, phenotypic and genetic profiles can be dramatically altered. Nevertheless, a recent report identifying IgG in breast cancer cells was unexpected, revealing differentiation features normally associated with B lymphocytes. To extend these findings, we focused on immunoglobulin variable (V) region gene analysis using well-defined breast cancer cell lines expressing the epithelial marker, epithelial cell adhesion molecule (EpCAM). VH gene transcripts were identifiable by nested reverse transcription-PCR either as single or dual V, diversity (D), and joining (J) rearrangements in four of six lines, most being potentially functional. V(D)J transcripts were observed in sequential cultures, indicating stable expression. To exclude coexisting lymphocytes, each cell line was shown to be EBV negative, with CD19/CD20 and cytoplasmic/surface immunoglobulin also absent by flow cytometry. Identified VH transcripts were then sought in individual tumor cells, isolated as EpCAM+ single cells by flow cytometry. Importantly, in three of three selected cell lines, VH genes were identifiable in a significant fraction (~32%) of single cells. In five of six identified VH genes, somatic mutations were apparent with no intraclonal variation, indicating cessation of mutational activity. VH transcripts were pre- and post-isotype switch, with activation of switch events evident from expressed germ-line switch transcripts in two of six lines. Strikingly, six of six cell lines expressed activation-induced cytidine deaminase (AID) essential for mutational and switch activity. These data suggest either a de novo rearrangement and modification of VH genes in epithelial tumor cells or assimilation of lymphocyte-derived chromatin. Constitutive AID activation in malignant epithelial cells further raises a potential for inducing aberrant mutational activity. (Cancer Res 2006; 66(8): 3996-4000)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Ongoing genetic modifications are a hallmark of tumor cells influencing survival and progression with the full capacity of malignant cells to induce such changes under scrutiny. Of interest in this regard, two studies of epithelial breast cancer reported that IgG can be identified in tumor cells directly based on assays specific for the heavy chain constant region (1, 2). These findings are unexpected, given that immunoglobulin gene expression normally associates with lineage fidelity in B lymphocytes. In breast cancer cells, however, it was not clear whether any rearranged immunoglobulin variable (V) region genes were being expressed (2). To address this, we examined several well-defined and commonly used breast cancer cell lines for VH gene expression coupled with an analysis of additional immunoglobulin heavy chain (IgH) locus events.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Cell lines. A previously described (3) panel of six well-characterized breast cancer cell lines (BT474, MDA-MB-231, MCF-7, SKBR3, T47D, and ZR75-1) were cultured in DMEM (Invitrogen, Paisley, United Kingdom) supplemented with 10% FCS (Autogen Bioclear, Wiltshire, United Kingdom).

RNA extraction, cDNA synthesis, and VH gene analysis. Confluent cultures (70%) were harvested and RNA extracted using RNABee reagent (Biogenesis, Inc., Poole, United Kingdom). Total RNA (1-2 µg/40 µL) was reverse transcribed to cDNA using Moloney murine leukemia virus reverse transcriptase (Promega, Southampton, United Kingdom) using oligo(dT) primers and amplified using a mixture of VH leader primers together with downstream CH primers as described (4). An aliquot of the first PCR (2 µL) was used for nested amplification using a mixture of FR1 VH and CH primers. Rescue of amplified DNA for cloning and sequencing before VH identification and analysis was as reported (4).

RAG gene expression. A nested reverse transcription-PCR (RT-PCR) assay was used to analyze RAG1 and RAG2 gene expressions. Primers were as follows: RAG1 (final product 635 bp), external 5'-GAGAGAGCAGAGAACACACTTTG (sense) and 5'-GTCCCCGACGGGCAGTGTTG (antisense) and internal 5'-CTTTGGCCAGGCAGCCTGC (sense) and 5'-CCTCGGGAAGTAAACCTCACATG (antisense); and RAG2 (final product 660 bp), external 5'-CTTTACAGTCAGCCTTCTGCTTGC (sense) and 5'-CCTCGGGAAGTAAACCTCACATG (antisense) and internal 5'-CAGCCCCTCTGGCCTTCAG (sense) 5'-GATAGCCCATCCTGAAGTTCTGG (antisense). RAG expression has been documented as a frequent event in follicular lymphoma (5), and cDNA from one such follicular lymphoma case was used as a positive control.

Activation-induced cytidine deaminase expression and germ-line switch transcript analysis. A nested RT-PCR approach to detect full-length and variant activation-induced cytidine deaminase (AID) transcripts was used based on the assay described previously (4). IH-CH germ-line transcripts were identified using published protocols by nested RT-PCR (4). Correct amplification of AID and sterile transcripts was confirmed by DNA sequence analysis in each instance.

Flow cytometry. Immunophenotypic analysis was done using a direct immunofluorescence technique by dual-color staining with a panel of monoclonal antibodies comprising anti-CD19, anti-CD20 and anti–epithelial cell adhesion molecule (EpCAM; Becton Dickinson, San Diego, CA), and anti-IgG and anti-IgM (Caltag Laboratories, Burlingame, CA). Background corrections were obtained by incubating cells with an appropriate isotype control antibody. Cytoplasmic staining was carried out to detect IgG, IgA, and IgM using the Fix & Perm Cell Permeabilization kit according to the instructions of the manufacturer (Caltag Laboratories). Data acquisition and analysis were done by flow cytometry (FACScan, Becton Dickinson) with CellQuest software (Becton Dickinson). In all cases, 10,000 events were acquired. The CD19+/CD20+/EpCAM– Ramos lymphoma cell line was used as a control. Preparation and analysis of single tumor cells were carried out as described (4).

EBV analysis. Four EBV-associated genes were analyzed by RT-PCR (30 cycles) using the following primers: EBNA-1 (481 bp), 5'-GGGTGGTTTGGAAAGCATCGTGG and 5'-CATCACCCTCCGCGGCAGCCCC (reverse); EBNA-2 (716 bp), 5'-GCTGCTACGCATTAGAGACC and 5'-TCCTGGTAGGGATTCGAGGG (reverse); BZLF-1 (608 bp), 5'-ATTGCACCTTGCCGCCACCTTTG and 5'-CGGCATTTTCTGGAAGCCACCCGA (reverse); and LMP-1 (635 bp), 5'-TCCTCCTCTTGGCGCTACTG and 5'-TCATCACTGTGTCGTTGTCC (reverse).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Gene assembly for lymphocyte antigen receptors is unique among mammalian genes in that multiple germ-line gene elements are recombined to generate a functional transcriptional unit. In immunoglobulin VH genes, these mechanisms yield a unique signature motif in the CDR3 domain, which can be used to track clonal progeny. Here, we have examined VH gene use and IgH locus events in breast cancer cell lines typically used as disease models. Use of these breast cancer cell lines obviated problems of contamination that can arise when analyzing primary tumors because these have a complex histology in situ with coexisting stromal and lymphocytic cells.

VH gene transcripts were analyzed in breast cancer cell lines using a protocol known to reproducibly delineate tumor-derived VH genes in lymphoid malignancies (4). Surprisingly, rearranged VH transcripts were identified in several (four of six) of the breast cancer cell lines examined (Table 1 ; Fig. 1 ). In two of these lines, SKBR3 and ZR75-1, dual VH rearrangements were apparent in each line with differing donor germ-line VH genes. In the other two cell lines, MDA-MB-231 and T47D, a single VH rearrangement was identified, both derived from the VH3 family. All transcripts were potentially functional, except in T47D where usage of the nonfunctional pseudogene V3-41 gene was identified (6).


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  		Table 1. Immunogenetic features in breast cancer cell lines at whole population and single-cell level

 

Figure 1
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  		Figure 1. Deduced amino acid sequences of VH-CH transcripts identified in breast cancer lines. Comparisons are made with the closest germ-line VH genes, of which V3-41 is a pseudogene carrying two stop codons in germ-line configuration. Uppercase, replacement mutations; lowercase, silent mutations.

 
To delineate the tumor-associated VH gene expression further, we chose to analyze these genes in individual tumor cells. In three selected cell lines (MDA-MB-231, T47D, and ZR75-1), single tumor cells were obtained by EpCAM+ cell sorting. Firstly, the functional integrity of cDNA was confirmed in each cell analyzed by amplification of a housekeeping gene. Importantly, we identified the same VH gene in single cells as identified previously in the bulk cell line population, and the frequency of single cells expressing VH genes was significant, averaging ~32% (Table 1). Furthermore, single-cell analysis clearly showed that the dual VH rearrangement observed in the bulk ZR75-1 population could be tracked to separate cells expressing only one gene and not the other, with each VH gene expressed at a frequency of ~13% in this cell line (Table 1). In this case, biallelic VH gene recombination seems unlikely. This finding revealed VH gene expression in a sizeable fraction of each cell line but not in all tumor cells, indicating events that are likely to have occurred in a minority of cells after transformation. Analysis at this level also clearly delineated tumor-associated VH genes and will be necessary to obviate any issue of contamination in the analysis of primary breast cancer. This has been highlighted in a study of >40 primary breast cancer tumors evaluated by DNA microarray profiles, where, although rearranged Ig{lambda} light chain and Igµ transcripts were frequently identified, the preparation of tumor samples by gross excision could not exclude lymphocytic infiltrate (7).

These observations are striking given the nature of normal VH gene assembly in B cells. VH gene rearrangement occurs early in B-cell development when V, diversity (D), and joining (J) genes located on chromosome 14 are functionally rearranged, requiring RAG1 and RAG2 catalysis (8). VH assembly is mediated by deletional DNA recombination between donor segment genes, which are straddled by recognition motifs to allow the RAG proteins to correctly orientate V(D)J segments (9). In addition, allelic exclusion ensures that only one functional V(D)J transcription unit is expressed in each B cell for subsequent differentiation. Several possibilities may then explain rearranged VH transcripts in breast cancer lines. The first of these suggests that malignant epithelial cells may have initiated the required cascade of complex molecular events to rearrange VH genes intrinsically, incorporating RAG expression. Such rearrangements seem to bypass normal lineage-restricted constraints of gene expression. To examine this, we analyzed each of the cell lines for RAG1 and RAG2 expressions and could not identify any gene transcripts using assays able to identify positive expression in control lymphoma cells (Fig. 2 ). However, this may not negate an earlier phase of RAG expression in epithelial tumor cells. Another possibility is that breast cancer tumor cells in vivo acquire extraneous genes from neighboring cells and maintain these in the tumor genome. The modified breast cancer cells could then be the source for derived cell lines. Some recent observations suggest a potential for such modifications. Surprisingly, endothelial cells lying adjacent to lymphoma or multiple myeloma cells have revealed genetic aberrations that are identical to the tumor clone, including entire IgH chromosomal translocations (10, 11). Furthermore, the genetic aberrations acquired by endothelial cells in these diseases were maintained following several cycles of cell culture (10). Although the underlying mechanisms for acquisition and maintenance of aberrant genes in endothelial cells are not clear, several possibilities exist, one of which suggests gene transfer of apoptotic bodies from tumor cells (10). Current work stemming from the present study is aimed at mapping chromosomal abnormalities in the breast cancer cell lines examined more fully.


Figure 2
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  		Figure 2. Expression of AID, sterile germ line, EBV-associated gene transcripts, and RAG1 and RAG2 in breast cancer cell lines. Gene-specific primers were used to amplify each gene and amplified bands of predicted size were resolved on agarose gels before elution and nucleotide sequence verification. The EBV genes analyzed were EBNA1, EBNA2, BZLF, and LMP1. For AID expression, Ramos cells constitutive for its expression were used as control. For RAG gene transcripts, cDNA from a follicular lymphoma case was used as a positive control. BT, BT474; MD, MDA-MB-231; MC, MCF-7; SK, SKBR3; T47, T47D; ZR, ZR75-1.

 
If, however, it is shown that the underlying mechanisms generating rearranged VH genes in breast cancer involve induction of a B-cell–associated genetic program, these findings would be a remarkable indicator of the "plasticity" of gene expression inherent in these tumor cells. The finding of the V3-41 pseudogene use in T47D is intriguing. Its use in normal B cells would lead to a dysfunctional cell that would be aborted, whereas any aberrant VH assembly in breast cancer tumor cells would not be subject to such controls.

The analysis of breast cancer VH genes revealed extensive somatic mutations in five of six of the rearranged genes (Table 1; Fig. 1), with one displaying complete germ-line homology. A further evaluation of mutational patterns for a role for antigen in selection was not undertaken, as such analysis is hindered by hotspots of mutations, which occur in the absence of any selection pressure, and uncertainty introduced by targeting of mutations to nonfunctional V(D)J rearrangements (12, 13). By cloning the amplified VH gene product, each cell line could be analyzed for any variation in VH sequence patterns. In five of five lines, identical mutational patterns were found in multiple (24-28) clones (Table 1) from each cell line, indicating intraclonal homogeneity due to cessation of mutational activity. Targeted V gene mutations are normally a feature exclusive to B cells. This generally occurs in the germinal center of secondary lymphoid follicles but can also be triggered at ectopic sites possibly also in B cells infiltrating breast cancer tissue (14). Although not all requirements that normally drive B-cell V gene mutations are clear, signals via antigen stimulation of surface immunoglobulin and those derived from activated T cells are important (15). It is unknown, however, to what extent transformed B cells require these signals, as in established lymphoma cell lines, mutational activity can be constitutive (16). Clearly, tumor cells can override normal constraints. It is tempting then to speculate that breast cancer tumor cells in these cell lines may have initiated de novo mutations in VH genes endogenously. In the ZR75-1 line specifically (Table 1; Fig. 1), some cells expressed the V5-51 gene in germ-line configuration, whereas, in other cells, the V3-23 gene displayed somatic mutations, suggesting a restriction of mutational activity to specific cells.

VH genes in the breast cancer cell lines were expressed as pre- and post-switched transcripts (Table 1), and in two cases, dual VH transcripts in each cell line were switched to different isotypes, to IgG/IgA in SKBR3 and IgM/IgG in ZR75-1 (Fig. 1). Isotype switch in B cells is preceded by induction of sterile germ-line transcripts from IH exons, which lie upstream of the coding region for the immunoglobulin heavy chain isotype (9). In one of three cell lines displaying switched VH transcripts (T47D), I{gamma}-C{gamma} sterile transcripts were also identifiable (Fig. 2). However, these were also observed in BT474 (Fig. 2), in which no VH gene rearrangement was detectable. This indicates a continual activation of the switch locus in some breast cancer cells, not always paralleling switched VH gene expression.

To assess immunoglobulin protein expression, antibodies directed against the immunoglobulin isotype were used in fluorescence-activated cell sorting (FACS) analysis of four selected breast cancer cell lines. Notably, there was no evidence for any intracellular or extracellular expression of immunoglobulin molecules (Fig. 3 ). It seems that the functional VH transcripts are being translated at a very low level in these breast cancer cells, if at all. As these transcripts were identified by nested RT-PCR, it is possible that the level of protein expression is below the detection sensitivity by FACS analysis. This observation seems to differ from the previous reports where intracellular IgG was reported in the MCF-7 cell line and primary breast cancer tumors using assays for the constant region domains (1, 2). It is feasible though that this may reflect instability in breast cancer lines, which alters phenotypic characteristics. Such instability has been shown to alter chromosomal content in these cell lines (17). Of note, a complete absence of CD19/CD20 expression and lack of any transcripts associated with EBV infection from four marker genes, EBNA1, EBNA2, BZLF, and LMP1, in the breast cancer cell lines (Figs. 2 and 3) indicated that any contamination by B lymphocytes seemed unlikely. As ~32% of tumor cells in breast cancer lines expressed VH transcripts (Table 1), any coexisting B cells would have been readily identified by flow cytometry for CD19/CD20. If any B cells are indeed present, they are below the detection sensitivities of the assays used.


Figure 3
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  		Figure 3. Expression of immunoglobulin, CD19/CD20, and EpCAM assayed by flow cytometry in four selected breast cancer lines and controls. A, extracellular and intracellular expression of IgM and IgG (B-cell lymphoma and myeloma cell lines as controls). B, dual expression of CD19 and CD20. C, dual expression of CD19 versus EpCAM.

 
For both somatic mutation of V genes and isotype switch in B cells, there is an absolute requirement for AID expression. This enzyme deaminates deoxycytosine in exposed ssDNA to initiate a series of molecular events that result in replacement nucleotides in V genes or in DNA double-strand breaks for IgH deletional recombination between switch regions (18). Interestingly, transcripts for AID were found constitutively in all six breast cancer lines examined at a level comparable with B-cell lymphoma Ramos cells (Table 1) and with no evidence for any truncated variants (4). This further argues for the possibility that VH gene mutations could be generated endogenously in these tumor cells and that B-cell specific factors may not be required. Indeed, prokaryote DNA can be hypermutated following AID transfection in Escherichia coli (19).

Importantly, identification of AID expression in epithelial tumor cells extends a very recent report, where AID transcripts were found to be present in pluripotent mammalian tissues, including oocytes and primordial germ cells, at a level comparable with lymphoid tissue (20). In that report, it was postulated that AID might play a role in epigenetic reprogramming. This suggests that AID activity in tumor cells may have widespread implications in maintaining the malignant phenotype. It has also been suggested that aberrant mutations and genomic instability are likely when AID is gratuitously expressed (21). Indeed, in a mouse model, AID is required to generate aberrant c-myc/IgH chromosomal translocations, although it is unclear whether its activity is required as an initiating or potentiating event (22, 23). An additional question raised is whether AID expression in epithelial tumor cells is triggered by the same signals operative in B cells (9) or whether additional pathways exist. Interestingly, expression of AID in these tumor cells is dissociated from any ongoing mutational activity in VH genes, as extensive cloning analysis revealed stable sequences (Table 1). We also consistently identified tumor cells expressing AID in the absence of any V(D)J rearrangements (Table 1). This reinforces the concept that AID expression in breast cancer cells could be a tumor-associated feature, where its activation in the absence of any apparent IgH locus activity frees its availability to potentially target the tumor genome.

These observations imply complex genomic events in the derivation of breast cancer cell lines. Whether these events are recapitulated in primary breast cancer cells is as yet not fully clear. They raise the possibility that some of the rearranged Ig{lambda} transcripts found in primary breast cancer by DNA microarrays may in fact have been tumor derived (7). In addition, in some lung cancer cases, rearranged VH genes have also been reported in microdissected tumor cells (2). Secretion of such immunoglobulin molecules, if translated in primary epithelial malignancy, would then invoke questions of a potential role in tumor escape and progression. In summary, these data have identified rearranged VH genes in breast cancer cell lines, and although their acquisition seems more likely by uptake of B-cell chromatin, the finding of pseudogene VH gene use in one line leaves the question of their origins open.


   Acknowledgments
 
Grant support: Leukaemia Research Fund, United Kingdom.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 10/13/05. Revised 2/ 3/06. Accepted 3/ 7/06.


   References
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 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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