NEOPLASIA

Expression of the AID protein in normal and neoplastic B cells

Laura Pasqualucci, Roberta Guglielmino, Jane Houldsworth, Jessica Mohr, Said Aoufouchi, Roberto Polakiewicz,
R. S. K. Chaganti, and Riccardo Dalla-Favera

Somatic hypermutation (SHM) targets pri-
marily the immunoglobulin variable re-
gion (IgV) genes in germinal center (GC)
B cells, thereby allowing antibody affinity
maturation. A malfunction of SHM, termed
aberrant somatic hypermutation (ASHM),
was found in about 50% of diffuse large
B-cell lymphomas (DLBCLs), leading to
mutations in the 5ⴕ sequences of multiple
genes, including oncogenes. Although
the SHM mechanism is largely unknown,
it was shown to require the activation-
induced cytidine deaminase (AID) gene.
AID mRNA is expressed in GC B cells and
GC-derived lymphomas, but the pattern
of expression of the AID protein is not
known. Using 2 specific antibodies, here
we show that the AID protein can be
detected in GC centroblasts and their
transformed counterpart (Burkitt lym-
phoma) but not in pre-GC B cells and
post-GC neoplasms, including B-cell
chronic lymphocytic leukemia and mul-
tiple myeloma. DLBCLs displayed vari-
able levels of AID expression, which did
not correlate with IgV ongoing hypermuta-
tion, ASHM, or disease subtype. Finally,
both in normal and malignant B cells
the AID protein appeared predominantly
localized in the cytoplasm. These results
indicate that the AID protein is specifi-
cally expressed in normal and trans-
formed GC B cells; nonetheless, its pre-
dominantly cytoplasmic localization
suggests that additional mechanisms may
regulate its function and may be altered
during lymphomagenesis. (Blood. 2004;
104:3318-3325)
© 2004 by The American Society of Hematology

Introduction
Somatic hypermutation (SHM) is a specialized process that takes
place in germinal center (GC) B cells in response to T cell–
dependent antigen stimulation.1,2 This process introduces single
nucleotide substitutions, with occasional deletions and duplica-
tions, primarily into the variable region of the immunoglobulin
(IgV) heavy and light chain genes, resulting in the production of
high-affinity antibodies and allowing affinity maturation of the
humoral immune response.3 SHM requires active transcription of
the target locus but is not IgV sequence specific and does not
depend on the V region promoter.4-7 In fact, at least 3 non-Ig genes,
including BCL6, the FAS/CD95 gene, and the genes encoding the 2
components of the BCR (B29 and mb1) have been shown to acquire
somatic mutations during the normal GC reaction, indicating that
this mechanism may target more genes than originally suspected.8-12
The molecular basis of SHM remains largely unknown. How-
ever, studies from the past 4 years have identified the AID gene,
encoding for activation-induced cytidine deaminase, as an absolute
requirement for both SHM and class switch recombination (CSR)
in humans and mice.13,14 Activation-induced cytidine deaminase
(AID) expression is also sufficient to initiate both events in
fibroblasts expressing transcribed artificial constructs.15,16 Because
of the high homology with the RNA-editing enzyme apolipoprotein
B editing catalytic subunit 1 (APOBEC-1), it has been proposed
that AID may function as a cytidine deaminase to modify a
preexisting mRNA into a new one, possibly encoding an endonucle-
ase.14,17 However, experimental evidence in Escherichia coli
indicated that AID may act directly on DNA and convert deoxycy-
tidines to uracils, which are then processed by uracil-DNA
glycosylase (UNG) and endonucleases.18 AID would thereby lead
to the creation of abasic sites, which may be repaired by base
excision repair and putative error-prone mechanisms. Indeed, in
vitro AID exhibits cytidine deamination activity on single-stranded
DNA, with a base specificity similar to that reported for SHM.19-22
Recently, the SHM process has been shown to malfunction in
about 50% of diffuse large B-cell lymphomas (DLBCLs) as well as
in about 20% of AIDS-related non-Hodgkin lymphomas (NHLs)
and in a significant fraction of primary central nervous system
lymphomas derived from non-HIV patients.23-25 In these tumors,
multiple somatic mutations are introduced into the 5⬘ region,
including coding sequences, of several genes that do not represent
physiologic SHM targets. These comprise the well-known proto-
oncogenes PIM1, PAX5, RhoH/TTF, and cMYC, all of which have

From the Institute for Cancer Genetics and the Departments of Pathology and
Genetics & Development, Columbia University, New York, NY; Laboratory of
Cancer Genetics and the Department of Medicine, Memorial Sloan-Kettering
Cancer Center, New York, NY; Cell Signaling Technology, Beverly, MA; and
INSERM U373 Faculté de Médecine Necker - Enfants Malades, Paris, France.
Two of the authors (R.P. and J.M.) are employed by a company (Cell Signaling
Technology, Inc, Beverly, MA) whose potential product was studied in the
present work.
The online version of this article contains a data supplement.

Submitted April 26, 2004; accepted June 28, 2004. Prepublished online as
Blood First Edition Paper, August 10, 2004; DOI 10.1182/blood-2004-04-1558.
Supported by a Leukemia and Lymphoma Society Specialized Center of
Research (SCOR) grant with contributions from the Cathy and Scott Zeilinger
Philanthropic Fund of the Jewish Community Federation of Cleveland, the Carl
and Ruth Shapiro Family Foundation, Lesley Goldwasser and Jonathan Plutzik
and Friends, and Marla Kaminski and Friends. L.P. is a Special Fellow of the
Leukemia and Lymphoma Society. Also supported by the Ligue Nationale
Contre le Cancer (Equipe labellisée) (S.A.).
An Inside Blood analysis of this article appears in the front of this issue.
Reprints: Laura Pasqualucci, Institute for Cancer Genetics, Columbia
University, 1150 St Nicholas Ave, New York, NY 10032; e-mail: lp171@
columbia.edu.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology

3318 BLOOD, 15 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 10

BLOOD, 15 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 10
been already implicated in the pathogenesis of lymphoid malignan-
cies. Analogous to the physiologic targets (IgV and BCL6),
mutations at these loci are scattered through the first approximately
2 kb from the promoter with characteristic hotspot (RGYW/
WRCY) motifs and exhibit a bias for transitions over transversions,
with higher frequency at G:C pairs.23,24 Such distinctive features
strongly support the hypothesis that this phenomenon, termed
“aberrant somatic hypermutation” (ASHM), results from an aber-
rant activity, possibly a loss of target specificity, of the physiologic
SHM process and therefore of AID itself. Because the mutations
affect both regulatory and coding sequences of the affected genes,
with several amino acid substitutions predicting a change in the
protein structure, ASHM may represent a major contributor to
DLBCL pathogenesis. Indeed, for some of the targeted genes, such
as cMYC, the oncogenic effect of some mutations has been
demonstrated.26-28
Based on RNA expression data, AID appears selectively
expressed in GC B cells and GC-derived malignancies14,29-31;
however, the pattern of expression of the AID protein has never
been investigated. Using 2 specific antibodies, we examined the
distribution and levels of the AID protein in purified normal B-cell
subpopulations (naive and centroblasts) as well as in B-lymphoid
tumors derived from GC and post-GC B cells. These results have
implications for the mechanism regulating AID function and for its
possible role in lymphomagenesis.
Materials and methods
Cell lines and primary lymphoma cases
All B-cell lines used were cultured in Iscove modified Dulbecco medium
(IMDM) or RPMI (Gibco, Grand Island, NY) supplemented with 10% fetal
calf serum (FCS), 100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM
L-glutamine. The lung carcinoma cell line H1299 was grown in Dulbecco
modified Eagle medium (DMEM)/10% FCS. Ramos and H1299 were
stably transduced with pBABE-puro retroviral vectors or with vectors
expressing a C-terminal Flag-hemagglutinin (HA)–tagged human AID
cDNA (AID-FH), followed by selection in 0.5 ␮g/mL puromycin. The 25
newly diagnosed DLBCL frozen tissue samples were obtained from the
Department of Pathology of the Memorial Sloan-Kettering Cancer Center.32
Only cases in which the fraction of neoplastic cells corresponded to more
than 70% (n ⫽ 21) were selected for the correlative analysis between AID
protein expression and DLBCL subgroup or ASHM. The B-cell chronic
lymphocytic leukemia (B-CLL) primary cases and their characterization for
the presence of IgV mutations are described in detail elsewhere.33
Isolation of normal human B-cell subpopulations from tonsils
Naive and centroblast (CB) B-cell subpopulations were isolated by
magnetic cell separation using the MidiMACS system (Miltenyi Biotec,
Auburn, CA) as described.34 Briefly, naive B cells were isolated by
depletion of GC B cells (CD10, CD27), memory B cells (CD27), plasma
cells/blasts (CD27), and T cells (CD3), followed by a positive enrichment
of IgD-positive cells. CBs were isolated in a single step by magnetically
labeling CD77⫹ cells. The purity of the isolated fractions was determined
on a FACSCalibur (Becton Dickinson, San Jose, CA) and by IgV region
gene analysis.34
Antibodies
The method used for the generation of the anti-AID rabbit polyclonal
antibody (Ab) is reported by Faili et al.35 The anti-AID mouse monoclonal
Ab (clone 7E7), an IgG1 ␬ light chain, was raised against a synthetic
peptide selected from the C-terminal half of the human AID protein.
Additional details are provided as Supplemental Materials (at the Blood
website, see the Supplemental Materials link at the top of the online article).
AID PROTEIN EXPRESSION IN B CELLS 3319
The following Abs were also used: anti-BCL6 (N3, Santa Cruz Biotechnol-
ogy, Santa Cruz, CA), anti-HA (3F10, Roche Diagnostics, Indianapolis,
IN), anti–␤-actin (AC15, Sigma, St Louis, MO), and anti–␣-tubulin (clone
B-5-1-2, Sigma).
Protein extracts, cell fractionation, and Western blot analysis
Total protein extracts were prepared from exponentially growing cell lines
and snap-frozen lymphoma biopsies as described previously.36 To isolate
nuclear and cytoplasmic fractions, cells were incubated for 15 minutes at
4°C in hypotonic buffer A (10 mM HEPES [N-2-hydroxyethylpiperazine-
N⬘-2-ethanesulfonic acid] pH 7.9, 10 mM KCl, 0.1 mM EDTA [ethylenedia-
minetetraacetic acid], 1 mM dithiothreitol [DTT], and protease inhibitors
[Sigma]); Nonidet P-40 (NP40) was added to a final concentration of 0.4%,
and the samples were centrifuged for 5 minutes at 3000 rpm. Supernatants
were used as cytoplasmic extracts after further centrifugation for 30
minutes at 14 000 rpm. The nuclear pellets were washed twice in buffer A,
resuspended in about 10 volumes of buffer C (20 mM HEPES pH 7.9, 400
mM NaCl, 1 mM EDTA, 0.4% NP40, 1 mM DTT, and protease inhibitors),
vortexed for 30 minutes at 4°C, and centrifuged for 30 minutes at 14 000
rpm. The final salt concentration in the nuclear extracts was adjusted to 300
mM by addition of buffer D (20 mM HEPES pH 7.9, 1 mM EDTA, 1 mM
DTT, and protease inhibitors). For Western blot analysis, protein extracts
(30 ␮g whole-cell extract or 10 ␮g subcellular fraction) were gel
electrophoresed on 4% to 20% gradient sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS-PAGE) gels (Invitrogen Life
Technologies, Carlsbad, CA), transferred to nitrocellulose membranes, and
immunostained according to standard methods. Proteins were detected
using the enhanced chemiluminescence (ECL) reagents (Amersham Bio-
sciences, Freiburg, Germany) as recommended by the manufacturer.
To compare the AID protein expression levels in DLBCL samples from
different experiments, equivalent amounts of GC centroblasts were loaded
in each gel and used as an internal reference. Band intensities were
measured using the NIH Image program 1.62, and AID expression was
normalized first for ␤-actin, as a control for loading, and then for the CB
sample loaded in the same gel.
RNA extraction, Northern blot analysis, and gene
expression profiling
Total RNA was prepared using the Trizol reagent (Invitrogen Life
Technologies) followed by RNeasy purification (QIAGEN, Valencia, CA),
and Northern blot analysis was performed according to standard proce-
dures, with radiolabeled probes corresponding to the full-length human
AID, BCL6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
cDNAs. The detailed characterization of normal B-cell subpopulations by
gene expression profiling has been reported previously.34 The protocol for
microarray hybridization of the DLBCL samples to the Affymetrix HG95Av2
oligonucleotide array, as well as the expression data analysis, has also been
reported in detail.32
PCR amplification, cloning, and sequencing analysis of PIM1,
PAX5, RhoH/TTF, and cMYC
Genomic DNA from 31 DLBCL samples, including 10 cell lines and 21
primary biopsies, was extracted according to standard methods. Mutational
analysis of PIM1, PAX5, RhoH/TTF, and cMYC was performed on selected
genomic regions previously shown to contain more than 90% of the
mutations found in DLBCL.23 Following polymerase chain reaction (PCR)
amplification, purified amplicons were sequenced directly from both
strands and compared with the corresponding germ line sequences as
described.23 All sequence variants were confirmed on independent PCR
products, and nucleotide changes due to previously reported polymor-
phisms or occurring more than once in separate cases were excluded from
the assessment of the mutation frequencies unless their somatic origin could
be proven. Cases were scored as ASHM-positive if they harbored 1 or more
somatic mutations distributed in 1 or more of the 4 proto-oncogenes.
3320 PASQUALUCCI et al BLOOD, 15 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 10

Analysis of IgV gene intraclonal variation
The rearranged IgVH genes were amplified from genomic DNA using the
Pfu Turbo polymerase (Stratagene, La Jolla, CA) together with a set of 6 VH
gene family–specific primers hybridizing to sequences in the framework
region (FR)I and a JH primer mix in separate reactions for each VH primer.8
PCR products were gel purified and cloned into pGEM-T vector (Invitrogen
Life Technologies) as described.23 For each PCR product, 25 to 58 clones
were sequenced and analyzed. Mutation frequencies were calculated by
dividing the total number of unique mutations (ie, changes observed in
individual subclones) by the total number of base pairs sequenced.
expected based on RNA data (not shown). Western blot analysis
with an anti-HA Ab, which recognizes only the tagged protein,
revealed the same 27-kDa band (Figure 1C, third panel), document-
ing its exogenous nature. Similar results were obtained using the
monoclonal and the polyclonal Abs, although the latter one gave
significantly higher background. The reactivity in cells expressing
AID mRNA or a transfected AID cDNA indicates that both the
monoclonal and polyclonal Abs can specifically recognize the
AID protein.
AID protein expression in normal B-cell subpopulations

To examine the expression pattern of the AID protein in normal

Results
Identification of the AID protein by specific antibodies
Two antibodies were used throughout the study: one is a rabbit
polyclonal Ab raised in the laboratory of one of the authors
(S.A.)35; the second is a monoclonal Ab obtained against a peptide
sequence selected from the C-terminal half of the human AID
polypeptide (see “Materials and methods”). To test the ability of
these Abs to recognize the human AID protein, we first used 4
B-cell lines that were documented by Northern blot (NB) analysis
to express (ODHI I, Ly10) or lack (ODHI III, SUDHL6) endoge-
nous AID mRNA (Figure 1A). Western blot analysis on total
protein extracts detected a specific band of the expected 24-kDa
molecular weight in ODHI I and Ly10 but not in the 2 RNA-
negative cell lines ODHI III and SUDHL6 (Figure 1B; the
polyclonal Ab is shown). We then engineered a human epithelial
cell line (H1299), lacking AID mRNA expression, and the B-cell
line Ramos, which expresses low levels of AID mRNA, to stably
express an exogenous, Flag-HA–tagged human AID protein (AID-
FH) under the control of pBABE retroviral sequences. Figure 1C
shows the presence of an approximately 27-kDa band (exo)-
consistent with the predicted size of the double-tagged fusion
protein—only in AID-FH–transfected cells (Figure 1C, top 2
panels, lanes 2 and 4) but not in control vector–transfected cells
(Figure 1C, lanes 1 and 3). In addition, a band of smaller molecular
weight was recognized in both AID-FH– and control vector–
transfected Ramos cells (Figure 1C, lanes 3 and 4); this smaller
band corresponded in size to the endogenous AID protein (endo)
and was not present in H1299 (Figure 1C, lanes 1 and 2), as
B-cell subpopulations, pre-GC naive B cells (IgD⫹ CD27⫺) and
GC CBs (CD77⫹) were purified from the reactive tonsil of normal
individuals, and Western blot analysis was performed on total
protein extracts as well as on nuclear and cytoplasmic cell
fractions. Elevated AID protein levels were detected in the CB
population (ie, the cells deputed to SHM) but not in the pre-GC
naive B cells, confirming RNA data generated by gene expression
profiling (Figure 2A-B). Unexpectedly for a protein that is
presumed to function as a DNA mutator, Western blot analysis of
GC B cells after cell fractionation revealed a predominantly
cytoplasmic localization of AID, with only traces being detected in
the nuclear fraction (Figure 2C). The purity of the extracts was
monitored using anti–␣-tubulin and anti-BCL6 Abs, respectively.
Thus, the AID protein is expressed in GC B cells, where it resides
predominantly in the cytoplasm.
AID protein expression in B-cell malignancies
In B-cell neoplasms, the AID protein was found restricted to cells
displaying a mature GC phenotype, including 10 of 10 group I
Burkitt lymphoma (BL) cell lines, where the levels were mostly
comparable to CBs (Figure 3, top left panel). AID expression was
also examined in 3 pairs of BL lines (KEM, MUTU, ODHI), each
one derived from the same tumor case but selected in vitro to
represent either a group I GC phenotype or a group III post-GC
immunoblastic phenotype.37 Elevated AID protein levels were
detected in the group I but not in the group III BL lines (Figure 3,
top left panel), suggesting that AID expression may be down-
regulated in this later stage of differentiation. Among DLBCL

Figure 1. Identification of the AID protein by specific antibodies. (A) Northern blot analysis of endogenous AID expression in 2 BL (ODHI I, ODHI III) and 2 DLBCL (Ly10,
SUDHL6) cell lines. An approximately 2.7-kb message, corresponding to the AID transcript, can be detected in ODHI I and Ly10 but not in ODHI III and SUDHL6. Filters were
stripped and sequentially hybridized with probes for BCL6, as marker of differentiation stage, and GAPDH as control for loading. (B) Western blot analysis of the same cell lines
with anti-AID rabbit polyclonal Abs shows a specific band of the predicted 24-kDa molecular weight in ODHI I and Ly10, but not in ODHI III and SUDHL6, consistent with the
RNA data in panel A. Immunoblotting with anti-BCL6 (N3) and anti–␤-actin Abs is shown in the bottom panels. (C) Western blot analysis of AID expression in H1299 and Ramos
cells, stably transduced with pBABE retroviral vectors (H1299-V, Ramos-V) or with vectors expressing an AID-Flag-HA protein (H1299-AID-FH and Ramos-AID-FH). Both the
rabbit polyclonal antiserum (poly) and the mouse monoclonal (7E7) Abs were used (top 2 panels). An approximately 27-kDa band corresponding to the size of the exogenous
protein (exo) is detected in lysates from AID-FH–transduced cells (lanes 2 and 4) but not in cells transduced with the control vector (lanes 1 and 3). In the B-cell line Ramos, the
endogenous AID protein (endo) can also be seen (lanes 3 and 4). Membranes were stripped and sequentially probed with anti-HA, which recognizes the exogenous protein,
and with anti–␤-actin as control for loading (bottom 2 panels).
BLOOD, 15 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 10 AID PROTEIN EXPRESSION IN B CELLS 3321

Figure 2. AID protein expression and subcellular localization in normal B lymphocytes. (A) AID mRNA expression in purified normal B-cell subpopulations (5 samples
each of N [naive], CB [centroblasts], CC [centrocytes], M [memory]). AID was represented by 2 probe sets in the Affymetrix U133Plus GeneChip array. BCL6, BCL7A, and the
proliferation-associated gene Ki67 are shown along with BCL2 and TOSO as markers for the identity of the purified subpopulations. (B) Western blot analysis of AID and ␤-actin
expression in whole cell extracts obtained from naive and CB populations. (C) Western blot analysis of AID expression in nuclear (nu) and cytoplasmic (cy) extracts from
tonsillar GC B cells. The purity of the fractions was monitored using anti-BCL6 and anti–␣-tubulin Abs, respectively.

cases, which included 25 primary biopsies and 10 cell lines, AID
expression was very heterogeneous, ranging from complete ab-
sence (3 of 10 cell lines and 10 of 25 biopsies; overall, 13 of 35
samples, corresponding to 37% of the cases) to levels comparable
to the ones observed in CBs (Figure 3, top 2 right panels, shows
representative results). No evidence of AID protein expression was
found in post-GC tumors such as MM (0 of 7 cell lines) and B-CLL
(0 of 12 samples, including 10 primary cases and 2 cell lines)
regardless of their IgV mutational status (Figure 3, bottom panels).
In all samples studied, NB analysis documented the complete
concordance between RNA and protein levels in terms of relative
distribution (data not shown). Collectively, these data indicate that
expression of the AID protein is restricted to B-cell malignancies
derived from GC cells.
Expression of the AID protein in DLBCL subgroups
Based on gene expression profiling studies, DLBCLs have been
classified into 3 biologically and clinically distinct subgroups: the
GC B-cell–like (GCB), the activated B-cell–like (ABC), and the
type 3 DLBCL.38,39 To investigate whether the AID protein was
differentially expressed among these phenotypic groups, we used
the Affymetrix U95Av2 oligonucleotide microarray to generate
gene expression profiles from 30 DLBCL samples, including 9 cell
lines and the 21 primary biopsies that contained more than 70%
tumor cells, and then measured the AID protein levels in each
group identified. The analysis classified 15 samples (50%) into the
GCB subgroup, 9 samples (30%) into the ABC subgroup, and 6
samples (20%) into the type 3 group. In all of them, expression of
the AID protein was very heterogeneous, with 9 of 15 (60%)
positive cases in the GCB-like, 7 of 9 (77.8%) positive cases in the
ABC-like, and 4 of 6 (66.6%) positive cases in the type 3 DLBCL
(Figure 4, top panel); however, when compared with the GCB type,
the ABC DLBCLs had on average higher expression levels (about
3-fold) both in primary biopsies and in cell lines (Figure 4, bottom
panel). Moreover, the overall higher levels of AID in ABC
DLBCLs tend to inversely correlate with the expression of BCL6, a
well-established marker of GC (Figure 1A-B and data not shown),
differently from what is observed in GC B cells or in BL, where the
2 proteins are coexpressed (see “Discussion”).
AID expression in DLBCL does not correlate with ongoing SHM
To examine whether the differential expression of AID in DLBCL
cases reflects the ongoing activity of the SHM machinery, we
investigated a number of samples for the presence of intraclonal
variation (ICV) in their IgV sequences, an indicator that SHM has
been active during clonal expansion. The rearranged IgV genes of 6
cell lines, representative of AID-positive (Ly3, Ly7, Ly10, RCK8)
and -negative (Ly8, SUDHL6) cases, were amplified from genomic
DNA using the proofreading Pfu Turbo polymerase and then
cloned and sequenced (n ⫽ 25 to 58 clones per sample). The BL
Ramos clone 6, which is known to undergo SHM in culture
constitutively, was used as positive control, while Ramos clone 1,
which has lost the ability to hypermutate, served as a negative
control.40 Indeed, detectable AID protein levels were observed in
Ramos clone 6 and were associated with significant intraclonal
variation in its IgV sequences (22 mutational events distributed in
14 of 27 clones, corresponding to a frequency of 0.239 ⫻ 10⫺2/bp;
P ⬍ .0001); conversely, only 1 nucleotide difference was found in
31 DNA clones sequenced from the AID-negative Ramos clone 1
(frequency, 0.009 ⫻ 10⫺2/bp, which is not significantly different

Figure 3. The AID protein is expressed in GC but not in post-GC–derived B-cell malignancies. Western blot analysis of AID expression in BL (top left panels), DLBCL (top
right panels), and post-GC–derived B-CLL and MM samples (bottom panels). Both IgV mutated and unmutated cases were included in the B-CLL filter. The top right panel
shows 2 pairs of isogenic BL cell lines representative of the group I/latency I and group III/latency III phenotype (see text for a detailed explanation); the B-cell lymphoma line
BJAB serves as a negative control, because it does not express AID mRNA (L.P., unpublished results, 2003). In each membrane, equivalent amounts of total protein extracts
from purified CBs were included; ␤-actin controls for loading.
3322 PASQUALUCCI et al
Figure 4. The AID protein is expressed in both GCB- and ABC-like DLBCLs.
(Top) AID protein levels, measured by Western blotting and normalized for ␤-actin as
described in “Materials and methods,” are shown in individual DLBCL samples
classified by gene expression profiling as GCB-like (E), ABC-like (F), and type 3 (O).
Two different scales are used for the primary biopsies (left) and the cell lines (right),
because lower AID levels were consistently observed in the former ones, likely due to
the presence of contaminating nontumor cells. (Bottom) Histograms show the
average AID protein expression levels in the 3 groups. Error bars indicate standard
deviations.
from the expected error rate of the Pfu polymerase) (Figure 5A; see
also Supplemental Table S1). Of the DLBCL cell lines analyzed,
only 1 (Ly7) displayed a significant number of IgV intraclonal
variants (11 of 19 426 bp, corresponding to a mutation frequency of
0.057 ⫻ 10⫺2/bp), while the frequency of mutations in the remain-
ing 5 lines was not significantly higher than the polymerase error
rate (range, 2 of 5957 bp to 5 of 23 142 bp sequenced) (Figure 5B
and Supplemental Table S1). These extremely low frequencies
were detected regardless of the AID protein levels and even in cell
lines, such as Ly3 and Ly10, which express protein amounts
comparable to GC B cells and higher than Ramos clone 6 (Figure
5B). Thus, differently from GC CB and BL cases, the heteroge-
neous expression of AID in DLBCLs does not reflect the level of
activity of the SHM machinery.
BLOOD, 15 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 10
AID expression levels do not correlate with ASHM in DLBCL
To investigate whether AID overexpression is involved in the
pathogenesis of DLBCL-associated ASHM, we comparatively
analyzed AID protein levels and the presence of somatic mutations
in 4 genes previously identified as aberrant targets: PIM1, PAX5,
RhoH/TTF, and cMYC.23 To this end, 31 DLBCL samples were first
subjected to PCR amplification and direct sequencing of selected
regions representing the hypermutable area in these genes.23
Because PCR products were sequenced directly in all experiments,
the contribution of Taq polymerase error to mutations in the final
sequence is insignificant. The overall mutation load in each case
was then plotted against the normalized AID protein levels,
measured by Western blotting, and quantitated using the NIH
Image software.
PCR products of PIM1, PAX5, RhoH/TTF, and cMYC were
obtained from all DLBCL cell lines and from 20 of 21 primary
cases (30 total evaluable samples). The analysis revealed the
complete absence of somatic mutations in 2 of 10 cell lines (20%)
and 5 of 20 primary biopsies (25%), while the remaining 23
samples, including 8 cell lines and 15 biopsies, carried 1 or more
somatic mutations distributed in 1 or more of the 4 target genes
(range, 1 to 28 mutational events per case in the cell lines; 1 to 18
mutational events per case in the primary biopsies) (Supplemental
Table S2). All the mutations, which included mostly single
nucleotide substitutions but also 4 deletions and 1 duplication, were
confirmed on independent PCR products. These results are analo-
gous to those originally reported for an independent cohort of
DLBCL samples.23 When we compared the mutation status with
the AID protein levels, we found that, both in cell lines and in
primary biopsies, AID expression did not correlate with the
presence of ASHM (Figure 6). In particular, AID was expressed in
3 of 7 (43%) unmutated and 17 of 23 (74%) aberrantly mutated
samples (Figure 6, top). Moreover, no direct correlation was
observed in the mutated samples between AID protein amount
and the total number of mutations (not shown), indicating that
a simple increase in AID expression levels is not sufficient to
explain ASHM.
Predominant cytoplasmic localization of the AID protein in
normal and transformed B cells
To examine whether abnormal subcellular localization, rather than
overexpression of AID, could be responsible for loss of target
specificity in DLBCL cases with ASHM, we performed Western
Figure 5. AID protein expression is associated with
ongoing SHM in Ramos but not in DLBCL cell lines. (A)
Total cell extracts were prepared from 2 Ramos clones that
express (cl.6) or lack (cl.1) AID mRNA expression40 and, from
each of them, 3 dilutions were analyzed by immunoblotting
using anti-AID Abs and ␤-actin as control (top). In the bottom
panel, the AID protein levels from the top panel, quantitated
using the NIH Image software and normalized for ␤-actin (f),
are compared with the levels of ICV in the corresponding
rearranged IgV genes (o), expressed as frequency of muta-
tions and calculated as described in “Materials and meth-
ods.” The mutation frequency in the consensus sequence,
defined as the sequence identified by direct sequencing and
common to all DNAclones analyzed, is indicated in parenthe-
ses below the chart (also see Supplemental Table S1). (B)
AID protein expression in 10 DLBCL cell lines (top). The
correlation betweenAID protein levels (f) and the level of IgV
genes ICV (o), assessed as in panel A, is shown in the
bottom panel for 6 representative lines. Numbers in parenthe-
ses indicate the mutation frequency in the IgV consensus
sequence.
BLOOD, 15 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 10
Figure 6. Lack of correlation between AID protein levels and ASHM in DLBCL.
(Top) AID protein expression levels in DLBCL samples displaying (F) or lacking (E)
ASHM, as defined by the presence of mutations in PIM1, PAX5, cMYC, and
RhoH/TTF. AID protein levels were measured by Western blot analysis and
normalized for ␤-actin as described in “Materials and methods.” (Bottom) Histograms
show the average expression levels in the 2 groups. Error bars indicate standard
deviations.
blot analysis on nuclear and cytoplasmic fractions prepared from
10 AID-expressing cell lines, including 7 DLBCL and 3 BL. The
DLBCL lines were representative of both the ABC (Ly3, Ly10) and
the GCB (Ly7, Ly1, Ly18, RCK8) type and displayed ASHM in 6
of 7 cases. The results shown in Figure 7 indicate that—with the
exception of the ABC-like, ASHM-positive Ly10 cell line, where
AID appears to be evenly distributed in the nuclear and cytoplas-
mic fraction—AID is mostly retained in the cytoplasm (more than
90%), and only trace amounts of the protein can be detected in the
nuclear fraction, analogous to what observed in normal GC B cells.
This distribution was observed regardless of the histologic type of
lymphoma (BL versus DLBCL) and also of the evidence for
ongoing SHM (see CB and Ramos clone 6). Thus, in steady state
conditions, the AID protein appears to be localized mostly in the
cytoplasm, both in normal and transformed B cells.
Discussion
In the past few years, the AID protein has been the object of
extensive studies due to its primary role in SHM and CSR. As a
mutator, AID has also been implicated in the pathogenesis of B-cell
lymphomas, and indeed its deregulated expression may represent a
good candidate to explain the loss of target specificity that seems to
Figure 7. Predominant cytoplasmic localization of the AID
protein in BL and DLBCL. Nuclear (N) and cytoplasmic (C)
fractions were prepared from 10 AID-positive lymphoma cell lines,
including 3 BL and 7 DLBCL, and immunoblotted with anti-AID
Abs, followed by anti-BCL6 (nuclear) and anti–␣-tubulin (cytoplas-
mic) as controls for the purity of the fractions. The presence or
absence of ASHM is also given; nd indicates not determined.
AID PROTEIN EXPRESSION IN B CELLS 3323
derange the SHM mechanism in DLBCL.23,24 Based on RNA data,
AID appears selectively expressed in GC B cells and GC-derived
malignancies. However, the expression pattern of the AID protein
has never been shown, mainly due to unavailability of specific Abs.
This is the first study to report the distribution of the AID protein in
normal and transformed human B cells, its relationship with IgV
SHM, and its correlation with ASHM. Our data have implications
for the understanding of the AID function in general and its specific
role in lymphomagenesis.
The results of our study complement available data on AID
mRNA expression by showing a close correlation between AID
transcripts and protein levels in GC B cells and GC-derived
malignancies29,30 (data not shown). Thus, no major control mecha-
nism appears to be involved in the expression of AID at the level of
RNA translation or protein stability. Nonetheless, neither RNA nor
protein studies on bulk cell populations can address the issue of
AID expression in cell subpopulations. Therefore, our results
cannot determine whether AID is expressed in all GC B cells or in
selected subpopulations, a question requiring the use of immunohis-
tochemistry techniques that, unfortunately, could not be performed
with the 2 Abs described.
Consistent with its distribution in normal B-cell subpopulations,
the AID protein was not detected in B-CLL samples. B-CLL
represents a post-GC–derived malignancy in at least 60% of the
cases, which carry somatically mutated IgV genes; moreover,
based on gene expression profiling analysis, all B-CLL cases
appear to originate from a post-GC memory B cell.41 Our data are
in apparent contradiction with recent studies reporting expression
of the AID mRNA, mostly in the IgV unmutated subgroup of the
disease.29,42,43 However, these studies were performed using re-
verse transcriptase (RT)–PCR methods, while more sensitive
quantitative assays, including limiting dilution assays, revealed that
only 0.01% to 0.2% of the B-CLL tumor cells are AID positive44
and that the total mRNA levels in B-CLL cases correspond to only
about 5% of those observed in GC B cells,45 the population where
AID typically exerts its activity. Thus, our data are consistent with
previous reports in that the bulk of the B-CLL cells do not express
AID. The biologic relevance of these low and very sparse AID
mRNA expression levels in B-CLL remains to be proven.
Our results show that the AID protein is expressed—in hetero-
geneous fashion—in all phenotypic subtypes of DLBCL (GC-like,
ABC-like, and type 3) and that the AID amounts are tendentially
higher in the ABC type. Analogous results have been obtained by
comparing AID mRNA levels in larger DLBCL databases gener-
ated by gene expression profiling at different institutions (http://
llmpp.nih.gov/lymphoma). The presence of AID—in some cases at
supraphysiological levels—in the ABC DLBCLs was somehow
unexpected. In fact, this tumor subtype has been typically defined
by the down-regulation of GC-specific expression markers (eg,
BCL6) and up-regulation of post-GC markers such as IRF4 and
XBP1, overall suggesting a derivation from a later, post-GC stage
of differentiation.38,46 In addition, both the SHM machinery and the
3324 PASQUALUCCI et al BLOOD, 15 NOVEMBER 2004 䡠 VOLUME 104, NUMBER 10

CSR mechanism have been presumably shut off in this disease
type, because the tumor cells lack intraclonal diversity in their
rearranged IgV genes (our data and Lossos et al47) and express high
levels surface IgM.38,39,48 Thus, the presence of AID in the ABC
DLBCL subgroup may suggest deregulation of AID expression;
alternatively, this may reflect an activation status or the origin from
a distinct B-cell subpopulation characterized by the indicated
phenotypic features, possibly at the exit of the GC. Because the
presently available Abs are not suitable for immunohistochemistry/
immunofluorescence studies of AID, this question remains
unanswered.
We could not find a correlation between AID expression levels
and evidence of ASHM in the DLBCL primary cases analyzed.
Considering that no structural abnormalities of the AID coding
sequence are present in DLBCL, based on DNA sequencing
data,23,29 several possibilities may explain this finding: (1) AID may
have acted at earlier stages, before the biopsy was taken; however,
tumors like BL consistently express high levels of AID but do not
show evidence of ASHM23; (2) because the extent of the ASHM
target is not known, it is possible that cases presently classified as
“unmutated” may in fact carry mutations in genes other than the
ones identified so far and therefore represent “false negatives”; and
(3) regulation of AID activity (eg, gene target recognition and
accessibility; shuttling of the protein to the target sequence) rather
than the absolute expression levels may be implicated in ASHM
(see next paragraph). For instance, the ASHM defect may reside
primarily in other molecules associated with AID.
One perhaps surprising finding of this study is represented by
the predominant cytoplasmic localization of the AID protein in all
samples analyzed, including CBs. This result is in agreement with
the original report by Rada et al, who used confocal microscopy to
detect an exogenous GFP-AID fusion protein stably transfected in
the Ramos B-cell line,49 and with 2 more recent studies also
performed by transduction of GFP-AID retroviral constructs in B
and non-B cells.50,51 However, the localization of AID in the
cytoplasm is in disagreement with its presumed activity as a
mutator, whether directly on DNA or on pre-mRNA, because the
RNA-editing process is also thought to take place in the nucleus.52
While we cannot exclude the possibility that the biochemical
procedure used may have allowed some leakage of AID into the
cytoplasm, it is also conceivable that very small amounts of AID in
the nucleus at any given time may be sufficient to exert its activity.
Nevertheless, one may wonder why these cells would produce such
a large amount of AID when not necessary. A more plausible
explanation is that nuclear/cytoplasmic shuttling of AID constitutes
a major point of regulation in its function, where sequestration of
AID in the cytoplasm would serve as an active mechanism to
protect genetic information from uncontrolled mutational activity.
Indeed, it has been recently reported that a GFP-AID fusion protein
can accumulate in the nucleus of stably transfected cells following
treatment with the inhibitor of chromosome region maintenance/
exportin 1 (CRM-1)–dependent nuclear export leptomycin B
(LMB).50,51 Notably, mutants that accumulate in the nucleus were
associated with higher mutation activity on cotransfected artificial
target constructs, while mutants that could not shuttle to the nucleus
upon LMB treatment lost their activities in both SHM and CSR.50,51
These results suggest that AID nuclear localization quantitatively
correlates with its biologic function. Further studies aimed at the
identification of signals controlling AID subcellular localization are
necessary in order to understand its regulation in GC B cells and its
possible deregulation in DLBCL.
Acknowledgments
We thank U. Klein for the isolation of the normal B-cell subsets, V.
Miljkovic and A. Babiac for assistance in DNA sequencing, Claude-
Agnès Reynaud for kindly providing us with the anti-AID polyclonal
antibody, and A. Martin for the Ramos clone 6 and clone 1.

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