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Expression of Recombination Activating Genes (RAG-l and RAG-2) in
Epstein-Barr Virus-Bearing B Cells
By Ingrid Kuhn-Hallek, David R. Sage, Lincoln Stein, Holly Groelle, and Joyce D. Fingeroth
SIgM-), CDlO+, and lackedterminal deoxynucleotidyl transRecombination activating genes 1 and 2 (RAG-1 and RAGferase. In EBV’ Burkitt’s lymphoma lines, transcription of
2). are the only lymphoid-specific genes requiredthe
forsitevirus latent membrane protein-l (LMP-1) was correlated
directed recombination reaction leadingto generation of Bwith downregulation of RAG-l and RAG-2. Conversely, abcell receptors and T-cell receptors (TCRs). RAGS are normally
sence of LMP-1 in clones of EBV’tumor lines was associated
expressed during narrow
a
window of precursorlymphocyte
with increasedRAGtranscription.Translocationofc-myc
development.RAG expressionwas examined in Epstein-Barr
into V(D)J locihasbeenobservedinendemicBurkitt’s
virus (EBV)-infected B cells. No steady-state RAG RNA was
found in EBV immortalized cells, including newly establymphomas, and heptamer-nonamer recombination signal
lished B lymphoblastoid cell lines derived
from precursor
sequences havebeen
identified at somechromosomal
lymphocytesthat transcribed RAGSat the time of infection.
breakpoints. Association of RAG transcriptionwith EBV inRAG RNAswere detected in some endemic (EBV’) and also
fection raises the possibility that, under certain conditions,
in some sporadic (EBV-) Burkitt‘s lymphoma lines that had
virus might predispose to aberrant V(D)J recombination rebeen infected with EBV in vitro. The RAG+, EBV+ Burkitt’s
actions.
0 1995 by The American Society of Hematology.
lines were unusual in that they were SIgM+ (one was SIgG’,
E
PSTEIN-BARR virus (EBV) was discovered 30 years
ago in tissue from patients with African Burkitt’s
lymphoma.’ Although in vitro EBV efficiently immortalizes
only normal B lymphocytes, the viral genome has been identified in a variety of B and T lymphoid, histiocytoid, and
epithelial neoplasms.’ African Burkitt’s lymphoma isthe
most common childhood tumor in endemic regions that are
characterized by proximity to the equator and a high incidence of malaria. The short latency period betweenviral
infection and malignancy, coupled with the observation that
virtually all of these tumors contain EBV genomes, suggests
virus contributes to early tumorigenic events.’
In normal B cells immortalized by EBV in vitro (referred
to as lymphoblastoid cell lines [LCLs]), at least 11 latent
viral genes encoding six nuclear proteins (EBNA-l, -LP,
-2, -3A, -3B, and -3C), three membrane proteins (latent
membrane protein [LMPI-1, -2A, and -2B), and two small,
nonpolyadenylated RNAs (EBER 1 and 2) are regularly tran~cribed.~.’Molecular genetic analyses indicate that the
EBNA-I, -LP, -2, -3A, and -3C and the membrane protein
LMP-l are absolutely required for immortalization.6 The
role(s) of the respective proteins in initiation and maintenance of the transformed state are variably understood.
EBNA-1 regulates replication of the EBV plasmid and is a
transcriptional transactivator of additional viral gene^.^,^,'"
The remaining nuclear proteins are believed to function in
regulating viral and/or growth regulatory gene^.^.','^," LMP1 promotes transformation of rodent fibroblasts,”.’3 inhibits
epithelial differentiati~n,’~
upregulates cellular activation-associated antigensIs (ie, CD21, CD23, CD40, CD54, LFA-l,
LFA-3, and others), and downmodulates specific cellular
genes, including CD10, CD77, insulin-like growth factor
receptor type l , and E-~adherin.’~”~
Studies indicate that
LMP-l transduces a signal that results in the activation of
nuclear factor kappa B (NFKB)-responsive elements.’8.’9
In contrast to LCLs, viral gene expression in endemic
lymphomas is more variable. With the exception of EBNA1, required for episomal replication of the virus, transcription
of latent genes may be undetectable.” Variable expression
of LMP-1 has been observed among the respective Burkitt’s
lymphoma lines, although, within a given Burkitt’s cell line,
the level of LMP-1 expression appears tobevery consistent.” All Burkitt’s lymphomas contain an 8/14 or related
Blood, Vol 85, No 5 (March I), 1995:
pp 1289-1299
chromosomal translocation.’*22In endemic Burkitt’s lymphomas, the c-myc protooncogene is commonly, though not invariably, juxtaposed to the region of the Ig heavy chain locus
transcribed during the V(D)J recombination pro~ess.”~’~
Heptamer-nonamer recombination signal sequences that
serve to target gene segments for V(D)J recombination have
been identified in the vicinity of several breakpoint region^.'^
The enzymes that mediate V(D)J recombination are collectively known as the recombinase c o m p l e ~ . *The
~ - ~indi~
vidual proteins that participate in this reaction are largely
unknown. Two closely linked genes, RAG-l and RAG-2,28.29
that together are both necessary and sufficient to mediate
V(D)J recombination of an artificial substrate after transfer
into nonlymphoid cells have been identified. These genes are
coordinately expressed during a narrow windowof precursor
lymphocyte development which in B lymphocytes spans the
late pro-B-cell through early immature B-cell stages. RAG1 and RAG-2 are highly conserved during vertebrate evolution,*‘ and gennline deletion of either gene in mice abrogates
lymphocyte maturation.’”.’’ A third lymphoid-specific protein, terminal deoxynucleotidyl transferase (TdT), is expressed early in development and also participates in V(D)J
recombination through addition of nucleotides to the coding
joint that is normally formed. However, TdT is not strictly
required for this site-directed recombination reaction.”
The observations that translocations involving V(D)J loci
From the Laboratory of Infectious Diseases, Dana-Farber Cancer
Institute, Boston; andthe Department of Pathology, Brigham and
Women’s Hospital, Boston, MA.
Submitted September 7, 1994; accepted October 27, 1994.
Supported by Grant No. R29 A126835 from the National Institutes
of Health (NIH). H.G. was supported by Grant No. P30 A128691
from the NIH and via the DFCI small grant program.
Address reprint requests to Joyce D. Fingeroth, MD, Laboratory
of Infectious Diseases, Dana-Farber Cancer Institute, 44 Binney St,
Boston, MA 02115.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1995 by The American Society of Hematology.
0006-4971/95/8505-002021$3.00/0
1289
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KUHN-HALLEK ET AL
1290
as well as the patternof EBV gene expression in the Burkitt's
m
m .m
c
c5 3
Y c5
A a
0
.c
lymphoma lines determine whether RAG genes are expressed.
0
CV
U
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MATERIALSAND
a
a z
m
!l
RAG-1
B
actin
J
0
-l
0
CV
0
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.-a
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N
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METHODS
Cell l i n e s . All cell lines weremaintained in RPMI. 108 heatinactivated fetal calf serum(FCS) (BioWhittaker, Walkersville. MD)
supplemented with 100 UlmL penicillin, I O 0 pglmL streptomycin.
and 20 lnmollL L-glutamine (BioWhittaker) in a humidified 5% CO2
incubator at 37°C. The following cell lines were used: B LCLs: B95
or B9S-8 (of marmoset derivation; a common source of virus), SB,
NAD 20, X50-7. JY. and all of the fetal lines derived at the Laboratory of Infectious Diseases. Dana-Farber Cancer Institute (Boston,
MA); acute lymphoblastic leukemia lines: NALM-6 and LAZ-221;
Namalwa.
EBV+ (endemic) Burkitt's lymphoma lines: Daudi.
Akata. Raji, MABA, Jijoye, P3HR-I (alsoa common source of
virus). and clone 13 of P3HR-I; EBV- (sporadic) Burkitt's lines:
BL4I: and in vitro-inBJAB (Burkitt's-like). Ramos.BL30,and
fectedEBV'
(sporadic) Burkitt's lymphomalines:BJABIB95,
BJABlP3, RamoslB95. RamoslP3, BL30lB95. BL30lP3. BL411B95.
and BL41lP3. In the latter group of cell lines, B95 and P3 designate
the strain of infecting virus derived from the producer lines noted
above (ie. B954 and P3HR-I). The B cell lines were obtained from
the American Type Culture Collection (ATCC; Rockville, MD) and
from the laboratories of Beverly Blazar (Wellesley College. Wellesley. MA) and Jack Strominger, Fred Wang, and Elliot Kieff(Harvard
University, Boston. MA). The Akata cell line" was obtained from
Kenzo Takada (Nihon University, Tokyo, Japan).
RAG-l
RAG-2
A
D
.-.m
U
m
CI
m
Y
0
(U
a
m
a z
c)
7-
cu
(U
0
N
c)
c)
J
-l
m
0
m m
RAG-2
28s RNA
B
Fig 1. Expression of RAG RNAs in endemic Burkitt's lymphoma
lines. (A) TotalRNA from five Burkitt's lines
(Akata, Daudi, Namalwa,
P3HR1, and Maba), a B-LCL (NADZO), and a B-ALL line (Nalm 6) was
hybridized with an RAG-l cDNA. (B) The identical RNA hybridized
with a cDNA to pactin. (C) Total RNA from three EBV' (endemic)
Burkitt's lymphoma lines (Raji, Namalwa, and Daudi) and an LCL
(NAD 201 was hybridized with RAG-l andRAG-2 cDNAs. LA2 221, a
B-ALL, was used as a control. (Dl The identical RNA hybridized with
an oligonucleotide t o 28s RNA.
occur in EBV' Burkitt's lymphomas and that structural elements associated with recombinase activity have been identifiedat some chromosomal breakpoints from these tumors
prompted us to examine whether RAG-l and RAG-2 were
transcribed in EBV-immortalized lymphocytes andEBV'
tumor lines. Wereportthat
RAG gene transcripts can be
detected in several virus-infected Burkitt's lymphoma lines.
Evaluation of these lines suggests that the presence of virus
TdT
C
:
7
Fig 2. Expression of RAG-2 and TdT RNAs in Burktt's lymphoma
lines compared with ALL lines. Total RNA from two endemic Burkitt's
lymphomas (Raji and Akata), two ALLs (Nalm-6 and LAZ 2211, one
LCL (NADZOI, one sporadic Burkitt's lymphoma(BWO), and two EBVinfected subclones of BL30 IBUOIB95 and BWO/PBI was hybridized
with (A)
an RAG-2 cDNAand (B1a TdT cDNA. (C)
An ethidium bromide
stain of the gel: RNA transfer was complete.
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RAG
G E N E S IN EBV’ B CELLS
1291
Tissue preparation. Discarded fetal tissue from anonymous donors was obtained from the Department of Pathology at the Brigham
and Women’s Hospital (Boston, MA). Fetal age was estimated on
the basis of crown-rump length and foot length.33Bone marrow was
flushed from intact femurs withRPM1 (BioWhittaker), and cells
were incubated on 60 X 15-mm plastic tissue culture dishes (FalconBecton Dickinson, San Jose, CA) for approximately 1 hour. Nonadherent cells were layered on a cushion of LSM (OrganonTeknikaCappel, Durham, NC) and centrifuged to remove nonlymphoid cells.
Single-cell suspensions from fetal liver, spleen, and thymus were
prepared by mincing the tissue through wire mesh. The cells were
then processed as described above.
Virus pur$cation and establishment of immortalized cell lines.
Marmoset cells immortalized with the prototype B95-8 strain of
EBV (ATCC) were suspended at 5 X 10’ to 1 X 10‘ cells per
milliliter and grown for 5 days. The cells were centrifuged at 400g
for I O minutes, and the supernatant was poured through a 0.22-pm
filter (Nalgene, Rochester, NY). The virus-containing supematant
was directly incubated with lymphocytes at IO3 to 10‘ cells per
milliliter for 2 hours at 37”C, washed once, and resuspended in
media at a concentration appropriate to each experiment.
Antibodies. The following murine monoclonal antibodies were
used for cytometric analyses of B cell lines: anti-IgM-HB57
(ATCC), anti-class I1 HLA-LB 3.1 (Jack Strominger), CD10-J5
(Coulter Immunology, Hialeah, FL), CD19-B4 (Coulter Immunology), CD20-B1 (Coulter Immunology), CD21-HB5 (ATCC), B7B7g (Lee Nadler and Arnold Freedman, Dana-Farber Cancer Institute), CD23-ME112 (The Binding Site Ltd, Birmingham, UK),
CD30-IOA30 (AMAC, Westbrook, ME), CD34-HPCA-l (BectonDickinson, San Jose, CA), CD39-AC2 (Biodesign, Kennebunkport,
ME), CD40-B-B20 (Biosourct- Camarillo, CA), CD54-HB20
(ATCC), CD58-HB205 (ATCC
td CD77-IOB77 (AMAC). The
monoclonal antibodies UPClO
2a; Organon Teknika-Cappel),
MOPC 21 (IgGl; Organon Teb
-Cappel), MOPC104E (IgM),
Iwn specificities wereusedas
and P3 (IgGI; ATCC) with UI
isotype-matched controls for the ‘I : bodies enumerated above. Antibodies were used at a concentrat) . , I of approximately 10 to 40 pg/
mL determined to be approximately 10-fold in excess of saturation
based on cytometric analysis of standard cell lines. For detection,
fluorescein isothiocyanate (FITC)-labeled goat F(ab’)* antimouse
IgG or, in some cases, antimouse IgGAM (Tagoimmunologicals,
Camarillo, CA) at a concentration of 10 pg/mL was used. For analysis of Ig light chains, affinity-isolated FITC-labeled goat F(ab’)*
anti-human lambda light chain and anti-human kappa light chain
antibodies were used (Tagoimmunologicals) with preimmune goat
FITC-labeled F(ab’ ), (Tagoimmunologicals) as a control.
Probes. The following DNAs were used as probes. Human Cp
and JH,34CA,” and C K ~ genomic
‘
clones were provided by Philip
Leder, Harvard University. Human RAG-l cDNA(H36)” was provided by Marjorie Oettinger, Massachusetts General Hospital, Boston, MA. A 707-bp EcoRI subfragment of human RAG-l was used
as a probe. The size of RAG-l RNA detected was 6.6 kb. RAG-2
CDNA’’ was provided by Craig Thompson, University of Michigan
Medical Center, Ann Arbor, MI. An approximately 650-bp Not ISal I subfragment of human RAG-2 was used as a probe. The size of
the major RAG-2 RNA was 2.2 kb, with additional species detected.
Human CD10 cDNA’~ was provided by Margaret Shipp, DanaFarber Cancer Institute. A 1,587-bp subfragment ofhuman CD10
was used as a probe. The size of the major CD10 RNAs were 5.7
and 3.7 kb, with additional species detected. Human TdT cDNA’~
was provided by Mary Sue Coleman, University of North Carolina,
Chapel Hill, NC. A 1,528-bp BamHI subfragment was usedas a
probe. The size of the TdT RNA was approximately 4 kb. BNLF1 genomic DNA39was provided by Bill Sugden, University of Wisconsin McCardle Laboratories, Madison, WI, and was used to detect
LMP-1. An approximately 500-bp NCOI-NCOI subfragment conI
7
taining LMP-l coding sequences was used as a probe. The size of
the LMP-I RNA was approximately 2.6 kb. A second genomic DNA
containing the BarnHI N fragment was obtained from Fred Wang.”
This probe, designated pSV2gptMTLM, detected the smaller and
less abundant LMP-2 RNAs of approximately 1.9 and2.1 kb, as
well as LMP-l. BamHI fragments from the EBV genome were provided by Jack Strominger.w Appropriate subfragments ofBamHI
C’: BamHI ’E: BamHI K
:
andBamHI W were usedto detect
EBERS, EBNA-SA, -3B, -3C, -1, and -LP. The EBNA-2 gene was
detected with c D N A ~provided by Fred Wang and Elliot Kieff. An
approximately 2-kb EcoRI subfragment of EBNA-2A cDNA was
used as probe. The size of the RNAs were approximately 2.6 to 2.8
kb. Glyceraldehyde-6-phosphate dehydrogenase (GAPDH) cDNA4’
and ,L-actin cDNA4‘ probes obtained from the ATCC were used as
controls. The probes were labeled with c~[’~P]d-cytidine
triphosphate
(dCTP; New England Nuclear, Boston, MA) to a specific activity
of 1 to 5 X 10’ c p d p g using the Random Primed DNA Labeling Kit
according to the manufacturer’s instructions (Boehringer Mannheim
Biochemicals, Indianapolis, IN). An antisense 17-base oligonucleotide to 28s RNA47was also used as a control in some experiments
and was c~[’~P]d-adenosinetriphosphate (dATP) radiolabeled at the
3’ end with TdT.4R
DNA blot hybridization. Genomic DNA was prepared from tissue culture cells as de~cribed.~’For analysis of Ig gene rearrangements, DNA was cut with the indicated restriction enzymes,
and DNA blothybridization was performed with thespecified probes
as described.“’
RNA blot hybridization. RNAwas prepared usingthemethod
of Chomczynski and Sacchi“ and quantitated by absorbance at 260
nm. Total RNA, 15 pg per lane, was subjected to electrophoresis
through a 1% agarose gel containing 2.2 molL formaldehyde and
blotted onto NitroPlus transfer membranes (MSI, Westboro, MA)
as de~cribed.~’
The RNA was fixedto the membrane using ultraviolet
light applied with the Stratalinker 2400 (Stratagene, La Jolla, CA)
at 120 mJ for 60 seconds to the RNA side of the membrane. Prehybridization, hybridization (approximately 10‘ cpm of specific probe
per milliliter), and washing was performed as described.” Two or
more final washes were performed with0.2 X standard sodium
citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) at 60°C to 65°Cfor
30 and then 20 minutes. The membrane was air-dried and exposed to
XAR-5 film (Kodak, Rochester, NY) with an intensifying screen at
-70°C for 1 to I O days.
RESULTS
Endemic Burkitt’s lymphomas express RAG-1 and RAG-2.
EBV-infected cells have not been reported to express RAG
genes~28.s~sl However, whenseveralwell-characterizedendemic Burkitt’s lymphoma liness8 were examined for
RAG
gene expression, RAG-l RNA was detected in three of
these
lines: Akata, Daudi, and Namalwa (Fig 1, A and B). RAG-l
RNA was not detected in P3HR1, MABA, or Raji (Fig 1, C
and D). Namalwa has been reported to lack RAG transcripts;
however,thecelllinehasbeenincultureformanyyears,
and several independent sources are
a~ailable.’~
Namalwa cells
obtained from the ATCC expressed both RAG-l and RAG-2
(Fig l , C and D). Transcription of
RAG-l in the central nervous
system has been detectedin the absence of RAG-260;however,
alllymphocytepopulationsthusfarstudiedtranscribeboth
genes.In the EBV+ Burkitt’s lymphoma lines, transcription
of RAG-l was always accompanied by RAG-2 (Fig 1, C and
D, and Fig 2). A single RAG-l mRNA of approximately 6.6
kb was detected, whereas a major
RAG-2 transcript of approximately 2.2 kb with one or two additional species were detected
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KUHN-HALLEK ET AL
1292
m D E
L
m 0 m
m0Z
W
A
-
U
U:
RAG-l
B
RAG-2
E
RAG-l
C
F
F-i
RAG-2
CD10
G
D
28s RNA
Fig 3. RAG gene expression in fetal lymphocyte populations in the presence and absence of EBV. (A through D) RAG gene expression in
cells immortalized by EBV at different stages of lymphoid differentiation.SB, adult blood; Cord A, cord blood; F.L/F.M/F.F, fetal bone marrow
of 19,21, and 20 weeks, respectively. Nalm-6 isa control pre-BALL line that is not
EBV-infected. Total RNA from therespective cell lines was
probed with an (A) RAG-l cDNA, (B) RAG2 cDNA, (C) CD10 cDNA, and (D) antisense oligonucleotide t o 28s RNA. (E through G) RAG gene
expression in fetal lymphocytes before immortalization with EBV. Total RNA prepared from purified lymphocytes from adult spleen, cord
blood, fetal bone marrow and fetal liver was probed
with an (E) RAG-l cDNA and (F) RAG-2 cDNA. (G) An ethidium bromide stain of thegel:
RNA transfer was complete.
(Figs 1 and 2). This was similar to control B-lymphoblastic
leukemia cell lines (Fig 2) and to normal fetal B cells (Fig 3).
Developmerltcrlchorcrcterizcrtior1 of RAG-]- oncl RAG-2e.upressirlg cell lines. Burkitt's lymphomas arise from B cells
with a resting (ie, nonactivated) phenotype."' The normal counterpart of this B lymphocyte is believed to reside in the bone
marrow and in the germinal center of lymph nodes. The majority of Burkitt's lymphomas and their derivative cell lines are
SI"'.''
These lymphocytes are thus similar to B cells spanning the stages between immature B [having recently completed V(D)J recombination], and mature B (resting before
switch recombination). Most B cells that actively express RAG
genesare less mature. These earlier pre-B cells most often
coexpress TdT, and many are CDIO'. The RAG' acute lym-
phoblastic lymphoma (ALL) cell lines Nalm 6 and LAZ 221
are believed to represent cells fixed at this early pre-B-cell
stage, and both are TdT' (Fig 2) and CDIO'"-5" (Fig 3). RAG
gene transcription was initially thought to cease at the appearance of SlgM; however, RAG RNAs have recently been detected in small numbers of SIgM' cells in murine transgenic
tumors, bone marrow. and neonatal spleen."'."'
The RAG' Burkitt's lymphomas Daudi and Namalwa were
SIg" (reportedlb.??.~'J.fd
and confirmed by flow cytometry) and,
therefore, resembled the murine SlgM'. RAG' tumor lines.
Akata, on the other hand. was SlgG'. SlgM-." (data not
shown), implying that switch recombination had occurred. In
this regard, Akata was distinct from the majority of EBV'
Burkitt's lymphomas and unlike any RAG' B cells that have
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RAG GENES IN EBV' B CELLS
1293
o e
Q
c
cy
cy
H
U
J
E E 0
n n
J
z
z
LI
LI
U
lili U. U.
RAG-1
RAG-2
I
GAPDH
Fig 3. (Cont'd) (H and I) Temporal expression of RAG RNAs were
assessed in lymphocytes from liver and from bone marrow of fetus
IIN immediately after isolation (d 0). after 4 days (d 4) in culture
without EBV, and after 2 weeks in culture with EBV, at which time
the cells were noted to be immortalized (F. IIN LCL). LA2 221 is a
control ALL line. Total RNA from the respective cells was probed
with (H) RAG-land RAG-2 cDNAs and (I) GAPDH cDNA as a control.
been described.sas6As with virtually all Burkitt's lymphomas,"'
the RAG' Burkitt's lines were CDIO' (data not shown). However, unlike the ALL cell lines, none
of the RAG' endemic
lymphomalinesexpressed
TdT RNA (Fig 2 anddatanot
shown).
EUV immortalized cells established from fetcrl U Ivnlphocytes, which actively transcribe RAG-l and RAG-2, c/o not
express RAGRNA. Because most LCLs originate from cord
or adult peripheral blood, transcription of RAG genes would
predictably be absent in these populations. In addition, many
EBV' lines hadbeen in culture for long periods, raising the
possibility that transcription of RAG genes could cease upon
prolongedpassage, as hasbeenobserved
in murineB-cell
lines."' Based on the findings
in the endemic lymphoma lines
and also on the observation that murine B cells immortalized
with
Abelson
leukemia
virus
frequently
expressed
RAG
B LCLswas
genes,"'.hs a panel of newlytransformedfetal
establishedtodeterminewhetherRAGtranscriptscould
be
detected.
B-lineage cells were obtained primarily from bone marrow
(femur), but also from liver, spleen, and thymus of SO fetuses
aged I I to 22 weeks and incubated with ERV. Twenty stably
immortalized fetal lines established from 14 to 22-week fetal
lymphocytes were further characterized. Predominantly
monoclonal, but also some polyclonal, populations were obtained. The surface phenotype of a l l the fetal lymphoblastoid
cells was identical to that of lines established from cord or
peripheral adult blood (ie, CD1 la', CD19'. CD20'. CD21'.
CD23', B7',CD30',CD39',
CD40'. CDS4',CDS8'.
CD34 , CDIO- , CD77 ; data not shown) andresembled
activated mature B cells. In some cases, however, no Ig was
present on thecell surface, while in others, heavy chain
alone could be detected. The majority of LCLs were SlgM'
with a lambdxkappa ratio of approximately I :I , reflecting
the surface Ig composition of normal fetal tissue. These results were in agreement withrecent descriptions ofpro-.
pre-, and immature B cells transformed by EBV.s".f"-6x The
state ofIg gene rearrangement in the respective lines was
confirmed by DNA blot hybridization (data not shown).
RAG- 1 and RAG-2 RNA expression by three newlyestablished fetal LCLs (4 to 6 weeks after incubation with virus)
wasfirst compared with expression by the established peripheral blood cell line SB"" and a newly established cord
blood cell line, Cord A (Fig 3, A through D). No RAG-l or
RAG-2 RNA was detected in any of the EBV-transformed
cells, including fetus L(p+, X+), fetus M ( p - ~ - h - ) and
fetus F(p+, K + ) (Fig 3, A through D). CDIO RNA, which
is normally expressed in fetal B cells and Burkitt's lymphoma~,~
but
" which is downmodulated by LMP-I was not detected in any of these lines (Fig 3, A through D). As expected, a B-acute lymphoblastic leukemialine,Nalm-6.7'
expressed all three RNAs.
When purified fetal B lymphocytes used to establish immortalized lines were evaluated, RAG transcripts were
readily detected (Fig 3, E through G), demonstrating that at
the time EBV was added, fetal lymphocytes transcribed
RAG-l and RAG-2. Interestingly, while RAG RNAs were
-Em
A
z
0
E
m
U
0
m
m
a
J 7
m m
.
.
RAG-l
B
Fig4. RAG-l expression in EBV-, sporadic Burkitt's lymphomas.
Total RNA from three EBV- sporadic Burkitt's lymphomas (Ramos,
BL41, and BL30). one Burkitt's-like lymphoma (BJAB), the endemic
Burkitt's lymphoma (Namalwa), and an ALL (Nalm-6) were probed
with (A) an RAG-l cDNA and (B) a /%actin cDNA for control.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
KUHN-HALLEK ET AL
1294
L
m
W
E
m
z
A
RAG- 1
B
LMP-1
C
a,v)
0
E
ClJ
c
U
J
U m
D
Fig 5. Comparison of RAG-l, RAG-2, and LMP-1 expression in EBV-infected B cells from different lineages. (A through D) Endemic Burkitt's
lymphoma lines. Total RNA from three lymphoblastoid cell lines
[F.F.(fetal), 8958. and NAD201and threeendemic Burkitt's lymphomas (Jijoye,
Namalwa, and Maba) was prepared and probed with (A) an RAG-l cDNA or (B) an LMP-l genomic subfragment. (C) An ethidium bromide
stain of the above gel: RNA transfer was complete. (D) A p-actin cDNA. (E through H) Sporadic Burkitt's lymphoma lines and EBV-infected
sublines. Total RNA was prepared from two sporadic Burkitt's lymphoma lines (Ramos and BL411 and six sporadic Burkitt's lymphoma lines
that had been infected with EBV in vitro (Ramos/B95, Ramos/P3, BL41/B95, BL41/P3, BL3O/P3, and BJAB/B95) and probed with (E) a RAG-l
cDNA or (F1 a subfragment of the MTLMgenomic clone encoding LMP-1. (G) An ethidium bromide stainof the above gels: RNA transfer was
complete. (H) A p-actin cDNA was used as a control.
always detected in lymphocytes from fetal bonemarrow
(Fig 3, E andF), spleen, and thymus (data not shown),
transcription in liver was more variable, often equal to (Fig
3, H and I) but sometimes less than (Fig 3, E through G)
RNAs detected in other lymphoid populations from the same
fetus. In the latter case, the hepatic lymphocytes were less
mature (predominantly early pro-B cells based on absence
of B- and pre-B-cell surface markers; data not shown). RAG
RNAs were not detected in mature lymphocyte populations
(Fig 3, E through G).
When fetal B cells were placed in culture without EBV,
RAG gene expression diminished over I week (Fig 3, H and
I), as many fetal cells died and others presumably differentiated? Virtually no RAG transcripts could be detected 4 days
after EBV was added. However, because of substantial RNA
degradation, particularly in the presence of virus, it was not
possible to conclusively demonstrate that virus accelerated
downmodulation of RAG genes in cultured B cells. In one
fetal cell line (F IIN LCL),which grew out from bone marrow lymphocytes less than 2 weeks after incubation with
EBV, no RAG-l or RAG-2 RNA could be detected (Fig 3,
H and I). None of the newly established fetal cell lines
expressed RAG- 1, RAG-2, or CD 10 RNA (Fig 3). TdT was
also absent from all lines studied (data not shown).
Sporodic Burkitt's lvmphomns do not express R A G I or
RAG-2, but may transcribe these genes qjier EBV infection.
Sporadic Burkitt's lymphomas are morphologically similar
to endemic tumors: however, they differ in some salient
aspects. They occur worldwide, uncommonly contain EBV
DNA (approximately 15% to 20%). vary in tissue distribution, peakat a later age, andcan be distinguished on a
molecular genetic basis.'.'' Virtually all Burkitt's lymphomas
contain an 8/14 or related chromosomal translocation..'.'? In
endemic Burkitt's lymphomas, the c-myc protooncogene is
often juxtaposed to the region of the Ig heavy chain locus
transcribed during the V(D)J recombination process.'.'."
Heptamer-nonamer signal sequences that serve to target gene
segments for V(D)Jrecombinationhave
sometimes been
identified in the vicinity of these breakpoints." In sporadic
Burkitt's lymphomas, c - m y ismore frequently joined to
loci transcribedduring switch recombination; however, these
distinctions are not absolute. Furthermore, in patients infected with
HIV,
an increased frequency of Burkitt's
lymphomas has been observed that occur sporadically, but
are often EBV' and resemble endemic lymphomas. Because
this has led to some confusion, in this report EBV+ will
always be equated with endemic lymphomas andEBVwith sporadic lymphomas.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
RAGGENES
1295
IN EBV' B CELLS
D
RAG1
A
E
RAG-l
LMP-1
B
LMP-1 F
C
actin
Fig 6. Comparison of RAG-l and LMP-1 RNAs in clonal derivatives of EBV+ Burkitt's lymphoma lines. (A through C) Total RNA from the
EBV', sporadic Burkitt's lymphoma line BL30/B95 and five clonal derivatives was probed with (A) an RAG-l cDNA (B) an LMP-1 genomic
subfragment, and (C) a P-actin cDNA. Total RNA from Raji, an endemic Burkitt's lymphoma and froman LCL was similarly probed forcontrol.
(D through F) Comparison of RAG-l and LMP-l RNAs in Daudi and two clonal derivatives by blot hybridization. Daudi, an EBV' Burkitt's
lymphoma, is deleted in EBNA-2IEBNA-LP. D.2, clone 2 of Daudi; D.5, clone 5 of Daudi. 8958, a marmoset LCL, and Laz 221, an ALL, are
controls. Total RNA was prepared from therespective cell lines and probed
with (D) an RAG-lcDNA and (E) a genomic subfragment from the
LMP-1 coding region. (F) An ethidium bromide stainof the above gel: transfer was complete.
When three EBV-, sporadic Burkitt's lymphomasRamos,N BL41," and BL307' (Fig 4)-were analyzed, no
RAG-I or RAG-2 RNA was detected, nor were these genes
detected in one EBV- Burkitt's-like lymphoma, BJAB.These data are consistent with the proposalthat sporadic
lymphomas arise from mature B cells at rest before switch
recombination, a stage at which RAGgenes are silent. Somewhat unexpectedly, when the sporadic Burkitt's lines that
hadbeen infected withEBV in vitro were assessed, RAG
gene transcription was observed in several infected lines
of the four sporadic
(Figs 2 and 5; E throughH).Each
Burkitt's lines had been infected in vitro either with transforming virusB958
or with the nontransforming strain
P3HR1,74giving riseto sets of three: ie, BL4l (parent),
BL4IIP3, and BL41/B95 (see Materials and Methods). These
sets have been used extensively to characterize EBV-induced
gene expression. The P3HRI strain is deleted in the EBNA2EBNA-LP viral transactivation genes required for immortalization, but can stably infect the tumor lines because they
do not require virus for growth. The EBV' sporadic Burkitt's
lines BL4IP3 (Fig 5) and BL30/P3 (Figs 2 and 5) and also
BL30/B958 (Figs 2 and 6 ) expressed RAG-I and RAG-2,
whereas others (BL41B958, RamodB958, RamosIP3,
BJAB/B958, and BJABP3) did not (Fig 5, Table l ) . Similar
to other lymphoid cells, RAG-l and RAG-2 expression was
coordinate (data not shown). The EBV',RAG'
sporadic
lines were SIgM'7s (data not shown), CDIO' (data not
shown), and TdT- (Fig 2 and data not shown), similar to
RAG' endemic lymphomas.
EBV' Burkitt's lymphomas that express LMP-I downmoduhte RAG-l and RAG-2. The complexpattern ofRAG
gene expression in EBV-infected B cells raised the possibility that RAG genes might be regulated by a viral product.
Whereas RAG-l and RAG-2 expression in endemic and particularly in EBV-infected sporadic Burkitt's lymphoma lines
suggested an association withvirus, the absence ofRAG
transcripts in fetalLCLsand
in some EBV' Burkitt's
lymphomas suggested that a viral gene product(s) that was
consistently expressed in LCLsbutvariablyexpressed
in
Burkitt's lymphomas might negatively regulate RAG RNAs.
Prior studies had shown that, in EBV' Burkitt's lymphoma,
transcription of viral lytic genes was rare and that transcription of latency associated genes, except for EBNA-I, was
variable and frequently absent." Evaluation of latent RNAs
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1296
KUHN-HALLEK ET AL
Table 1. Summary of RAG-l, RAG-2, and LMP-l RNA Expression in
EBV-Infected B-Cell LinesDetected by Blot Hybridization
RAG-l
RAG-2
EBV’ Burkitt‘s lymphoma lines
+
t
Namalwa
Daudi
+
Akata
Raji
Jijoye
MABA
P3HR1
Lymphoblastoid cell lines
B958
All LCLs
EBV Burkitt’s lymphoma lines and their EBV-infected sublines
Ramos (EBV-)
Ramos/B95
Ramos/P3 (EHRA)
BJAB (EBV-)
BJAB/B95
BJAB/P3
BL30 (EBV-)
BL3O/B95*
+
+
BL30/P3
EL41 (EBV-)
BL41/B95
BL41/P3
ALL lines (EBV , controls)
+
Nalm 6
Laz 221
+
+
+
+
-
-
-
-
-
-
-
LMP-l
+
+
+
+/-
+
+
When the I O remaininglatency-associatedtranscripts
were assessed, no EBV genes other than LMP-I correlated
with absence of RAG RNAs. However, a role for additional
EBV genes cannot be absolutely excluded. In particular, the
reciprocal pattern of RAG/LMP-l expression in the naturally
occurring mutant tumor lines [ie, P3HR-I, Daudi. and Namalwa(Table 1) deleted in EBNA-2EBNA-LP(P3HR-I
and Daudi) and in LMP-2 (Namalwa)] suggested that negativeregulation by thesegenes independent of effects on
LMP- I was unlikely. Transcription of EBNA-3 genes in the
Burkitt’s lines was variable and did not correlate with absence of RAG gene expression (datanot shown). The EBV’
cell lines, irrespective of RAG expression, transcribed the
EBNA- I protein4.’ (data not shown).
-
+
+
DISCUSSION
These experiments show that the lymphoid-specific genes
RAG-l and RAG-2 may be
expressed in B cells infected
+
with EBV. RAG transcripts were readily detected
in three
+
of sevenendemic (EBV’) Burkitt’slymphomacell lines.
Developmentalanalysisshowed thattheseBurkitt’slines
+/were more mature than previously characterized RAG’ hu+
+
man Bcells, as theywereuniformly SIg’ and TdT . The
importance of TdT fornormal execution of the V(D)J recom+
binationreaction in vivo is notfullydefined.
In mice,a
+
+
subgroup of Eu-myctransgenicB-celltumorsas
well as
+
smallpopulations of normalB cells in bone marrowand
+
neonatal spleen have been
found to coexpress RAG genes
Each of the cell lines was independently analyzed between two and and SIgM. Preliminary experiments in the laboratory indi10 times for expression of RAG-l,
RAG-2, and LMP-l RNAs. Many
cate thatthispopulation
is also present in human fetalB
of the lines were subsequently clonedby limiting dilution. Variation
cells,
which
suggests
that
RAG’ endemic Burkitt’s
(relative to the parental line) was sometimes detectedin the clones.
lymphoma linesrepresent the counterpartof a normal human
The inverse correlation between LMP-l and RAG genes expression,
B-cell population.
however, was most clearly observed in the clones.
Interestingly, the RAG’ endemicBurkitt’slymphoma
line, Akata, was SIgG’, SIgM-, indicatingthatswitchrecombination hadoccurred. This finding was unusual because
RAGRNAs have not beendetectedatthis
latestage of
in the endemic Burkitt’s lymphomas and alsoin the infected
maturation.s4~shThepossibilitythat RAGgenes might be
sporadic Burkitt’s lymphomas that did and that did not exexpressedtransiently
during B-cellontogeny(ie,
during
press RAG genes showed an association
between expression
switching or during DNA repair) has often been raised, but
of LMP- 1 and the absence of RAG-l and RAG-2 RNAs
has never, in fact, been demonstrated. Detection of RAG
(Fig 5 , Table l), with the possible exception of BL3043958
RNAs in Akata thus raised the possibility that prolongation
(Fig 6).
or de novo induction of RAGgenes might in some way
Cloning of Burkitt’s lymphoma cell lines confirms the inrelate to EBV infection. This was supported by the further
verse relationship between RAG gene and LMP-I expresobservation that among well-characterized RAG- sporadic
sion. In initial studies BL30B958 appeared to expressboth
Burkitt’s lines, RAG RNAs were sometimes detected after
RAG genes and LMP-1 (Fig 6, A through C), albeit at low
in vitro infection withEBV. Thesefindings raisethe possibillevels. As manyof the Burkitt’s lines had
been in continuous
ity thatvirus may alter the developmental window during
culture, it was possible that variation had developed among
which RAGgenes can be expressed. BecauseEBV effiindividual cells. Thus, to more precisely define the relationciently immortalizes mature and immature B cells, the inship between viral and RAG gene expression, BL30IB958
creased frequency of myc translocations that appear to be
was cloned by limiting dilution. The derived clones all exrelated to the RAG gene-dependent recombinase isunexpressed RAG-l, and no LMP-I could be detected (Fig6, A
plained. Evidence has recently been presented which shows
through C). WhenDaudi Burkitt’s lymphomacellswere
that virus can infect cells that have already developed the
similarly cloned, comparison of five newly derived clones
characteristic myc translo~ation.’~Although we have not
demonstrated a reciprocal relationship between the level of
demonstrated functional recombinase activity,thecurrent
LMP-1 and RAG-l steady-state RNA expression (Fig 6, D
findings raise the possibility that EBV may contribute more
through F) that was similar to that reported for CD10 and
directly to aberrant recombination by supporting or inducing
LMP-1. Five clones from the Namalwa line all expressed
RAG transcripts in certain cellular environments.
LMP-1, and no RAG-l or RAG-2 RNA was detected in
Because RAG RNAs could be detected in several EBV+
these clones (data not shown).
-
-
-
-
-
-
-
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RAG GENESIN
1297
EBV’ B CELLS
Burkitt’s lines, it seemed plausible that failure to detect these
genes in LCLs might simply reflect inadequate study of immortalized precursor B cells. However, when a panel of fetal
LCLs derived from hematopoietic tissues was established,
none of the fetal lines expressed RAG-l or RAG-2. Two
LCLs from patients withcommon variable immunodeficiency and one from a patient with X-linked agammaglobulinemia (provided byDr Jerrold Schwaber, Hahnemann
Medical College, Philadelphia, PA) also did not express detectable RAG genes (personal observation, March 1994).
Based on Ig configuration, these fetal lines represented a
spectrum of precursor B lymphocytes that included cells
predicted to express RAG genes. In the mouse, fetal B cells
transformed in vitro with Abelson leukemia virus frequently
express RAG- 1 and RAG-2. Moreover, murine lymphocytes
transformed by Abelson virus continue to rearrange in vitro,
whereas it has been reported that precursor B cells transformed by EBV do
Although the possibility that
EBV selectively immortalizes RAG- fetal cells cannot be
completely excluded, taken together, these findings suggest
that a viral gene consistently expressed in LCLs but variably
expressed in Burkitt’s lymphoma lines downmodulates RAG
gene expression.
Previous studies showed that LMP-1, a viral membrane
protein required for imm~rtalization$~.~~
was able to downmodulate expression of CD10. As with RAG-I and RAG2, CD10 RNA was detected in fetal B cells before but not
after EBV immortalization. Not only the presence but the
level of LMP- 1 RNA was shown to be important in eliciting
effective downmodulation of CDIO,l5 and this was similarly
observed when single clones from the Daudi lymphoma line
were studied. Clones derived from additional EBV’, RAG’
Burkitt’s lines have further confirmed this relationship. Because LMP-I is required for immortalization of B cells, no
LCLs would be predicted to express RAG-l or RAG-2.
If LMP-1 is highly expressed in LCLs, how could RAG
genes become expressed and putatively contribute to aberrant recombination reactions? The host factors that regulate
LMP-1 expression in B cells in vivo are currently unknown.
It is clear that LMP-I is often undetectable in Burkitt’s
lymphomas, which presumably arise from LCLs. If LMP-1
were downmodulated in vivo in immortalized B cells (ie, by
unknown factors inthe host microenvironment), then survival of the cell would depend on acquisition of genetic
changes that independently enhanced cell growth. Recently,
it has been observed that expression of LMP-I (as well as
other latency-associated genes) is decreased in SCID mice
that develop EBV+ B-cell tumors after injection with human
LCLs.’* Study of fetal LCLs in the SCID environment might,
therefore, help to clarify how RAG genes are regulated in
these cells.
As previously reported, EBNA-l is detected in all EBV+
Burkitt’s lymphoma line^,^^,'^ and this included the RAG+
lines. EBNA-I is a multifunctional protein that controls transcription of additional EBV gene^^,^ and is itself regulated
in a complex manner in different cell lineage^^,^"-^,*' and
during the viral life cycle.82Thus, it is possible that EBNA1 or, alternatively, an as yet undetected viral product(s), or
the presence of viral DNA itself could alter RAG transcription. A preliminary report suggesting that EBNA-l augments
transcription of c-myc and Ig genes,” is intriguing, as enhanced transcription also characterizes exons undergoing
V(D)J rec~mbination.’~
The current experiments show that the lymphoid-specific
genes RAG-I and RAG-2, required for V(D)J recombination, maybe expressed inEBV’ Burkitt’s lymphomas and
demonstrate that RAG genes are differentially regulated in
distinct virus-infected B cells. The extent to whichRAG
transcription correlates with efficient V(D)J recombinase activity in these cells is under investigation. These results establish the possibility of a link between EBV infection and
the development of chromosomal translocations involving
immunoglobulin VDJ loci in Burkitt’s lymphomas.
ACKNOWLEDGMENT
We thank David Weaver, Evelyn Kurt-Jones, Jeff Bergelson, and
Robert Finberg for helpful discussions and critical evaluation of the
manuscript.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1995 85: 1289-1299
Expression of recombination activating genes (RAG-1 and RAG-2) in
Epstein-Barr virus-bearing B cells [see comments]
I Kuhn-Hallek, DR Sage, L Stein, H Groelle and JD Fingeroth
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