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Znt. J. Cancer: 67,86-94 (1996)
0 1996 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de I’llnion Internationale Contre le Cancer
EXPRESSION OF EPSTEIN-BARR VIRUS LATENT INFECTION GENES
AND ONCOGENES IN LYMPHOMA CELL LINES DERIVED FROM
PYOTHORAX-ASSOCIATED LYMPHOMA
Hiroyuki K A ” O ’ , Yutaka YASUNAGA’,
Masahiko OHSAWA’,
Masafumi TANIWAKI?,
Keiji IUCH13, Norifumi NAKA’,
Masanori SHIMOYAMA“
and Katsuyuki AOZASA’,~
Kei TORIKAI’,
‘Department of Pathology, Osaka University Medical School, Suita 565; 2Department of Internal Medicine, Kyoto Prefectural
Universiy of Medicine, Kyoto 602; 3Department of Suvery, National Kinki-Chuo Hospital, Sakai 591; and 4National Cancer
Center Hospital, Tokyo 104, Japan.
Malignant lymphomas frequently develop in the pleural cavity
of patients with long-standingpyothorax. The term pyothoraxassociated lymphoma (PAL) has been proposed for this type of
tumor. Most PALs are diffuse lymphomas of the B-cell type and
contain Epstein-Barr virus (EBV) DNA. We have established 2
lymphoma cell lines from biopsy specimens of PAL cases, OPL- I
and OPL-2. and examined their growth characteristics and the
expression of EBV latent infection genes and oncogenes. OPL-2
exhibited a more rapid growth and higher saturation density
than OPL- I, and only OPL-2 exhibited colony-forming activity in
soft agar. OPL- I and -2 were positive for B-cell differentiation
markers and showed clonal surface immunoglobulins. Both lines
contained a single predominant form of episomal EBV DNA,
indicating clonal cellular proliferation of an EBV-infected progenitor cell. OPL- I and -2 contained type B and A EBV genome,
respectively. Expressionof EBV nuclear antigen (EBNA)2 mRNA
and protein was detected by Northern and Western blot
analysis in OPL-I, but not in OPL-2. On the other hand, the
expression of latent membrane protein (LMP) I mRNA in both
OPL-I and -2 was extremely weak and detectable only by
reverse transcription-polymerasechain reaction. Proteinexpression of LMPl was not observed by Western blot analysis or
immunocytochemistry. Both lines expressed c-myc mRNA.
Only OPL-I expressed mRNA of c-fgr, an oncogene whose
expression is upregulated by EBNA2. Both OPLs expressed
bcl-2 mRNA without detectable expression of LMPl protein.
o 1996 Wiley-Liss,Inc.
Epstein-Barr virus (EBV) has been identified as an etiological agent of the African type of Burkitt’s lymphoma and of
nasopharyngeal carcinoma (NPC) in southern China (Wolf et
al., 1993). Latent infection genes of EBV, including EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs),
are expressed in B cells during latent infection and lead to
immortalization of B cells (Rickinson et al., 1992). Burkitt’s
lymphoma cells express only EBNA1, which is not recognized
as a target by virus-specific T cells, and thus can evade immune
surveillance in vivo (Rickinson et al., 1992;Wolf et al., 1993). In
the course of in vitro culture, however, most of them express all
latent infection genes (Rickinson et al., 1992). Since EBNA2
and 3s, as well as LMPs, serve as target viral antigens for the
elimination of infected cells by cytotoxic T cells (CTLs)
(Rickinson et al., 1992), Burkitt’s lymphoma cells expressing
latent infection antigens other than EBNAl are thought to be
eliminated by CTLs in vivo (Rickinson et al., 1992).
EBV infection in immunosuppressed patients, such as human immunodeficiency virus-infected patients or allograft
recipients receiving long-term immunosuppressive therapy,
leads to polyclonal B-cell proliferative disorders, frequently
resulting in development of monoclonal malignant lymphomas
(Rickinson et ab, 1992;Wolf et al., 1993). These lymphomas are
most often diffuse lymphomas of the B-cell type and contain
EBV DNA (Rickinson et al., 1992). Suppressed immune
functions in these patients are thought to cause incomplete
elimination of the cells expressing EBV latent infection genes
(Rickinson et aL, 1992).
Genetic lesions including chromosomal translocation and
point mutation play an important role in EBV-related B-cell
tumors (Gaidano and Dalla-Favera, 1993). The activation of
c-myc in Burkitt’s lymphoma is induced by chromosomal
translocation, not by EBV infection (Battey et al., 1983).
Diffuse large cell lymphoma (DLCL) frequently develops in
the pleural cavity of patients with long-standing pyothorax
(pyothorax-associated lymphoma; PAL) (Iuchi et al., 1989).
Although no systemic immunosuppressive condition has been
identified, almost all these lymphomas contain EBV DNA
(Fukayama et al., 1993). Among EBV latent infection genes,
EBNA2 and LMPl are essential for the transformation of B
cells (Wolf et a l , 1993), which is partly explained by oncogene
activation, i.e., EBNA2 transactivates c - f ~ (Knutson, 1990),
and LMPl transactivates bcl-2 (Henderson et al., 1991).
Previous immunohistochemical studies have described the
expression of EBNA2 and LMPl in PAL cells (Fukayama et
al., 1993), although the relationships among EBV latent gene
expression, oncogene expression and the growth of lymphoma
cells as well as the contribution of EBV infection to the
development of PALs remain unknown.
In the current study, we established 2 lymphoma cell lines
from patients with PAL and examined the growth characteristics and the expression of EBV latent infection genes along
with the oncogenes c-myc, c-fgr and bcl-2. The expression of
EBNA2 varied in the 2 lymphoma cell lines. However, a
restricted expression of LMPl was common.
MATERIAL AND METHODS
Establishment of cell lines
Small pieces of the biopsy specimens were minced with
stainless steel mesh and washed several times with RPMI 1640
medium medium (Nissui, Tokyo, Japan) supplemented with
300 U/ml of penicillin and 300 pg/ml of streptomycin. Subsequently, culture (5 x lo6 cells/ml) was started in RPMI 1640
supplemented with 20% heat-inactivated FCS (ICN, Lisle, IL)
at 37°C in 5% C 0 2 in air. No heterologous feeder layers were
used for cell line establishment. Once cell growth was established, the concentration of FCS in the medium was reduced
to 15%.
Zmmunophenotypic analysis
Three micrometer sections of formalin-fixed, paraffinembedded tissue and cytocentrifuged specimens of cultured
cells were studied by immunochemistry for expression of cell
surface marker proteins and EBV latent infection gene products. Appropriate tissue sections (lymphoid tissue and lymphoma) or cytospin slides (lymphoblastoid (LCL) and lymphoma cell lines) were also subjected to immunostaining as a
positive control. The polyclonal antisera and the monoclonal
’To whom correspondence and reprint requests should be sent:
Department of Pathology, Osaka University Medical School, 2-2
Yamada-oka, Suita 565, Japan. Fax: (81)6-879-3719.
Received: October 27,1995 and in revised form March 1,1996.
87
PAL CELL LINES
antibodies (MAbs) used in our study, together with their
respective clusters of differentiation and EBV antigens, are
shown in Table I.
Cytogenetic analysis
Chromosomal study was performed on samples obtained
from the cell lines OPL-1 and -2 by the trypsin-Giemsa
banding method. Well-spread met aphases were photographed
and arranged according to the I-ecommendations of ISCN
(1991) for cancer cytogenetics. Fluorescence in situ hybridization (FISH) with C, and VH segments of the IgH gene locus
was performed as described previclusly (Taniwaki et al., 1994).
Cells
EBV-positive or -negative Burkitt’s lymphoma cell lines,
Raji or Ramos, respectively, were (obtained from the Japanese
Cancer Research Resources Bank. An LCL was established by
infection of peripheral blood lyinphocytes from a healthy
donor with EBV derived from B95-8 cells (a generous gift from
Dr. K. Takada, Sapporo, Japan). Cells were grown in RPMI
1640 medium supplemented with 10% heat-inactivated FCS
and collected at 3 x lo5 to S x lo5cells/ml (more than 95%
viability) and used for nucleic acid and protein extraction.
DNA extraction and Southern blot
Total cellular DNA was extracted from cells and tissue
specimens as described previously (Kanno et al., 1993). DNA
was quantitated by measuring OD260and stored at -20°C until
use. Ten micrograms of DNA were digested with Bam HI,
Hind 111 or Pst I (GIBCO-BRL, Gaithersburg, MD), electro-
phoresed in 0.7% agarose gels and Southern blotted as
described previously (Kanno et al., 1993). The filters were
hvbridized with human JHprobe or LMPl exon 2 oligonucleotrde probe (described below) for checking clonality of B cells
or EBV genome, respectively. LMPl exon 2 is located in the
Bam HI-NJhetfused terminal fragment, which includes about
500 bp terminal repeat (TR) of EBV (Fennewald et aL, 1984).
Since reduplication of TR varies among EBV clones, the
presence of a single predominant band of Barn HI-NJhet
fragment represents clonal proliferation of EBV-infected cells
(Raab-Traub and Flynn, 1986).
mRNA extraction and Northern blot
Total cellular RNA was extracted from cells by the acid
guanidium-phenol chloroform method. mRNA was purified by
using oligo-dT latex (Takara, Kyoto, Japan), quantitated by
measuring OD260and ethanol-precipitated at -80°C until use.
Five micrograms of mRNA were electrophoresed in 1%
formaldehyde-agarose gels, and Northern blotted as previously described (Kanno et af., 1993).
Preparation ofprobes and hybridization
Human immunoglobulin JH (Nishida et al., 1982), human
c-myc (Battey et al., 1983) and human c-fgr (Tronick et al.,
1985) DNA probes were obtained from the Japanese Cancer
Research Resources Bank. Human bcl-2 (Tsujimloto and
Croce, 1986) DNA probe or murine actin DNA probe were
kindly provided by Dr. Y. Tsujimoto or Dr. M. k3tagawa
(Osaka University, Suita, Japan), respectively. These probes
were labeled with C K - [ ~ ~ P ] - ~by
C Trandom
P
priming. EBNAl
TABLE I - IMIVIUNOPHENOTfPE OF PAL CELL LINES O p t - 1AND OPL-2 AND OF THE ORIGINAL
TISSUE SAMPLES
Antigen
Antiserum (AS) or
monoclonal antibody
AS
AS
AS
AS
CD3
CD4
CDS
CD8
CDlO
CDlla
CDllc
CD15
CD19
CD20
CD21
CD22
CD23
CD24
CD30
CD43
CD45R
CD45RO
CD54
CD57
CD74
CDw75
EBNA2
LMPl
AS
AS
AS
SK7
OPD4
SK3
L17F12
SK1
SS2136
25.3.1
S-HCL-3
MMA
4G7
L26
H299
1F8
SHC1-I
BU38
BA-1
Ber-H2
MTI
4KB5
MB1
UCHL-1
84H10
HNK-1
LN2
LN1
PE2
CS1-4
Source
D
D
D
D
D
D
D
BD
D
BD
BD
BD
D
IMT
BD
BD
BD
KM
C
D
BD
D
BMB
D
BGL
D
BGL
D
IMT
BD
T
T
D
D
Case I
OPL-I
++-
ND
-
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
+
ND
ND
ND
ND
ND
ND
-
+
+ND
ND
++
+
-
++-
OPL-2
-
-
+-
+
-
-
ND
-
-
ND
ND
ND
ND
ND
ND
ND
ND
-
-
+
-
ND
+
+
+
+
ND
ND
ND
ND
ND
ND
ND
-
-
-
-
-
ND
ND
ND
ND
+
ND
ND
-
-
+
ND
-
+-
+
-
+
ND
ND
ND
ND
ND
ND
+-
++-
Case 2
++
-
+
+
+
+
+
+
++
+-
ID, Dako (Glostrup, Denmark); BD, Becton Dickinson (Mountain View, CA); IMT, Immunotech (Marseille, France); KM, Kyowa Medix (Tokyo, Japan); C, Coulter (Hialeah, FL); BMB,
Boehringer Mannheim (Indianapolis, IN); BGL, BioGenex (San Ramon, CA); T, Techniclone
(Santa Ana, CA); PTD, not done.
88
KA”0 E T A L
oligonucleotide probe (5’-TCCTAGGCCATTTCCAGGTCCTGTACCTGG-3’, in K exon) (Sample et al., 1986), EBNA2
oligonucleotide probe (5’-CCTATGTAACGCAAGATAGAATGTAGGCAT-3’ in YH exon) (Sample et ab, 1986) and
LMPl oligonucleotide probe (5’-GATGAACAGCACAATTCCAAGGAACAATGC-3’, in exon 2) (Fennewald et a/., 1984)
were 5-end-labeled with Y-[~~P]-ATP
by T4 polynucleotide
kinase (New England Biolabs, Beverly, MA).
For the check of clonality of B cells using the human JH
probe, Southern-blotted filters were prehybridized for 4 hr at
42°C in 6 x SSPE (0.9 M NaCI, 0.06 M Na phosphate, 6 mM
EDTA), S X Denhardt’s solution, 0.1% SDS, 200 pg/ml
denatured salmon testes DNA (Sigma, St. Louis, MO), 50%
formamide and hybridized in a solution of the same composition containing the probe at 2 x lo6 cpm/ml. Filters were
hybridized for 16 hr at 42”C, washed twice for 30 min at room
temperature with 1x SSPE, 0.1% SDS and twice for 15 min at
50°C with 0.1X SSPE, 0.1% SDS and exposed at -80°C. For
detection of oncogenes and actin mRNA, Northern-blotted
filters were prehybridized, hybridized and washed in the same
manner as described above with the exception of an additional
15 rnin washing at 60°C with 0 . 1 ~
SSPE, 0.1% SDS for actin
mRNA.
For checking clonality of the EBV genome and for detection
of EBV latent infection gene expression using oligonucleotide
probes, filters were prehybridized for 4 hr at 42°C in 6 x SSC
(0.9 M NaCI, 0.09 M Na3 citrate), 5 X Denhardt’s solution,
0.5% SDS, 20 pg/ml denatured salmon testes DNA and
hybridized in a solution containing the probe at 3 x lo6
cpmiml. Filters were hybridized for 16 hr at 42”C,washed with
2 x SSC, 0.1% SDS twice for 10 rnin at room temperature and
once for 20 rnin at 50°C and exposed at -80°C.
PCR ampliJication of EBNAZ gene and Southern blot analysis
for the typing of EBVgenome
For PCR amplification of the EBNA2 region of the EBV
genome, 1 pg of DNA was diluted to a SO pl solution
containing 0.2 mM dNTP, 3 mM MgCI2, 1.25 U of Taq
polymerase (Promega, Madison, WI), 1X Taq buffer supplied
by manufacturer and 1.5 JLM both primers. Primers are
designed to amplify the 89 bp segment, both ends of which are
conserved and the middle portion of which varies between type
A and B EBV (Sixbey et al., 1989). Primer sequences were
5 ’-CCACCAGCAGCACCAGCACA-3’and 5 ‘-GGTGGCCACCATGGTGGCCC-3’. PCR was performed (1 cycle at 94°C
for 3 min, 60°C for 1 min, 72°C for 1 rnin and 39 cycles at 94°C
for 1 min, 60°C for 1 min, 72°C for 1 min) and amplified
products were electrophoresed in 2% agarose gels and Southern blotted as previously described (Kanno et a[., 1993).
EBNA2A- or -2B-specific oligonucleotide probes (EBNA2Aspecific,5 ’-TTACATCATCTACCCTCG-3’, EBNA2B-specific,
S‘-GCACTTCCTCCAACTCCA-3’)
were designed to hybridize to the variable region between the 2 primers (Sixbey eta/.,
1989) and 3’-end-labeled with fluorescein-11-dUTP by using
the ECL 3’-oligolabeling system (Amersham, Aylesbury, UK).
Filters were hybridized with the labeled probe, washed and
incubated with anti-fluorescein horseradish peroxidase conjugate. Signals were generated with ECL detection reagents
(Amersham) and exposed at room temperature by following
the procedures suggested by the manufacturer.
Reverse transcription (RT)-PCR analysis
For RT-PCR amplification of LMPl and actin mRNA, 1 pg
of mRNA was converted to cDNA with 200 U of Moloney
murine leukemia virus reverse transcriptase (GIBCO-BRL) in
the presence of 5 mM MgC12, 1 mM dNTP, 20 U of RNase
inhibitor (Promega), 500 nM downstream primer and 1X R T
reaction buffer supplied by the manufacturer in 20 pl reaction
volume. After incubation at 37°C for 60 min, the R T mixture
was boiled for 5 min and subjected to PCR amplification. Ten
microliters of the RT mixture (cDNA sample) was diluted to
50 p1 containing 0.2 mM dNTP, 1.5 mM MgCI2, 1.25 U of Taq
polymerase, I X Taq buffer and 500 nM of both primers.
Primer sequences were 5 ' -TGGAGCCCTTTGTATACTCC-3'
(exon 1) and 5’-GATTCATGGCCAGAATCATC-3‘
(exon 3)
for LMP1, amplifying the 405 bp segment of mRNA and the
intervening 154 bp introns (Fennewald et al., 1984); and
5‘-ATCATGTTTGAGACCTTCAA-3’and 5’-CATCTCTTGCTCGAAGTCCA-3’ for human actin, amplifying the 318 bp
segment of mRNA. PCR was performed (1 cycle at 94°C for 3
min, 58°C for 1 min, 72°C for 2 rnin and 34 cycles at 94°C for 1
min, 58°C for 1 min, 72°C for 2 min) and amplified products
were electrophoresed, Southern blotted and hybridized with
non-radiolabeled LMPl oligonucleotide probe; signals were
then generated in the same manner as described in the
previous section. In the case of actin, amplified products were
examined in ethidium bromide-stained gels with an ultraviolet
transilluminator.
Western blot analysis
Cells were directly lysed with Laemmli sample buffer and
boiled for 5 min. Lysates were electrophoresed in SDS 7.5%
polyacrylamide gels and blotted onto nitrocellulose membranes as described previously (Kanno et al., 1993). After
incubation in 5% dried milk in PBS at room temperature
overnight, membranes were incubated at room temperature
for 3 hr with a 1/100 dilution of anti-EBNA2 (PE2) or with a
1/25 dilution of anti-LMP1 (CS1-4) mouse MAbs. After
washing with PBS supplemented with 0.05% of Tween 20,
membranes were incubated at room temperature for 2 hr with
1/400 dilution of horse anti-mouse immunoglobulins (Vector,
Burlingame, CA), washed and subsequently incubated for 1 hr
with 1/4,000 dilution of peroxidase-labeled protein A (Boehringer Mannheim, Indianapolis, IN). Signals were generated
with ECL detection reagents by chemiluminescence.
RESULTS
Patients
The cell lines OPL-1 and OPL-2 were derived from 2 patients
(case 1 or 2). They had suffered from chronic pyothorax
resulting from pulmonary tuberculosis and were followed at
the National Kinki-Chuo Hospital, Sakai, Japan. During the
course of pyothorax, a tumor developed in the thoracic wall,
and open biopsy was performed. Histopathologic examination
and immunophenotypic analysis of biopsy materials revealed
B-cell lymphoma. Case 1 entered into complete remission with
chemotherapy and has remained alive for more than 1%years
since biopsy. Aggressive chemotherapy was not effective in
case 2, and the patient died approx. 2 months after biopsy. The
major clinical characteristics of each patient are summarized
in Table 11.
TABLE I1 -CLINICAL AND BIOLOGICAL CHARACTERISTICS OF THE
PAL CELL LINES AND OF THE PATIENTS FROM WHICH THEY
WERE DERIVED’
~
~~
Case 1
(OPL-I)
Case 2
(OPL2)
Clinico-pathologicalfeatures of
original cases
76
67
Age Gears)
M
M
Sex
Duration of Dvothorax hears)
46
40
Artificial pneimothora;
’
Done
Done
Histologic diagnosis
DIB
DIB
CR
Died
Prognosis
Growth characteristics
Doubling time (hours)
48
24
Saturation density (cells/rnl)
5 x 10s
1 x 106
Colony formation on soft agar
(-)
(+I
IDIB, diffuse lymphoma of immunoblastic cell type; CR, complete remission.
PAL CELL LINES
Establishment of cell lines
Two PAL cell lines, OPL-1 arid OPL-2, were established
from biopsies of PAL cases 1 and 2, respectively. In both cases,
the biopsy samples obtained prior to antineoplastic chemotherapy were diagnosed as diffuse lymphoma of the immunoblastic
type ( D W
After cultures were started, cells growing in suspension were
visible as small clusters at day 14 in OPL-1 and day 10 in
OPL-2. Once established, OPL-1 and OPL-2 cells were passed
at a density of 1.5 x lo5 and 1 :< 10s cells/ml every 3 days,
respectively. The growth characteristics of the 2 lines are
summarized in Table 11.
Immunophenotypic analysis
Table I shows the results of the comparative immunophenotypic analysis on cell lines OPL-1 and OPL-2 and the original
tissue samples. The B-cell origin of OPL-1 and OPL-2 was
demonstrated by the expression of surface immunoglobulins
together with the presence of several B-cell-restricted markers, irrespective of negative reactivity for anti-CD19 antibody
in both OPLs. Analysis of immunoglobulin light chains showed
the monoclonal nature of OPL-1 and OPL-2 and was in
agreement with a clonal derivation from the respective tumor
biopsy specimens. The expression of B-cell activation markers
(CD23 and/or CD30) in OPLs suggests an LCL-like character
rather than a Burkitt’s lymphoma-like character. Many tumor
cells in both cases 1 and 2 were: positive for EBNA2 (Fig.
la, b). On the other hand, reactivity for LMPl was weak and
was found in only a small number of tumor cells in case 1, but
89
tumor cells in case 2 were LMPl negative (Fig. lc, d). As for
cytocentrifuged cells from the established cell lines, more than
30% of OPL-1 cells showed a much stronger expression of
EBNA2 protein, but OPL-2 cells were EBNA2 negative by
immunocytochemistry (Table I and Fig. 2u, b). The expression
of LMPl was not detected in OPLs by immunocytochemistry
(Table I and Fig. 2c, d).
Cytogenetic analysis
Eighteen cells (OPL-1) and 24 cells (OPL-2) were analyzed.
Both cell lines showed a highly complicated karyotype, with
numerical and multiple structural rearrangements. Chromosomal numbers ranged from 46 to 207 in OPL-1, and from 53 to
129 in OPL-2. Although it is difficult to define a modal number
of chromosomes in both cell lines, common structural abnormalities were identified only in OPL-2 as follows: del(l)(q21) x
1-2, add(2)(pll.l), add(7)(q32 x 2, der(ll)t(ll;l4)(pl5;qll) x
2, add (15)(p13), der(l9)t(1;19)(q21;p13.3) X 2. Band 14q32.33
was not involved in structural rearrangements.
Using FISH with the IgH probe, 3 co-localized signal of C,
and VH segments were observed, one on the normal chromosome 14 and the others on the telomeric region of duplicated C
group-sized chromosome, possibly der(1l)t(ll;l4)(pl5;qll).
The split signal of C, and VH,which indicates 14q32 translocation commonly found in B-cell malignancies, was not detected.
Immunoglobulin gene rearrangement analysis
DNA extracted from the biopsy specimens and OPLs were
digested with Hind 111 or Pst I and subjected to Southern blot
FIGURE
1 - Immunostaining for EBNA2 and LMPl of biopsy specimens of cases 1 and 2. Signals were generated with the alkaline
phosphatase anti-alkaline hosphatase (APAAP) method (a, 6) or with the avidin-biotin-complex(ABC) method (c, d). (a, b) EBNA2staining of cases 1 (a) and (6). Positive staining is visible in nuclei of many tumor cells in both cases. (c, d) LMP1-stainingof cases 1 (c)
and 2 (d). Scattered and weak staining is visible only in case 1 (arrow heads), but no staining is visible in case 2. Scale bar = 17 p.m.
!
90
KANNO ETAL.
FIGURE
2 - Immunostaining for EBNA2 and LMPl of cytocentrifuged slides of OPL-1 and OPL-2. Signals were generated with the
avidin-biotin-complex (ABC) method. (a, b) EBNA2-staining of OPL-1 (a) and 2 (b). Positive staining is visible in nuclei of
approximately 30% of OPL-1 cells, but no staining is visible in OPL-2. (c, d) LMP1-stainingof OPL-1 (c) and 2 (d). No staining isvisible
in OPL-1 and 2. Scale bar = 17 pm.
analysis with human JH probe. OPL-2 displayed clonal rearrangements of IgH gene identical to those detectable in the
biopsy specimen from which OPL-2 originated (Fig. 3). On the
other hand, no clear band was observed in OPL-1 and its
original biopsy specimen probably because of the heteroploid
pattern of its chromosomes (results not shown).
EBVgenome analysis
In the Southern blot analysis of Bam HI-digested DNA
samples probed with LMPl oligonucleotide probe, both cell
lines and their original biopsy specimens showed a slightly
broad signal. However, they contained a predominant terminal
repeat in fused form, and their size was identical between each
cell line and the original biopsy specimen (8 kb in case 1 and
OPL-1. and 10 kb in case 2 and OPL-2) (Fig. 4a). Thus, OPLs
were identical to their original lymphoma tissues at the level of
episomal EBV DNA and showed a monoclonal cellular proliferation of an EBV-infected progenitor cell. The type of EBV
in OPL-1 and OPL-2 was type B and type A, respectively, by
PCR-Sout hern blot analysis (Fig. 46).
Expression of EBNA2 and LMPl in OPLs
Since EBV genes EBNA2 and LMPl are known to alter the
growth phenotype of human B cells (Rickinson et al., 1992;
Wolf et al., 1993), we examined the expression of these
molecules in OPLs at both mRNA and protein levels.
By Northern blot analysis of mRNA from OPL-1 with an
EBNA2 oligonucleotide probe, we observed 2 bands (Fig. 5a).
Ten
FIGURE 3 - Southern blot analysis for kH
micrograms of DNA were digested with Hind IrI (a) or Pst I @),
electrophoresed in 0.7% agarose gel, Southern blotted and hybridized with 32P-labeled JH DNA probe (5 days of exposure at
-80°C). DNA size markers are indicated o n the right. Arrows
indicate rearranged bands of case 2 and OPL-2.
PAL CELL LINES
91
By Northern blot analysis of mRNA from cells with an
LMPl oligonucleotide probe, we observed a band of about 2.6
kb in LCL and Raji cells. No band was observed in mRNA
sample from OPLs (Fig. 56), though low-level expression of
LMPl mRNA in OPLs was found by RT-PCR, which is much
more sensitive than Northern blot analysis (results not shown).
We could not observe LMPl protein expression in OPLs by
Western blot (Fig. 5e).
These results taken together clearly show that EBNA2
mRNA and protein were expressed in OPL-1 cells at a higher
level than in LCL and Raji cells but were not detectably
expressed in OPL-2 cells. On the other hand, OPLs showed
little expression of LMPl mRNA, and the expression of LMPl
protein was not observed in OPLs.
Oncogene expression in OPLs
Since EBV infection of B cells is known to up-regulate the
expression of c-&r (Knutson, 1990), 6cl-2 (Henderson et a/.,
1991) and c-myc (Wennborg et al., 1987), we examined OPLs
for the expression of these genes. Expression of c-myc mRNAs
was noted in both OPL-1 and -2 and that of c-fgr in OPL-1
alone (Fig. 6). Expression of 6cl-2 was observed in both OPL-1
and OPL-2, with much stronger expression in OPL-2 than in
OPL-1 (Fig. 6).
DISCUSSION
F~CURE4-Southern blot analysis for the clonality of EBV
genome (a) and PCR analysis for typing of EBV genome (6).
(a) Ten micrograms of DNA were digested with Bam HI,
electrophoresed in 0.7% agarose gel, Southern blotted and hybridized with 32P-labeledLMPl oligonucleotide probe. DNA size
markers are indicated on the left (10 days of exposure at -80°C).
OPLs and their original bio sy specimens contain a predominant
terminal repeat (arrowheag). (b) One microgram of DNA was
amplified by PCR, electrophoresed in 2% agarose gels, Southern
blotted and hybridized with non-radiolabeled EBNA2A- or
EBNA2B-specific oligonucleotide probe. Signals were generated
by chemiluminescence 4 min of exposure at room temperature).
EBNA2 sequences amp ified b PCR in biopsy specimen of case 1
and OPL-1 hybridize only with EBNA2B-specific probe. Amplified EBNA2 products in bio sy specimen of case 2, OPL-2 and
Raji cells hybridize with EB&2A-..pc ecific probe.
I
One was approximately 3.0 kb, the same size as that observed
for EBNA2 in LCL and Raji cells. The other band, unique to
OPL-1 cells, was approximately 0.5 kb smaller in size. By
Western blot analysis, we observe,d a strong expression of the
approximately 73 kDa EBNA2 protein in OPL-1 (Fig. 5 d ) , but
only a weak expression in Raji cells (Fig. 5d, arrowhead).
EBNA2 protein in OPL-1 cells was approximately 7 kDa
smaller in size than that in Raji cells. Since the sizes of
EBNA2 protein in type A or type B EBV-infected cells are
82-87 or 72 kDa, respectively (Dambaugh et al., 1984), the
difference in size of EBNA2 protein between OPL-1 (type B
EBV) and Raji (type A) reflected the type of EBV genome
they contained. On the other hand, neither mRNA nor protein
for EBNA2 was detected in OPL-2 by Northern or Western
blot analysis.
Among the several EBV genes associated with latent infection, expression of EBNAl is observed in all EBV-associated
malignancies since it is essential to the replication of EBV
genome (Rickinson et al., 1992). OPLs also expressed EBNA1RNA by Northern blot analysis (results not shown). On the
other hand, EBNA2 and LMPl activate a variety of cellular
genes including oncogenes (Wennborg et al., 1987; Knutson,
1990; Henderson et al., 1991), adhesion molecules (Rickinson
et al., 1992) and activation markers (Rickinson er al., 1992;
Wolf et al., 1993), suggesting that these genes have direct
effects on cellular transformation. However, the level of
expression of EBNA2 and LMPl varies among these malignancies (Rickinson et al., 1992; Wolf et al., 1993), as shown in
Table 111. Therefore, analysis of the pattern of EBV latent
infection gene expression, especially EBNA2 and LMPl,
might shed light on lymphomagenesis in PAL.
In our current study, we showed that the expression of EBV
latent infection genes varied among PAL cell lines, although
restricted expression of LMPl was a constant finding. As
reported previously (Fukayama et al., 1993), however, the
immunohistochemical study revealed some LMP1-positive cells
in the original tumor specimen of case 1. Several explanations
are possible: 1) cross reactions of anti-LMP1 antibody might
have occurred; 2) LMP1-positive cells in the PAL tissue
specimen might not be the most rapidly growing cells, and thus
only the LMPl negative cells grew in culture; or 3) LMP1expressing cells in vivo might become LMPl negative during in
vitro culture. In this regard, conversion from LMPl negative to
positive has been reported during in vitro culture of Burkitt’s
lymphoma cells (Rickinson et al., 1992). Although we cannot
address the specific likelihood of these options, restricted
expression of LMPl might be a feature characteristic of PAL.
LMPl is recognized as a target molecule by EBV-specific
CTLS (Rickinson et al., 1992). Restricted expression of LMPl
in PALS might enable transformed cells to evade immunological surveillance in non-immunocompromisedhosts. Furthermore,
induction of cellular adhesion molecules, such as LFA-1, LFA-3
and ICAM-1, which participate in CTL recognition (Rickinson et
aL, 1992), might be weak due to restricted expression of LMPl.
Immunocytochemical procedures revealed the expression of
ICAM-1 (CD54) alone in OPL-2 but both LFA-1 (CDlla) and
ICAM-1 in OPL-1. These findings suggest a role of induction
mechanism other than those mediated by LMPl.
92
KA”0 ETAL.
FIGURE
5 - Northern and Western blot analysis of EBV latent infection genes. Northern blot for EBNA2 (a), LMPl (b) and actin (c)
mRNAs. Five micrograms of mRNAs were applied to each well, Northern blotted and hybridized with 32P-labeledEBNA2 or LMPl
oligonucleotide probes or with actin DNA probes. RNA size markers are indicated on the left (7 (a), 10 (b) or 1 (c) days of exposure at
-80°C). OPL-1 mRNAs hybridize with EBNA2 probe only, but not with LMPl probe. OPL-2 mRNA sample contains no detectable
amount of EBNA2 and LMPl transcripts. Western blot for EBNA2 (d) or LMPl (e). Cells (3 x lo7) were lysed with 400 p,l of Laemmli
sample buffer, and a 40 pl aliquot of each sample was applied to each well. After electrophoresis, proteins were transferred to a
nitrocellulose membrane, incubated with anti-EBNA2 (d) or anti-LMPl (e) antibodies and visualized as described in Material and
Methods. The positions of molecular size markers are indicated (in kDa) on the left (20 min of exposure at room temperature).
FIGURE6 -Northern blot analysis of the expression of oncogene mRNAs. Five micrograms of mRNA samples were applied to each
well, Northern blotted and probed with 32P-labeledc-myc (a), c-fgr (b) and bcl-2 (c) genomic DNA probes and with mouse actin DNA
probe (d). RNA size markers are indicated on the left (3 (a, c), 4 (b) or 1 (d) days of exposure at -80°C).
PAL CELL LINES
TABLE III - PATTERNS OF EBV LATENT GEYE EXPRESSION IN
HUMAN MALIGNANCIES
Burkitt’s lymphoma
Hodgkin’s disease
ML/ICH
OPL-1
OPL-2
EBNAl
EBNAZ
LMPl
+
+
+
+
+
-
+
+
+-
-
+
+/+/‘ML/ICH, malignant lymphoma developed in immunocompromised hosts; +/-,weak mRNA expression detectable by RT-PCR
only.
LMPl transactivates the oncogene bcl-2 (Henderson et af.,
1991). OPL-2 contained bcl-2 mRNA without detectable
expression of LMPl protein. ‘his finding suggested bcf-2
activation by a non-LMP1-mediated mechanism. On the other
hand, the expression of [email protected], which correlated with the
expression of EBNA2, was strong in the EBNA2-expressing
OPL-1 but undetectable in the EBNA2-negative OPL-2.
Because the growth rate of the OPL-1 was much lower than
that of OPL-2, EBNA2-mediated c-f’ expression might contribute little to the growth of PAL cells. Therefore, the
differences in growth rate and clncogene expression between
OPL-1 and OPL-2 could not be adequately explained by the
pattern of EBV latent gene expression. The latent infection of
EBV might be important in the initial phase of PAL lyrnphomagenesis, and subsequent genetic lesions might be necessary for
the development of overt lymphoma.
EBNA2 can also be a target molecule for host CTL response
(Rickinson et af., 1992). The EBNA2-positive cells were
present in the original biopsy specimen of OPL-2, but not in
the cells derived in culture. Did the EBNA2-positive cells turn
EBNA2 negative during in vitro culture? A previous study
reported the reverse phenomenon, i.e., from EBNA2 negativity to EBNA2 positivity (Rickinson et al., 1992). We assume
that the EBNA2-negative cells were the most rapidly growing
cells in the original tumor tissue of OPL-2 and could escape
host immune surveillance. On the other hand, EBNA2positive cells must be the most rapidly growing cells in the
original tumor tissue of OPL-1. Because the EBNA2 may work
as a target molecule for CTL, transformed cells in case 1might
require some immunosuppressive circumstances to survive
against host immune surveillan~x.In fact, only OPL-1, not
OPL-2, expressed mRNA of an immunosuppressive cytokine,
interleukin-10 (data not shown).
OPL-2 had type A EBV, and OPL-1 had type B. The
difference in EBV subtype in OPLs might influence their
growth characteristics since EBNA2A induces a much more
enhanced growth phenotype in 13 cells than EBNAZB in vitro
93
(Rickinson et al., 1987). In fact, OPL-2 with type A EBV
showed a more enhanced growth rate than that of OPL-1 with
type B. However, OPL-2 did not express EBNA2. These
findings also suggest that genetic lesions other than EBV
infection are essential for the development of overt lymphoma.
The profile of expression of EBV latent infection genes in
OPL-1 also provided an interesting issue in the regulation of
EBV gene expression. EBNA2 transactivates LMPl in B cells
(Wang et al., 1990); however, LMPl expression in OPL-1 was
restricted in spite of the higher expression of EBNA2 protein.
This might not be anecdotal since the expression of LMPl in B
cells is regulated by various factors (Boos et af., 1987). Since 2
different-sized EBNA2 mRNAs were observed in OPL-1,
some alterations in EBNA2 transcripts might affect subsequent LMPl expression. The YH exon of EBNA2 in type B
EBV is approximately 100 bp smaller than that in type A EBV
(Dambaugh et al., 1984; Sample et al., 1986), which could not
be distinguished in agarose gels. Thus, the larger EBNA2
mRNA in OPL-1 may represent the transcripts processed in
the same manner as that in LCL and Raji cells containing type
A EBV, and the smaller one might contain some alterations.
Further analysis may provide interesting information not only
about EBV-mediated lymphomagenesis, but also in the regulation of EBV gene expression.
Before the development of PAL, patients suffer from
long-standing pyothorax with a mean duration of more than 30
years (Iuchi et al., 1989). Biologically active metabolites produced in chronic inflammatory sites might induce cellular
DNA aberrations, the accumulation of which might be essential for the development of overt lymphoma.
In conclusion, our present study suggests that the restricted
expression of LMPl is common in PAL cells. This may
contribute to the escape of EBV-infected cells from host
immune surveillance. However, additional events other than
the latent infection of EBV might be essential for causing a
growth advantage sufficient for the development of overt
lymphoma. Our cell lines may provide a useful model for
studying lymphomagenesis in PAL, the EBV-positive B-cell
lymphoma developing in non-immunocompromised hosts with
long-standing chronic inflammation.
ACKNOWLEDGEMENTS
We thank Dr. M.E. Bloom for critically reviewing the
manuscript; Ms. Y.Kanno and Y. Matsumoto for preparing it;
and Ms. I. Kuroki for technical assistance. This work was
supported in part by a grant-in-aid for cancer research (4-5)
from the Ministry of Health and Welfare and grants from the
Ministry of Education, Science and Culture (05152111,
06770125), Japan.
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