[CANCER RESEARCH 53, 127-132, January 1, 1993]
Theophylline Induced Differentiation Provides Direct Evidence for the Deregulation
of c-myc in Burkitt's Lymphoma and Suggests Participation of Immunoglobulin
Enhancer Sequences1
J. T. Sandlund, L. M. Neckers, H. E. Schneller, L. S. Woodruff, and I. T. Magrath
Pediatrie Branch. Clinical Oncolo%\ Program, Division of Cancer Treatment. National Cancer Institute. NIH, Bethesda. Maryland 20892 ¡J.T. S.. L M. N., I. T. M.¡:Department
of Henuitohgv/Oncologv.
St. Jude Children's Research Hospital. Memphis. Tennessee 38101 ¡J.T. S., H. E. S.. L S. W.¡:and Department of Pediatrics, University of Tennessee.
College of Medicine. Memphis. Tennessee 38103 ¡J.T. S.I
ABSTRACT
Most of the evidence that supports the hypothesis that the c-myc gene is
abnormally regulated in Burkitt's lymphoma (BL) is indirect. The puta
tive abnormal expression of c-myc is likely, at least in part, to be a
consequence of the usurpation of its regulatory sequences by immunoglobulin enhancer elements, which are invariably juxtaposed to c-myc by the
translocations associated with this tumor (C. M. Croce, J. Erikson, A.
Ar-Rushdi, D. Aden, and K. Nishikura, Proc. Nati. Acad. Sci. USA, 81:
3170-3174, 1984). We have developed a differentiation induction model
system to examine this issue more directly. In a variety of non-BL cell lines,
differentiation
induction results in the down-regulation
of c-myc
(G. P. Studzinski, A. K. Bhandal, and Z. S. Brelvi, Proc. Soc. Exp. Biol.
Med., ¡79:288-295, 1985; Y. Matsui, R. Takahasi, K. Minara, T. Nakagawa, T. Koizumi, Y. Nakao, T. Sugiyama, and T. Eugita, Cancer Res., 49:
1366-1371, 1985; T. Mitchell, E. Sariban, and D. Kufe, Mol. Pharmacol.,
30: 398-402,1986;
Z. S. Brelvi, and G. P. Studzinski, J. Cell. Physiol., 128:
171-179, 1986). Since BL is of B-cell origin, differentiation is associated
with persistent or increased expression of immunoglobulin genes. There
fore, if c-myc and c-u are coregulated in BL via immunoglobulin enhancer
sequences, persistent or increased expression of the c-myc gene, rather
than down-regulation, should occur in differentiated BL cells. Differenti
ation was induced in four BL cell lines with theophylline (7 x Kl' M),and
mRNA was examined by Northern blot analysis. In all four BL lines
studied (JD38, AG876, KK124, and Daudi), there was persistent or in
creased expression of both c-u and c-myc genes (detected with a third exon
c-myc probe), in contrast to the decreased expression of the c-myc gene
observed in the three Epstein-Barr virus transformed lines studied
(A3317, TC84, and CB34). In the BL cell line, JD38, the c-myc gene is
truncated (the second and third exons are translocated to chromosome 14
while the first exon remains on chromosome 8). In this line, we demon
strated that theophylline induced differentiation results in down-regula
tion of the first exon while the level of expression of the translocated
second and third exons remains unchanged or increases. Thus, in JD38,
the c-inyc genes adjacent to the immunoglobulin constant region are ab
normally regulated, while the first exon remaining on chromosome 8 is
regulated as is the normal c-myc gene in other non-BL differentiationinduction model systems. These data directly demonstrate that c-myc is
abnormally regulated in BL and strongly support the hypothesis that the
translocated c-»iv<-gene is regulated by sequences present in the juxta
posed immunoglobulin gene. Furthermore, we propose that the structural
alterations present in the translocated c-myc alÃ-eleare permissive for
immunoglobulin enhancer usurpation of transcriptional control.
INTRODUCTION
BL 2 is invariably associated with one of three characteristic chro
mosomal translocations [t2;8(pl3;q24), t8;14(q24;q32), and t8;22(q24;qll)], in all of which the c-myc oncogene situated on band q24
Received 7/13/92; accepted 10/20/92.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by Grants CA-20180 and CA-21765 from the National Cancer
Institute, the American Lebanese Syrian Associated Charities, and the Cancer Research
Foundation of America.
2 The abbreviations used are: BL, Burkitt's lymphoma; EBV, Epstein-Barr virus; SSC.
standard saline-citrate; SDS, sodium dodecyl sulfate.
of chromosome 8 has been shown to be involved (1-4). Each chro
mosomal abnormality results in the juxtaposition of an immunoglob
ulin gene constant region to the c-myc gene, as well as structural
changes in the c-myc gene ranging from point mutations to major
truncations. It has been demonstrated that it is the c-myc gene in
volved in the translocation that is expressed in BL and not the normal
alÃ-ele(5). The steady state level of c-myc mRNA in BL cells, however,
is similar to that of nonmalignant rapidly dividing B-cells (6). More
over, the c-myc protein is virtually always unaltered since the coding
sequences of c-myc, largely derived from the second and third exons,
are only rarely mutated (7). Nonetheless, the expression of the struc
turally altered c-myc alÃ-eleinvolved in the translocation circumstan
tially implicates it in the pathogenesis of BL, via presumptive dereg
ulation of the gene.
Although c-myc has been shown to be abnormally regulated in
hybrids between mouse myeloma and human Burkitt's lymphoma cell
lines (8), there has been no direct demonstration, to date, of abnormal
c-myc regulation in nonhybrid Burkitt's lymphoma model systems.
Several possible mechanisms leading to deregulation of c-myc in BL
have been proposed. One suggestion is that structural alteration in the
5' region of the gene leads to constitutive expression (7). This could
be a consequence of failure of one or more transcriptional repressor
proteins to bind to this region or other regions of the gene (9, 10), in
some cases reducing or abrogating the block to elongation (11), or
separation of the coding region from the regulatory region of the gene.
An alternative mechanism that has been proposed is alteration in
posttranscriptional processing of c-myc mRNA (12-14). Although
lack of a first exon [not an infrequent consequence of the t(8;14)] has
been shown to increase the half-life oÃ-c-myc mRNA (12-14), there is
evidence that separation from regulatory sequences in the 5' flanking
region, first exon, and 5' intronic regions does not, per se, lead to
constitutive expression of c-myc (7). Nor are truncated c-myc con
structs able to induce tumors in transgenic mice (15). Moreover, the
suggestion that truncation of c-myc is the only structural abnormality
needed is not an adequate explanation for the juxtaposition of c-myc
with an immunoglobulin constant region. The latter is best explained
by postulating that immunoglobulin locus elements positively influ
ence transcription of the juxtaposed c-myc gene (8). This is a very
attractive possibility since several enhancer elements have been de
scribed within the immunoglobulin loci (16, 17). Moreover, c-myc
genes with or without the first exon coupled to an immunoglobulin
enhancer are able to induce B-cell lymphomas in transgenic mice (15).
To investigate the role of immunoglobulin enhancer elements in the
deregulation of c-myc, we have examined c-myc expression in BL cell
lines induced to undergo differentiation. Because immunoglobulin
synthesis is an essential characteristic of maturing B-cells, continued
or increased expression of immunoglobulin genes would be expected
to occur in BL cells (a B-cell tumor) undergoing differentiation.
Indeed, we have previously demonstrated this to be so ( 18). Therefore,
if c-myc is brought under the control of immunoglobulin sequences in
BL, one would expect to observe continued or increased expression of
c-myc after differentiation induction in contrast to the down-regulation
127
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THEOPHYLLINE
INDUCED
DIFFERENTIATION
of c-myc observed in other non-BL differentiation induction systems.
Thus, persistent expression of c-myc, a gene involved in cellular
proliferation, in differentiated BL cells would both demonstrate an
abnormality of its expression and provide supporting evidence for
coregulation of c-u and c-myc genes. In this paper we describe c-myc
expression in BL cell lines induced to undergo differentiation with
theophylline as previously reported (7 X IO"3M) (19).
MATERIALS
LYMPHOMA
CELLS
performed with a panel of monoclonal antibodies (anti-B4, anti-Bl, ami-B2,
anti-J5, anti-TIO, anti-PCA-1, anti-IgG, anti-IgM, anti-IgA, anti-IgD, anti-x,
anti-X, anti-TAC, anti-T9, and anti-DR).
Morphological Changes. Cytocentrifuged preparations of both control and
theophylline treated BL cells were made and stained with hematoxylin and
eosin. Transmission electron microscopic evaluation of these cells was per
formed after fixation in glutaraldehyde.
RESULTS
AND METHODS
Cell Proliferation. Cell growth was arrested with theophylline
treatment, but cell viability remained at 70-90% (trypan blue) at 48 h
when RNA was extracted. The untreated cell line, JD38, demonstrated
a DNA histogram characteristic of cells in log phase growth. Incuba
tion of JD38 with theophylline resulted in G, arrest (Fig. 1).
Marker Studies. Both control and theophylline treated BL cells
were screened for reactivity with a panel of monoclonal antibodies
(B-cell panel, immunoglobulin panel, anti-TAC, and anti-T9) but no
Cells. Four BL cell lines (JD38, AG876. KK124, and Daudi) were studied.
Three EBV transformed lymphoblastoid cell lines (A3317, TC84, and CB34)
were included as B lineage controls. JD38 and KKI24 are American (sporadic)
BL cell lines; AG876 and Daudi are African (endemic) BL cell lines. All BL
cell lines contain an 8;14 translocation except for KK124 which contains an
8;22 translocation. The chromosome 8 breakpoint in AG876 and Daudi occurs
5' of the first exon while in JD38 it occurs in the first intron, and in KK124 it
occurs 3' of the third exon.
The cells were maintained at 37°Cby subculture every 3 to 4 days in RPMI
significant changes were observed after theophylline treatment (data
not shown).
Morphological Changes. With both light (Fig. 2) and electron
microscopy (Fig. 3), plasmacytoid changes were observed in the theo
phylline treated BL cells (i.e.. reduction in the nucleus/cytoplasm
1640 containing 20% fetal calf serum, 100 units/ml penicillin, and 100 ug/ml
streptomycin. For theophylline treatment the cells were diluted to a concen
tration of 0.5 x IO6 cells/ml in the same culture medium and cultured in a
160-ml volume in 650-cm2 plastic flasks (Costar, Boston, MA) at 37°C.
Theophylline (Sigma) was diluted in RPMI 1640 and added to the diluted BL
cells to a final concentration of 7 X IO"3 M at 0 h.
RNA Transfer. Cytoplasmic RNA was extracted at 48 h in guanidine
isothiocyanate over a cesium chloride gradient as described (20). Twenty ug of
total cellular RNA were loaded in each lane, electropheresed in a 1% agarose
gel, transferred to Nytran (Schleicher & Schuell). prehybridized, and hybrid
ized to the appropriate 12P-labeled probe in 50% formamide at 42°C.The
Nytran blots were washed in 6 x SSC/0.5% SDS at 21°Cfor 15 min x 2, then
in 1 x SSC/0.5% SDS at 37°Cfor 15 min X 2, and then in 0.1 x SSC/1.0%
SDS at 65°Cfor 60 min x l.
Probes. DNA probes used included a Cla-Ec'oRI fragment containing the
third exon of c-myc (21), a Pvull-Pvull fragment containing the first exon of
c-myc (22), an EcoRl-EcoRl fragment containing a large portion of the
c-u region (23), an EcoRI-EcoRI fragment containing C-K (24), and a comple
OF BURKITTS
eoo
JO3B - Control
100
DNA Content
mentary DNA clone for actin (25).
Flow Cytometry
DNA contents of both control and theophylline treated
JD38 cells were measured by flow cytometry. Surface marker analysis was also
JD38 - Theophylline
200
100
DNA Content
200
Fig. 1. DNA histograms of both control and theophylline treated Burkitt's lymphoma
cells (cell line, JD38).
B
Fig. 2. Light microscopy of control (A) and theophylline treated (B) Burkitt's lymphoma cells (cell line, JD38) demonstrating
plasmacytoid changes induced by theophylline.
128
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THF.OPHYLLINE
INDUCED
DIFFERENTIATION
OF BURKITTS
LYMPHOMA
CELLS
« .
jftft« i
mìSI
-v
I
Fig. 3. Electron microscopy of control (A] and theophylline treated (fl) Burkitt's lymphoma cells (cell line. AG876) demonstrating plasmacytoid changes induced by theophylline.
ratio, eccentricity of the nucleus, development of a single prominent
nucleolus). By electron microscopy, a variable increase in cytoplasmic
organdÃ-es (i.e., mitochondria and golgi apparatus) was also observed.
c-myc Expression. In the four BL lines examined, unchanged or
increased steady state levels of c-myc mRNA (hybridized with a third
exon c-myc probe) were demonstrated in cells incubated with theo
O
OC
o
u
co
co
phylline as compared to the reduced levels observed in the three EBV
transformed lymphoblastoid cell lines used as controls (Fig. 4). In the
BL cell line JD38. the translocation results in a truncation of the c-myc
gene; i.e., the first exon remains on chromosome 8 and is expressed as
an approximately 900-base pair message readily distinguishable from
the 2.3-kilobase message arising from the remaining part of the c-myc
IOLillI
o.0LUXHsoi-1oocOu*
o
oc
X
(L
O
o
o
X
CO
CD
00
00
I
I
K
CL
O
l_r^.COCO<1_JOocI—zOOsui-1i
o
o
o
cocou1XQLOLUX1-300u1co
co oo
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LU
8
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S
§
s¿ <
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Fig. 4. c-mvc expression (using third exon
probe) in both control and theophylline treated Burkilt's lymphoma cells (JD38, AG876. KKI24, and
Daudn and EBV transformed lymphoblastoid cells
(A3317. TC84, and CB34). ß-Actincontrols are
also shown. Theophylline treatment resulted in un
changed or increased steady state levels of c-myc
mRNA in Burkitt's lymphoma cells as compared to
reduced c-mvr mRNA levels in EBV transformed
lymphoblastoid cells.
«M»-Â
'actirr
129
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THEOPHYLLINE
INDUCED
DIFFERENTIATION OF BURKITTS
LYMPHOMA CELLS
reduction in the level of c-myc mRNA (third exon c-myc probe; Fig.
£ *
DC u.
£o
Ik LU
O
O
x
h-
CO
(Ó
h. r«.
oo oo
o
x
ce ¿
£o
^
LLi
O x
ü
hob oó
co co
O
O
-3
-3
6). demonstrating that de novo protein synthesis is not required for
continued expression of c-mvc in differentiated BL cells.
Immunoglobulin Gene Expression. JD38 (BL) was examined for
modulation of immunoglobulin gene expression. The levels of both
c-\i and C-K mRNA remained unchanged in the theophylline treated
g
E *°£
oLU
S
O
O
H-
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il
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cells (Fig. 7).
o
-
ü
+
l •¿
II
C-myc
3rd Exon
hT-
c-myc
C\J
CO
ü hIO
<O
-
(3rd exon)
probe
ß-Actin -
C-myc
1st Exon
Fig. 6. c-myc expression in JD38 cells treated with theophylline alone (Lane 2),
theophylline and actinomycin D {Lane 4), cycloheximide alone (Lane 5). iheophylline and
cycloheximide (Lane 6), and actinomycin D alone (Lane 3). Lane 1, untreated JD38.
ß-Actincontrols are also shown.
Fig. 5. c-m\r expression as delected with both first and third exon c-mvc probes in both
control and theophylline treated Burkitt's lymphoma cells (AG876. JD38. and Daudi). In
o
O
x
OC Du
JD38. which contains a truncated c-wvr gene, theophylline induced differentiation results
in a reduced level of expression of the first exon, while the level of expression of the
translocated second and third exons remains unchanged or increased.
t
oc
o
o I-X
o
regulated (i.e., to 55% of the level in untreated cells as determined by
densitometric analysis), while the expression of the RNA fragment
containing the second and third exons, detected with a third exon
probe, was unaffected or slightly increased (Fig. 5). The absence of a
2.3-kilobase mRNA species detectable with the first exon c-myc probe
indicates that the c-myc alÃ-elein the normal chromosome 8 was not
induced by the theophylline treatment.
c-myc Half-Life. RNA was extracted from 48-h cultures of control
and theophylline treated JD38 cells after preincubation in actinomycin
D (0.5 ng/ml) for 3 h. The level of c-myc message was equally reduced
(third exon c-mvc probe) in both the control and theophylline treated
cells incubated with actinomycin D, demonstrating that the persistent
or increased expression of c-mvc in theophylline treated BL cells was
not secondary to posttranscriptional message stabilization (Fig. 6).
Forty-eight-h cultures of control and theophylline treated JD38 cells
were also incubated in cycloheximide (20 ng/ml) for 2 h, with no
O X
ü
oo oo
co co
o o
oo co
co co
gene translocated to chromosome 14. In Northern blots of RNA ob
tained from theophylline treated JD38 cells hybridized with a first
exon probe, we demonstrated that the separate first exon is down-
Pactir
O
-3
O
-3
I
I
oLU
I
I
ßactin
Fig. 7. c-p and e K expression in both control and theophylline
ß-Actincontrols are also shown.
treated JD38 cells,
130
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THEOPHYLLINE
INDUCED
DIFFERENTIATION
OF BURKITTS
LYMPHOMA
CELLS
DISCUSSION
an immunoglobulin enhancer region but not in mice transgenic for the
same structurally altered c-mvc gene (i.e., lacking a 5' region) not
The induction of differentiation in BL cells with theophylline, as
evidenced by plasmacytoid morphological changes, has enabled us to
directly demonstrate that the translocated c-mvc gene in BL does not
respond to normal regulatory signals which result in the down-regu
lation of c-mvc in other differentiation systems that have been ex
plored (26-29). Indeed, transfection of c-myc into Friend erythroleu-
coupled to an immunoglobulin enhancer, further support the hypo
thesis that the constitutive expression of the translocated c-mvc gene
in BL may be a consequence of its juxtapostition to the immunoglo
bulin constant region (8. 15). More specifically, the expression is
likely to be the result of cis regulation by either the known J-C intron
immunoglobulin enhancer or by another enhancer element within the
immunoglobulin constant region (8, 15). In this context, the recent
identification of an enhancer element in the 3' region of the rat
kemia cells has been shown to prevent differentiation (30). The
differentiation of BL cells is associated with persistent expression of
immunoglobulin genes, as would be expected in cells of the B lineage.
We reasoned that if the translocated c-myc gene in BL is brought under
immunoglobulin locus strongly suggests that a similar enhancer is
present in humans (31). Such an enhancer could influence c-mvc
expression regardless of where the breakpoint is in the heavy chain
immunoglobulin locus.
In summary, the induction of differentiation in BL cells with theo
phylline, as evidenced by plasmacytoid morphological changes, pro
vides a model system which clearly demonstrates that the translocated
c-mvc gene in BL is abnormally regulated, almost certainly via an
immunoglobulin enhancer element.
the control of one or more enhancers present in the juxtaposed im
munoglobulin locus, the expression of the translocated c-myc gene
would remain unchanged in differentiated BL cells, in contrast to the
down-regulation of c-myc observed in many differentiated non-BL
malignant cell lines. When the four BL cell lines (JD38. AG876,
KK124, and Daudi) were induced to undergo differentiation with
theophylline, c-myc expression was not reduced but rather paralleled
the ongoing expression of c-u as predicted. In some cell lines, a small
but reproducible increase in the expression of c-myc was observed.
Thus, in BL cells, c-mvc is not down-regulated in circumstances
ACKNOWLEDGMENTS
where it would normally be so. That this finding was not the result of
posttranscriptional message stabilization was demonstrated by the
equivalent reduction in the level of c-myc message in both control and
differentiated BL cells incubated in actinomycin D.
Additional confirmation of altered regulation of the translocated
c-myc alÃ-elewas obtained in the BL cell line, JD38. This line contains
a truncated c-mvc gene; the first exon remains on chromosome 8 while
the second and third exons are translocated to chromosome 14. With
a first exon probe, we demonstrated that in contrast to the persistent
expression of c-myc transcripts detected with either first or third exon
probe in the two BL lines with grossly intact c-mvc genes (AG876,
Daudi), the separately expressed first exon in JD38 was variably
down-regulated (i.e., an average of a 55% reduction by densitometric
analysis) with theophylline, while the expression of its second and
third exons, as detected with a third exon probe, was unaffected or
slightly increased. Thus, only the c-mvc exons which are juxtaposed to
We are indebted to Donna Davis for technical expertise in electron micro
scopic studies and to Peggy Vandiveer for assistance in the preparation of the
manuscript.
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Our data are remarkably consistent with those of Croce et al. (8)
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THKOPHYI.l.INt
17. Garcia. J. V., Bich-Thug.
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INDUCED
DIFFERENTIATION
L. T.. Haft'ord. J . and Queen. C. Synergism between
OF BURKITTS
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1.32
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research.
Theophylline Induced Differentiation Provides Direct Evidence
for the Deregulation of c- myc in Burkitt's Lymphoma and
Suggests Participation of Immunoglobulin Enhancer Sequences
J. T. Sandlund, L. M. Neckers, H. E. Schneller, et al.
Cancer Res 1993;53:127-132.
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