Download Evidence for RNA Tumor Viruses in Human

[CANCER RESEARCH 33,1515 1526, June 1973]
Evidence for RNA Tumor Viruses in Human Lymphomas Including
Burkitt's Disease1
S. Spiegelman,2 D. Kufe, R. Hehlmann, and W. P. Peters
Institute of Cancer Research and the Department
New York, New York 10032
of Human
Genetics and Development,
Summary
Human lymphomas (including Burkitt's disease) contain
an RNA related in sequence to that of the Rauscher leu
kemia virus. The viral-related RNA found in these lym
phomas is a 70 S component encapsulated with RNA-instructed DNA polymerase in a particle possessing a den
sity characteristic of the RNA tumor viruses. Further
more, the DNA synthesized by the lymphoma particles hy
bridizes specifically to the RNA of the Rauscher leukemia
virus. The particles identified in 81% of human lymphomas
and in 87% of Burkitt's tumors thus contain four features
characteristic of a known oncogenic RNA agent.
The relation of these RNA particles to the DNA-containing Epstein-Barr virus, an etiological candidate for
Burkitt's lymphoma, remains to be established. Both or
neither may be necessary, or one may be the true causa
tive agent. Relevant to the ultimate resolution of these
issues is the demonstration (reported herein) that the
presence of Epstein-Barr virus information in nonneoplastic
cells, such as in infectious mononucleosis, is not accompan
ied by the appearance of RNA particles.
We have used molecular hybridization to detect virusspecific RNA in tumors (2) and have found that corre
sponding neoplasms of murine and human origin exhibit
remarkable similarities. Thus, human breast carcinomas
(3) contained RNA possessing sequence homology to
RNA of MMTV.3 This type of RNA was unique to the
malignant adeno- and medullary carcinomas, being undetectable in normal breast tissue and in such benign
pathologies as fibrocystic disease and fibroadenoma. In
keeping with the known unrelatedness of the murine leukemogenic and mammary tumor viruses, we found that
breast cancer RNA did not hybridize to DNA comple
mentary to the RNA of RLV. Finally, and more compelling
was the demonstration that human leukemic cells (10)
1Presented at the American Cancer Society Conference on Herpesvirus and Cervical Cancer, December 8 to 10, 1972. Key Biscayne, Fla.
This research was supported by the NIH, National Cancer Institute.
Special Virus Cancer Program Contract 70-2049. and Research Grant
CA-02332.
2Presented by.
3The abbreviations used are: MMTV, mouse mammary tumor virus:
RLV, Rauscher leukemia virus; AMV, avian myeloblastosis virus;
EBV, Epstein-Barr virus; pRNA, polysomal RNA: TNE buffer, 0.01 M
Tris-HCl (pH 8.3), 0.15 M NaCl, 0.01 M EDTA; RSV, Rous sarcoma
virus.
College of Physicians
and Surgeons
of Columbia
University,
and human sarcomas (15) both contain RNA with homol
ogy to RNA of RLV, not to that of MMTV.
The pursuit of this intriguing concordance between the
neoplasms of mice and humans was of obvious interest.
From the viewpoint of etiology and cellular pathology,
lymphomas in mice are linked to the leukemias and sar
comas. In addition, some human lymphomas are accom
panied by the clinical appearance of a peripheral leukemia.
In any event, if human neoplasms parallel those observed
in mice, one might expect that human lymphomas would,
like the leukemias and sarcomas, contain RNA uniquely
homologous to that of a mouse leukemia agent. Of par
ticular interest is Burkitt's lymphoma, a disease as
sociated with a DNA-containing herpes-like agent.
Viral-related RNA in Hodgkin's Disease and Other
Human Lymphomas
We present evidence herein that human lymphomas, in
cluding Hodgkin's disease, lymphosarcomas, and reticulum
cell sarcomas, contain RNA exhibiting homology to the
RNA of RLV, but not to the unrelated RNA's of either
MMTV or AMV (11). As summarized in Chart 1, 69% of
the RNA's derived from lymphomas yielded positive hy
bridizations with RLV DNA, whereas none of the 48 con
trol tissues was positive. Furthermore, the RNA de
rived from Hodgkin's lymphoma showed no significant
reactions with either AMV or MMTV DNA.
RNA Related to That of a Murine Leukemia Virus in
Burkitt's Tumors
Discovery of RNA homologous to that of the murine
leukemia virus in human lymphomas immediately raises
the issue of Burkitt's disease, a malignant lymphoma
found in children living in a geographical belt extending
across Central Africa. There exists serological (12) and
nucleic acid hybridization (23) evidence linking this dis
ease to EBV, a herpes-like virus that contains DNA de
tected in (6) and isolated from (7) Burkitt's lymphoma
cells grown in culture. Since a causal relation between EBV
and Burkitt's tumor has not been conclusively established,
it is pertinent to inquire whether this lyphoma, like the
others examined here, contains RLV-related RNA.
A study of 21 African Burkitt's lymphomas clearly
demonstrated that 76% contained RNA related to that of
the RLV but not to that of MMTV or AMV. The pRNA
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1515
Spiele/man, Kufe, Hehlmann, and Peters
Tumor
Tissue
buffer. The cytoplasmic pellet is then treated with NP-40
to disrupt possible virus particles and is used in a standard
endogenous reverse-transcriptase
reaction to generate
DNA-3H after 15 min of synthesis at 37°.The reaction
product was freed of protein by treatment with sodium
dodecyl sulfate and phenol and was subjected to sedimen
tation analysis in a 10 to 30% glycerol gradient with suit
able markers for determination of the apparent size dis
tribution of the DNA synthesized. In a reaction mediated
by a B- or C-type virus, DNA-3H will sediment in a 70 S
region of the gradient representing the 70 S RN A/DN A-3H
early reaction product.
Features such as sensitivity to RNase and the require
ment for all 4 deoxyribonucleoside triphosphates can also
be used to demonstrate that the appearance of the 70 S
RNA:DNA-3H complex is in fact the result of a reversetranscriptase reaction. However, due to the possibility of
nontemplated, end-addition reactions, the most definitive
proof finally demands that the DNA-3H synthesized be
hybridizable to an RNA derived from a known oncogenic
virus.
The successful use of the simultaneous detection
technique to detect RNA viruses in mouse and human
Normal Human Tissues
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Chart I. Results of hybridization reactions with RLV DNA-3H and
pRNA from human lymphomas and normal human cells. The normal
tissues tested are: normal white blood cells (X); PHA-stimulated lym
phocytes (B): human lymphocyte cell line NC-37 (®);lymph nodes,
spleens, and other adult tissues [including liver (A), intestine (O), and
striated muscle (D)]; and fetal tissues [including liver (A), lung (V).
limbs (O), and placenta (D)]. The reactions were then subjected to Cs2SO4
equilibrium density centrifugation as described by Axel el al. (2). The
amount of RLV DNA-3H hybridized to cell RNA is expressed as de
scribed by Hehlmann el al. (11). i, S.D.
extracted from a variety of 51 normal human tissues, in
cluding African biopsies and an EBV-containing lymphoblastoid cell line (NC-37), did not show significant an
nealing to the RLV DNA. These results have been de
scribed in detail (16) and are summarized in Chart 2.
An Analysis of the Significance
Human Neoplasia
of Viral-related
Tumor
Tissue
with Rauscher
DNA
Normal Human Tissues
•¿10-8-M•x
14},
706-Q.0
Cx
5-fO4-3-2-1
RNA in
The existence of viral-related RNA in human neoplasms
does not of course establish a viral etiology for this disease.
One must perform experiments designed to answer the
following questions, (a) How large is the RNA being de
tected? (b) How much homology does it in fact have to the
RLV RNA? (c) Is the viral-related RNA associated with a
reverse transcriptase (RNA-instructed DNA polymerase),
and is it located in structures characteristic of incomplete
or complete virus particles? The requisite techniques re
solving these issues were developed (10, 20-22) and were
applied to extracts of human tumors and normal tissues in
a search for evidence of oncogenic RNA viruses.
The enrichment of possible virus particles is accomplished
by disruption of the cells in the presence of EDTA, to
destroy the ribosomal structures. After removal of
nuclei and mitochondria, the cellular supernatant is centrifuged at 98,000 x g through 20% glycerol in the TNE
1516
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Chart 2. Results of hybridization reactions with RLV DNA-3H and
pRNA from Burkitt's tumors and from normal human cells. The pRNA's
of normal tissues were derived from normal white blood cells (X);
PHA-stimulated lymphocytes (gj); human lymphocyte cell line NC-37
(®). spleens; and other adult tissues [including liver (A), intestine
(O), and striated muscle (D)]; fetal tissues [including liver (A), lung (VK
limbs (O). placenta (D)]; and African control tissues [tonsillitis (A) and
benign mandibular cyst (D)]. The reactions were then subjected to
Cs »SO
4equilibrium density centrifugation. as described by Kufe et al. (16).
CANCER RESEARCH
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RNA Tumor Viruses and Human Lymphomas
milk (21, 22) led to the demonstration of complexes of 70
S RNA and reverse transcriptase in peripheral white
blood cells of 95% of leukemic patients (4) and in 79% of
human breast cancers examined (1). In both of these can
cers, high-molecular-weight RNA was found to be en
capsulated with reverse transcriptase in a particle posses
sing the density characteristic of the RNA tumor viruses.
The Simultaneous Detection of 70 S RNA and Reverse
Transcriptase in Human Lymphomas
Chart 3A shows a representative outcome of a 70 S
RNA: DNA complex synthesized by the pellet fraction
from an involved spleen of a patient with Hodgkin's dis
ease. In certain samples, additional
80r
peaks have been
B
60RNAase
40
70 S RNÄ
52 S RNÄ
!2
10
Fraction
Number
15
30
Fraction Number
100
80
60r
ijO|-
60
70SRNA
40
70 S RNA
O 20
X
f
I
2
10
Fraction
15
20
Number
23
Fraction Number
Chart 3. Detection of 70 S RNA DNA-3H in human lymphoma tissue. A and B, Hodgkin's disease (RP and 263, respectively), C, normal
spleen (24414), D, uninvolved spleen from Hodgkin's disease patient (JG). Five g of tissue were finely minced and disrupted with a Silverson homogenizer at 4°in TNE buffer. This suspension was centrifuged at 4000 x g for IO min at 2°.The resulting supernatant fluid was then layered on a 13-ml
column of 20% glycerol in TNE buffer and spun at 98.000 x g for I hr at 4°in a Spinco SW-27 rotor. The resulting pellet was resuspended in 0.5 ml
0.01 MTris-HCI (pH 8.3), brought to 0.19 Nonidet P-40 (Shell Chemical Co.. New York. N. Y.). and incubated at 0°for 15 min. DNA was synthe
sized in a reverse-transcriptase reaction mixture (final volume, I ml) containing: 50 Amólesof Tris-HCI (pH 8.3): 20 Amóles0.09% NaCl solu
tion; 6 AmólesMgCI2; 100 jumóleseach of dATP, dGTP, and dCTP; and 50 /¿moles
dTTP-3H (50 Ci/mmole). Actinomycin D (50 /jg/ml) was added
to inhibit DNA-instructed DNA synthesis. After incubation at 37°for 15 min, the reaction was adjusted to 0.2 M NaCl and 1% sodium dodecyl
sulfate. An equal volume of a phenol:cresol (7:1) mixture containing 8-hydroxyquinoline (0.2 g/100 ml of mixture) was added, and the final
mixture was shaken for 5 min at 25°.The aqueous phase was then layered over a linear glycerol gradient (10 to 30% in TNE buffer) and centrifuged
at 40,000 rpm for 180 min at 2°.Fractions were collected from below and assayed for trichloroacetic acid-precipitable radioactivity. As shown
in Chart Iß,one aliquot of the product was run directly on the glycerol gradient, while the other aliquot was incubated in the presence of RNase A (50
Mg/ml) and RNase T, (50 jug/ml) for 15 min at 37°prior to sedimentation analysis.
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1973
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Spiegelman,
Kufe, Hehlmann,
and Peters
observed at positions 52 S and 35 S. As shown in Chart 35,
peaks are detected at both the 70 S and 52 S positions. It
is further demonstrated that these complexes are due to
an RNA-dependent reaction, as prior treatment with
RNase eliminates both of the peaks. When equivalent
quantities of either a normal or an uninvolved spleen from
a patient with Hodgkins' disease are analyzed by the same
technique, no incorporation of TTP-3H is detected in a
rapidly sedimenting structure (Chart 3, C and D). Table 1
summarizes the findings with 36 human lymphomas, which
include 28 Hodgkin's disease specimens, 6 lymphosarcomas, and 2 reticulum cell sarcomas (D. Kufe, R. Hehl-
Table 1
Tests for viral-specific RNA in human lymphomas
S"Hodgkins
70
RXN"
Sensitivity'+
disease133109344123660149159112947492206197112622295622142424812516161165913109L
1.2.3.4.5.6.7.8.9.10.11.12.13.14.15.16.17.18.19.20.21.22.23.24.25.26.27.28.1.2.3.4.5.6.I.2.1.2.3.4.5.Tumor249234
++
NT"+
++
++
NT+
node)263274218223241270245252227269218SZ253259229248281283293286284AK2282442972242622542440526829
(lymph
NT-14.4
4_+
NT+
4—4
4+
NT_+
+4
4+
NT+
NT+
NT+
NT4
+__4
4+
NT_+
NT4
vmphosarcoma29542158118524128Reticulum
++
4+
NT+
NT—+
NT+
sarcoma83112av.
cell
80.6%7
positive of 36 =
19.4%MiLvVfWi173Acpm
negative of 36 =
4+
+av.
cpmHypersplenism1741756X199av.
= 302
cpmav.
of negatives = 16
ofcpm4
positives = 371
44
4+
4—= 89 cpmRNase
" Acid-precipitable radioactivity found in the 70 S region of the glycerol gradients, as described in the
legend to Chart 3.
6 RXN, the positivity or negativity of a reaction (designated as positive if the cpm exceed 30).
'Degradation by RNase A and T, of acid-precipitable radioavtivity in the 70 S region of the glycerol
gradients, as described in the legend to Chart 3.
" NT, not tested.
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RNA Tumor Viruses and Human Lymphomas
Table 1—Continued
Diagnosis
Specimen
cpm 70 S°
RXN"
spleens\.2.3.4.5.6.7.8.9.10.11.12.13.14.GlioblaslomaMalignant
Control
melanomaCancer
pancreasMultiple
of
myelomaCancer
lungAnemiaMyocardial
of
infarctionArteriosclerotic
diseaseHodgkins heart
spleen)Myocardial
disease (noninvolved
infarctionMeningiomaMyocardial
infarctionCarcinoma
breastHodgkins
of
disease (noninvolved spleen)2440924408244072440424406244122441324414JG2443524417244322441525014451522194611292
= 14 cpm_-----------—-
mann, W. Peters, A. Fjelde, and S. Spiegelman. Particles
Encapsulating RNA-instructed DNA Polymerase and
High Molecular Weight Virus-related RNA in Human
Lymphomas, in preparation). Included also are 14 uninvolved spleens, with the diagnosis at the time of autopsy,
and 5 cases of hypersplenism. The number of cpm in the
70 S region of the glycerol gradient, as determined by
external size markers, was taken as a measure of the pres
ence and extent of the reaction. The average cpm in the
70 S region for the control series was 14 whereas, in con
trast, the malignant tissues yielded an average of 302. In
view of the low value of the controls, we have arbitrarily
designated as positive any reaction that yielded more than
30 cpm in the 70 S region. With these criteria, all of the
control samples were negative and 80.6% of the malignant
tissues were positive. Fifteen of the positive tumors were
tested for RNase sensitivity of the 70 S DNA complex,
and in each of them the complex was degraded. Note that
3 cases of hypersplenism yielded positive responses,
which were sensitive to RNase. The significance of this
fact and its possible relation to premalignant conditions
requires further investigation.
The demonstration of a 70 S RNA: DNA complex that
is sensitive to RNase argues for the presence of a RNAdependent reaction. However, due to the possibility of
nontemplated end-addition reactions, a more definitive
proof demands that the DNA-3H synthesized be hybridizable to the relevant "oncogenic" RNA template.
One approach to the question is to compare the an
nealing of the DNA-3H with pRNA isolated from tumors
and normal tissues. If the synthesized DNA-3H is specific,
then only lymphomas should contain the complementary
template and possess hybridizable RNA.
Another approach is to hybridize the DNA-3H with 70 S
RNA prepared from RLV or RD-114 virus. Positive
outcomes would be expected from the earlier demonstra
tions that human lymphoma pRNA hybridizes to syn
thetic DNA complementary to RLV RNA (11) and to
RD-114 RNA. Furthermore, on the basis of these DNA:
RNA hybridization studies, the DNA-3H prepared from
lymphoma tissue extracts should possess greater homology with RD-114 RNA than with RLV RNA and, if it
is specific, the DNA-3H should not hybridize to the 70 S
RNA's of the unrelated AMV or RSV.
In order to avoid the loss that accompanies the isola
tion of the DNA synthesized from its 70 S complex in a
glycerol gradient, the total DNA product of an endoge
nous reaction was deproteinized and subjected to Sephadex G-50 column chromatography, and the appropriate
fractions were alcohol precipitated. The resulting nucleic
acid pellet was then extensively alkali digested to destroy
RNA, and the DNA-3H was recovered. The isolation of
cellular pRNA has been described previously (2).
Chart 4A shows a Cs2SO4 equilibrium gradient profile
of an annealing reaction between human DNA-3H product
synthesized from a lymphoma spleen (Table 1, Tumor 228)
and pRNA isolated from the same tumor. It is evident that
approximately 7.5% of the DNA-3H is shifted from the
DNA to the RNA region of the gradient. Upon an
nealing Tumor 228 DNA-3H to an equivalent amount of
pRNA isolated from a normal spleen, we noted no signifi
cant amount of hybridization (Chart 4B), and only 0.5%
of the DNA-3H was detectable in the RNA region of the
gradient.
Chart 5A shows a Cs2SO4 equilibrium gradient profile
of an annealing reaction between DNA-3H from
Hodgkin's disease spleen (Tumor 211) and the RLV 70 S
RNA. It is clear that approximately 10% of the DNA-3H
has shifted to the RNA region of the gradient. Upon an
nealing an equivalent amount of AMV 70 S RNA to the
same DNA-3H, we observed no significant shift to the
RNA or hybrid region (Chart 5B). In view of the finding
that RD-114 DNA-3H hybridizes to pRNA from human
lymphomas to a greater degree than does RLV DNA3H,
it was interesting to determine whether DNA-3H synthe
sized from a lymphoma spleen would hybridize to RD-114
RNA. Chart 5C shows a profile of an annealing reaction
between DNA-3H from a lymphoma spleen (Tumor 228)
and RD-114 RNA. Approximately 36% of the DNA-3H
has shifted to the RNA and hybrid region of the gradient,
over 3 times that observed in similar reactions with RLV
RNA, whereas no significant shift is observed when an
nealing to an equivalent amount of RSV RNA (Chart 5D).
The results obtained from annealing reactions of DN A-3H
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1519
Spiegelman,
Kufe, Hehlmann,
and Peters
20OO r
-.1.8
20
Fraction
Chart
(Tumor
4. Cs2SO« density
profiles
228) (A), and to normal
reactions
24432) pRNA
30
Fraction Number
Number
of annealing
spleen (Tumor
25
of human
lymphoma
(B). A standard
(Tumor
RNA-instructed
228 DNA-3H
to human
DNA polymerase
lymphoma
reaction
spleen
pRNA
was performed
as de
scribed in the legend to Chart 3. except that the glycerol gradient sedimentation step was omitted. The aqueous phase was instead subjected to Sephadex
G-50 column chromatography,
and the DNA-3H was isolated and precipitated with 2 volumes of ethanol. The precipitate was digested in 0.4 M
NaOH for 24 hr at 43°and neutralized.
The DNA-3H product was then annealed to 300 pg of pRNA isolated from the lymphoma spleen and the normal spleen. The hybridization
reac
tion (50 /¿I)was performed, in the presence of 50% formamide and 0.4 M NaCl. After annealing for 24 hr at 37°,the reaction mixture was subjected
to Cs2SO4 density analysis.
synthesized in human lymphomas with cellular and viral
RNA's are summarized in Table 2.
The Simultaneous
Detection
of 70 S
Reverse Transcriptase in Burkitt's Tumors
RNA
and
The data described thus far indicate that human lym
phomas contain (a) particles that encapsulate reverse
Chart 7, A, B, and C shows representative 70 S RNA:
transcriptase and (b) a 70 S RNA related to that of RLV DNA complexes synthesized by the pellet fractions of
and RD-114. In order to determine whether this particle biopsy specimens of Burkitt's tumors (17). In certain
possessed the density characteristic of an RNA tumor samples, additional peaks have been observed at po
virus, we prepared the pellet fraction from human lym sitions of 35 S. It is further demonstrated (Chart 1C) that
phomas and subjected it to sucrose equilibrium centrifuga- these complexes contain a 70 S RNA molecule, since prior
tion on a linear gradient of 20 to 60% sucrose. The gradient treatment with RNase eliminates the peak. When equiv
was then divided into 10 equal fractions that were diluted alent quantities of peripheral white cells of a patient with
to 15% sucrose and again spun at 100,000 x g for 1hr.
infectious mononucleosis were analyzed by the same tech
We then carried out simultaneous detection tests on the nique, we detected no incorporation of TTP-3H into a
pellet from each fraction to determine the distribution in rapidly sedimenting structure (Chart ID).
Table 3 summarizes the findings with 11 Burkitt's tu
the density gradient of 70 S RNA-instructed DNA-synthesizing activity. Chart 6 shows that the particles mors and indicates the site of involvement, whether the
possessing the reverse transcriptase and its 70 S RNA tumor was primary or secondary, and the modality of
template localize at a density of 1.23 g/ml, the density chemotherapy. The 70 S region of the glycerol gradient
characteristic of cores of the oncogenic viruses. Six lym was located with the aid of external size markers, and the
phomas were studied in this manner (R. Hehlmann, D. number of cpm included in it was taken as a measure of
the presence and extent of the reaction. The Burkitt's tu
Kufe, and S. Spiegelman. The Density of RNA-containing
Particles in Human Lymphomas, in preparation), and mors as a group yielded an average of 304 cpm in the 70 S
the 70 S RNA reverse-transcriptase activity was localized region, compared with an average of 11 cpm for nonBurkitt control material listed in Table 4. On the basis of
at either the 1.16 or 1.19 g/ml density region (charac
teristic of the complete virus particle), or at the "core" the low average for negatives, we have arbitrarily desig
nated any reaction yielding more than 30 cpm in the 70 S
density region of 1.23 to 1.26 g/ml.
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RNA Tumor Viruses and Human Lymphomas
5
10
15
Fraction
20
25
30
5
Number
10
15
Fraction
20
25
SO
Number
300
DNA
240
240
120
5
IO
15
Fraction
Chart 5. Annealing
obtained
and hybridized
reaction of lymphoma
as described
20
25
30
RNA.
in the legends to Charts 2 and 3.
Lymphoma
DNA-3H
pRNA1.
Tumor
+4.241
+5. 224
tested."RD-114
NT, not
RNA.CRSV
RNA.Normalspleen
JUNE 1973
20
25
Number
equilibrium
was
gradient centrifugation.
With these criteria,
2 of the Burkitt's
tumors listed in Table 3 were negative, yielding an average
of 14 cpm, and 9 were positive, with an average of 364
cpm in the 70 S region. Six of the positive tumors were
tested for RNase sensitivity of the 70 S RNA: DNA
complex, and in every instance the complex was destroyed.
Four more Burkitt's tumors were examined (Table 6) and
pRNA_NT—_._-RLV
RNA++NTNTNTNT+
RNA__NTNTNTNTc
+2. 234
NT"3.
211
+6.229
+7.286
+0
228
15
reactions were analyzed by CsjSOt
region as positive.
Hybridization of lymphoma DNA-3H product to tumor pR NA and
normal spleen pRNA, to RL V and AMV RNA, or lo RD-I 14
and RSV
Fraction
DNA-3H to I jig of 70 S RNA from (A) RLV, (B) AMV, (C) RD-I 14, and (D) RSV. The DNA-'H
in the legend to Chart 4. The annealing
Table 2
As described
IO
Number
"AMV
found to be positive. Thus, 87% of the Burkitt samples
included in this survey gave clear positive responses.
Table 4 lists a set of 6 non-Burkitt samples examined
by the simultaneous detection test. These were chosen as
controls for examination, since they are particularly rele
vant to the relation of EBV to the RNA particles detected
in the Burkitt's tumors. The 1st is derived from the
peripheral white blood cells of a patient with infectious
mononucleosis,
a self-limiting
nonneoplastic
condition
in which prospective studies (13, 19) have strongly impli-
1521
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Spiegelman, Kufe, Hehlmann, and Peters
-x^^-XM2
S
o40
ÃŽ3330
<"20
S?IO0
4XE
o
Its behavior in glycerol gradients and its response to
RNase demonstrate the RNase-sensitive synthesis of an
RNA DNA-3H complex, with a sedimentation coefficient
21/1.27r~1.25°=l.23->-1.201.17
1 1.14
1.0712345678-,60---«.X1.01950 [
cated the EBV. The 2nd is a cell line derived from the
lymphocytes of an infectious mononucleosis patient. This
line and the remaining 4 lymphoblastoid lines listed are
known to contain the EBV genome, on the basis of the
presence of virus-associated antigens (14). All of these lines
were negative for the 70 S RNA-directed DNA polymerase. It is clear that the existence of EBV information is not
mandatorily linked to the detectable presence of the RNA
particles found in Burkitt's tumors.
,
II
Chart 6. Localization of 70 S RNA reverse-transcriptase complexes
isolated from Hodgkin's spleen on a sucrose density gradient. The amounts
of DNA-3H (cpm) sedimenting in the 70 S region of each glycerol gra
Fractions
dient are plotted in the form of a histogram.
28S I8S
lOO
ÃŽOOrA
50
200
100
50
70S
70S
l
Froction
Fraction
Number
Number
200r C
8Or D
100
60
50
40
20
—¿
»•^•«^f^»
IO
Froction
15
20
Number
«»f »«•¿
«^
15
25
Fraction
20
25
30
Number
Chart 7. A, B, and C, detection of 70 S RNA:DNA-3H in Burkitt's lymphoma tissue: D, peripheral white blood cells from a patient with acute
mononucleosis. The procedure is as described in the legend to Chart 3. In C, one aliquot of the product was run directly on the glycerol gradient, while
the other aliquot was incubated in the presence of RNase A (50 jig/ml) and RNase T, (50 Mg/ml) for 15 min at 37°prior to sedimentation analysis.
1522
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VOL. 33
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RNA Tumor Viruses and Human Lymphomas
Table 3
Simultaneous detection of 70 S RNA and reverse transcriptase in Burkitt's lymphomas
Preparation of tumor pellet fractions and assay for synthesis of 70 S RNA: DNA-3H complex is as described in the legend to Chart 3. The sum of
the cpm in the 70 S position monitored by external size markers is recorded and is designated as positive if the cpm exceeds 30. RNase sensitivity de
picts abolition by RNase A and T, of acid-precipitable radioactivity in the 70 S region of the glycerol gradients, as described in the legend to Chart 3.
The clinical data, including modality of chemotherapy at the time of biopsy, are self explanatory.
RNase70
or
RXN1239
S
Sensitivity
Name1.2.3.4.5.6.7.8.9.10.11.AnNaluLuIrKeAmAlPaPwNaAge55757441912SexFFMFMMFFFMFSiteOvaryOvaryOrbitOvaryAscitesMesenteryOrbitOvaryRight
secondaryInitialInitialRelapseRelapseInitialInitialRelapseInitialInitialInitialInitialTreatmentNoneNoneCTX,°
+196
+786
VCRCTXNoneNoneCTXNoneVCR,
MTX,
+243
+100
+111652
+224
+430
+48
ORTNoneNonecpm
mandibleCervical
lymphnodeOvaryPrimary
+
+
+
+
+
+
NT
NT
NT
av. = 304 cpm
av. of positives = 369 cpm
av. of negatives = 14 cpm
9 positive of 11 =
2 negative of 11 = 18%
"The
tested.
abbreviations
used are: CTX,
+
cyclophosphamide;
MTX,
methotrexate;
VCR,
vincristine: ORT,
orthomerphalan;
NT,
not
Table 4
Simultaneous detection of 70 S RNA and reverse transcriptase in peripheral white blood
cells from a patient (SL) with infectious mononucleosis and in lymphoblasloid cell lines
The lymphoblastoid cell lines have been shown to contain the EBV genome on the basis of ex
pression of early antigen, viral capsid antigen, or membrane antigen ( 14). Preparation of cellular
pellet fractions and assay for synthesis of 70 S RNA:DNA-3H complex are as described in the
legend to Chart 3. The sum of the cpm in the 70 S position, determined by external size markers,
is recorded and is designated as positive if the cpm exceeds 30.
70S111268719RXN_-___
mononucleosisInfectious
1.2.3.4.5.6.CellsSL833-LF265L33-I-I6-I9303-LNC-37DonorInfectious
mononucleosisNormalNormalNormalNormalcpm
av. cpm = 11
of 70 S in extracts of Burkitt's tumors. However, due to the
possibility of nontemplated end-addition reactions, more
definitive evidence again demands that the DNA-3H
synthesized be hybridizable to its presumed RNA tem
plate.
One approach to the resolution of such issues is to com
pare the annealing of the DNA-3H with cytoplasmic pRNA
isolated from Burkitt's tumors and normal tissue. If the
DNA-3H synthesized by the Burkitt's particles is tumor
specific, then Burkitt's tumors should (and normal tissue
should not) possess hybridizable RNA. Another pathway
depends on our previous (16) findings that Burkitt's tu
mors contain RNA homologous to that of the RLV. Con
sequently, if the DNA-3H synthesized by the Burkitt's tu
mor particles is instructed by an RNA related to that of the
RLV, hybridization should occur with the RLV RNA
and not with the unrelated AMV RNA.
JUNE 1973
We prepared DNA-3H product complexed to 70 S
RNA by performing an endogenous RNA-instructed
DNA polymerase reaction with the pellet fraction from a
Burkitt's tumor. Following velocity centrifugation of the
reaction product, the DNA-3H:RNA
complex sedimenting in the 70 S region of the glycerol gradient was
isolated by being pooled, precipitated with alcohol, and
then analyzed on a Cs2SO4 gradient (Chart 8/4). Some
DNA is released during the manipulation, but most of the
product bands in the RNA density region, which indicates
that most of the DNA molecules remain complexed to
large RNA molecules. Following alkali digestion to re
move the RNA, the resulting purified DNA-3H was
annealed to 70 S RNA's prepared from RLV and AMV.
The outcomes of the annealing reactions were analyzed
in Cs2SO4 gradient (Chart 8). It is clear from Chart 85
that the Burkitt's DNA-3H is unable to hybridize to
1523
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
Spiegelman, Kufe, Hehlmann, and Peters
,
50
Ë
IO
Fraction
Number
100
IO
Fraction
15
20
25
30
35
Number
25
Fraction
30
Number
Chart 8. Characterization of 70 S RNA: DNA-3H complex from abstracts of Burkitt's tumors. A simultaneous detection assay was performed on
the pellet fraction from a Burkitt's tumor (An) and analyzed by glycerol velocity centrifugation as described in the legend to Chart 3. Aliquots of the
glycerol gradient fractions were assayed for trichloroacetic acid-precipitable counts, and the 70 S RNA: DNA-3H complex was pooled and precipitated
with 2 volumes of ethanol in the presence of yeast-carrier RNA (10 ng/ml final concentration). After centrifugation at 10,000 x g for 30 min at -20°,
the precipitate was resuspended in TNE buffer and divided into 3 aliquots. Aliquot I (A), was added directly to 11 ml of half-saturated cesium sulfate
(Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, N. Y.) in 0.003 M EDTA (p = 1.52 g/ml) and centrifuged in a Spinco 50 Ti rotor at 44,000
rpm for 60 hr at 15°.Aliquots 2 and 3 were digested in 0.4 MNaOH at 43°for 24 hr, neutralized with HC1 in the presence of 0.01 MTris-HCl, pH 7.4,
and brought to 50% formamide (Eastman Organic Chemicals. Rochester, N. Y.). After heat denaturation for 10min at 80°.Aliquot 2 was hybridized to
0.1 j£g
AMV RNA (A), and Aliquot 3 was hybridized to O.I /¿gRLV RNA (C) in the presence of 50% formamide and 0.4 M NaCI for 18 hr at 37°.
The annealing reactions were then analyzed by Cs2SO4 density analysis.
Table 5
Hybridization of Burkin's tumor DNA-3H produci lo 300 fig of
Burkill's tumor pRNA, to 300 ng of normal Ivmph node pRNA,
to O.I ng ofRLV RNA, and to O.I ng of AMV RNA
The hybridization reaction (50 ¿¡1)
was performed in the presence of
50% formamide and 0.4 M NaCI. Annealing conditions and Cs2SO4
gradient analysis are described in the legend to Chart 4.
tumorpRNAAn
BurkittDNA-3H1.
node
RLV
RNA-pRNA
RNA+NT
An2.
Na3.
Ny's
++NT"NTLymph
; Na
+NT
+AMV
" NT, not tested.
AMV RNA but does complex to RLV RNA (Chart 8C).
Table 5 summarizes the results of annealing reactions
with DNA-3H's synthesized by particles from 3 different
Burkitt's tumors. In all cases, specific hybridizations
occur only with RLV RNA or Burkitt's tumor RNA. This
outcome is logically consistent with our earlier demon
1524
stration (16) that Burkitt's tumor RNA hybridizes to DNA
complementary to RLV RNA but not to DNA comple
mentary to AMV RNA.
The data thus far described indicate that Burkitt's tu
mors contain both particles that encapsulate reverse
transcriptase and a 70 S RNA related in sequence to
that of the RLV. It was of interest to see whether the par
ticles possessed the density characteristic of an RNA tumor
virus. To this end, a pellet fraction was prepared from a
Burkitt's tumor and subjected to equilibrium centrifuga
tion in a linear gradient of 15 to 55% sucrose. The gradient
was then divided into 10 equal fractions that were diluted
to 15% sucrose and again spun at 100,000 x g for l h r.
Simultaneous detection tests were then carried out on the
pellet from each fraction to determine the distribution
in the density gradient of 70 S RNA-instructed DNA syn
thesizing activity. Chart 9 shows that the particles pos
sessing 70 S RNA-instructed DNA polymerase localize
within a density of 1.16 to 1.19, the density range charac
teristic of the oncogenic RNA viruses. As summarized in
Table 6, 3 Burkitt's tumors and 1 African histiocytic lym-
CANCER
RESEARCH
VOL. 33
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RNA Tumor Viruses and Human Lvmphomas
1.25.
120
1.15-^
LIO :
~|2
4
o
(O
,X11
A
1Ev\i—s,Nk\
IIi11///I»
E
Q.
\1\\ni
^^' A
—¿
y
i a su E x
I
Fraction
Number
Chart 9. Sucrose gradient localization of 70 S RNA and RNA-instructed DNA polymerase activity in extracts of Burkitt's tumors. A pellet
fraction was prepared from Burkitt's tumor (Ny), as in the legend to
Chart 3. The pellet was resuspended in TNE buffer and layered on
linear gradient of 15 to 55% sucrose in TNE and spun in a Spinco SW-27
rotor at 4°for 210 min. The gradient was dripped from below through a
recording Gilford spectrophotometer
at A2<0. and 10 equal fractions
were collected. Each fraction was diluted with TNE to a sucrose con
centration of less than 15% and then was spun at 100,000 x g. The pel
let obtained from each of the 10 fractions was then subjected to the simul
taneous detection assay (as described in the legend to Chart 3), and the
amount of 70 S RNA:DNA-3H
synthesized from the 10 different den
sity regions was determined
by glycerol velocity centrifugation.
phoma were analyzed in a similar manner, and all of them
gave the same results.
Discussion
As in our previous studies with human leukemia (4)
and breast cancer (1), the experiments described herein
Sucrose gradient localization
The amount
of 70 S RNA:DNA-3H
were performed to elucidate the possible etiological sig
nificance of our earlier (11) detection in human lymphomas,
including Burkitt's tumors (16), of RNA uniquely homolo
gous to that of the RLV. The data obtained show that at
least a portion of the RNA we found exists in the form of
a 70 S RNA associated with an RNA-instructed DNA
polymerase in a particle with a density between 1.16 and
1.19 g/ml. The particles thus identified in the cells from
both of these cancers have at least 3 of the biochemical
and physical features diagnostic of the RNA tumor viruses.
Of further interest was the demonstration that the
DNA-3H synthesized by the lymphoma or the Burkitt's
tumor reverse transcriptase on its own endogenous tem
plate hybridizes specifically to lymphoma pRNA, RLV
RNA, or RD-114 RNA. The lack of response to AMV
RNA and RSV RNA argues against the possibility that
complexing is due to polyadenylate stretches found in
RNA tumor viruses (5, 8, 18). The successful specific
hybridization to RLV RNA or RD-114 RNA by the
DNA-3H synthesized by the tumor enzyme and its RNA
template are consonant with the reasoning of the earlier ex
periments (9, 11) in which DNA synthesized from RLV
RNA or RD-114 RNA were used as probes to find
viral-related information in human lymphomas and in
Burkitt's disease.
Twenty-nine of the 36 lymphomas examined gave evi
dence of the presence of the 70 S RNA:reverse-transcriptase complexes, whereas none of the 14 uninvolved
spleens yielded positive responses. It should be empha
sized that negative outcomes with the neoplastic or control
samples cannot be accepted as evidence for the absence of
the relevant reaction. In fact, 3 cases of hypersplenism
with no overt evidence of cancer yielded positive 70 S re
actions. On the basis of our studies thus far, it appears
that certain nonmalignant states may show evidence of a
positive reaction leading to the formation of a 70 S RNA:
DNA complex. It is relevant to recall that we reported (21)
the presence of 70 S RNA and reverse transcriptase in
the milk of normal cancer-free women.
Of the 15 Burkitt's tumors examined, 13 (or 87%) gave
unambiguous evidence of the presence of 70 S RNA : RNAinstructed DNA polymerase complexes. Of particular inter
est are the negative results with the 6 types of cells reported
of 70 S RNA
Table 6
and RNA-instructed
Burkitt's tumors
synthesized
by an endogenous
DNA polymerase
RNA-instructed
activity in extracts of
DNA
polymerase
was determined
by glycerol velocity centrifugation
for each of 10 sucrose density fractions, as described in the legend to Chart 3. The density
fraction or fractions yielding the maximum number of cpm in the 70 S region are listed. The reaction is designated as posi
tive if the cpm in the 70 S region exceeds 30.
The clinical data, including modality of chemotherapy
at the time of biopsy, are self explanatory.
Name1.
Na2.
II3.
Ny4.
Oy"Age1110128SexFFFMSiteRetroperitonealOvaryOvarySpleenPrimaryorsecondaryResistantInitialInitialInitialTreatmentCTX"NoneNoneNonecpm70S13216016347633Densityregi
' CTX. cyclophosphamide.
1 Histiocytic lymphoma.
JUNE 1973
1525
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Spiegelman, Kufe, Hehlmann, and Peters
logical and Biological Studies on a Virus in Cultured Lymphoblasts
in Table 4. Numbers 2 to 6 are known to contain EBV in
from Burkitt's Lymphoma. J. Exptl. Med., 121: 761-770, 1965.
formation, on the basis of hybridization or the possession of
8.
Gillespie,
D.. Marshall, S., and Gallo, R. C. RNA of RNA Tumour
early, virus coat, or membrane antigens associated with EBV
Viruses
Contains
Poly A. Nature, 236: 221 231, 1972.
(14). The 1st 2 control cells listed were derived from pa
9. Gulati, S. C., Axel. R., and Spiegelman, S. Detection of RNA-in
tients with infectious mononucleosis, a nonneoplastic
structed DNA Polymerase and High Molecular Weight RNA in
disease implicated with the EBV (R. Hehlmann, D.
Malignant Tissue. Proc. Nati. Acad. Sei. U. S., 69: 2020 2024, 1972.
Kufe, and S. Spiegelman. The Density of RNA-con- 10. Hehlmann, R., Kufe, D., and Spiegelman. S. RNA in Human
taining Particles in Human Lymphomas, in preparation).
Leukemic Cells Related to the RNA of a Mouse Leukemia Virus.
Proc. Nati. Acad. Sei. U. S., 69: 435-439, 1972.
If a more extensive study confirms this pattern in infec
tious mononucleosis, evidence would be provided iden 11. Hehlmann. R., Kufe, D., and Spiegelman, S. Viral-related RNA in
Hodgkins' Disease and Other Human Lymphomas. Proc. Nati.
tifying the RNA particles as unique components of neoAcad. Sei. U. S., 69: 1727 1731, 1972.
plastic tissues. In any case, it is evident from the results
shown in Table 4 that EBV information can be present in 12. Henle, G., Henle, W., Clifford. P., Diehl, V.. Kafuko, G., Kirya, B.,
cells that lack the type of RNA particles found in Burkitt's
Klein, G. Morrow, R., Munube, G., Pike. P.. Tukel, P., and Ziegler,
J. Antibodies to Epstein-Barr Virus in Burkitt's Lymphoma and
tumors, leukemic cells, and breast cancer.
Control Groups. J. Nati. Cancer Inst., 43: 1147 1157, 1969.
The presence in human lymphomas (11), including 13. Henle, G„Henle. W., and Diehl, V. Relation of Burkitt's TumorBurkitt's tumors (16), of RNA related to that of the RLV
suggested the involvement of a viral agent in these diseases.
The data presented here further strengthen this implica
tion by showing that cells from both of these diseases con
tain a viral-related 70 S RNA associated with a reverse
transcriptase, 2 uniquely identifying characteristics of
the RNA tumor viruses. Thus for leukemia (4), breast
cancer (1) and, now, for human lymphomas including
Burkitt's disease, the evidence for involvement of an RNA
tumor virus in human neoplasia is becoming more com
pelling.
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CANCER
RESEARCH
VOL. 33
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Evidence for RNA Tumor Viruses in Human Lymphomas
Including Burkitt's Disease
S. Spiegelman, D. Kufe, R. Hehlmann, et al.
Cancer Res 1973;33:1515-1526.
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