Download Synthesis of a Viral Protein with Molecular

[CANCER RESEARCH 42, 681-686.
0008-5472/82/0042-OOOOS02.00
February 1982]
Synthesis of a Viral Protein with Molecular Weight of 30,000 (p30) by
Leukemic Cells and Antibodies Cross-Reacting with Simian Sarcoma
Virus p30 in Serum of a Chronic Myeloid Leukemia Patient1 2
Jos P. A. Derks,3 Lilian Hofmans, Hans W. Bruning, and Jon J. v. Rood
Department of Immunohaematology.
Bloodbank, Building 23. University Hospital, Riinsburgerweg
ABSTRACT
We studied a patient with a Philadelphia chromosome-posi
tive chronic myeloid leukemia, who died in relapse after multiple
transfusions and grafting with bone marrow from his monozygotic twin brother (referred to as "donor" in this paper). We
present data indicating that this patient may have had a retrovirus infection and that this virus is related to the group of
exogenous primate type C retroviruses.
Antibodies to simian sarcoma virus (SSV) M.W. 30,000 pro
tein (p30) but not endogenous feline virus RD-114 could be
found in patient but not donor serum. Patient but not donor
cells were able to actively synthesize a p30 protein that could
be precipitated with patient serum and rabbit anti-SSV p30 but
not with donor serum or rabbit anti-RD-114 p30. Patient p30
resembles SSV p30 but not RD-114 p30 in peptide mapping
by limited proteolysis and subsequent slab gel electrophoresis.
Patient but not donor cells were able to actively synthesize a
M.W. 78,000 protein that could be precipitated with goat antiSSV. An enzyme with properties of reverse transcriptase was
increased 30-fold in patient cells when compared with donor
and other control cells.
Related to the presence of widespread infectious agents may
be the finding that, in the course of the patient's disease, donor
serum showed increasing amounts of possibly ¡mmunoregulatory (Cancer Research, submitted for publication) antibodies,
reactive with autologous and, more effectively, with patientderived cell membrane M.W. 80,000 protein (a possible idiotypic receptor structure) and M.W. 94,000 protein (a T-cell
alloantigen).
INTRODUCTION
Various lines of evidence indicate that a retrovirus may be
involved in at least some human cancers. Important contribu
tions in the field of immunology came from studies with antisera
to retroviruses and viral structural proteins. Special attention
was paid to viral p30," which is coded for in the gag region of
Received Septembers, 1980; accepted October 23, 1981.
' This study was supported by the Queen Wilhelmina Foundation, the Dutch
Cancer Research Fund (Project LUKC-l-76.33).
2 This article is one of a series of two. Paper 2, submitted to Cancer Research,
is entitled, "Molecular Characterization of the TCA-1 Alloantigen on T-Cells of a
Leukemic Patient with Serum from a Monozygotic Twin Brother."
3 To whom requests for reprints should be addressed.
" The abbreviations used are: p30, a viral protein with a molecular weight of
30,000; gp70, a viral glycoprotein with a molecular weight of 70,000; SSV,
simian sarcoma virus; p23,30, a B-cell antigenic complex containing protein
subunits with a molecular weight of 23,000 and 30,000 respectively; SPA,
Sepharose-CL-4B
Protein-A (Pharmacia, The Hague, The Netherlands); RD,
endogenous feline virus RD-114; NGS, normal goat antiserum; NHuS, normal
human serum; NRS, normal rabbit serum; FCS, fetal calf serum; RPMI, Roswell
Park Memorial Institute medium; p12, p45, p75, p78, p80, p94, membrane
FEBRUARY
1982
10, 2333 AA Leiden, The Netherlands
the viral RNA, and gp70, coded for in the env region, because
they possess, among others, interspecies-specific
determi
nants. The occurrence of these antigenic determinants in hu
mans is possible since some antigenic determinants of the p30
polypeptide found in different Old World Monkey species have
not undergone extensive evolutionary divergence (21). Re
cently, it was shown that antibodies cross-reacting with type C
viral p30 can be found in human sera and exúdate fluids (12).
Conflicting data as to whether (23) or not (see reviews in Refs.
8 and 9) anti-gp70 antibodies occur in human sera can be
found in the literature. High titers of precipitating antibodies to
SSV gp70 have been reported to occur in human sera of
healthy individuals and patients with neoplastic diseases (23).
However, it was convincingly shown that these anti-gp70 anti
bodies are formed in response to cellular modification of glycoproteins rather than as a consequence of exposure to virus
(2, 22). Human sera in those studies appeared not to recognize
viral gp70 produced in human cells. Nevertheless, the possi
bility that gp70 may be found in virally infected human cells
with the help of heterologous anti-gp70 antisera is not ruled
out. It has been reported that, using heterologous antisera,
mammalian type C RNA viral gp70, gp45, and p30 were
isolated from normal and leukemic bone marrow culture supernatants (18).
Related to a viral etiology may be the finding that antibodies
cytotoxic for human leukemia cells have been found in un
treated leukemia patients (6, 14) and in healthy individuals (for
both related and unrelated donors) (5, 7).
We performed immunological and chemical studies to eluci
date the antigenic differences between a chronic myeloid leu
kemia patient and his monozygotic twin brother. The possibility
of a viral etiology of the disease was considered. Therefore,
antisera to retroviruses and viral structural proteins and both
cells and sera of the patient and his twin brother were thor
oughly investigated with a variety of techniques. A difference
in the antigenic profiles of the cell membranes was shown to
be associated with a difference in outgrowth of functional Tlymphocytes.2 In this paper, we show that other antigenic
differences may have resulted from a retrovirus infection in at
least some of the leukemic cells of this particular patient.
MATERIALS
AND METHODS
Sera. Alloantisera to HLA-A, B, and DR antigens which are used in
routine HLA typing were also used in radioimmunoprecipitation
exper
iments. Rabbit anti-p23,30, kindly provided by Dr. J. L. Strominger
(Biology Laboratory Harvard University, Cambridge, Mass.), was pre
pared by immunizing rabbits with the purified B-cell-specific antigen
proteins with molecular weights of 12,000. 45.000,
and 94,000, respectively.
75,000.
78,000,
80.000,
681
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J. P. A. Derksetal.
complex p23,30 (24). This antiserum recognizes the total B-subset of
lymphocytes. A rabbit antiserum to HSB cells (a lymphoblastoid T-cell
line) is a strong anti-T-cell antiserum containing only small amounts of
B-cell-specific
antibodies. A rabbit antiserum to human /52-microglobulin (was supplied by Dako, Copenhagen, Denmark). Goat antiSSV was obtained from the National Cancer Institute (Bethesda, Md.)
(Serum 3S-0172). It had been prepared by immunizing a goat with
disrupted SSV particles propagated in the NC37 cell line. According to
the National Cancer Institute, the serum showed only one single band
when tested in immunodiffusion against SSV antigens. It showed 2
bands when tested against NC37 cellular antigens. The SPA-binding
lgG2 fraction of the absorbed serum did not contain reactivity toward
SSV p30 in a radioimmunoprecipitation.
We absorbed the serum twice
with cell membranes of 10s uninfected NC37 cells per ml antiserum to
remove all aspecific activity in radioimmunoprecipitation.
Rabbit
SSV p30 and rabbit anti-RD p30 were prepared by immunizing
Zealand White rabbits with purified viral p30s. The rabbit anti-SSV
and rabbit anti-RD p30 antisera were kindly provided by Dr. P.
antiNew
p30
Her-
brink, State University of Leiden, Leiden, The Netherlands (12). NGS,
NHuS, NRS, and PCS were from Flow Laboratories, Irvine, Scotland.
Cells. Mononuclear cells were prepared from fresh heparinized
blood by centrifugaron
on Ficoll-lsopaque and by washing the interphase cells 3 times with 10% FCS-RPMI (v/v). These leukocytes were
either used directly for radiolabeling with [35S]methionine or stored in
liquid nitrogen for later experiments.
The conditions
for cryopreserva-
tion are those described by van Lambalgen ef al. (28), using 10%
dimethylsulfoxide-25%
FCS-RPMI as a freezing medium.
Preparation of [35S]Methionine-labeled Antigens. Antigen prepa
rations were obtained by culturing 5 x 106 to 5 x 107 leukocytes in
dependent voltage settings (26).
Calibrating proteins to determine molecular weights were bovine
serum albumin (M.W. 67,000), ovalbumin (M.W. 45,000), a-chymotrypsinogen (M.W. 25,000), and lysozyme (M.W. 14,000).
Fluorography. For fluorography, the gels were consecutively equil
ibrated with dimethyl sulfoxide, impregnated with PPO by immersion in
4 volumes 20% (w/w) PPO in dimethyl sulfoxide, soaked in water,
dried onto Whatman No. 3MM chromatography paper (0.33 mm thick),
and exposed to preflashed (15) Kodak XR-1 X-Omat R film at 70°by
the procedure described by Bonner and Laskey (3). Development was
performed in Kodak DX-80 developer. The films were then scanned in
a densitometer. We did not quantitate the radioactivity that was precip
itated with the different antisera since the amounts of specifically
precipitated radioactivity were very small, and a sample for counting
had unacceptably high error levels.
Peptide Mapping. Peptide mapping was performed according to the
technique described by Cleveland et al. (4). Shortly, radioactive protein
bands of interest were cut from a preparative gel and subsequently
applied directly onto a 15% polyacrylamide slab gel in the presence of
a proteolytic enzyme. Partial proteolysis occurs during reelectrophoresis, thereby generating characteristic banding patterns. Chymotrypsin (65 units/mg) and pepsin (2500 units/mg) were purchased from
Worthington Biochemical Corp., Freehold, N. J. Staphy/ococcus aureus V8 protease (500 units/mg) was obtained from Miles Laboratories
(Slough, England).
Reverse Transcriptase Assay. The levels of reverse transcriptase
present in particles with the density of retroviral cores were assayed
as described by Muyen (30). Briefly, the amount of [3H]TTP incorpo
rated by particles denser than 1.22 g/cu cm, derived from 107 blood
10 ml S-Minimal Essential Medium (GIBCO, Paisley, Scotland) without
methionine, with 20 ID penicillin, 20 fig streptomycin,
20 jug glucose,
and 1 ml heat-inactivated
dialyzed FCS, in the presence of 1 mCi
[35S]methionine (The Radiochemical Centre. Amersham, United King
leukocytes by detergent treatment, was calculated,
thymidylate-polyriboadenylate
as a template.
dom) for 20 hr. The cells were then washed and lysed by freezing and
thawing 3 times in the presence of DNase and 2500 klU Trasylol, and
subsequently centrifuged at 600 x g to remove the cellular debris. The
plasma membranes and small particles were isolated by ultracentrifu-
quent testing for antiviral p30 activity were performed by Herbrink (12).
Briefly, antiviral antibodies were concentrated by affinity chromatog
raphy using disrupted SSV particles that were immobilized on Sepha-
gation at 70,000 x g of the resulting supernatant and extracted in 1 ml
0.5% (w/v) NP-40 for 0.5 hr at 0°in the presence of 250 klU Trasylol
and 10% (v/v) FCS-RPMI. The extract was clarified
gation at 70,000 x g.
lodination Procedure.
by ultracentrifu-
125Iradiolabeling was performed using the
chloramine-T technique (10), starting with 2 jug of purified viral p30s or
NP-40 extracts of 5 x 106 leukocytes and 200 jiCi ' 25I(The Radiochem
ical Centre). The inorganic reactants after radiolabeling were removed
with small quantities of Dowex 1-X2-100 on small columns, followed
by overnight dialysis. The specific activity was in range of 5 x 105 to
107 cpm//ig of protein.
Immunoprecipitation.
The NP-40 lysates were pretreated with SPA-
bound IgG of NGS, NHuS. and NRS to absorb the radiolabeled proteins
that might interact aspecifically with the IgG of the antisera. Specific
precipitation of radiolabeled antigens with antisera was carried out by
mixing the pretreated NP-40 lysate of 2 to 5 x 10s cells with the SPAbound IgG of 5 to 10 /ul of an antiserum.
The antibody-antigen
com
plexes were eluted from the SPA beads with diluted acetic acid, pH
2.9. The eluate was lyophilized and subsequently resuspended in 50
/jl Laemmli sample buffer. Some aspecific binding of radioactive ma
terial to IgG could not, however, always be prevented. Experiments
with normal control sera indicate the level of this aspecific binding.
Gel Electrophoresis. The antibody-antigen preparations were ana
lyzed by sodium dodecyl sulfate-polyacrylamide
slab gel electrophoresis according to
were heated for 5
onto 10 to 12.5%
experiments were
(17-20°), protein
the method of Studier (26). The antigen preparations
min in a boiling water bath and subsequently applied
polyacrylamide slab gels (80 x 140 x 2 mm). The
carried out under standard conditions of temperature
load (overloading was prevented by not using more
than 10 fil of serum for precipitation),
682
pH, salt concentration,
using oligodeoxy-
Detection of Antiviral p30 Antibodies in Human Sera. Concentra
tion of antibodies cross-reacting with type C viral antigens and subse
rose beads. Bound antibodies were eluted and quantitated by incuba
tion with '"l-labeled
viral p30s and precipitation of antigen-antibody
complexes with S. aureus Cowan I. The viral p30s used in this study
were obtained from and described by Herbrink (12). The p30 prepa
rations were pure as determined by NH2-terminal amino acid analysis
and sodium dodecyl sulfate-polyacrylamide
gel electrophoresis. Upon
aging after radiolabeling, these preparations showed decreasing antigenicity (12) and increasing amounts of low-molecular-weight
degra
dation components.
RESULTS
Monozygocity was established by phenotyping patient and
brother for 21 polymorphic gene markers (P < 0.006). The
HLA phenotype was A2, Aw33, B14, B15.2, W6, Cw3, DR5,
DR7. Granulocyte antigens 5B, NA1, NA2, NB, and NCI could
be detected only on donor cells.
The reverse transcriptase activity in the cells was assayed
according to and by van Muyen ef al. (30), who described and
Table 1
Reverse franscr/pfase activity: amount of fHJTTP incorporated in the
trichloroacetic acid-precipitable material during 30 min at 37°by the 1.22 g/cu
cm fraction of 10' blood leukocytes calculated, using o/igodeoxythymidylatepolyriboadenylate
as a template
Total no. of leukocytes/cu mm
Patient in blast
crisisPatient
remissionPatient
in
relapseDonor23,0001,4001
in
% of blasts
cpm/10'
cells
70,000700900112,0003,00075,0001,500
and time-
CANCER
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VOL. 42
p30 Protein and Antiviral p30 Antibodies in Chronic Myeloid Leukemia
Table 2
Anti-type C viral p30 antibodies in eluates obtained from SSV Sepharose beads
precipitatedChronic
% of input counts
centra
tion fac
tor815
p3021.2
myeloid leukemia patient
Bone marrow donor
BackgroundSSV
Concentration
9.07.0
250
9.04.6
±1.4CCon
±0.8°RDp3011.7
/il starting serum
factor
jil eluate
X [efficiency of concentration
procedure (40%)]
" Average ±S.D. of 40 experiments.
c Average ±S.D. of 29 experiments.
gens. Using NRS as a negative control and the aspecifically
bound actin (M.W. 45,000) as a positive internal standard, we
were able to show that rabbit anti-SSV p30, but not rabbit antiRD p30, was able to precipitate an actively synthesized p30
from patient but not donor cell extract (Chart 1). We must note
that the proteins recognized by NRS in these experiments
probably are coprecipitated with actin that apparently was not
effectively removed from the radiolabeled preparation by
preabsorption with SPA-bound NRS, NHuS, and NGS.
In another set of experiments, where actin contamination
was more effectively removed from the extracts, we were able
to show that, using the same NRS and NHuS as negative
controls and rabbit anti-y82-microglobulin and rabbit anti-p23,30
as positive controls (Chart 2, C and F), patient serum also
reacted with a p30 protein that was actively synthesized by
patient but not donor cells (Chart 2, B and £).
Peptide mapping of electrophoretically
purified 125l-labeled
proteins with proteolytic enzymes and subsequent slab gel
electrophoresis according to the technique described by Cleve
land et al. (4) revealed that patient p30 indeed has the char
acteristics of SSV p30 and not RD p30. Pepsin and chymotrypsin, although used in a metabolically active concentration
(Fig. 1, J and K), were not able to digest patient p30, SSV p30
or RD p30 substantially (Fig. 1, A, B, D, E, G, and H). V8
protease, however, was able to split patient p30 and SSV p30
into fragments of similar size (Fig. 1, C and F), whereas RD
p30 was affected otherwise (Fig. 1,1).
The p80 and p94 precipitated by donor serum in autologous
donor (Chart 2£) and also in patient cell extracts in much
higher amounts (Chart 26) are antigens which are probably
involved in the process of immunoregulation2 (see also "Dis
cussion").
e
migration—
PATIENT
e
e
migration-»
e
Using a goat anti-SSV antiserum that was extensively
ab-
DONOR
Chart 1. A 12.5% polyacrylamide slab gel which used [35S]methionine-labeled
NP-40 lysates of patient and donor cells and immunoprecipitation with several
antisera. Left, radioactive antigen profiles of patient cell membrane extracts when
reacted with several antisera; right, those of donor cell membrane extracts. Both
sets of profiles have been put onto the same scale by using the actin contami
nation (M.W. 45.000) as an internal standard and NRS as a negative control. The
profile of the NRS (which is the same NRS as used in Chart 2, C and F) is
explained under "Results." Numbers over peaks, molecular weights in units of
1000. 72, running front of the gel, containing proteins with molecular weights of
12,000 and lower. Ra, rabbit antiserum to.
evaluated the assay in full. The reverse transcriptase activity in
donor cells was at control level (Table 1). The reverse tran
scriptase activity in patient cells was 30 times higher before
transplantation, reverted to about normal in the remission, but
rose again in the relapse phase of the disease. The presence
of the Philadelphia chromosome followed the same pattern.
Antibodies cross-reacting with SSV p30 but not RD p30
could be found in patient but not donor serum, as is shown
(Table 2) by immunoprecipitation of radiolabeled SSV p30 and
RD p30 with antibodies that are derived from patient and donor
serum by affinity chromatography using immobilized SSV par
ticles on Sepharose beads (12). Despite a much higher con
centration factor for the antibodies from donor serum, a sig
nificant amount of antibodies cross-reacting with SSV p30, but
not with RD p30, could be found only in patient serum.
Immunochemical investigations on proteins present in the
leukemic cells were performed by immunoprecipitation
and
polyacrylamide slab gel electrophoresis of radiolabeled anti
FEBRUARY
1982
e
migration
—¿
PATIENT
Chart 2. A 12.5% polyacrylamide
e migration
—¿â€¢
$
DONOR
slab gel which used [35S]methionine-labeled
NP-40 lysates of patient (PAT) and donor (DON) cells and immunoprecipitation
with several antisera. Left, radioactive antigen profiles of patient cell membrane
extracts when reacted with several antisera; right, those of donor cell membrane
extracts. Both sets of profiles have been put onto the same scale by using
heterologous rabbit antiserum to human /J2-microglobulin (Raß2M) and rabbit
anti-p23,30 (RaP23,30) as positive reagents and NRS as a negative control.
Numbers over peaks, molecular weights in units of 1000. ) 2, running front of the
gel, containing proteins with molecular weights of 12,000 and lower. GaSSV.
goat anti-SSV.
683
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J. P. A. Derks et al.
loid leukemia patient. The evidence presented is: (a) a high
level of an enzyme with some properties of reverse transcriptase in the leukemic cells (Table 1). Reverse transcriptase can
be regarded as a marker for the presence of retroviruses, the
RNA tumor viruses (30); (D) the presence of antibodies to SSV
p30 in patient serum (Table 2); (c) the active synthesis of a
particle or membrane-bound p30 protein by patient cells that
can be precipitated by rabbit anti-SSV p30 (Chart 1/4) and
patient serum (Chart 28); (d) the resemblance of patient p30
with SSV p30 in peptide mapping by limited proteolysis and
subsequent slab gel electrophoresis (Fig. 1); (e) the active
synthesis of a p78 protein by patient cells that can be precipi
tated by goat anti-SSV (Chart 2/4); ( f) an increasing amount of
antibodies in the donor during the course of the patient's
ABC
DEF
CHI
J
K
L M
Fig. 1. Peptide maps on 15% polyacrylamide slab gels. Vertically, the molec
ular weights are expressed in units of 1000. Digestion of '2sl-labeled proteins
that were cut from a sodium dodecyl sulfate-polyacrylamide
gel was performed
during reelectrophoresis in the presence of 0.5 fig pepsin (A, D. G, K), 0.5 fig
chymotrypsin (B, E, H. M), or 0.2 fig S. aureus protease V8 (C, F, /). A to C.
digestion of patient p30; D to F. digestion of SSV p30: G to /, digestion of RD
p30; J. native p45, cell membrane-derived protein of unrelated donor; K, diges
tion of p45 by pepsin; L. native p75, cell membrane-derived protein of unrelated
donor; M. digestion of p75 by chymotrypsin. Patient p30, SSV p30. and RD p30
were run on separate gels, which is seen in a slight shift of the specific bands.
However, when set onto the same scale of gel dimensions, and therewith using
the molecular weight markers per gel, no difference in position is seen in A to C
versus D to F. D, E, and F are on the same scale with G, H. and /, thereby
indicating a molecular weight difference between SSV p30 and RD p30 in
addition to a different digestion pattern.
disease, reactive with autologous and, more effectively, with
patient-derived cell membrane proteins p80 and p94 (Charts
2, ßand E, and 3), which are probably antigens involved in the
process of immunoregulation.? Patient antibodies were not
directed to the interspecies-specific
determinants of RD p30,
as became clear from a negative radioimmunoprecipitation
assay with 125l-labeled RD p30, which is derived from a cat
virus, RD-114 (Table 2). Patient p30 probably is lacking those
RD p30 cross-reacting
interspecies-specific
determinants,
since concomitantly it could be precipitated by rabbit anti-RD
p30.
sorbed with uninfected NC37 host cells, a p78 could be pre
cipitated from patient but not donor cell extracts (Chart 2, A
and D).
The donor serum was investigated for antiidiotypic activity
towards SSV p30, because of the alleged tumor-suppressive
aspects of such activities (11). Therefore, we tried to isolate
the idiotypic SSV p30-specific IgG present in patient serum
(see Table 2) by affinity chromatography using immobilized
donor serum immunoglobulin. Absorption and elution of patient
serum on a column of Sepharose-coupled donor serum immu
noglobulin thus resulted in 5.25 mg immunoglobulin ("pat-id"),
which was resuspended in 200 jul Dulbecco's phosphatebuffered saline. This preparation was tested for idiotypic activ
ity towards SSV p30 and was found to be negative (Chart 3F).
In concordance with Table 2, Chart 3 gives further evidence
that donor serum does not contain antibodies to SSV p30
(Chart 3C), whereas patient serum does (Chart 3D). In addition,
Chart 3 shows that donor serum is not antiidiotypic to SSV p30
because absorption of patient serum on immobilized donor
serum immunoglobulin does not affect its capability to react
with SSV p30 (Chart 3£),while concomitantly, the eluate ("patid" preparation) had no reactivity toward SSV p30 (Chart 3F).
Using S. aureus as a matrix to precipitate according to the
technique described by Herbrink (12), it was confirmed that
the "pat-id" preparation did not contain specific antibodies
against and is therefore not idiotypic to freshly prepared SSV
p30, Rauscher leukemia virus p30, or murine leukemia virus
p30. Consequently, donor serum cannot be considered anti
idiotypic to viral p30.
pat-id
e ^w^
t- ssv-pao-f
1 front —¿
Chart 3. A 9.0% polyacrylamide
slab gel which used an aged '"l-labeled
DISCUSSION
SSV p30 preparation and immunoprecipitation with several antisera. A, MRS; ß,
rabbit anti-SSV (RaSSV); C, donor serum (DOW); D, patient serum (PAT); E,
patient serum depleted of the "Pat-id" preparation (Pat-id depl. PATI; F. "Patid" (described under "Results" and "Discussion").
The electrophoretic
front
Our experiments provide evidence for the presence of a
retrovirus in at least some cells of this particular chronic mye-
contains degraded components and contaminants of SSV p30. The specific
reactivity of the sera to SSV p30 is seen as a shoulder to the electrophoretic
front.
684
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VOL. 42
p30 Protein and Antiviral p30 Antibodies in Chronic Myeloid Leukemia
Differences between SSV p30 and patient p30 on one hand
and RD p30 on the other, like those found in peptide mapping
(Fig. 1), may be the molecular basis of these immunological
reactivities.
Two lines of evidence indicate an exogenous nature of the
eventual virus involved: (a) it is related to SSV, which belongs
to the group of exogenous primate type C viruses, and it does
not resemble RD, which showed partial relationship to the
endogenous baboon virus at least in its p30 antigenic deter
minants (25). (o) it is reported that a humoral immune response
generally does not occur naturally against the endogenous viral
structural proteins p30 and p12 in mice (13, 17), and an antip30 antibody response was observed only upon active immu
nization of mice with allogeneic murine leukemia virus-infected
leukemia cells (20).
p30-antigenic specificities have been found in human tumor
cell cultures (31) and in leukocytes of leukemic (19, 27) and
preleukemic patients (27). Antibodies to virus p30 have been
found in sera of healthy individuals and patients with various
diseases (12, 18, 23). These data are compatible with our
findings in one single patient. Our data further indicate that a
humoral immune response to viral p30 does not result in
effective protection against the disease. It is unclear whether
this is due to the fact that only a small percentage of the
malignant cells contain p30 or that p30 is an internal protein
and thus that the anti-p30 antibodies cannot be virolytic or
virus neutralizing.
In the course of the patient's disease, the donor showed an
increasing amount of possibly immunoregulatory2 antibodies,
specific for autologous and patient p80 and p94. The fact that
the process of immunoregulation was activated in this closely
related monozygous twin may indicate the presence of a wide
spread infectious agent. p94 can be precipitated with a T-cell
specific antiserum, rabbit anti-HSB (Chart 2, A and D); evidence
that p94 is the T-cell alloantigen TCA-1 is presented in another
article and is partly based on a strong reactivity of donor serum
on enriched T-cell fractions when tested in the TCP test ac
cording to a technique presented by van Leeuwen (29).
At this stage, we can exclude that p80 as recognized by
donor serum is surface membrane IgM, an Fc receptor, the
equivalent of viral gp70 or a leukemia antigen.2 We cannot
exclude, however, that p80 is an idiotypic T-cell receptor,
because that could not be tested due to lack of knowledge of
the specific antigen that it should recognize. Idiotypic T-cell
receptors may have a molecular weight of about 70,000 (16).
Idiotypic structures on tumor cells do occur and antiidiotypic
antisera were shown to control growth of those tumors (11). A
receptor structure, M.W. 70,000, idiotypic to murine leukemia
viruses, has been shown in virus-induced lymphomas (1). We
have concluded, however, that p80 is not idiotypic for viral
p30, because donor serum is not antiidiotypic to idiotypic
antivirus p30 patient antibodies (Chart 3).
Combined with the data reported by other authors, our
findings suggest that a retrovirus was present in this particular
patient. This virus might be involved in at least some human
cancers.
ACKNOWLEDGMENTS
We gratefully thank Dr. P. Herbrink and Dr. G. Muyen. Department of Pathol
ogy, for their generous cooperation and for providing the antiviral antisera. We
thank Dr. S. Warnaar. Department of Pathology, for reading this manuscript; Dr.
FEBRUARY
1982
F. Zwaan, Department of Hematology, for providing patient material; R. P.
Schuitemaker for donating so much blood; and Dr. J. J. van Rood for establishing
the essential contacts, stimulating discussions, and for reading the manuscript.
REFERENCES
1. Baird, S. M. Antigenic properties and molecular weights of murine leukemia
virus-binding proteins. J. Immunol.. 122: 1389-1396,
1979.
2. Barbacid, M., Bolognesi, D., and Aaronson, S. A. Humans have antibodies
capable of recognizing oncoviral glycoproteins: demonstration that these
antibodies are formed in response to cellular modification of glycoproteins
rather than as consequence of exposure to virus. Proc. Nati. Acad. Sei. U.
S. A., 77: 1617-1621,
1980.
3. Bonner. W. M., and Laskey, R. A. A film detection method for tritium labeled
proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem., 46: 8388, 1974.
4. Cleveland, D. W , Fischer, S. G., Kirschner, M. W., and Laemmli, U. K.
Peptide mapping by limited proteolysis in sodium dodecyl sulfate and
analysis by gel electrophoresis. J. Biol. Chem., 252: 1102-1106,
1977.
5. Cohen. E., Gregory. S. G., Weinreb, N., and Minowada, J. Further appraisal
of antibodies to leukemia" associated antigen in sera of plasmapheresis
donors. Prog. Clin. Biol. Res., 16: 109-114, 1977.
6. Day, N. K., Winfield, J. B., Gee. T., Winchester, R.. and Teshima. H.
Evidence for immune complexes involving anti-lymphocytic antibodies as
sociated with hypocomplementaemia
in chronic lymphocytic leukaemia
(CLL). Clin. Exp. Immunol., 26: 189-195. 1976.
7. Doré.J. F.. Bertoglio, J.. and Guibot. C. Anticorps cytotoxiques pour des
cellules leucémiques humaines dans des serums humains normaux. Ann.
Immunol., 728: 155-157, 1977.
8. Gallo. R. C. Cellular and virological studies directed to the pathogenesis of
human myelogenous leukemias. In: R. Neth, R. C. Gallo. P. H. Hofschneider,
and K. Mannweiler (eds.). Modern Trends in Human Leukemia III, pp. 7-24.
Berlin: Springer-Verlag, 1977.
9. Gardner, M. B., Rasheed, S., Shimizu. S.. Rongey, R. W., Henderson, B. E..
McAllister, R. M., Klement, V., Charman, H. P., Gilden, R. V.. Heberling. R.
L.. and Huebner, R. J. In: H. H. Hiatt, J. D. Watson, and J. A. Winsten (eds.),
Origins of Human Cancer, pp. 1235-1251. Cold Spring Harbor, N. Y.: Cold
Spring Harbor Laboratories. 1977.
10. Greenwood, F. C.. Hunter. W. M , and Glover, J. S. The preparation of I3'l
labeled growth hormone of high specific radioactivity. Biochem. J., 89:114123, 1963.
11. Haughton, G., Lanier, L. L., Babcock, G. F.. and Lynes, M. A. Antigeninduced murine B-cell lymphomas. II. Exploitation of the surface idiotype as
tumor specific antigen. J. Immunol.. 121: 2358-2362.
1978.
12. Herbrink, P., Moen, J. E. T., Brouwer, J., and Warnaar, S. O. Detection of
antibodies cross-reactive with type C RNA tumor viral p30 protein in human
sera and exúdate fluids. Cancer Res.. 40: 166-173. 1980.
13. Huebner, R. J., Sarma, P. S., Kelloff, G. J., Gilden, R. V., Meier. H., Meyers,
D. D., and Peters. S. L. Immunological tolerance to RNA tumor virus genome
expression: significance of tolerance and prenatal expressions in embryoneogenesis and tumorigenesis. Ann. N. Y. Acad. Sci., 181: 246-250,
1971.
14. Jose. D. J.. Ehert, H., Colebatch. J.. Waters, K., Wilson, F., and O Keefe, D.
Immune function at diagnosis in relation to responses to therapy in acute
lymphocytic leukemia of childhood. Blood. 47: 1011-1021,
1976.
15. Laskey. R. A., and Mills. A. D. Quantitative film detection of aH and MC in
polyacrylamide gels by fluorography. Eur. J. Biochem., 56: 335-341, 1975.
16. Lea. T., Forre. O. T., Michaelsen. T. E., and Natvig, J. B. Shared idiotypes
on human peripheral blood B and T lymphocytes. J. Immunol., 122: 24132417, 1979.
17. Lee, J. C.. and Ihle, J. N. Autogenous immunity to endogenous RNA tumor
virus: reactivity of natural immune sera to antigenic determinants of several
biologically distinct murine leukemia viruses. J. Nati. Cancer Inst., 55: 831838. 1975.
18. Louie, S., Curtis. J., Till, J. E., and McCulloch. E. A. Antibodies in human
sera to oncornavirus like proteins from normal or leukemic cell cultures. J.
Exp. Med., 144: 1243-1253,
1976
19. Metzgar, R. S., Mohanakumar. T., and Bolognesi, D. P. Relationships
between membrane antigens of human leukemic cells and oncogenic RNA
virus structural components. J. Exp. Med.. 143: 47-63. 1976.
20. Nowinski, R. C.. and Watson, A. Immune response of the mouse to the major
core protein (p30) of ecotropic leukemia virus. J. Immunol., /1 7: 693-696,
1976.
21. Sherr, C. J.. Benveniste. R. E., and Todaro, C. J. Type C viral expression in
primate tissues. Proc. Nati. Acad. Sei. U. S. A.. 71: 3721-3725.
1974.
22. Snyder. H. W.. Jr., and Fleissner, E. Specificity of human antibodies to
oncovirus glycoproteins: recognition of antigen by natural antibodies di
rected against carbohydrate structures. Proc. Nati. Acad Sei. U. S. A., 77:
1622-1626,
1980.
23. Snyder. H. W., Jr., Pincus, T.. and Fleissner, E. Specificities of human
immunoglobulins reactive with antigens in preparations of several mamma
lian type-C viruses. Virology. 75: 60-73. 1976.
685
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1982 American Association for Cancer Research.
J. P. A. Derks et al.
24. Springer, T. A., Kaufman, J. F., Giphart, M. J., Man, D. U., Terhorst, C., and
Strominger, J. L. Origins of lymphocyte diversity. Cold Spring Harbor Symp.
Quant. Biol., 41: 387-396, 1976.
25. Stephenson, J. R., Reynolds, R. K., and Aaronson, S. A. Comparisons of
the immunological properties of two structural polypeptides of type C RNA
viruses endogenous to Old World Monkeys. J. Virol., 17: 374-384, 1976.
26. Studier, F. W. Analysis of bacteriophage early RNAs and proteins on slabgel.
J. Mol. Biol., 79. 237-248, 1973.
27. Toth, F. D., Madar. Z.. Kiss, J., Jako, J., Vaczi, L.. Rak, K., and Redai. I.
Expression of oncornavirus antigens in peripheral leucocytes of patients
with chronic myeloid leukaemia. Acta Microbiol. Acad. Sci. Hung., 26: 4753. 1979.
686
28. van Lambalgen, R., Farrant, J., and Bradley, B. A. Curve-shift analysis of
cryopreserved killer T cell function. J. Immunol. Methods, 27. 327-338,
1979.
29. van Leeuwen, A. Alloantibodies that react with subsets of human T-cells.
Tissue Antigens, 14: 437-443, 1979.
30. van Muyen, G. N. P.,te Velde, J.. den Ottolander, G. J.. Brand, A., KoopmanBroekhuyzen, N.. Schaberg, A., and Warnaar, S. 0. On the presence of
reverse transcriptase in myelo- and lymphoproliferative disorders. Cancer
(Phila.), 43: 1682-1688,
1979.
31. Zürcher, C.. Brinkhof, J., Bentvelzen. P.. and de Man, J. C-type virus
antigens detected by immunofluorescence in human bone tumour cultures.
Nature (Lond.), 245. 457-459. 1975.
CANCER
RESEARCH
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VOL.
42
Synthesis of a Viral Protein with Molecular Weight of 30,000
(p30) by Leukemic Cells and Antibodies Cross-Reacting with
Simian Sarcoma Virus p30 in Serum of a Chronic Myeloid
Leukemia Patient
Jos P. A. Derks, Lilian Hofmans, Hans W. Bruning, et al.
Cancer Res 1982;42:681-686.
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