Expression of a transformation-related protein (p53) in the malignant stage of Friend virus-induced diseases.

Abstract

Stage 1 (pre-malignant) and stage 2 (malignant) cells derived from mice infected with Friend murine leukemia virus or polycythemia-inducing Friend virus complex were examined and compared for the expression of a transformation-related cellular protein, p53. Stage 2 cells were found to express high levels of p53, whereas stage 1 cells did not express detectable levels of this protein. These results indicate that p53 may be a marker for transformed cells present in the second stage of diseases induced by Friend murine leukemia virus or polycythemia-inducing Friend virus complex.

Full text

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JOURNAL OF VIROLOGY, June 1983, p. 1022-1026
0022-538X/83/061022-05$02.00/0
Copyright © 1983, American Society for Microbiology
Vol. 46, No. 3
Expression of a Transformation-Related Protein (p53) in the
Malignant Stage of Friend Virus-Induced Diseases
SANDRA K. RUSCETTI* AND EDWARD M. SCOLNICKt
Laboratory of Tumor Virus Genetics, National Cancer Institute, Bethesda, Maryland 20205
Received 13 December 1982/Accepted 24 February 1983
Stage 1 (pre-malignant) and stage 2 (malignant) cells derived from mice infected
with Friend murine leukemia virus or polycythemia-inducing Friend virus com-
plex were examined and compared for the expression of a transformation-related
cellular protein, p53. Stage 2 cells were found to express high levels of p53,
whereas stage 1 cells did not express detectable levels of this protein. These
results indicate that p53 may be a marker for transformed cells present in the
second stage of diseases induced by Friend murine leukemia virus or polycythe-
mia-inducing Friend virus complex.
Both Friend murine leukemia virus (F-MuLV)
and the polycythemia-inducing F-MuLV-spleen
focus-forming virus complex (FVP) induce
erythroleukemias in susceptible mice (8, 28). It
has been suggested that these diseases can be
divided into multiple stages (13, 15, 16, 19, 26,
27, 30). Stage 1 (pre-malignant) is characterized
by the rapid hyperplasia of actively differentiat-
ing erythroid precursor cells induced by the
virus infection. These cells, occurring early in
the disease, have a limited proliferative capacity
and are not serially transplantable. Stage 2 (ma-
lignant), occurring late in the course of the
disease, is characterized by the appearance of
transplantable malignant erythroblasts that can
be grown as permanent cell lines in culture. The
relationship between the cells present in stage 1
and stage 2 of these diseases is not known.
In an effort to characterize biochemical differ-
ences between cells obtained from the different
stages of Friend diseases, we examined various
cells for the expression of a transformation-
related phosphoprotein, p53. Elevated levels of
this protein have been detected in spontaneously
transformed cells, as well as in cells transformed
by viruses, chemicals, or X rays (1, 3, 11, 14,
23). Our results indicate that p53 may be a
marker for the transformed cells present in the
second stage of diseases induced by F-MuLV or
FVP.
For these studies, spleen cells (stage 1) were
obtained from NIH Swiss mice infected 2 weeks
earlier as newborns with molecularly cloned
F-MuLV clone 57 (20) or DBA/2 mice infected 2
weeks earlier as adults with the Lilly-Steeves
strain of the FVP complex (29). Erythroleuke-
t Present address: Virology and Cell Biology Division,
Merck, Sharp & Dohme Research Laboratories, West Point,
PA 19486.
mia cell lines (stage 2) used in these studies
included: 57-TP-1 and 57-TP-3, derived from
NIH Swiss mice infected with F-MuLV (19);
DS19, derived from DBA/2 mice infected with
FVP (25); and HFL/d, derived from BALB/c
mice infected with FVP (7). In some experi-
ments, these cell lines were chemically induced
to differentiate by previously described proce-
dures (9, 22; F. Ruscetti, S. Ruscetti, and A.
Oliff, submitted for publication). AV-1 and AV-2
cells, derived from in vitro infection of DBA/2
bone marrow cells with FVP (5), and 416B
cells, derived by in vitro infection of BDF1 bone
marrow cells with FVP (4), were included as
hematopoietic cell lines that are not known to be
tumorigenic.
Monoclonal
against p53 (6), 3T3 cells transformed with simi-
an virus 40 (SV40), and Meth A cells (2) were
generously provided by Gilbert Jay, National
Cancer Institute, Bethesda, Md. Goat antiserum
to Rauscher MuLV gp7O was obtained through
the Division of Cancer Cause and Prevention,
National Cancer Institute. Goat antiserum spe-
cific for mink cell focus-inducing virus gp7O has
been previously described (24). As a negative
control for the p53 monoclonal antibody, condi-
tioned medium was obtained from SP2 mouse
myeloma cells grown in Dulbecco minimal es-
sential medium with 10% fetal calf serum. Pulse-
labeling, immune precipitation, and polyacryl-
amide gel electrophoresis were carried out by
previously described procedures (24).
When erythroid cells obtained from stage 1 or
stage 2 spleens after in vivo infection with either
F-MuLV or FVP were examined for the expres-
sion of viral proteins, no significant differences
could be detected (Fig. 1). Spleen cells obtained
from F-MuLV-infected mice (stage 1), as well as
cell lines derived from these mice (stage 2),
expressed both ecotropic (gPr85en') and mink
antibody
prepared
1022

Page 2

NOTES
1023
A
B
1 2
3
1 2
3
.a
U...
gPr85
A.
env
gPr8O
"
-92K
env
a
gPr85I
lo
68K
_ -43K
C
D
13
1 3
-92K
...I,
-68K
gp52 W
*M
-K43K
FIG. 1. Viral protein expression in stage 1 and stage 2 cells derived from mice infected with F-MuLV or FVP.
Spleen cells (stage 1) obtained from NIH Swiss mice infected 2 weeks earlier as newborns with F-MuLV (A) or
DBA/2 mice infected 2 weeks earlier as adults with FVP (C); erythroleukemia cell lines (stage 2) 57-TP-1 (B) and
DS19 (D) were pulse-labeled for 60 min. with [35S]methionine. Cell lysates were immune precipitated with goat
antiserum to Rauscher MuLV gp7O (lanes 1), goat antiserum specific for the gp7O of mink cell focus-inducing
murine leukemia viruses (lanes 2), and normal goat serum (lanes 3). Antigen-antibody complexes were collected
after binding to Staphylococcus aureus, and the precipitated proteins were separated by electrophoresis on 7%
sodium dodecyl sulfate-polyacrylamide gels. Molecular weight markers were phosphorylase b (92,000), bovine
serum albumin (68,000), and ovalbumin (43,000).
cell focus-inducing virus-related (gPr80e'l') enve-
lope precursors (Fig. 1A and B); spleen cells
obtained from FVP-infected mice (stage 1), as
well as cell lines derived from these mice (stage
2), expressed both ecotropic envelope precur-
sors (gPr85eW') and the 52,000-dalton envelope
A
BC
12
r2r
_
.....
.:j::..48.
.::
*:::
,, Y}
..
*:_.
:,. :
....
::
::
*,e
ps3> s_ * *
*.:
.::
_.^i,..:::E^:::::.
2
-'92K
-68K
-'43 K
Fig. 2. p53 expression in various cell lines. SV40-
transformed 3T3 cells (A), Meth A cells (B), 57-TP-1
cells (C) were pulse-labeled for 60 min with [35S]meth-
ionine. Cell lysates were immune precipitated with a
monoclonal antibody preparation to p53 (lanes 1) or
control SP2 supernatant (lanes 2). Antigen-antibody
complexes were collected after binding to S. aureus,
and the precipitated proteins were separated by elec-
trophoresis on 7% sodium dodecyl sulfate-polyacryl-
amide gels.
glycoprotein encoded by the spleen focus-form-
ing virus (Fig. 1C and D).
As previously shown, monoclonal antibody to
p53 will specifically precipitate a 53,000-dalton
protein from 3T3 cells transformed by SV40
(Fig.
2A) or methylcholanthrene
When this monoclonal antibody was reacted
against extracts from the 57-TP-1 erythroleuke-
mia cell line, a 53,000-dalton protein was also
specifically precipitated (Fig. 2C). To determine
whether this protein was expressed in all stages
of erythroleukemia induced by either F-MuLV
or FVP, various cells were radiolabeled, and
extracts were immune precipitated with anti-p53
monoclonal antibody. No p53 could be detected
in the stage 1 cells from mice infected with
F-MuLV (Fig. 3A and B) or FVP (Fig. 3C and
D). In contrast, significant levels of this protein
could be detected in stage 2 cell lines derived
from mice infected with F-MuLV (Fig. 3E and
F) or FVP (Fig. 3G and H). Other stage 2 cell
lines derived from mice infected with F-MuLV
or FVP were also shown to express high levels
of p53 (data not shown).
Like the p53 expressed in other transformed
cells, the p53 expressed in stage 2 erythroleuke-
mia cell lines is phosphorylated and can be
detectedin both stationary and logarithmic
phases of the growth cycle (data not shown).
Stage 2 cell lines derived from mice infected
with either F-MuLV or FVP can be induced to
differentiate
into mature erythrocytes when
grown in the presence of certain inducers (9, 22;
Ruscetti, Ruscetti, and Oliff, submitted for pub-
lication). When the cells were examined for
(Fig.
2B).
VOL. 46, 1983

Page 3

1024
NOTES
A
B
1 2
1 2
C
D
1
2
1 2
E
F
12
1
2
G
H
1
2
1 2
-92K-
-92K-
-68K-
-43K-
-68K-
-43K-8
p53kl
_*.-_o
FIG. 3. p53 expression in stage 1 and stage 2 cells derived from mice infected with F-MuLV or FVP. Spleen
cells (stage 1) obtained from individual mice infected with F-MuLV (A and B) or FVP (C and D) and
erythroleukemia cell lines (stage 2) derived from mice infected with F-MuLV (E, 57-TP-1; F, 57-TP-3) or FVP (G,
DS19; H, HFL/d) were pulse-labeled with [35S]methionine for 60 min. Cell lysates were then immune
precipitated with monoclonal antibody to p53 (lanes 1) or control SP2 supernatant (lanes 2). Antigen-antibody
complexes were collected after binding to S. aureus, and the precipitated proteins were separated by
electrophoresis on 7% sodium dodecyl sulfate-polyacrylamide gels.
changes in the levels ofp53, no differences could
be found between uninduced 57-TP-1 cells and
57-TP-1 cells induced to differentiate with hemin
(Fig. 4A and B, respectively) or between unin-
duced DS19 cells (Fig. 4C) and DS19 cells
induced to differentiate with either dimethyl
sulfoxide or hexamethylenebisacetamide (Fig.
4D and E). In all cases, >70% of the induced
cells were benzidine positive at the time they
A
B
1 2
1
2
were examined for levels of p53, indicating that
the majority ofthe cells had undergone erythroid
maturation.
Cell lines derived by in vitro infection of
hematopoietic cells with FVP were also exam-
ined for expression of p53. Neither AV-1 (Fig.
5A), AV-2 (Fig. SB), nor 416B (Fig. SC) cells, all
derived from in vitro infection of bone marrow
cells with FVP (4, 5), expressed detectable lev-
C
1
2
D
E
1 2
1
.
2
(
92 K
.
p53)0-
I
ps3
"I"..
a
_f,"
68 K
j-
_b
-tp53
43 K
i
FIG. 4. p53 expression in stage 2 F-MuLV and FVP erythroleukemia cell lines before and after treatment
with inducers of differentiation. Erythroleukemia cell lines derived from mice infected with F-MuLV (A and B,
57-TP-1) or FVP (C to E, DS19) were labeled for 60 min with [35S]methionine either uninduced (A and C) or after
induction with hemin (B), dimethyl sulfoxide (D), or hexamethylenebisacetamide (E). Cell lysates were immune
precipitated with monoclonal antibody to p53 (lanes 1) or control SP2 supernatant (lanes 2). Antigen-antibody
complexes were collected after binding to S. aureus, and the precipitated proteins were separated by
electrophoresis on 7% sodium dodecyl sulfate-polyacrylamide gels.
J. VIROL.

Page 4

NOTES1025
A
2
13
B
2
C
2 3
1
3
1
do
d
D
2 3
<92K
gPr85env
e
lo
-'68K
gp52a- _
p53).*
FIG. 5. p53 expression in cell lines derived by in vitro infection of bone marrow cells with FVP. AV-1 (A),
AV-2 (B), and 416B (C) cells were pulse-labeled for 60 min with [35S]methionine. Cell lysates were immune
precipitated with goat anti-Rauscher MuLV gp7O serum (lanes 1), monoclonal antibody to p53 (lanes 2), and
control SP2 supernatant (lanes 3). Antigen-antibody complexes were collected by binding to S. aureus, and the
precipitated proteins were separated by electrophoresis on 7% sodium dodecyl sulfate-polyacrylamide gels. (D)
shows the p53 in 57-TP-1 cells for comparison.
els of p53. (57-TP-1, an in vivo-derived cell line
which expresses high levels of p53, is shown in
Fig. SD for comparison.) All of these cell lines,
however, expressed high levels of viral envelope
proteins (Fig. 5A to C, lanes 1). These results
indicate that a high level of p53 expression is not
a general property of rapidly proliferating hema-
topoietic cell lines. In contrast to the in vivo-
derived FVP cell lines, AV-1, AV-2, and 416B
cells have not yet been shown to be tumorigenic
when injected intraveneously into syngeneic ir-
radiated mice. Studies are in progress to deter-
mine whether these lines can become tumorigen-
ic, p53-expressing cells.
Our results indicate that p53 may be a marker
for transformed erythroblasts present in the sec-
ond stage of diseases induced by F-MuLV or
FVP. Since high levels of p53 are associated
with transformation by a variety of oncogenes,
this result could indicate that a cellular oncogene
has been activated in hematopoietic cells ofmice
infected with F-MuLV or FVP and is responsi-
ble for the transformed phenotype of the stage 2
spleen cells. It has been proposed that lympho-
mas induced in birds by the avian leukosis
viruses are the result of the promotion of the
transcription of a particular cellular oncogene
(18, 21); such a model may explain the stage 2
transplantable cell lines from F-MuLV- or FVP-
infected mice. Studies are currently being car-
ried out to determine whether any new RNAs
reactive with probes either to known cellular
oncogenes or to viral long terminal repeats are
expressed in stage 2, but not stage 1, hematopoi-
etic cells after in vivo infection with either
F-MuLV or FVP.
It has previously been shown that T-antigen is
physically complexed with p53 in SV40-trans-
formed cells (10, 12, 17). It is interesting that
monoclonal antibody to p53 coprecipitates a
protein in stage 2 cells with a molecular weight
of approximately 70,000 (see especially Fig. 4).
We are currently carrying out studies to further
characterize this protein. If such a protein plays
an important role in erythroid cell differentia-
tion, binding of p53 to such a protein could
modify its biological function and have profound
effects on erythroid differentiation.
We thank John Feild for expert technical assistance during
the course of this work.
LITERATURE CITED
1. Crawford, L. V., D. C. Pim, E. G. Gurney, P. Goodfellow,
and J. Taylor-Papadimitriou. 1981. Detection of a com-
mon feature in several human tumor lines-a 53,000
dalton protein. Proc. Natl. Acad. Sci. U.S.A. 78:41-45.
2. DeLeo, A. B., G. Jay, E. Appella, G. C. Dubois, L. W.
Law, and L. J. Old. 1979. Detection of a transformation-
related antigen in chemically induced sarcomas and other
transformed cells of the mouse. Proc. Natl. Acad. Sci.
U.S.A. 76:2420-2424.
3. DeLeo, A. B., H. Shiku, T. Takahashi, M. John, and L. J.
Old. 1977. Cell surface antigens of chemically induced
sarcomas of the mouse. J. Exp. Med. 146:720-734.
4. Dexter, T. M., T. D. Allen, D. Scott, and N. M. Teich.
1979. Isolation and characterization of a biopotential
haematopoietic cell line. Nature (London) 277:471-474.
5. Dexter, T. M., T. D. Allen, N. G. Testa, and E. Scolnick.
1981. Friend disease in vitro. J. Exp. Med. 154:594-608.
6. Dippold, W. G., G. Jay, A. B. DeLeo, G. Khoury, and
L. J. Old. 1981. p53 transformation related protein: detec-
VOL. 46, 1983

Page 5

1026
NOTES
tion by monoclonal antibody in mouse and human cells.
Proc. Natl. Acad. Sci. U.S.A. 78:1695-1699.
7. Freedman, H. A., and F. Lilly. 1975. Properties of cell
lines derived from tumors induced by Friend virus in
BALB/c and BALB/c-H-2b mice. J. Exp. Med. 142:212-
223.
8. Friend, C. 1957. Cell-free transmission in adult Swiss mice
of a disease having the character of a leukemia. J. Exp.
Med. 105:307-318.
9. Friend, C., W. Scher, J. G. Holland, and T. Sato. 1971.
Hemoglobin synthesis in murine virus-induced leukemic
cells in vitro: stimulation of erythroid differentiation by
dimethylsulfoxide. Proc. Natl. Acad. Sci. U.S.A. 68:378-
382.
10. Harlow, E., D. C. Pim, and L. V. Crawford. 1981. Com-
plex of a simian virus 40 large-T antigen and host 53,000-
molecular weight protein in monkey cells.
37:564-573.
11. Jay, G., A. B. DeLeo, E. Appella, G. C. Dubois, L. W.
Law, G. Khoury, and L. J. Old. 1980. A common transfor-
mation-related protein in murine sarcomas and leukemias.
Cold Spring Harbor Symp. Quant. Biol. 44:659-664.
12. Lane, D. P., and L. V. Crawford. 1979. T antigen is bound
to a host protein in SV40-transformed cells. Nature (Lon-
don) 278:261-263.
13. Levy, S. B., L. A. Blankstein, E. C. Vinton, and T. J.
Chambers. 1979. Biologic and biochemical characteristics
of Friend leukemic cells representing different stages of a
malignant process, p. 409-428. In Y. Ikawa and T. Odaka
(ed.), Oncogenic viruses and host cell genes. Academic
Press, Inc., New York.
14. Linzer, D. I. H., and A. J. Levine. 1979. Characterization
of a 54K dalton cellular SV40 tumor antigen present in
SV40 transformed cells and uninfected embryonal carci-
noma cells. Cell 17:43-52.
15. Mager, D., M. E. MacDonald, I. B. Robson, T. W. Mak,
and A. Bernstein. 1981. Clonal analysis of the late stages
of erythroleukemia induced by two distinct strains of
Friend leukemia virus. Mol. Cell. Biol. 1:721-730.
16. Mager, D. L., T. W. Mak, and A. Bernstein. 1981.
Quantitative colony method for tumorigenic cells trans-
formed by two distinct strains of Friend leukemia virus.
Proc. NatI. Acad. Sci. (U.S.A.) 78:1703-1707.
17. McCormick, F., and E. Harlow. 1980. Association of a
murine 53,000-dalton phosphoprotein with simian virus 40
large-T antigen in transformed cells. J. Virol. 34:213-224.
18. Neel, B. G., W. S. Hayward, H. L. Robinson, J. Fang, and
S. Astrin. 1981. Avian leukosis virus-induced tumors have
common proviral integration sites and synthesize discrete
new RNAs: oncogenesis by promoter insertion. Cell
23:323-334.
19. Oliff, A., S. Ruscetti, E. C. Douglass, and E. Scolnick.
J.
Virol.
1981. Isolation of transplantable erythroleukemia cells
from mice infected with helper-independent Friend mu-
rine leukemia virus. Blood 58:244-254.
20. Oliff, A. I., G. L. Hager, E. H. Chang, E. M. Scolnick,
H. W. Chan, and D. R. Lowy. 1980. Transfection of a
molecularly cloned Friend murine leukemia virus DNA
yields a highly leukemogenic helper-independent type C
virus. J. Virol. 33:475-486.
21. Payne, G. S., S. A. Courtneidge, L. B. Crittenden, A. M.
Fadly, J. M. Bishop, and H. E. Varmus. 1981. Analysis of
avian leukosis virus DNA and RNA in bursal tumors: viral
gene expression is not required for maintenance of the
tumor state. Cell 23:311-322.
22. Reuben, R. R., R. Wife, R. Breslow, R. Rifkind, and P.
Marks. 1976. A new group of potent inducers of differenti-
ation in murine erythroleukemia cells. Proc. Natl. Acad.
Sci. U.S.A. 73:862-866.
23. Rotter, V., M. A. Boss, and D. Baltimore. 1981. Increased
concentration ofan apparently identical cellular protein in
cells transformed by either Abelson murine leukemia
virus or other transforming agents. J. Virol. 38:336-346.
24. Ruscetti, S. K., D. Linemeyer, J. Feild, D. Troxler, and
E. M. Scolnick. 1979. Characterization of a protein found
in cells infected with the spleen focus-forming virus that
shares immunological cross-reactivity with the gp7O found
in mink cell focus-inducing virus particles.
30:787-798.
25. Singer, D., M. Cooper, G. Maniatis, P. Marks, and
R. Rifkind. 1974. Erythropoietic differentiation in colonies
of cells transformed by Friend virus. Proc. Natl. Acad.
Sci. U.S.A. 71:2668-2670.
26. Tambourin, P., F. Wendling, F. Moreau-Gachelin, M.
Charon, and P. Bucau-Varlet. 1980. Friend leukemia: a
multi-step malignant disease, p. 127-138. In G. Rossi
(ed.), In vivo and in vitro erythropoiesis. Elsevier/North-
Holland Publishing Corp., Amsterdam.
27. Tambourin, P. E., F. Wendling, C. Jasmin, and F. Smadja-
Joffe. 1979. The physiopathology of Friend leukemia.
Leukemia Res. 3:117-129.
28. Troxler, D., and E. Scolnick. 1978. Rapid leukemia in-
duced by cloned strain of replicating murine type-C virus:
association with induction of xenotropic-related RNA
sequences contained in spleen focus-forming virus. Virol-
ogy 85:17-27.
29. Troxler, D. H., W. P. Parks, W. C. Vass, and E. M.
Scolnick. 1977. Isolation of a fibroblast nonproducer cell
line containing the Friend strain of the spleen focus-
forming virus. Virology 76:602-615.
30. Wendling, F., F. Moreau-Gachelin, and P. Tambourin.
1981. Emergence of tumorigenic cells during the course of
Friend virus leukemias. Proc. Natl. Acad. Sci. U.S.A.
78:3614-3618.
J. Virol.
J. VIROL.