Download Equivalent Expression of Endogenous Murine

[CANCER
RESEARCH
38, 566-569,
March 1978]
Equivalent Expression of Endogenous
Genes in C3H/10T/2
Cells1
Murine Leukemia Virus-related
Cells and Chemically Transformed
Derivative
Michael J. Getz,2 Paula K. Elder, and Harold L. Moses
Department
of Pathology
and Anatomy,
Mayo Clinic and Mayo Foundation,
ABSTRACT
The possibility that chemical carcinogens may induce
enhanced expression of endogenous C-type RNA tumor
virus genes in the absence of intact virus particle produc
tion has been partially tested in a model system. This
was accomplished by measuring the abundance and di
versity of murine leukemia virus-related RNA sequences
associated with the polyribosome fraction of nontransformed C3H/10T1 2 clone 8 cells and a 3-methylcholanthrene-transformed derivative clone. Although both
clones are virus nonproducers, they were found to contain
significant amounts of polyadenylate-containing murine
leukemia virus-related RNA sequences; however, both
the types and quantities of such sequences appear indis
tinguishable in both clones. These results suggest that
expression of the corresponding gene sequences into
RNA is not related to the maintenance of the transformed
state in these chemically transformed cells.
INTRODUCTION
The interaction between certain chemical carcinogens
and endogenous C-type RNA tumor virus genes is a poorly
understood process. Although no evidence exists that di
rectly implicates C-type viral gene expression in chemically
induced transformation, an association of virus particles
with some naturally occurring and chemically induced tu
mors as well as with certain chemically transformed cells
in culture has been noted (9, 20). This raises the possibility
that chemical carcinogens can activate endogenous C-type
viral genes in the cellular genome. The relationship, how
ever, between this capability and the mechanism of chemi
cally induced neoplastic transformation is unclear. Two
important aspects are involved: what the specificity of such
activation is in those cell types in which it is readily apparent
and whether viral gene function can ever be completely
uncoupled from the transformed phenotype. Earlier work
in our laboratory demonstrated that transformation of a
clone of virus-negative AKR mouse embryo cells in culture
by a variety of chemical carcinogens [benzo(a)pyrene, 3MCA,3 5,6-dibenzanthracene, and 7,12-dimethylbenzanthracene] resulted in the appearance of MuLV gs antigens and
intact virus particle production (15). In a recent study we
1 This work was supported
by Grant NP-192 from the American
Cancer
Society, by Grant CA 16816 from the National Cancer Institute, Department
of Health, Education, and Welfare, and by the Mayo Foundation.
2 To whom requests for reprints should be addressed.
3 The abbreviations
used are: 3-MCA, 3-methylcholanthrene;
MuLV,
murine leukemia virus; gs, group-specific;
cDNA, complementary
DNA.
Received August 26, 1977; accepted November 30, 1977.
566
Rochester,
Minnesota
55901
have also shown that a 3-MCA-transformed derivative AKR
clone exhibits a 150- to 200-fold increase in the amounts of
MuLV-related RNA sequences in the polyribosome fraction
relative to the parent nontransformed clone (8). This large
increase is not accompanied by detectable changes in
either the types or the abundance of nonviral polyadenylatecontaining mRNA, suggesting some degree of selectivity in
the large enhancement of MuLV-specific RNA sequence
content; i.e., it does not appear to result from a random
derepression of a large number of cellular genes.
Chemically transformed cell lines exist, however, that
show no evidence of C-type RNA viral antigen synthesis or
particle production. Several cell lines have been derived
from a C3H mouse embryo cell line designated C3H/10T'/2
(16). Studies have shown no evidence for MuLV or mouse
mammary tumor virus gs antigens, MuLV cell surface anti
gens, DNA polymerase-containing virus particles, or infec
tious virions in chemically transformed C3H/10TV2 cells
(14). It does not necessarily follow, however, that all viral
gene functions have been uncoupled from the transformed
phenotype. The protein product(s) of viral oncogenes has
not been conclusively identified, and expression of 1 or
more proviral genes may often occur in the absence of
complete virus production (4, 10, 17, 19). In addition,
different proviral gene products may be regulated inde
pendently of each other (3), and there is often a lack of
coordination between the amounts of viral-specific RNA
and virus-specific proteins in cells (3, 5). The experiments
described in the present article are concerned with whether
or not the types and amounts of MuLV-related RNA se
quences in cells bear a relationship to the transformed
phenotype.
MATERIALS
AND METHODS
Chemicals. Tissue culture media (McCoy's Medium 5A
and Eagle's basal medium) were purchased from Grand
Island Biological Co., Grand Island, N. Y. Fetal calf serum
was purchased from Reheis Chemical Co., Phoenix, Ariz.
Penicillin G and streptomycin were purchased from Eli Lilly
& Co., Indianapolis, Ind. Radioisotopes were purchased
from Amersham/Searle Corp., Arlington Heights, III. Oligodeoxythymidylate cellulose Grade T-2 was purchased from
Collaborative Research, Waltham, Mass. Hydroxyapatite
(DNA grade) was purchased from Bio-Rad Laboratories,
Richmond, Calif. S, nuclease was purchased from Miles
Biochemicals, Elkhart, Ind.
Cell Culture. AKR-2B and AKR-MCA cells are grown in
McCoy's Medium 5A supplemented with 10% heat-inacti
vated fetal bovine serum, 100 units penicillin G per ml, and
CANCER
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Chemical-Viral Cocarcinogenesis
100 /¿gstreptomycin
per ml under a 5% C0;,:95% air
environment at 36°.C3H/10T'/2 and C3H/MCA-58 cells are
grown in Eagle's basal medium supplemented
with 10%
fetal calf serum, 100 units penicillin G per ml, and 50 /J.Q
streptomycin per ml. For these studies cells were grown in
490-sq cm plastic roller bottles (Corning Glass Works,
Corning, N. Y.).
All cell lines have been assayed for growth in soft agar
and for tumorigenicity
in athymic "nude" mice as previously
described (8). The presence or absence of MuLV gs anti
gens was determined
by the membrane immunofluorescence technique described
in detail elsewhere (11, 12).
Briefly, cells were stained with goat anti-Tween ether-dis
rupted MuLV followed by counterstaining
with fluorescein
isothiocyanate-conjugated
rabbit anti-goat -/-globulin. The
cells were then examined by fluorescence
microscopy.
AKR-MCA cells scored 100% positive, whereas all other
cell lines used in this study were negative.
Cell Fractionation and Purification of Polysomal RNA.
Cells were harvested by a brief treatment with trypsin:EDTA
and lysed by the Nonidet P40 technique
as previously
described (7, 8). Polyribosomes were prepared by centrifugation of the postmitochondrial
supernatant through 2 M
sucrose; RNA was purified by phenol:chloroform:isoamyl
alcohol extraction and polyadenylate-containing
RNA iso
lated by 2 passages over oligodeoxythymidylate
cellulose
columns as described elsewhere (7, 8).
Purification of AKR-MuLV and Isolation of 70S RNA.
AKR-MuLV (L, strain) was propagated in roller bottle cul
tures of mouse embryo SC-1 cells, harvested at 12-hr
intervals, and purified by a combination
of velocity sedi
mentation and equilibrium
density centrifugation
in 15 to
60% sucrose gradients as described elsewhere (8). Viral
RNA was extracted from purified virions and 70S RNA
isolated by sedimentation
in 10 to 40% sucrose gradients
as previously described (8).
Synthesis and Purification of cDNA. The synthesis and
characterization
of cDNA from purified AKR-MuLV 70S RNA
has been described in detail elsewhere (8). Briefly, a ran
dom mixture of oligodeoxyribonucleotides
(generated by
DNase digestion of calf thymus DNA) was used to prime
cDNA synthesis from purified 70S RNA by avian myeloblastosis virus reverse transcriptase. The cDNA was isolated by
chromatography
on Sephadex G-50 and purified further by
sedimentation
in 4 to 11% alkaline sucrose gradients. The
[3H]dCTP-labeled cDNA had a specific activity of 3734 dpm/
ng. Titration analysis indicated that 50 to 60% of the viral
RNA sequences are transcribed into cDNA with a frequency
closely proportional to their frequency in 70S RNA (8).
RNA-cDNA Hybridization. The general RNA-cDNA hybrid
ization procedures including S, nuclease assay of hybrid
formation are described in detail elsewhere (8). The titration
analysis used to measure the concentration
of MuLV-specific sequences in cellular RNA's is based on titrating a
constant amount of cDNA with increasing
amounts of
cellular RNA, driving all reactions to a D,t value sufficient
to ensure reaction completion
of a 1:1 mixture of cDNA
and 70S RNA, and measuring the fraction of cDNA rendered
S, nuclease resistant. This method is independent of kinetic
considerations
and has been rigorously justified (21).
Thermal Denaturation of Hybrids. The thermal stability
MARCH
of RNA-cDNA hybrids was determined by thermal elution of
hydroxyapatite-bound
hybrids in 0.14 M neutral phosphate
buffer as previously
described
(8).
RESULTS
The parent and chemically transformed derivative clones
of AKR cells used in these studies are referred to as AKR2B and AKR-MCA, respectively. The origin and properties
of these cells have been described previously (8, 15). AKRMCA cells are tumorigenic
in 100% of injected newborn
AKR or athymic nude mice and produce C-type RNA virus
particles as assayed by electron microscopy, XC-cell assay,
and indirect immunofluorescence
assay with gs MuLV antisera (8). AKR-2B cells are negative for tumorigenicity,
MuLV
gs antigens, and virus particle production (8). The parent
and chemically transformed derivative clones of C3H cells
are designated C3H/10T'/2 and C3H/MCA-58, respectively,
and have been extensively described elsewhere (14, 16).
These cells exhibit properties similar to the AKR cells
except that both clones are virus nonproducers
(14). Re
pressed endogenous MuLV can, however, be induced from
both nontransformed
and transformed clones of C3H cells
by treatment with 5-iododeoxyuridine,
demonstrating
that
the complete viral genome is present in the DNA of both
C3H clones (13, 14). It has also been shown that a cDNA
probe synthesized
from AKR-MuLV
hybridizes
almost
equally well with DNA from AKR and C3H mice (2).
A highly representative
cDNA probe was used for the
analysis of viral-specific
RNA in AKR and C3H cells. The
probe was synthesized from purified MuLV 70S RNA (AKR
L, strain) by exogenous reverse transcriptase
in the pres
ence of random oligodeoxyribonucleotides
as primer (18).
Fifty to 60% of the viral 70S RNA can be hybridized by an
equal amount of this cDNA (8). Since the distribution
of
primer binding sites and, hence, the initiation of reverse
transcriptase should be random on 35S RNA templates, it
is unlikely that the expression of a significant region of the
endogenous
viral genome would escape detection with
this probe. Quantitation of the amounts of complementary
sequence in purified polyadenylate-containing
RNA isolated
from the polyribosome fraction of AKR and C3H cells was
accomplished
by titration analysis (8, 21).
Chart 1 illustrates results obtained by titrating a constant
amount of cDNA with increasing amounts of the test RNA's
and driving
centrations
all reactions to completion.
of MuLV-related sequences
The relative con
in the test RNA's
are reflected by the amount of RNA necessary to achieve
equivalent probe hybridization,
whereas qualitative differ
ences in sequences are reflected by the fraction of the
probe hybridized at saturation. As shown previously a large
quantitative difference exists in the concentration
of MuLVrelated sequences in the test RNA's isolated from AKR-2B
and AKR-MCA cells. The total fraction of cDNA hybridized,
however, is similar (68 to 76%). Since titration of the probe
with purified MuLV 70S RNA also results in 70 to 75%
probe hybridization
(8), these results indicate that the
majority of MuLV 70S RNA sequences are present in both
AKR clones, although they are at greatly different concen
trations. In contrast to these results, only about 35% of the
probe is hybridized at apparent saturation with RNA's iso-
1978
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
567
M. J. Getz et al.
lated
sents
Chart
when
from C3H/10T1/2 and C3H/MCA-58 cells. This repre
about 50% of the hybridizable cDNA sequences.
1 also shows that the same results are obtained
a 1:1 mixture of C3H/10T'/2 and C3H/MCA-58 RNA's
is used for titration, indicating that the same subset of
MuLV-related RNA sequences is present in both clones.
Little or no difference has been detected in the initial
slopes of C3H RNA titration curves, indicating that these
sequences are also present in very similar concentrations
in both C3H clones.
Thermal denaturation profiles of MuLV 70S RNA, C3H/
10T/2 RNA, and C3H/MCA-58 RNA hybrids with cDNA
confirm the close homology between sequences present in
the C3H cells and a fraction of sequences present in
purified MuLV 70S RNA (Chart 2). Hybrids formed with the
234567
RNA/cDNA
10
x10'3(w/w)
Chart 1. Titration of MuLV cDNA with polysomal polyadenylate-containing RNA from AKR and C3H cells. Each hybridization point contained 2.0 ng
cDNA, 1.0 ¿¿g
Escherichia coli rRNA, and the indicated amounts of the test
RNA's in a total volume of 5 Ml hybridization buffer (0.5 M NaCI. 25 HIM/V-2hydroxyethylp¡perazine-rV'-2-ethane sulfonic acid, and 0.5 M EDTA (pH 7.8):
formamide; 1:1). Hybridization mixtures were sealed in glass capillaries,
denatured, and incubated for 125 hr at 53°to a DJ of 0.6 [D,,, initial cDNA
concentration (M): (. time of annealing in sec]. The fraction of cDNA in
hybrid formation was determined by S nuclease digestion. •AKR-MCA
RNA; O, AKR-2B RNA; •C3H/MCA-58 RNA; D, C3H/10T'/a RNA; A, 1:1
mixture of C3H/MCA-58 and C3H/10T'/2 RNA; A. E. coli rRNA.
MCA-58 RNA exhibit a slightly reduced 7,,, but the signifi
cance of this has not been fully assessed. Similar results
were previously obtained for viral RNA sequences detected
in the 2 AKR clones (8). It cannot be ascertained, however,
whether such intracellular sequences belong exclusively to
the mouse-tropic MuLV or whether they reflect the expres
sion of closely related endogenous viral genomes (1).
DISCUSSION
The results of these analyses suggest that carcinogeninduced enhancement of MuLV-specific RNA levels in
mouse cells in culture may be dependent on the strain of
the target cell and irrelevant to the transformed phenotype.
This conclusion extends to the level of viral RNA sequence
content, previous demonstrations that the genome of the
cell and not the transformed state is the major determinate
of the degree of virus inducibility by chemical carcinogens
(14). However, it should be realized that transforming
events could involve subtle changes in the expression of a
small fraction of an endogenous viral genome or the acti
vation of transforming sarcoma virus genes that do not
share homology to the MuLV genome and thus may be
refractory to these analyses. In this context, however,
recent studies (6) have failed to detect Moloney sarcoma
virus-specific RNA sequences in 3-MCA-induced tumors in
BALB/c mice. It would seem therefore that current data do
not lend strong support to endogenous viral gene activation
as a causative and universally applicable basis for chemi
cally induced neoplastic transformation.
ACKNOWLEDGMENTS
We are grateful to Dr. Charles Heidelberger for the original seed stocks
of C3H/10T'/z and C3H/MCA-58 cells and for helpful discussions. We are
also indebted to Dr. G. Pearson for performing the membrane immunofluorescence studies, to Dr. W. Rowe for the original clone of AKR-2B cells, to
Dr Janet Hartley for AKR MuLV, and to Dr. J. W. Beard for avian myeloblastosis virus reverse transcriptas«
100
90
REFERENCES
80
7060-
]Ü
50
C
ï
40-
3020-
1060
70
80
90
-100
Temperature
Chart 2. Thermal stability of RNA-cDNA hybrids. cDNA (4.5 ng) and £.
coli rRNA (2.5 ^9) "ere mixed with either 45 ng AKR-MuLV 70S RNA (•),
13.5 M9 C3H/10TV»RNA (D). or 13.5 n9 C3H/MCA-58 RNA (A) in a total
volume of 5 /J hybridization buffer The reactions were annealed to a Dj of
0.6 and harvested with ice-cold 0.14 M sodium phosphate buffer (pH 6.8).
The mixture was absorbed to a 0.5-ml hydroxyapatite column equilibrated in
0.14 M sodium phosphate buffer at 60°.and the nonhybridized cDNA was
removed by washing The hybrid was sequentially eluted by raising the
temperature of the column in 5°increments, allowing 5 min for equilibration,
and removing the cDNA rendered single stranded by washing with (5) 2.5-ml
portions of 0.14 M sodium phosphate buffers. For the 70S RNA hybrid, T,„
is
81°;for the C3H/10P/2 RNA hybrid. T,„
is 81°;for C3H/MCA-58 RNA hybrid,
T„
is 79°.
568
1. Callahan, R., Benveniste, R. E., Lieber, M., and Todaro, G. J. Nucleic
Acid Homology of Murine Type-C Viral Genes. J. Virol. 14: 1394-1403,
1974.
2. Chattopadhyay. S. K., Lowy, D. R., Teich, N. M.. Levine. A. S., and
Rowe. W. P. Qualitative and Quantitative Studies of AKR-Type Murine
Leukemia Virus Sequences in Mouse DNA. Cold Spring Harbor Symp.
Quant. Biol.,39: 1085-1101, 1974.
3. Chen, J. H.. Hayward, W. S., and Hanafusa, H. Avian Tumor Virus
Proteins and RNA in Uninfected Chicken Embryo Cells. J. Virol., 14:
1419-1429, 1974.
4 Del Villano, B. C., Nave, B., Croker, B. P., Lerner, R. A., and Dixon, F.
J. The Oncornavirus Glycoprotein gp69/71 : A Constituent of the Surface
of Normal and Malignant Thymocytes. J. Exptl. Med., 141: 172-187.
1975.
5. Fan. H., and Mueller-Lantzsch. N. RNA Metabolism of Murine Leukemia
Virus. III. Identification and Quantitation of Endogenous Virus-Specific
mRNA in the Uninfected BALB/c Cell Line JLS-V9. J. Virol., 18: 401410, 1976.
6. Frankel, A. E., and Fischinger, P. J. Nucleotide Sequences in Mouse
DNA and RNA Specific for Moloney Sarcoma Virus. Proc. Nati. Acad.
Sci. U. S., 73. 3705-3709, 1976.
7. Getz. M. J., Elder, P. K.. Benz, E. W., Jr., Stephens, R. E., and Moses,
H. L. Effect of Cell Proliferation on Levels and Diversity of Poly(A)Containing mRNA. Cell, 7: 255-265, 1976.
8. Getz, M. J., Reiman, H. M., Slegai, G. P., Quinlan, T. J., Proper, J.,
Elder, P. K., and Moses, H. L. Gene Expression in Chemically Trans
formed Mouse Embryo Cells: Selective Enhancement of the Expression
of C-Type RNA Tumor Virus Genes. Cell, 11: 909-921, 1977.
CANCER
RESEARCH
VOL. 38
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
Chemical-Viral
9. Heidelberger, C. Chemical Carcinogenesis. Ann. Rev. Biochem., 44.
49-121,1975.
10. Lerner, R. A., Wilson, C. B., Del Villano, B. C., McConahey, P. J., and
Dixon, F. J. Endogenous Oncornaviral Gene Expression in Adult and
Fetal Mice: Quantitative, Histologie, and Physiologic Studies of the
Major Viral Glycoprotein, gp70. J. Exptl. Med., 143: 151-166, 1976.
11. Pearson, G., Orr, T., Redmon, L., and Bergs, V. Membrane Immunofluorescence Studies on Cells Producing Rat C-Type Virus Particles. Intern.
J. Cancer, 70: 14-19, 1972.
12. Pearson, G. R., Redmon, L. W., and Bass, L. R. Protective Effect of
Immune Sera against Transplantable Moloney Virus-induced Sarcoma
and Lymphoma. Cancer Res., 33: 171-178, 1973.
13. Rapp, V. R., and Nowinski, R. C. Endogenous Ecotropic Mouse Type-C
Viruses Deficient in Replication and Production of XC Plaques. J. Virol.,
78: 411-417, 1976.
14. Rapp, V. R., Nowinski, R. C., Reznikoff, C. A., and Heidelberger. C.
Endogenous Oncornaviruses in Chemically Induced Transformation.
Transformation Independent of Virus Production. Virology, 65. 392409,1975.
15. Reiman, H. M., Branum, E. L., and Moses, H. L. Effects of Chemical
Carcinogens and C-Type RNA Viruses on Transformation of Mouse
Embryo Cells. J. Cell Biol., 67: 358a, 1975.
MARCH
Cocarcinogenesis
16. Reznikoff, C. A., Bertram, J. S., Brankow, D. W., and Heidelberger, C.
Quantitative and Qualitative Studies of Chemical Transformation of
Cloned C3H Mouse Embryo Cells Sensitive to Postconfluence Inhibition
of Cell Division. Cancer Res., 33. 3239-3249, 1973.
17. Strand, M., Lilly, F., and August, J. T. Host Control of Endogenous
Murine Leukemia Virus Gene Expression: Concentrations of Viral Pro
teins in High and Low Leukemia Mouse Strains. Proc. Nati. Acad. Sci.
U. S., 77: 3682-3686, 1974.
18. Taylor, J. M., Illmensee, R., and Summers, J. Efficient Transcription of
RNA into DNA by Avian Sarcoma Virus Polymerase. Biochim. Biophys.
Acta, 442: 324-330, 1976.
19 Tung, J-S., Vitetta, E. S., Fleissner, E., and Boyse, E. R. Biochemical
Evidence Linking the GK Thymocyte Surface Antigen to the gp69/71
Envelope Glycoprotein of Murine Leukemia Virus. J. Exptl. Med., 747:
198-205, 1975.
20 Weinstein, B., Yamaguchi, N., and Gebert, R. Use of Epithelial Cell
Cultures for Studies on the Mechanism of Transformation by Chemical
Carcinogens. In Vitro, 77: 130-141, 1975.
21 Young, B. D., Harrison, P. R., Gilmour, R. S., Birnie, G. D., Hell, A.,
Humphries, S., and Paul. J. Kinetic Studies of Gene Frequency: II.
Complexity of Globin Complementary DNA and Its Hybridization Char
acteristics. J. Mol. Biol., 84: 555-568, 1974.
1978
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1978 American Association for Cancer Research.
569
Equivalent Expression of Endogenous Murine Leukemia
Virus-related Genes in C3H/10T½ Cells and Chemically
Transformed Derivative Cells
Michael J. Getz, Paula K. Elder and Harold L. Moses
Cancer Res 1978;38:566-569.
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