http://cancerres.aacrjournals.org/content/46/7/3574.full.pdf

[CANCER RESEARCH 46, 3574-3579, July 1986]
Selective Killing of Simian Virus 40-transformed Human Fibroblasts by
Parvovirus H-l1
Yong Quan Chen, Françoisede Foresta, Jacqueline Hertoghs, Bernard L. Avalosse, Jan J. Cornelis, and
Jean Rommelaere2
Laboratory of Biophysics and Radiobiology, UniversitéLibre de Bruxelles, B-1640 Rhode St. Genèse,Belgium [Y. Q. C., F. d. F., J. H., B. L. A., J. J. C., J. RJ, and
Laboratory of Molecular Oncology, 1NSERM UI86, Institut Pasteur de Lille, F-59019 Lille, France [B. L. A., J. J. C.. J. RJ
ABSTRACT
A normal strain of human foreskin fibroblasts, two SV40-transformed
derivatives with finite and infinite life spans, and an established line of
SV40-transformed newborn human kidney cells are compared for their
susceptibility to infection with parvovirus H-l. H-l inocula, which do not
detectably alter the growth of normal cells, cause a progressive degen
eration of all three SV40-transformed cultures. The resistance of normal
cells is not a membrane phenomenon since they adsorb and take up H-l
as efficiently as the transformants. Moreover, the fraction of infected
cells supporting the synthesis and nuclear migration of H-l proteins is
similar in normal and SV40-transformed cultures. On the other hand,
the enhanced H-l sensitivity of transformed cells correlates with a 5- to
30-fold increase in their accumulation of newly synthesized parvoviral
DNA, as compared with normal cultures. This stimulation of H-l DNA
replication is most pronounced for the amplification of duplex replicative
forms, although the conversion of parental single-stranded DNA to
replicative forms is also enhanced to a smaller extent. In addition, SV40-
transformed cells support productive H-l infection and release a burst of
infectious virus, whereas no H-l production can be detected in the normal
cell strain. The latter difference was confirmed for another series of 7
normal and 16 SV40-transformed strains of human skin fibroblasts.
Altogether, these results indicate that intracellular limitations on H-l
DNA replication are associated with the abortive nature of the parvoviral
life cycle in normal human fibroblasts and are overcome after SV40
transformation, resulting in the selective killing of the transformants.
This observation raises the possibility that oncolysis might contribute to
the oncosuppressive activity displayed by parvoviruses in vivo.
INTRODUCTION
Parvoviruses are nuclear replicating, single-stranded DNA
viruses of small size and low genetic complexity, which have
been isolated, in particular, from a number of avian and mam
malian species including humans (1). Parvoviruses have a lytic
replicative life cycle and are devoid of detectable oncogenic
activity, but they have the intriguing ability to suppress cancer
formation in laboratory animals and there is speculation about
their similar effect in humans (for reviews, see Refs. 2 and 3).
Vertebrate parvoviruses can be divided into two subgroups,
nondefective (so-called autonomous) and defective (so-called
adeno-associated) members, respectively. The antineoplastic
spectrum of autonomous parvoviruses is broad and includes
spontaneous (4) as well as \ ¡rally(3) and chemically (5) induced
cancers, whereas adeno-associated viruses specifically inhibit
helper virus oncogenicity (2). The cellular and molecular bases
of oncosuppression by parvoviruses are poorly understood.
Parvoviruses might interfere with complex physiological proc
esses conditioning tumor development (5). On the other hand,
adeno-associated (6, 7) and autonomous (8) parvoviruses have
Received 11/12/85; revised 3/5/86; accepted 3/26/86.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported by an Action de Recherche Concertéefrom the Ministère de la
Politique Scientifique and by grants from the Caisse Généraled'Epargne et de
Retraite, Loterie Nationale, and Fonds de la Recherche Scientifique Médicale.
2To whom requests for reprints should be addressed.
both been shown to inhibit in vitro transformation of cells in
culture, although they might act by different mechanisms. Thus,
direct interactions between parvoviruses and transformed cells
or their precursors, as occurring in vitro, might also contribute
to oncosuppression.
We reported previously that the autonomous parvovirus
MVM3 prevents the tumor virus SV40 from transforming in
vitro mouse cells which had been selected for their resistance to
MVM infection (8). This inhibition could be ascribed to the
specific lysis of SV40 transformants by MVM. Together with
the known requirements of parvoviruses for the proliferation
(3) and appropriate differentiation (1) of their host cells, this
observation led us to hypothesize that malignant transforma
tion may render normally resistant cells permissive to the
replication of these viruses. Hence, transformed cells would be
a preferential target for the lytic action of autonomous parvo
viruses. The possible involvement of such an oncolysis in cancer
suppression by nondefective parvoviruses remains to be deter
mined.
The work presented in this paper was aimed at analyzing the
mechanism by which SV40 transformation sensitizes cells to
the killing effect of autonomous parvoviruses and at determin
ing the generality of this phenomenon. Since it was known that
the nondefective parvovirus H-l can be propagated in an estab
lished line of SV40-transformed newborn human kidney cells
(9), it was decided to compare a normal diploid strain of human
foreskin fibroblasts and two SV40-transformed derivatives with
finite and infinite life spans for their susceptibility to H-l
infection. Consistent with the oncolysis hypothesis, SV40 trans
formation was found to correlate with an enhanced cell permis
siveness to H-l.
MATERIALS AND METHODS
Cells. Human cell strains were grown as monolayers in Eagle's
minimal essential medium supplemented with 10% fetal calf serum.
NB-E is an established line of S\ 40 transformed newborn human
kidney cells (10). VII 10 is a normal finite-life strain of foreskin human
fibroblasts from which nonestablished (VH-10 1SV) and established
(VH-10 SV), SV40 T-antigen-positive clones were independently de
rived by transformation with the early region of SV40 6-17 mutant
deleted within the replication origin. The VH-10 series was prepared
by B. Klein and kindly provided by A. van der Eb (Leiden University,
Leiden, The Netherlands). Cells were counted with a hemocytometer
after trypsinization and their viability was determined by their ability
to exclude 0.07% (w/v) trypan blue stain.
Virus. H-l parvovirus (wild type) was propagated in NB-E cells and
purified according to the method of Tattersall et al. (11). 3H- and 32P-
labeled virus was produced by incubating infected cells with [3H]thy-
midine (5 Ci/mmol, 25 ^Ci/ml) and 32P¡(carrier free, 0.1 mCi/ml), as
described by Rhode (12) and Rommelaere and Ward (13), respectively.
Conditions for cell infection, virus harvesting, and titration by plaque
assay have been described previously (14).
H-l Uptake. Cells were incubated for 60 min at 37°Cor 4°Cin the
3The abbreviations used are: MVM, minute virus of mice; SS, single-stranded;
RF, replicative form.
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CELL TRANSFORMATION AND SUSCEPTIBILITY TO PARVOVIRUSES
Table 3 H-l protein synthesis in normal and SV40-transformed human
fibroblasto
H-1-infected cells were tested for viral protein synthesis by an immunoradi-
ometric assay or immunoenzymatic staining. Background radioactivity due to the
immunoradiometric method (-350 cpm) and to input virus proteins (-150 cpm)
was measured 2 h postinfection and was subtracted. Nuclear staining was quan-
titated in 200 cells and its positiveness and intensity were determined after similar
background (A = 6) subtraction. The relative synthesis of viral proteins is referred
to the corresponding VH-10 value considered as 1.0.
Immunoradiometric assay Immunoenzymatic staining
% of Absorbance/
Relative positive positive Relative
Cells cpm/10* cells synthesis nuclei nucleus synthesis
VH-10VH-10
SVVH-10
1SVNB-E12422420325348471.01.952.63.926.5292628111621211.01.61.92.0
012345
Time postinfection (days)
Fig. 4. H-l virus production in normal and SV40-transformed human fibro
blasts. Cultures (5 x 10s cells) were infected with H-l virus (multiplicity of
infection, IO"2) and analyzed at intervals for the production of infectious virus.
Average values for 2 experiments (SD less than 15%). PFU, plaque-forming units.
Same symbols as for Fig. 1.
2) synthesis correlated, although the former was much lower
than the latter.
Production of Infectious H-l Particles. Cultures of normal
VH-10 cells were unable to amplify the H-l inoculum; the titer
of infectious virus harvested from these cultures rather declined
with time (Fig. 4). Thus, most normal fibroblasts did not appear
to sustain a productive H-l infection. In contrast, SV40-trans-
formed strains were all productively infected and released a
burst of infectious H-l (Fig. 4). NB-E and transformed VH-10
cells displayed high and intermediate degrees of permissiveness
compared to normal VH-10 cultures, respectively, which cor
related with their relative abilities to support H-l DNA repli
cation (Table 2) and susceptibilities to the lytic action of H-l
(Fig. 1). The induction of permissiveness to H-l as a result of
SV40 transformation is not unique to VH-10 cells. As shown
by Table 4, a similar effect was observed for a number of other
human skin fibroblasts. None of the normal cultures tested were
able to replicate H-l whereas SV40-transformed strains were
the hosts of a productive H-l infection.
Table 4 H-l virus replication in human fibroblasts
Cultures of human fibroblasts (5 x 10* cells) were infected with H-l (multi
plicity of infection, 10~2),incubated for 4 days, and tested for the production of
infectious virus. Increases in tiler of at least 3-fold over the input virus were
scored as positive. The virus titer in nonproducing cells actually decreased with
time postinfection. Input virus was titered 2 h postinfection.
CellsVH-10VH-10SVGM2149
GM2149SVGM1061
GM1061SVGM1085
GM1085SVAT5BI
AT5BIVA2BI
46BRXP30RO
GM0637 B
NB-ENo.
of SV40 H-l
strains transfer- produc-
Ref. tested mationtion38
139 2 ++139
3 ++140
2 ++140
8 ++41
a41
4241a10+-:
:
" National Institute of General Medical Sciences Human Genetic Mutant Cell
Repository.
DISCUSSION
Abortive Interaction between Parvovirus H-l and Normal Hu
man Fibroblasts
A series of nonpermanent cultures of normal human skin
fibroblasts were found to resist productive infection with par-
vovirus H-l. Resistance could be ascribed to the failure of an
intracellular stage of the replicative life cycle of H-l, resulting
in a lack of amplification of the virus inoculum. Similar results
have been reported for human amnion, embryo kidney (25),
and lung (9) cells. A quantitative comparison of H-l replication
was undertaken between normal skin fibroblasts and corre
sponding permissive cultures, namely SV40 transformants de
rived therefrom (see below). A striking difference was observed
at the level of the replication of RF DNA, which was much
enhanced in transformed cultures and correlated with their
ability to produce infectious virus. The defect of the H-l life
cycle in diploid human fibroblasts might concern RF DNA
replication itself. Alternatively, another step of the viral cycle
conditioning the extent of RF DNA replication might be im
paired, although our data suggest that virus uptake, nuclear
translocation, and parental DNA conversion are unlikely to be
primarily involved.
The inability of normal human fibroblasts to support RF
DNA replication efficiently might result from their failure to
express nonessential host functions usurped by H-l for its own
growth. In addition, H-l DNA replication appears to involve
virus-encoded functions (26). Nonpermissive cells might also
fail to synthesize or modify these viral products, although no
major impairment of H-l capsid gene expression was detected
in diploid human fibroblasts. Our data indicate that the limi
tation placed by normal cells on H-l replication can be reversed
as a result of their in vitro transformation by SV40. A parallel
can be drawn between this observation and other reports show
ing that cellular changes can render normally resistant cultures
susceptible to autonomous parvoviruses. Permissiveness to par-
voviruses has been shown to be dependent on host cell prolif
eration (27,28) and differentiation (29-31). Various physiolog-
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CELL TRANSFORMATION AND SUSCEPTIBILITY TO PARVOVIRUSES
ical states can lead to the arrest of the parvoviral life cycle at
different steps, both on the surface and on the inside of the cell
(32). Therefore, there appear to be multiple conditional host
factors which determine permissiveness to parvoviruses. It fol
lows that the specific abortive interaction reported here for
diploid human fibroblasts cannot be necessarily generalized to
other cell types.
Induction of 11-1 Sensitivity by SV40 Transformation
The acquisition of permissiveness to H-l replication by SV40
transformants correlated with their sensitization to parvoviral
lytic action. H-l inocula which had little effect on the growth
of normal cultures caused a progressive degeneration of trans
formed derivatives. Thus, the preferential killing of transformed
cells is likely to result from their propensity to replicate H-l
rather than their lower resistance to its cytopathic effect. A
similar sensitization of SV40 transformants to the autonomous
parvovirus MVM was reported previously for mouse cells which
had been selected in vitro for their resistance to this virus (8).
Therefore, SV40 transformation overcomes both natural and
selected cellular protections against parvoviral attack.
The enhanced susceptibility of SV40 transformants to H-l
infection does not require their establishment into a permanent
culture. Thus, SV40 transformation appears to act directly and
not merely by facilitating cell immortalization. Since the expres
sion of the SV40 genome in transformants is restricted to the
early region-encoding T-antigens (33), the latter protein(s) is
likely to mediate sensitization to H-l. Large T-antigen is known
to activate cellular gene expression (33, 34). As stated above,
cell permissiveness to parvoviruses involves host functions,
some of which are not permanently expressed and might con
ceivably be induced by SV40. On the other hand, a direct
interaction between the H-l genome and SV40 T-antigen(s)
might also potentiate H-l replication. Another tumor virus,
adenovirus, was found to provide helper functions to H-l in
coinfected human cells (35) and encodes a tumor antigen which
could possibly interact directly with the DNA of the defective
parvovirus adeno-associated virus (7). Whatever mechanism it
involves, sensitization to H-l is apparently not specific for
tumor virus-transformed cells. Indeed we have recently ex
tended the present observation to human diploid fibroblasts
transformed by 7-irradiation (36). Nonvirally transformed cells
therefore appear to supply helper functions similar to those
encoded or induced by SV40.
Altogether, our data point to a positive interrelation between
two complex cellular phenotypes each composed of multiple
components, i.e., permissiveness to autonomous parvoviruses
and /// vitro malignant transformation. This result raises two
intriguing possibilities, (a) The lytic life cycle of parvoviruses
may provide specific markers for cell transformation. We report
here that SV40 transformation bypasses a limitation to H-l
DNA replication. In contrast, a postreplicative step of the
growth of parvovirus MVM was found to be stimulated as a
result of the transformation of rat cells with the retrovirus avian
erythroblastosis virus (37). An interesting issue is whether the
overcoming of distinct barriers to parvovirus replication re
quires the expression of different transformation traits, (b) The
suppresion of cancer development by autonomous parvoviruses
may result, at least in part, from oncolysis. The preferential
cytolytic replication of autonomous parvoviruses in trans
formed cells accounts for the ability of these viruses to specifi
cally inhibit in vitro malignant transformation (8). Whether
cancer cells provide a similar target for destruction by parvo
viruses in vivo remains to be determined.
ACKNOWLEDGMENTS
The authors are greatly indebted to Drs. A. van der Eb and B. Klein
for their generous gift of VH-10 and derived cells, to C. Kumps for
technical assistance, and to Dr. P. Tattersall for critical reading of the
manuscript. Some human mutant cells were kindly provided by Drs. C.
Arieti, A. Lehmann, and D. Bootsma.
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