Journal o f General Virology (1992), 73, 2003-2009.

2003

Printed in Great Britain

Cooperation of oncogenes in cell transformation and sensitization to killing by the parvovirus minute virus of mice C. Legrand, 1 S. Mousset, ~* N. Salom~ 2 and J. Rommelaere ~,2 ~Department of Molecular Biology, Universit~ Libre de Bruxelles, 67 rue des Chevaux, B 1640 Rhode-St-Genbse, Belgium and 2Molecular Oncology Unit, Institut Pasteur de Lille, C N R S URA1160, 59019 Lille C~dex, France

The established line of normal Fisher rat fibroblasts (FR3T3) is naturally resistant to the parvovirus minute virus of mice (MVM), and was used as a model system to study the influence of stepwise transformation on the susceptibility of cells to this virus. When transformed with genes encoding the class I nuclear oncoproteins large T antigen of polyomavirus (PyLT) or v-myc, cells retained a normal appearance, but acquired some ability to form colonies in soft agar. On the other hand, the class II transforming oncogenes encoding the middle T antigen of polyomavirus (PyMT) and c-Ha-ras-1 induced both morphological alterations and a high capacity for anchorage-independent growth in transfected cells. The concomitant expression of oncogenes from both classes (PyLT+PyMT; v-myc+c-Ha-ras-1) induced a supertransformed phenotype characterized by the piling-up of

cells into poorly adherent foci, even in low density cultures. The progressive transformation of this cellular system was found to coincide with a gradual increase in its susceptibility to M V M p (MVM prototype strain) infection. Compared to parental cells, class I, class II and double transformants proved to be sensitized to killing by M V M p to a low, moderate and large extent, respectively. Thus, oncogenes from different functional classes appeared to cooperate in the responsiveness of cells to parvovirus attack. Interestingly, this cooperation exacerbated both the killing of infected cells and their capacity to produce viral non-structural (NS) proteins, in agreement with the reported cytotoxic activity of NS polypeptides. Therefore, in this system, parameters of the parvovirus life cycle may serve as indications of the overall progression of the transformation process.

Introduction

normal cells generally resist virus attack. This sensitization of transformed cells correlates with a stimulation of parvoviral gene expression, suggesting that some viral proteins may be cytotoxic (Rhode, 1987; Osawa et al., 1988; Cornelis et al., 1988b; van Hille et al., 1989; Brandenburger et al., 1990). It has been demonstrated recently that the intracellular accumulation of MVMencoded regulatory non-structural (NS) proteins is toxic for neoplastic human cells (Caillet-Fauquet et al., 1990). It has been shown that tumour viruses, cellular or viral oncogenes, and chemical or physical carcinogens may sensitize normally resistant cells to parvovirus-induced killing (Mousset & Rommelaere, 1982; Mousset et al., 1986; Cornelis et al., 1988a; van Hille et aL, 1989; Salem6 et al., 1990; Guetta et al., 1990). However, it is unclear whether cell sensitization to parvoviruses correlates with the expression of a specific facet(s) of the transformed phenotype. We have shown previously that abortive transformation induced by infection with the tumour virus simian virus 40 or by treatment with the tumour promoter TPA is not sufficient to sensitize mouse cells to killing by MVMp (prototype strain of MVM) (Mousset & Rommelaere, 1988). In this work, we took

Parvoviruses are small, non-enveloped, nuclear-replicating viruses with a linear ssDNA genome of about 5000 nucleotides. The low coding capacity of parvoviruses implies that they rely strongly upon exogenous helper factors for their replication. In the case of autonomous parvoviruses like the minute virus of mice (MVM), these factors are provided by the host cell and are expressed as a function of cell proliferation and differentiation (Cotmore & Tattersall, 1987). Often isolated from tumour tissues, tumour cell lines or stocks of oncogenic viruses (Siegl, 1984), parvoviruses were suspected of being oncogenic. Nevertheless, they proved to be devoid of such an activity and, on the contrary, to inhibit both spontaneous and induced cancers in laboratory animals (reviewed in Rommelaere & Cornelis, 1991). Therefore, the frequent association of parvoviruses with tumours is likely to reflect an opportunistic relationship instead of a causal one (Rommelaere & Tattersall, 1990). This oncotropism is consistent with the fact that a number of neoplastic or transformed cells cultivated in vitro are permissive to the lytic parvovirus life cycle, whereas 0001-0911 © 1992 SGM

2004

C. L e g r a n d and others

a d v a n t a g e o f the fact t h a t in vitro cell t r a n s f o r m a t i o n c a n be a c h i e v e d in a s t e p w i s e f a s h i o n b y using c o o p e r a t i n g o n c o g e n e s (Cuzin, 1984). T h i s p r o p e r t y is r e l a t e d to the s o m e w h a t artificial g r o u p i n g o f o n c o g e n e s into funct i o n a l classes I a n d II, d e p e n d i n g o n the p r i m a r y effect ( i m m o r t a l i z a t i o n versus t r a n s f o r m a t i o n ) a n d l o c a l i z a t i o n (nucleus versus c y t o p l a s m or p l a s m a m e m b r a n e ) o f c o r r e s p o n d i n g o n c o p r o t e i n s ( H u n t e r , 1991). T w o p r o t o t y p e class I [ p o l y o m a v i r u s large T ( P y L T ) a n d v-myc] a n d class I I [ p o l y o m a v i r u s m i d d l e T ( P y M T ) a n d c-Ha-ras-1] o n c o g e n e s were used, on t h e b a s i s o f t h e i r p r e v i o u s l y r e p o r t e d a b i l i t y to act in c o o p e r a t i o n in n e o p l a s t i c t r a n s f o r m a t i o n o f n o r m a l cells (Ruley, 1990). C o n s i s t e n t l y , we f o u n d t h a t P y L T o r v - m y c c a u s e d little p h e n o t y p i c a l t e r a t i o n in the e s t a b l i s h e d b u t o t h e r w i s e n o r m a l r a t f i b r o b l a s t line F R 3 T 3 g r o w n in l i q u i d m e d i u m . H o w e v e r , these o n c o g e n e s c o n f e r r e d o n corres p o n d i n g P y M T - or c - H a - r a s - l - i n d u c e d m o r p h o l o g i c a l t r a n s f o r m a n t s the c a p a c i t y to pile up into p o o r l y a d h e r e n t foci, e v e n a t low cell density. A l l single a n d d o u b l e t r a n s f o r m a n t s a c h i e v e d a g r e a t e r c l o n i n g effic i e n c y in semi-solid m e d i u m to a c e r t a i n extent. T h e r e f o r e , this cellular s y s t e m offered a s p e c t r u m o f g r a d u a l p h e n o t y p i c a l t e r a t i o n s to be c o m p a r e d w i t h the s u s c e p t i b i l i t y to M V M p infection. O u r results i n d i c a t e t h a t e x p r e s s i o n o f the class I or class I I o n c o g e n e s s t u d i e d resulted in e n h a n c e d killing o f F R 3 T 3 cells b y M V M p , a l t h o u g h t h e l a t t e r o n c o g e n e s p r o v e d to be m o r e efficient. T h e c o o p e r a t i o n o f b o t h classes o f o n c o g e n e s at t h e level o f cell t r a n s f o r m a t i o n is reflected in a m o d e s t s y n e r g i s m o f t h e i r s e n s i t i z a t i o n o f cells to M V M p infection. It is c o n c l u d e d that, in this system, s e n s i t i v i t y to M V M p m a y be i n d i c a t i v e o f the p r o g r e s s i o n o f the t r a n s f o r m a t i o n process r a t h e r t h a n c o r r e l a t e d w i t h a specific step.

Methods Cells, virus andplasmids. The established line of Fisher rat fibroblasts FR3T3 (Seif & Cuzin, 1977), its FR4 subclone (Guetta et al., 1990) and corresponding transformed derivatives were grown in Dulbecco's modified Eagle's medium supplemented with 10~ foetal calf serum (FCS) and 1~ sodium pyruvate. The mouse cell line A9 was cultivated in Eagle's MEM supplemented with 5% FCS. MVMp was propagated in A9 cells and purified according to Tattersall et al. (1976). The transformed cell lines FRLT1, FRMC14 and FREJ4 have been described previously by Rassoulzadegan et al. (1982), Salom6 et al. (t990) and van Hille et al. (1989), respectively. FR4 derivatives transformed by PyMT (FRMT) or both PyMT and PyLT (FRMLT) were isolated as geneticin (0-5 mg/ml)-resistant foci of morphologically altered cells, after cotransfection with plasmid pSV2neo (Southern & Berg, 1982) and pPyMT1 (Treisman et al., 1981) or pPyMLT97 (Gelinas et al., 1989). FRMCEJ clones were obtained by transfection of FRMCI4 cells with plasmid pSVneoEJ, which carries the c-Ha-ras-1 oncogene from the human bladder carcinoma cell line EJ (McKay et al., 1986), followed by the selection of geneticin (0-5 mg/ml)-resistant

colonies. Transfections were carried out by the calcium phosphate precipitation technique described by Brandenburger et al. (1990). Detection of oncoproteins. After cell lysis, oncoproteins were immunoprecipitated (Salom~ et al., 1990) with either the ras-specific monoclonal antibody YIJ-259 (Oncogene Science) or polyclonal antibodies that recognized both PyMT and PyLT antigens, obtained by intraperitoneal injection of polyomavirus-transformed PyB4A cells into BN rats. Oncoproteins were labelled either in vivo by prior incubation of cells for 1 h (PyLT) or 18 h (c-Ha-ras-1) with [35S]methionine (200 to 500 ~tCi/106 cells, 800 Ci/mol; Amersham), or after immunoprecipitation (PyMT) by an in vitro kinase reaction in the presence of [?-3zP]ATP (5 laCi/106 cells, 3000 Ci/mmol; Amersham) according to the method of Getinas et at. (1989). Proteins were fractionated by 7.5 to 15~ SDS-PAGE and visualized by autoradiography. The immunodetection of the v-myc protein in this system has been reported previously (Salom6 et aL, 1990). Clonogenicity in semi-solid medium. For the measurement of anchorage-independent growth, suspensions of 102 to 105 cells in 3 ml Dulbecco's modified MEM containing 10~ FCS and 0.33 ~ agar were seeded onto 60 mm diameter dishes containing 7 ml of the same medium. Colonies were counted by microscopic examination between 3 and 5 weeks after plating. Parvovirus cytotoxicity. Parvovirus-induced cell killing was determined by clonal growth assays, as described by Mousset & Rommelaere (1988). Between 200 and 1000 cells were plated in 60 mm plastic dishes and allowed to form colonies which were stained after 10 days. The plating efficiency of all cell lines ranged from 80 tO 100%. Cell survival was calculated from the number of colonies formed by MVMp-infeeted cells and expressed as a percentage of those formed by mock-infected cells. The m.o.i, was defined as the number of p.f.u, virus inoculated per cell. Paroovirus life cycle parameters. Total viral DNA was measured by hybridization using dispersed cell assays as described previously (Mousset & Rommelaere, 1988). Viral DNA amplification was calculated as the ratio of cell-associated viral DNA at 30 h to that at 2 h after infection at an m.o.i, of 1 p.f.u./cell. For the detection of the viral NS-1 protein, cultures were infected at an m.o.i, of 2 p.f.u./cell, incubated for 18 h and further labelled for 1 h with [35S]methionine (200 to 500 ~tCi/ml, 800 Ci/mol; Amersham). Cells were lysed and NS-1 protein was immunoprecipitated with a monospecific rabbit antiserum raised against a bacterial fusion protein which contains an MVMp NS-1 protein-specific amino acid sequence (Cornelis et al., 1988b). Proteins were fractionated by 10~ SDS-PAGE and visualized by fluorography.

Results Characterization o f the cell lines

T a b l e 1 s u m m a r i z e s the p r o p e r t i e s o f the cells used in this work. F R 3 T 3 a n d its s u b c l o n e F R 4 a r e e s t a b l i s h e d lines o f n o r m a l r a t fibroblasts w h i c h e x h i b i t s t r i n g e n t c o n t a c t i n h i b i t i o n a n d a n c h o r a g e - d e p e n d e n t growth. T h e P y L T a n d v - m y c - t r a n s f o r m e d d e r i v a t i v e s r e t a i n e d b o t h flat m o r p h o l o g y a n d g r o w t h a r r e s t at loose confluence, b u t c o u l d be d i s t i n g u i s h e d f r o m p a r e n t a l cells b y t h e i r significant, a l t h o u g h low (1 to 2%), c l o n i n g efficiency in soft agar. T h e P y M T a n d c-Ha-ras-1 t r a n s f o r m a n t s were characterized by their small and rounded appearance,

2005

Oncogene-promoted cell killing by M V M p

Table 1. Phenotypic properties of the FR3T3 cell line and its oncogene-transformed derivatives* Susceptibility to M V M p

Transformation parameters

Cell line

Oncogene

Morphological alteration

Saturation density (cells × 10-s)t

Clustered

growth

Cloning efficiency in soft agar (%)

M V M p cytotoxic effect MVMp DNA amplification:~

Cell survival (%)§

Lethal hits/celll[

FR4 FRLT1 FRMT1 FRMT4 FRMLT1 FRMLT2 FRMLT4

-

-

PyLT PyMT PyMT PyLT + P y M T PyLT + P y M T PyLT + P y M T

+ + + + +

10 91 ND¶ ND 150 ND

-+ + +

1 55 11 12 25 13

12 78 51 122 98 74

92 84 47 52 20 24 27

0-1 0.2 0.75 0.65 1.6 1.4 1-3

FR3T3 FRMC14 F R E J4 FRMCEJ1 FRMCEJ10 FRMCEJI5

v-myc EJ-ras v-myc + EJ-ras v - m y c + EJ-ras v - m y c + EJ-ras

+ +

9.4 9.1 91 ND ND 160

+ + +

< 10-4 2 45 48 40 52

11 10 110 71 57 35

89 65 47 16 14 4

0'1 0"4 0.75 1"8 2 3"2

+ +

9-4

-


'4 o :~:"

Fig. 1. Morphological appearance of normal F R 3 T 3 cells and transformed derivatives; phase contrast microscopy of cells in culture. (a) FR3T3, (b) F R M C 1 4 , (c) F R E J 4 and (d) F R M C E J 1 5 . Bar marker represents 200 Ixm.

2006

C. Legrand and others

(a)

p105 PyLT P

(b)

(c)

o

~

a~

~

g

~

g

g

g

g

g

a~

g

g

g

a~

tL

tL

~

tL

~

tL

I.h

~h

tr-

I-h

gh

~

tL

tL

68KO

68KO

25.7KO p21 ras I1~

p56 PyMT 18.4K0

43Kg

43KQ

Fig. 2. Production of oncoproteins. 10~ SDS-PAGE of proteins from parental cells (FR3T3 and FR4) and clonal derivatives transformedby (a) PyLT, (b) PyMTor (c) c-Ha-ras-1. (a and c) Fluorogramsof immunoprecipitatesfrom extracts containingequivalent amounts of [35S]methionine-labelledproteins. (b) Autoradiogramof in vitrophosphorylatedPyMT immunoprecipitatesprepared from equivalent amounts of total proteins and incubated in the presence of D,-32p]ATP. induced cell killing. Survival was determined as the residual clonogenicity of MVMp-infected versus mockinfected cells. Table 1 shows that the parental FR4 and FR3T3 lines were very resistant to MVMp infection (about 9 0 ~ survival), whereas oncogene-transformed derivatives displayed a broad spectrum of sensitivities to the virus cytopathic effect. The class I oncogenes PyLT and v-myc sensitized cells to MVMp only poorly (84 and 6 5 ~ survival, respectively), whereas the class II oncogenes PyMT and c-Ha-ras-1 were more efficient in this respect (52 and 2 3 ~ survival, respectively). Interestingly, the simultaneous expression of both types of oncogenes (PyLT + P y M T or v-myc+ c-Ha-ras-1) resulted in a further increase in MVMp-induced killing of double (compared to either class of single) transformants. Assuming a Poisson distribution, it was calculated that the average number of lethal hits per double transformant was greater than the sum of those inflicted on the corresponding single transformants, suggesting a modest synergistic effect of class I and II oncogenes on sensitization of cells to MVMp.

Oncogene-induced modulation of M V M p replication To unravel the role of parameters of the M V M p life cycle in cytotoxicity, viral D N A replication and expression were measured in the normal parental cells and in their transformed derivatives. It has been reported previously that some transformed cells have a greater capacity for replicating parvoviral D N A than their normal progenitors (Mousset et al., 1986; Avalosse et al., 1987; Cornelis et al., 1988a). This prompted us to investigate whether the enhanced toxicity of MVMp for FR3T3 fibroblasts transformed by

different oncogenes was associated with a stimulation of viral D N A replication. The amplification of input MVMp D N A was measured by hybridization at 30 h after infection with a multiplicity of 1 p.f.u./cell. As shown in Table 1, FR4 and FR3T3 parental lines as well as PyLT and v-myc transformants sustained only a low level of MVMp D N A synthesis. By contrast, PyMT, c-Ha-ras-1 and double transformants replicated viral D N A very efficiently. However, there was no quantitative correlation between the extent of MVMp D N A amplification and cell killing after infection. The NS-1 protein of autonomous parvoviruses is a multifunctional phosphoprotein, the expression of which has been associated with cell killing (Rhode, 1987; Osawa et al., 1988; Brandenburger et al., 1990; CailletFauquet et al., 1990). Therefore it was of interest to determine whether the varying sensitivity of the transformants tested to MVMp infection was related to the differential expression of this viral polypeptide. The synthesis of NS-I protein was assessed by metabolic labelling and immunoprecipitation. Fig. 3 shows that the parental cell lines FR4 and FR3T3 produced only minute amounts of this protein after infection. Oncogeneinduced transformation was accompanied by an increase in NS-1 production to a small (PyLT), moderate (PyMT, v-myc) or high (c-Ha-ras-1, PyLT + PyMT, v-myc + c-Ha-ras-1) extent. This gradient of NS-1 production (Fig. 3) paralleled that of virus cytotoxicity (Table 1), suggesting that the latter may reflect, at least in part, the capacity of cells for expressing the M V M p NS genes. A similar correlation has been reported recently for a series of related ras-transformed rat fibroblasts (van Hille et al., 1989). Together, these data indicate that various oncogenes or combinations thereof are able to interfere in a distinct and positive way with the control of NS protein production in parvovirus-infected cells.

Oncogene-promoted cell killing by M V M p

1

2

3

4

5

6

M

7

8

9

10

11

2007

12

97KO NSI[

68KO Fig. 3. Production of the MVMp NS1 protein in infected parental cells (FR3T3 and FR4; lanes 1 and 7) and derivatives transformed by v-myc (lane 2), c-Ha-ras-1 (lane 3), v-myc + c-Ha-ras-1 (lanes 4 to 6), PyLT (lane 8), PyMT (lane 9) or PyLT + PyMT (lanes 10 to 12). [3sS]Methionine_labelled cell extracts were immunoprecipitated with anti-NS-1 antiserum, and analysed by 10 % SDS-PAGE followed by fluorography. Lane M, mock-infected cells. Lanes 2 to 6, F RMC 14, FREJ4, FRMCEJ 1, FRMCEJ l0 and FRMCEJ15 cells; lanes 8 to 12, FRLTI, FRMT1, FRMT4, FRMLT2 and FRMLT4.

Discussion Use was made in this work of two pairs of viral or cellular oncogenes belonging to the so-called functional classes I (PyLT and v-myc) and II (PyMT and c-Ha-ras-1), and which act in cooperation to cause the neoplastic conversion of normal ceils (Cuzin, 1984; Ruley, 1990). Although the FR3T3 line used as the recipient for oncogene transformation had already acquired one of the class I-associated changes, namely immortality, the combination of either PyLT and PyMT or v-myc and cHa-ras-1 had a synergistic transforming action on these cells: double transformants grew in poorly adherent clusters of piled-up cells, in contrast with the parental line and either type of single transformant, which formed regular monolayers. The double transformants were also most susceptible to MVMp-induced killing. Unlike clustered growth, sensitization to MVMp was detectable to some extent even in the single transformants, and was only slightly greater than additive in the double transformants. Thus it appears that, in this system, sensitivity to MVMp is an indicator of overall cell progression into the transformation process, rather than a marker of a specific step thereof. However, it remains to be determined whether further neoplastic progression beyond stages simulated in vitro can also be distinguished by parvovirus parameters. Transformed cell lines that were sensitized to MVMp also produced greater amounts of viral DNA and proteins after infection, showing that at least one reason why normal cells resist MVMp consists of their poor ability to replicate the virus. It has been shown previously that in this and a number of other systems normal cells are fully competent in virus uptake (Chen et al., 1986; Cornelis et a/., 1988a). Thus transformation appears to up-modulate subsequent intracellular step(s) of the MVMp life cycle. The stimulation of NS protein production did not parallel that of viral DNA replication, implying a genuine effect of transformation on

MVMp gene expression, in addition to its influence on the amplification of viral DNA templates. The activity of promoter P4 programming of the NS transcription unit has recently been reported to be enhanced in rastransformed, compared with normal, rodent fibroblasts (Spegelaere eta/., 1991). Interestingly, the sensitivity of the different transformants tested to MVMp-induced killing varied with their capacity for NS protein, but not viral DNA, production. This result may be taken together with recent studies showing that the expression of transfected NS genes is cytotoxic (Caillet-Fauquet et al., 1990). Thus, the correlation observed between transformation-associated increases in NS protein synthesis and cell killing may not be fortuitous but indicative of a causal relationship. However, our data do not rule out the up-modulation of parvovirus cytotoxicity by host cell transformation involving additional controls besides that maintained over NS gene expression. Indeed, normal cells appear to resist the forced production of NS protein at levels that kill transformed derivatives (Mousset et al., 1991). Both class I and class II oncogene-induced transformants exhibited a distinct enhancement of their susceptibility to MVMp expression and cytotoxic activity. There was no simple correlation between the extent of this effect and the display of more conventional transformation traits, such as morphological alterations, reduced growth factor requirements or anchorage-independent growth. The class I and class II oncogenes tested differ in the subcellular location (intra- versus extranuclear) and primary molecular actions (regulation of DNA metabolism versus membrane or cytosolic activities) of the corresponding oncoproteins. However, many cytoplasmic oncoproteins do modulate the expression of cellular genes, including nuclear-acting proto-oncogenes. This is the case with PyMT, which has been shown to induce cmyc expression (Rameh & Armelin, 1991). Thus, oncogenes from different classes participate in interconnected regulatory circuits whose ultimate targets are

2008

C. Legrand and others

nuclear genes that are controlled at the level of their expression (Hunter, 1991). The cooperation of various oncogenes in neoplastic transformation suggests that the signalling pathways involved are at least partly distinct. This is supported, for instance, by the fact that some transcription factors are activated by transforming (class II) but not immortalizing (class I) oncoproteins (Wasylyk et al., 1989). The MVMp early promoter contains several putative binding sites for cellular transcription factors (Bodnar, 1988). Therefore it may not be too surprising that both classes of oncogenes have distinct effects on the expression of parvoviral genes encoding cytotoxic proteins, and cooperate in cell sensitivity to MVMp infection. The class I oncogenes analysed appeared to be much less efficient than their class II cooperators in sensitizing cells to MVMp infection. It is unknown whether this variation reflects the involvement of distinct cellular effectors of the parvovirus life cycle, or the differential modulation of a common effector. It would be interesting in this respect to determine whether MVMp responsiveness to oncogenes of a given class can be selectively abolished by site-directed mutagenesis of viral promoter sequences. We thank Drs F. Cuzin (Nice) and M. Bastin (Quebec) for giving plasmids pPyLT1 and pMLT97 respectively. We are grateful to Dr B. van Hille (Lille) for providing the FREJ4 line and plasmid pSVneoEJ, and to Dr T. Benjamin (Boston) for his gift of PyB4A cells. M. Tuynder is acknowledged for photographic illustrations. This work was supported by the Commission of European Communities, Caisse G6n6rale d'Epargne et de Retraite (Belgium) and Institut National de la Sant6 et de la Recherche M6dicale (France). C. Legrand and N. Salom6 were the recipients of fellowships from the Institut pour l'Encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture (Belgium) and the Association pour la Recherche sur le Cancer (France), respectively. S. Mousset is Chercheur Qualifi6 du Fonds National de la Recherche Scientifique (Belgium).

References AVALOSSE,B., CHEN, Y. Q., CORNELIS, J., DUPONCHEL, N., BECQUART, P., NAMBA, i . & ROMMELAERE, J. (1987). Amplification of parvoviral DNA as a function of host-cell transformation. In Accomplishment in Oncology, vol. 2, pp. 140-152. Edited by H. zur Hausen and J. R. Schlehofer. Philadelphia: J. B. Lippincott. BODNAR, J. W. (1988). Sequence organization in regulatory regions of DNA of minute virus of mice. Virus Genes 2, 167-182. BRANDENBURGER,A., LEGENDRE, D., AVALOSSE,B. & ROMMELAERE,J. (1990). NS-1 and NS-2 proteins may act synergistically in the cytopathogenicity of parvovirus MVMp. Virology 174, 576-584. CAILLET-FAUQUET, P., PERROS, i . , BRANDENBURGER, A., SPEGELAERE, P. & ROMMELAERE,J. (1990). Programmed killing of human cells by means of an inducible clone of parvoviral genes encoding non-structural proteins. EMBO Journal 9, 2989-2995. CHEN, Y. Q., DE FORESTA, F., HERTOGHS, J., AVALOSSE,B., CORNELIS, J. • ROMMELAERE, J. (1986). Selective killing of simian virus 40transformed human fibroblasts by parvovirus H-1. Cancer Research 46, 3574-3579.

CORNELIS, J. J., BECQUART, P., DUPONCHEL, N., SALOME, N., AVALOSSE,B., NAMBA, M. & ROMMELAERE,J. (1988a). Transformation of human fibroblasts by ionizing radiations, a chemical carcinogen, or simian virus 40 correlates with an increase in susceptibility to the autonomous parvoviruses H1 virus and MVM. Journal of Virology 62, 1679-1686. CORNELIS, J. J., SPRUY'r, N., SPEGELAERE, P., GUETTA, E., DARAWSHI, T., COTMORE,S. F., TAL, J. & ROMMELAERE,J. (1988b). Sensitization of transformed rat fibroblasts to killing by parvovirus minute virus of mice correlates with an increase in viral gene expression. Journal of Virology 62, 3438-3444. COTMORE, S. F. & TATTERSALL, P. (1987). The autonomously replicating parvoviruses of vertebrates. Advances in Virus Research 33, 91-159. CUZIN, F. (1984). Coordinated functions of three distinct proteins in the transformation of rodent cells in culture. Biochimica et biophysica acta 781, 193-204. GELINAS, C., SCHAFFHAUSEN,B., BOCKUS,B., RATIARSON,A. & BASTIN, M. (1989). Mutations in polyomavirus middle T antigen affecting tumorigenesis. Virology 170, 193-200. GUETTA, E., MINCBERG, M., MOUSSET, S., BERTINCHAMPS, C., ROMMELAERE,J. & TAL, J. (1990). Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production. Journal of Virology 64, 458-462. HUNTER, T. (1991). Cooperation between oncogenes. Cell 64, 249-270. MCKAY, I. A., MALONE, P., MARSHALL, C. J. & HALL, A. (1986). Malignant transformation of murine fibroblasts by a human c-Haras-1 oncogene does not require a functional epidermal growth factor receptor. Molecular and Cellular Biology 6, 3382-3387. MOUSSET, S. & ROMMELAERE,J. (1982). Minute virus of mice inhibits cell transformation by simian virus 40. Nature, London 300, 537-539. MOUSSET, S. & ROMMELAERE, J. (1988). Susceptibility to parvovirus minute virus of mice as a function of the degree of host cell transformation: little effect of simian virus 40 infection and phorbol ester treatment. Virus Research 9, 107-117. MOUSSET, S., CORNELIS, J. J., SPRUYT, N. & ROMMELAERE, J. (1986). Transformation of established murine fibroblasts with an activated cellular Harvey-ras oncogene or the polyomavirus middle T gene increases cell permissiveness to parvovirus minute virus of mice. Biochimie 68, 951-955. MOUSSET, S., CAILLET-FAUQUET, P. & ROMMELAERE, J. (1991). The cytotoxicity of MVM non-structural proteins is modulated by neoplasic transformation. 4th Parvovirus Workshop, Elsinore (Denmark), August 18-22 1991. Abstract P18. OSAWA, K., AYUB, J., KAJIGAYA,S., SHIMADA,T. & YOUNG, N. (1988). The gene encoding the nonstructural protein of B19 (human) parvovirus may be lethal in transfected cells. Journal of Virology 62, 2884-2889. RAMEH, L. & ARMELIN, i . (1991). T antigens' role in polyomavirus transformation: c-myc but not c-fos or c-jun expression is a target for middle T. Oncogene 6, 1049-1056. RASSOULZADEGAN, i . , COWlE, A., CARR, A., GLAICHENHAUS, N., KAMEN, R. & CUZlN, F. (1982). The role of individual polyomavirus early proteins in oncogenic transformation. Nature, London 300, 713718. RHODE, S. L., III (1987). Construction of a genetic switch for inducible trans-activation of gene expression in eucaryotic cells. Journal of Virology 61, 1448-1456. ROMMELAERE, J. & CORNELIS, J. (1991). Antineoplasic activity of parvoviruses. Journal of Virological Methods 33, 237-251. ROMMELAERE, J. & TA'rrERSALL, P. (1990). Oncosuppression by parvoviruses. In Handbook ofParvoviruses, vol. 2, pp. 41-57. Edited by P. Tijssen. Boca Raton: CRC Press. RULEY, E. (1990). Transforming collaborations between ras and nuclear oncogenes. Cancer Cells 2, 258-268. SALOMf/, N., VAN HILLE, B., DUPONCHEL, S., MENEGUZZI, G., CUZIN, F., ROMMELAERE, J. 86 CORNELIS, J. J. (1990). Sensitization of transformed rat cells to parvovirus MVMp is restricted to specific oncogenes. Oncogene 5, 123-130. SELF, R. & CUZlN, F. (1977). Temperature-sensitive growth regulation in one type of transformed rat cells induced by the tsa mutant of polyomavirus. Journal of Virology 24, 721-728.

Oncogene-promoted cell killing by M V M p

SIEGL, G. (1984). Biology and pathogenicity of autonomous parvoviruses. In The Parvoviruses, pp. 297-348. Edited by K. I. Berns. New York & London: Plenum Press. SOUTHERN, P. d~ BERG, P. (1982). Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. Journal of Molecular and Applied Genetics 1, 327-341. SPEGELAERE, P., VANHILLE, B., SPRUYT, N., FAISST,S., CORNELIS, J. & ROMMELAERE, J. (1991). Initiation of transcription from the minute virus of mice P4 promoter is stimulated in rat cells expressing a c-Haras oncogene. Journal of Virology 65, 4919-4928. TATTERSALL, P., CAWTE, P. J., SHATKIN, A. J. & WARD, D. C. (1976). Three structural polypeptides coded for by minute virus of mice, a parvovirus. Journal of Virology 20, 273-289.

2009

TREISMAN, R., NOVAK, U., FAVALORO, J. A, KAMEN, R. (1981). Transformation of rat cells by an altered polyomavirus genome expressing only the middle T protein. Nature, London 292, 595-600. VAN HILLE, B., DUPONCHEL, N., SALOME, N., SPRUYT, N., COTMORE, S. F., TATTERSALL, P., CORNELIS, J. J. & ROMMELAERE, J. (1989). Limitation to the expression of parvoviral nonstructural proteins may determine the extent of sensitization of EJ-ras-transformed rat cells to minute virus of mice. Virology 171, 89-97. WASYLYK, C., FLORES, P., GLrrMAN, A. & WASYLYK, B. V. (1989). PEA3 is a nuclear target for transcription activation by non-nuclear oncogenes. EMBO Journal 8, 3371-3378.

(Received 11 February 1992; Accepted 16 April 1992)