JOURNAL
OF
Vol. 64, No. 1
VIROLOGY, Jan. 1990, p. 458-462
0022-538X/90/010458-05$02.00/0 Copyright © 1990, American Society for Microbiology
Selective Killing of Transformed Rat Cells by Minute Virus of Mice Does Not Require Infectious Virus Production ESTHER
GUETTA,1 MICHAL MINCBERG,l SUZANNE MOUSSET,2 CLAIRE BERTINCHAMPS,2
JEAN ROMMELAERE,2'3 AND JACOV TAL1* Biology Department, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel1; Department of Molecular Biology, Universite Libre de Bruxelles, B 1640 Rhode St. Genese, Belgium2; and Molecular Oncology Unit, Institut National de la Sante et de la Recherche Me'dica!e U186 and Centre National de la Recherche Scientifique URA 0156, Institut Pasteur de Lille, 59019 Lille Cedex, France3 Received 6 July 1989/Accepted 1 October 1989
Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.
strain of MVM [MVM(p)]. MVM(p) infection caused a transient and slight cytopathic effect at days 1 and 2, after which the culture recovered and a normal growth rate was resumed. In contrast, the polyomavirus-transformed derivative cell lines were destroyed completely by MVM(p) infection within 24 h (Fig. 1). The total yield of infectious virus produced in polyomavirus-transformed cells was 102to 103-fold higher than that produced in similarly infected untransformed cell lines (Table 1). This difference range in virus yield is comparable with those of other permissive/ restrictive systems for MVM (9, 20, 21, 24). A subline of mouse L-cells, A9 [which is used as a standard permissive cell line for MVM(p)], was also included in all our experiments for comparison. Table 1 shows that virus production in A9 cells was only slightly lower than in F-111/Py cells. To investigate the relationship between the killing effect and virus production, cell killing during a single cycle of infection was quantitated by using an infectious center assay. This assay was performed as follows. At 4 h postinfection the cells were washed twice with phosphate-buffered saline to remove unattached and unabsorbed virus, trypsinized, suspended in Dulbecco modified Eagle medium containing 25 mM HEPES (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid) buffer (pH 7.3) supplemented with 10% fetal calf serum, and counted. Serial dilutions were prepared in the above medium, poured onto 60-mm dishes containing monolayers of 5 x 105 A9 indicator cells, and incubated for 4 to 8 h to allow all cells to attach. The monolayers were overlaid with agar, incubated for 5 to 6 days at 37°C, and then stained with 0.2% neutral red in Hanks salts solution containing 50 mM HEPES buffer, pH 7.3, and 0.45% Bacto-Agar (Difco Laboratories). Plaques were counted 20 h later. These assays consistently showed that the MVM(p) permissive fraction in F-111 cells was nearly identical to that found in the polyomavirus transformants (Table 1), although the lethal effect of MVM(p) was much more pronounced on the latter. Furthermore, immunofluorescence staining revealed similar percentages of cells
Certain parvoviruses possess the intriguing capability to destroy oncogenically transformed cells selectively in culture while having little or no effect on the viability of the parental, untransformed cells. This preferential toxicity to transformed cells may account for the inhibition of induced tumorigenesis (oncosuppression), reported to occur in vivo (2, 6, 8, 11, 22, 25-27). A variety of cells have been used to study the preferential killing effect of autonomous parvoviruses. These include mouse 3T3 cells (13-15), several rat fibroblast cell lines (13), and human fibroblasts and epithelial cells (3, 4). A variety of transforming agents have also been used, including simian virus 40 (1, 4, 10, 14, 15), avian erythroblastosis virus (5), human ras oncogene (13), and radiation and chemical carcinogens (1, 4). These studies show that (i) the killing effect of transformed cells is dependent neither on the type of cells nor on the transforming agent used and (ii) the preferential killing of transformed cells is correlated with elevated expression of the viral genome. In this study, we investigated the question of whether the production of infectious virus was required for the selective killing effect of minute virus of mice (MVM). The Fischer rat embryo fibroblast cell line (F-111) chosen for this study is subject to a stringent contact inhibition, its anchorage-independent growth is extremely low, and it has a very typical fibroblastic appearance. These properties allow a relatively easy isolation of cells with transformed phenotypes (7). The F-111 and F-111/Py (polyomavirus-transformed F-111) cell lines used in this study were obtained from T. Benjamin. Additional polyomavirus-transformed cell lines, established in our laboratory by using the method of Fluck and Benjamin (7), were also examined and yielded essentially identical results. All the transformed cell lines were shown to express the polyomavirus middle-T antigen. The F-111 cell line was useful for this study because of its exceptionally tight resistance to killing by the fibrotropic *
Corresponding author. 458
VOL. 64, 1990
NOTES
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FIG. 1. Cell killing by MVM(p). Cells were seeded at a low density (5 x 104 cells per 60-mm dish) and infected with 1 PFU of MVM(p) cell on the next day (day 0). (A) F-111 cells. (B) F-111/Py cells. In each panel, the photographs on the left are of mock-infected cultures and the ones on the right are of infected cultures. per
expressing MVM coat antigens in MVM-infected cultures of F-ill and F-lll/Py and also in A9 cultures (Table 1). Immunofluorescence staining was done at 24 h postinfection, which, under the experimental conditions used, was only a short time before the death of the highly susceptible Flll/Py culture. Thus, the majority of the F-111/Py cells were killed in the absence of a detectable level of capsid proteins. The synthesis of viral nonstructural proteins in cells which do not express viral capsid proteins remains to be determined. To show that the plaques originated from a round of infection in the plated cells, a control experiment was done. In this, a monolayer of 2 x 105 cells in a 60-mm dish was overlaid with 1 ml of 5 x 10-6 M trimethylpsoralen (tri-
oxsalen) in phosphate-buffered saline, incubated at 37°C for 10 min, and irradiated with two tubular fluorescent "black ray" lamps held in a model XX-15 reflector (UV Products, Inc., San Gabriel, Calif.) for 5 min at room temperature. The maximal emission of the lamps was about 360 nm, and the light intensity delivered to the sample was 2 to 4 mW/cm2. The drug was washed twice with phosphate-buffered saline, and the cells were infected with MVM(p) as described above. These conditions, shown to arrest cellular DNA and RNA synthesis (23), have no effect on the uptake of viral single-stranded DNA but reduce MVM production by over 99% (M. Mincberg and J. Tal, manuscript in preparation) and abolished infectious center production with all three cell lines (Table 1). Taken together, the above findings indicate
460
J. VIROL.
NOTES TABLE 1. Interaction of MVM(p) with untransformed and transformed rat cells
Cell line
infectious centers in': PFU of infectious virus per cell (avg)a'b Controlsd cells cells cells
F-111 1.1, 1.35, 1.47 (1.3) F-111/Py 1,150, 1,060, 1,230 (1,150) A9 1,030, 960, 890 (960)
Fluorescent nucleic cells ( fcls )
OF
Vol. 64, No. 1
VIROLOGY, Jan. 1990, p. 458-462
0022-538X/90/010458-05$02.00/0 Copyright © 1990, American Society for Microbiology
Selective Killing of Transformed Rat Cells by Minute Virus of Mice Does Not Require Infectious Virus Production ESTHER
GUETTA,1 MICHAL MINCBERG,l SUZANNE MOUSSET,2 CLAIRE BERTINCHAMPS,2
JEAN ROMMELAERE,2'3 AND JACOV TAL1* Biology Department, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel1; Department of Molecular Biology, Universite Libre de Bruxelles, B 1640 Rhode St. Genese, Belgium2; and Molecular Oncology Unit, Institut National de la Sante et de la Recherche Me'dica!e U186 and Centre National de la Recherche Scientifique URA 0156, Institut Pasteur de Lille, 59019 Lille Cedex, France3 Received 6 July 1989/Accepted 1 October 1989
Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.
strain of MVM [MVM(p)]. MVM(p) infection caused a transient and slight cytopathic effect at days 1 and 2, after which the culture recovered and a normal growth rate was resumed. In contrast, the polyomavirus-transformed derivative cell lines were destroyed completely by MVM(p) infection within 24 h (Fig. 1). The total yield of infectious virus produced in polyomavirus-transformed cells was 102to 103-fold higher than that produced in similarly infected untransformed cell lines (Table 1). This difference range in virus yield is comparable with those of other permissive/ restrictive systems for MVM (9, 20, 21, 24). A subline of mouse L-cells, A9 [which is used as a standard permissive cell line for MVM(p)], was also included in all our experiments for comparison. Table 1 shows that virus production in A9 cells was only slightly lower than in F-111/Py cells. To investigate the relationship between the killing effect and virus production, cell killing during a single cycle of infection was quantitated by using an infectious center assay. This assay was performed as follows. At 4 h postinfection the cells were washed twice with phosphate-buffered saline to remove unattached and unabsorbed virus, trypsinized, suspended in Dulbecco modified Eagle medium containing 25 mM HEPES (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid) buffer (pH 7.3) supplemented with 10% fetal calf serum, and counted. Serial dilutions were prepared in the above medium, poured onto 60-mm dishes containing monolayers of 5 x 105 A9 indicator cells, and incubated for 4 to 8 h to allow all cells to attach. The monolayers were overlaid with agar, incubated for 5 to 6 days at 37°C, and then stained with 0.2% neutral red in Hanks salts solution containing 50 mM HEPES buffer, pH 7.3, and 0.45% Bacto-Agar (Difco Laboratories). Plaques were counted 20 h later. These assays consistently showed that the MVM(p) permissive fraction in F-111 cells was nearly identical to that found in the polyomavirus transformants (Table 1), although the lethal effect of MVM(p) was much more pronounced on the latter. Furthermore, immunofluorescence staining revealed similar percentages of cells
Certain parvoviruses possess the intriguing capability to destroy oncogenically transformed cells selectively in culture while having little or no effect on the viability of the parental, untransformed cells. This preferential toxicity to transformed cells may account for the inhibition of induced tumorigenesis (oncosuppression), reported to occur in vivo (2, 6, 8, 11, 22, 25-27). A variety of cells have been used to study the preferential killing effect of autonomous parvoviruses. These include mouse 3T3 cells (13-15), several rat fibroblast cell lines (13), and human fibroblasts and epithelial cells (3, 4). A variety of transforming agents have also been used, including simian virus 40 (1, 4, 10, 14, 15), avian erythroblastosis virus (5), human ras oncogene (13), and radiation and chemical carcinogens (1, 4). These studies show that (i) the killing effect of transformed cells is dependent neither on the type of cells nor on the transforming agent used and (ii) the preferential killing of transformed cells is correlated with elevated expression of the viral genome. In this study, we investigated the question of whether the production of infectious virus was required for the selective killing effect of minute virus of mice (MVM). The Fischer rat embryo fibroblast cell line (F-111) chosen for this study is subject to a stringent contact inhibition, its anchorage-independent growth is extremely low, and it has a very typical fibroblastic appearance. These properties allow a relatively easy isolation of cells with transformed phenotypes (7). The F-111 and F-111/Py (polyomavirus-transformed F-111) cell lines used in this study were obtained from T. Benjamin. Additional polyomavirus-transformed cell lines, established in our laboratory by using the method of Fluck and Benjamin (7), were also examined and yielded essentially identical results. All the transformed cell lines were shown to express the polyomavirus middle-T antigen. The F-111 cell line was useful for this study because of its exceptionally tight resistance to killing by the fibrotropic *
Corresponding author. 458
VOL. 64, 1990
NOTES
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FIG. 1. Cell killing by MVM(p). Cells were seeded at a low density (5 x 104 cells per 60-mm dish) and infected with 1 PFU of MVM(p) cell on the next day (day 0). (A) F-111 cells. (B) F-111/Py cells. In each panel, the photographs on the left are of mock-infected cultures and the ones on the right are of infected cultures. per
expressing MVM coat antigens in MVM-infected cultures of F-ill and F-lll/Py and also in A9 cultures (Table 1). Immunofluorescence staining was done at 24 h postinfection, which, under the experimental conditions used, was only a short time before the death of the highly susceptible Flll/Py culture. Thus, the majority of the F-111/Py cells were killed in the absence of a detectable level of capsid proteins. The synthesis of viral nonstructural proteins in cells which do not express viral capsid proteins remains to be determined. To show that the plaques originated from a round of infection in the plated cells, a control experiment was done. In this, a monolayer of 2 x 105 cells in a 60-mm dish was overlaid with 1 ml of 5 x 10-6 M trimethylpsoralen (tri-
oxsalen) in phosphate-buffered saline, incubated at 37°C for 10 min, and irradiated with two tubular fluorescent "black ray" lamps held in a model XX-15 reflector (UV Products, Inc., San Gabriel, Calif.) for 5 min at room temperature. The maximal emission of the lamps was about 360 nm, and the light intensity delivered to the sample was 2 to 4 mW/cm2. The drug was washed twice with phosphate-buffered saline, and the cells were infected with MVM(p) as described above. These conditions, shown to arrest cellular DNA and RNA synthesis (23), have no effect on the uptake of viral single-stranded DNA but reduce MVM production by over 99% (M. Mincberg and J. Tal, manuscript in preparation) and abolished infectious center production with all three cell lines (Table 1). Taken together, the above findings indicate
460
J. VIROL.
NOTES TABLE 1. Interaction of MVM(p) with untransformed and transformed rat cells
Cell line
infectious centers in': PFU of infectious virus per cell (avg)a'b Controlsd cells cells cells
F-111 1.1, 1.35, 1.47 (1.3) F-111/Py 1,150, 1,060, 1,230 (1,150) A9 1,030, 960, 890 (960)
Fluorescent nucleic cells ( fcls )
