VIROLOGY
65, 446-454
Polyoma
(1975)
Virus-Transformed
Rat Cell Lines Inducible
Capsid Antigen
Synthesis
MARIAN Department
of Genetics,
Weizmann
for Viral
FOGEL Institute
Accepted January
of Science, Rehouot,
Israel
27, I975
A number of polyoma virus (PV)-transformed lines of rat, mouse and hamster origin established in the course of this study were investigated for virus induction by mitomycin C. Neither infectious virus nor V-antigen was detected in six mouse and four hamster lines. Out of 14 rat lines, three were found inducible for infectious virus, and one could be induced only for V-antigen synthesis. Two lines, designated as RPA and RPB differ from the others with respect to their response to mitomycin C and elevated temperature (40”). The proportion of V-antigen-containing cells increased 50- and go-fold in the RPA and RPB lines, respectively, when the mitomycin C-treated cultures were subsequently incubated at 40” for 24 hr, as compared to the cultures kept continuously at 37”. No infectious virus was detected in the mitomycin C-treated RPA cultures incubated at either temperature. The RPB line can be induced for infectious virus production at a cell frequency of l/100 V-antigen-containing cells when incubated at 37”. Though the proportion of V-antigen-containing cells increased in the heated cultures of this line 60.fold, the number of plaques produced by the cell extracts and the proportion of virus-producing cells were similar both in the heated and unheated cultures. Fifty percent of clones isolated from the RPA line and about 80% of RPB clones are inducible, exhibiting, except for one RPB clone, a similar pattern of response to the inducing agents as the parental lines. In heterokaryon cultures of untreated mouse embryo cells and RPA or RPB cells pretreated with mitomycin C and heat, a proportion of multinucleated cells was shown to contain V-antigen. However, virus maturation was not detected in the heterokaryon cultures with RPA cells and was not significantly enhanced in the cultures with RPB cells. We also studied the effect of elevated temperature (40”) on PV induction by mitomycin C in highly inducible PV-transformed lines. In contrast to the RPA and RPB lines, in the highly inducible lines, the elevated temperature, though exerting a relatively weak enhancing effect on induction by mitomycin C, increased the proportion of V-antigen-containing cells and the virus yields to an almost equal extent. These results would indicate .that the lack of virus production in the V-antigen-containing RPA and in most RPB cells is due to a deficiency of the viral genome persisting in these cells and that a temperature-sensitive factor is involved in the process of V-antigen induction in these lines.
formed lines has been rarely observed (Fogel and Sachs, 1969; Vogt, 1970; Saito The presence of viral genome, or a part of et al., 1970). We have described a PVrat cell line which can be it, in polyoma virus (PV)-transformed cells transformed induced for PV synthesis by various physihas been revealed by nucleic acid hybridical and chemical agents known to affect zation studies (Axelrod et al., 1964; Benjamin, 1966; Westphal and Dulbecco, 1968; DNA (Fogel and Sachs, 1969, 1970; Fogel, 1972, 1973). Selective damage of the celluManor et al., 1973). Though viral genome seems to persist in all PV-transformed cell lar DNA seems to be involved in the lines (Sjijgren et al., 1961; Defendi et al., process of PV induction in the LPT (large cells (Fogel, 1972, 1964; Habel, 1965; Fogel et al., 19671, plaque-transformed) 1973). In the present study, three PVinduction of virus synthesis in PV-transINTRODUCTION
446 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
POLYOMA VIRUS CAPSID ANTIGEN INDUCTION
transformed rat cell lines which are inducible for PV synthesis and one line inducible only for viral capsid (V)-antigen were established. Two of these lines are exceptional with respect to their response to mitomycin C and elevated temperature. One line produces upon induction V-antigen only, while the second preferentially produces V-antigen but also small yields of infectious virus. In these lines and their progeny clones, elevating the incubation temperature to 40” increased up to 120-fold the inducibility of V-antigen but not of infectious virus by mitomycin C. An SV40-transformed line inducible for Vantigen and not for infectious virus by heat treatment has been described (Margalith et al., 1970). The question arises whether heat may enhance induction by mitomycin C by an additive effect on the cellular DNA similar to the case of gamma irradiation and elevated temperature in Escherichia coli (Achey and Pollard, 1967), in which the initial rate of DNA degradation rose sharply as the postirradiation temperature increased from 30 to 41”. Alternatively, a temperature-sensitive factor, either of a viral or cellular nature may account for V-antigen induction in these PV-transformed lines. MATERIALS
AND METHODS
Cell cultures and PV-transformed lines. All cell lines except the LPT (Fogel and Sachs, 1969) were established in the course of these experiments (see Results, Table 1). The hamster and rat lines, except the RPB line, were obtained by infecting primary or secondary monolayer cultures with 100 PFU (plaque forming units) of virus/cell (Winocour and Sachs, 1960). The RPA and RPB lines were derived from the same rat primary cultures; one part of the cultures was infected with 100 PFU/cell (RPA) and another with 1,000 PFU/cell (RPB). The mouse lines were derived from secondary cultures infected with 10’ PFU/cell. The cultures were grown in a lo%-CO, atmosphere at 37” in Eagle’s medium (EM) containing a fourfold concentration of amino acids and vitamins. For the rat cultures. 10% horse serum and, for the hamster and
447
mouse cultures, 5% calf serum was added to the medium. At 3-5-day intervals, the cultures were passaged by a 1:4 split. Thirty to fifty days following infection, depending on the type of culture, all cells stained for PV-T (tumor) antigen (Fogel et al., 1967). Thereafter, all cultures were grown in EM supplemented with 5% fetal calf serum instead of the horse and calf sera. Neither polyoma virus capsid (V)antigen nor infectious virus was detected in any of the transformed cultures. V-antigen was also not detected in these cultures when superinfected with 100 PFU of PV/ cell. Virus stocks. Polyoma virus SP and IL11 strains are small and large plaque strains, respectively, which are routinely propagated in this laboratory (Medina and Sachs, 1961). The RLP virus is a large plaque strain from an inducible clone of the LPT line (Fogel and Sachs, 1969). Virus was grown in mouse kidney cells (Winocour, 1963) and was titrated by a plaque assay as previously described (Winocour and Sachs, 1960). Results were read after 12-13 days. Sendai virus was kindly supplied by Dr. A. Loyter, The Hebrew University, Jerusalem. Determination of proportion of uirusyielding cells. The infective-centre assay procedure used was as previously described (Winocour and Sachs, 1960). Cells at concentrations of 5 x 104, 1 x lo5 and 2 x lo5 in 0.1 ml of medium were plated on normal mouse embryo monolayers. In each experiment, 2 x lo6 cells obtained from two dishes were tested, unless otherwise stated. The cells were added to the medium of the assay plates, and, after incubation for 16 hr, to allow attachment of the cells to the monolayer, the medium was removed and agar-nutrient overlay was added. Plaques were counted after 13-14 days. Cloning of transformed cells. Clones from the original lines were isolated in soft agar (Macpherson and Montangier, 1964). For the cloning procedures, samples containing about lo5 cells in 0.1 ml of medium were incubat.ed in a water bath at 37” for 60 min with equal amounts of anti-PV serum that, at the dilution used, reduced a virus titer of 1 x lo5 PFU by about 99%. The
448
MARIAN
cells were then cultured for clone isolation in the presence of anti-PV serum sufficient to neutralize about 2 x lo5 PFU of virus. Cell fusion with Sendai virus. The fusion of cells by Sendai virus was performed in a manner similar to that described by Watkins and Dulbecco (1967). Transformed cells (2 x 106) and 4-day-old primary mouse embryo cells, mixed at a ratio of 1: 1, were suspended in 1.6 ml of Earle’s saline, and 0.4 ml of ultraviolet-inactivated Sendai virus, containing 6,400 hemagglutinating units, was added. The suspension was allowed to stand for 15-20 min at 4” then transferred to a water bath at 37” for a further 15 min. The tubes were then slightly agitated, and the contents of each distributed into four 35mm petri dishes containing 2 ml of EM with 5% fetal calf serum. The medium was changed after 20 hr of incubation at 37”. staining. Immunofluorescent Cells grown on glass coverslips were fixed in an acetone-methanol mixture (7:3) at -20” for 10 min. Anti-polyoma virus capsid (V)-antigen serum was prepared as described previously (Fogel et al., 1967); nonspecific staining of the fluorescein-conjugated antiserum was eliminated as described (Fogel, 1972). The anti-V-antigen serum, after conjugation with fluorescein and purification, stained V-antigen at a dilution of 1:16 when tested on PV-infected mouse embryo cells. Staining for the PV nuclear tumor (T)-antigen and preparation of anti-T serum was done was previously described (Fogel et al., 1967). The proportions of fluorescent cells that stained for V-antigen were calculated by counting a total of about 2 x lo4 cells in 100 microscopic fields on two coverslips. Treatment with vated temperature
mitomycin
C and ele-
(40”). Replicate cultures of each tested line were treated with mitomycin C obtained from the Nutritional Biochemical Corporation, Cleveland, OH) at concentrations of 0.25, 0.5 and 1.O pg/ml of medium for 1 hr at 37”. Four lines which were found inducible either for V-antigen or for both V-antigen and infectious virus at 37” were also subjected to heat treatment at 40 f 0.5” as follows. The cultures were treated with
FOGEL
mitomycin C for 1 hr as indicated. The medium was then changed to one not containing mitomycin C, and the cultures were kept at 37” for an additional 3 hr. A portion from each line was then transferred to an incubator at 40” for 24 hr. The remaining cultures were kept continuously at 37”. Control cultures, untreated with mitomycin C were incubated under identical conditions. The cultures were assayed for V-antigen 48 hr after the addition of mitomycin C and for infectious virus after 96 hr. RESULTS
Induction of PV-Antigen and Virus Synthesis in PV-Transformed Lines We investigated 14 PV-transformed lines of rat origin as well as four hamster and six mouse lines. Induction of PV or V-antigen synthesis was detected in only four lines of rat origin (Table 1). Two of the inducible lines (RPA and RPB) exhibited a different response to mitomycin C when the cultures were subsequently incubated at 40” than when they were kept continuously at 37” (Table 2). The proportions of V-antigen-containing cells in the RPA and RPB cultures preincubated at 40” is 50- to go-fold higher than in corresponding cultures kept at 37”. Infectious virus was not detected in the RPA line at either 37 or 40”; the RPB line, however, produced similar low yields of virus at both temperatures. From the data in Table 2, it is evident that even in the unheated RPB cultures a part of the Vantigen-containing cells do not produce infectious virus. The number of virusyielding cells, determined by the infective-centre assay, was about 5 x lo-’ in RPB cultures incubated at both temperatures. Since l/5,000 cells produce V-antigen in the unheated cultures, l/100 V-antigen-containing cells in these cultures produce infectious virus. In the heated cultures, the ratio of cells containing V-antigen to the cells producing virus increased by a factor of 120. In the control cultures not exposed to mitomycin C, whether incubated at 37 or 40”, neither V-antigen nor infectious virus was detected.
POLYOMA
VIRUS
CAPSID
ANTIGEN
TABLE
449
INDUCTION
1
POLYOMA VIRUS (PV)-TRANSFORMED LINES ASSAYED FOR PV INDUCTION BY MITOMYCIN C Cell species Rat Type of culture infected Infected with PV strain Number of PV-transformed linesb Number of lines inducible for PV or V-antigen
Hamster Primary RLP” 8
Secondary SP” IL110 3 3 0
1
Mouse
Secondary IL11 RLP 2 2
3
0
0
a SP, small plaque; IL11 and RLP are large plaque strains. b In the transformed lines all cells contained PV-T (tumor) antigen. Neither infectious virus was detected in these lines untreated with mitomycin C. TABLE
SP 2
Secondary IL11 2
0
RLP 2
0
0
viral capsid (V) antigen nor
2
PV-TRANSFORMED RAT CELL LINES INDUCIBLE BY MITOMYCIN C AT 37 AND 40” C” Transformed by PV strain
Cell line gbW72 V-antigenb
PFU/ 1 x 106 cells’
Plaqueforming cells/ 1 x 106 cells?
% Cells with V-antiger?
IL11
RPA RPB RP-78 RL
0.01 0.02 0.04
65, 446-454
Polyoma
(1975)
Virus-Transformed
Rat Cell Lines Inducible
Capsid Antigen
Synthesis
MARIAN Department
of Genetics,
Weizmann
for Viral
FOGEL Institute
Accepted January
of Science, Rehouot,
Israel
27, I975
A number of polyoma virus (PV)-transformed lines of rat, mouse and hamster origin established in the course of this study were investigated for virus induction by mitomycin C. Neither infectious virus nor V-antigen was detected in six mouse and four hamster lines. Out of 14 rat lines, three were found inducible for infectious virus, and one could be induced only for V-antigen synthesis. Two lines, designated as RPA and RPB differ from the others with respect to their response to mitomycin C and elevated temperature (40”). The proportion of V-antigen-containing cells increased 50- and go-fold in the RPA and RPB lines, respectively, when the mitomycin C-treated cultures were subsequently incubated at 40” for 24 hr, as compared to the cultures kept continuously at 37”. No infectious virus was detected in the mitomycin C-treated RPA cultures incubated at either temperature. The RPB line can be induced for infectious virus production at a cell frequency of l/100 V-antigen-containing cells when incubated at 37”. Though the proportion of V-antigen-containing cells increased in the heated cultures of this line 60.fold, the number of plaques produced by the cell extracts and the proportion of virus-producing cells were similar both in the heated and unheated cultures. Fifty percent of clones isolated from the RPA line and about 80% of RPB clones are inducible, exhibiting, except for one RPB clone, a similar pattern of response to the inducing agents as the parental lines. In heterokaryon cultures of untreated mouse embryo cells and RPA or RPB cells pretreated with mitomycin C and heat, a proportion of multinucleated cells was shown to contain V-antigen. However, virus maturation was not detected in the heterokaryon cultures with RPA cells and was not significantly enhanced in the cultures with RPB cells. We also studied the effect of elevated temperature (40”) on PV induction by mitomycin C in highly inducible PV-transformed lines. In contrast to the RPA and RPB lines, in the highly inducible lines, the elevated temperature, though exerting a relatively weak enhancing effect on induction by mitomycin C, increased the proportion of V-antigen-containing cells and the virus yields to an almost equal extent. These results would indicate .that the lack of virus production in the V-antigen-containing RPA and in most RPB cells is due to a deficiency of the viral genome persisting in these cells and that a temperature-sensitive factor is involved in the process of V-antigen induction in these lines.
formed lines has been rarely observed (Fogel and Sachs, 1969; Vogt, 1970; Saito The presence of viral genome, or a part of et al., 1970). We have described a PVrat cell line which can be it, in polyoma virus (PV)-transformed cells transformed induced for PV synthesis by various physihas been revealed by nucleic acid hybridical and chemical agents known to affect zation studies (Axelrod et al., 1964; Benjamin, 1966; Westphal and Dulbecco, 1968; DNA (Fogel and Sachs, 1969, 1970; Fogel, 1972, 1973). Selective damage of the celluManor et al., 1973). Though viral genome seems to persist in all PV-transformed cell lar DNA seems to be involved in the lines (Sjijgren et al., 1961; Defendi et al., process of PV induction in the LPT (large cells (Fogel, 1972, 1964; Habel, 1965; Fogel et al., 19671, plaque-transformed) 1973). In the present study, three PVinduction of virus synthesis in PV-transINTRODUCTION
446 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
POLYOMA VIRUS CAPSID ANTIGEN INDUCTION
transformed rat cell lines which are inducible for PV synthesis and one line inducible only for viral capsid (V)-antigen were established. Two of these lines are exceptional with respect to their response to mitomycin C and elevated temperature. One line produces upon induction V-antigen only, while the second preferentially produces V-antigen but also small yields of infectious virus. In these lines and their progeny clones, elevating the incubation temperature to 40” increased up to 120-fold the inducibility of V-antigen but not of infectious virus by mitomycin C. An SV40-transformed line inducible for Vantigen and not for infectious virus by heat treatment has been described (Margalith et al., 1970). The question arises whether heat may enhance induction by mitomycin C by an additive effect on the cellular DNA similar to the case of gamma irradiation and elevated temperature in Escherichia coli (Achey and Pollard, 1967), in which the initial rate of DNA degradation rose sharply as the postirradiation temperature increased from 30 to 41”. Alternatively, a temperature-sensitive factor, either of a viral or cellular nature may account for V-antigen induction in these PV-transformed lines. MATERIALS
AND METHODS
Cell cultures and PV-transformed lines. All cell lines except the LPT (Fogel and Sachs, 1969) were established in the course of these experiments (see Results, Table 1). The hamster and rat lines, except the RPB line, were obtained by infecting primary or secondary monolayer cultures with 100 PFU (plaque forming units) of virus/cell (Winocour and Sachs, 1960). The RPA and RPB lines were derived from the same rat primary cultures; one part of the cultures was infected with 100 PFU/cell (RPA) and another with 1,000 PFU/cell (RPB). The mouse lines were derived from secondary cultures infected with 10’ PFU/cell. The cultures were grown in a lo%-CO, atmosphere at 37” in Eagle’s medium (EM) containing a fourfold concentration of amino acids and vitamins. For the rat cultures. 10% horse serum and, for the hamster and
447
mouse cultures, 5% calf serum was added to the medium. At 3-5-day intervals, the cultures were passaged by a 1:4 split. Thirty to fifty days following infection, depending on the type of culture, all cells stained for PV-T (tumor) antigen (Fogel et al., 1967). Thereafter, all cultures were grown in EM supplemented with 5% fetal calf serum instead of the horse and calf sera. Neither polyoma virus capsid (V)antigen nor infectious virus was detected in any of the transformed cultures. V-antigen was also not detected in these cultures when superinfected with 100 PFU of PV/ cell. Virus stocks. Polyoma virus SP and IL11 strains are small and large plaque strains, respectively, which are routinely propagated in this laboratory (Medina and Sachs, 1961). The RLP virus is a large plaque strain from an inducible clone of the LPT line (Fogel and Sachs, 1969). Virus was grown in mouse kidney cells (Winocour, 1963) and was titrated by a plaque assay as previously described (Winocour and Sachs, 1960). Results were read after 12-13 days. Sendai virus was kindly supplied by Dr. A. Loyter, The Hebrew University, Jerusalem. Determination of proportion of uirusyielding cells. The infective-centre assay procedure used was as previously described (Winocour and Sachs, 1960). Cells at concentrations of 5 x 104, 1 x lo5 and 2 x lo5 in 0.1 ml of medium were plated on normal mouse embryo monolayers. In each experiment, 2 x lo6 cells obtained from two dishes were tested, unless otherwise stated. The cells were added to the medium of the assay plates, and, after incubation for 16 hr, to allow attachment of the cells to the monolayer, the medium was removed and agar-nutrient overlay was added. Plaques were counted after 13-14 days. Cloning of transformed cells. Clones from the original lines were isolated in soft agar (Macpherson and Montangier, 1964). For the cloning procedures, samples containing about lo5 cells in 0.1 ml of medium were incubat.ed in a water bath at 37” for 60 min with equal amounts of anti-PV serum that, at the dilution used, reduced a virus titer of 1 x lo5 PFU by about 99%. The
448
MARIAN
cells were then cultured for clone isolation in the presence of anti-PV serum sufficient to neutralize about 2 x lo5 PFU of virus. Cell fusion with Sendai virus. The fusion of cells by Sendai virus was performed in a manner similar to that described by Watkins and Dulbecco (1967). Transformed cells (2 x 106) and 4-day-old primary mouse embryo cells, mixed at a ratio of 1: 1, were suspended in 1.6 ml of Earle’s saline, and 0.4 ml of ultraviolet-inactivated Sendai virus, containing 6,400 hemagglutinating units, was added. The suspension was allowed to stand for 15-20 min at 4” then transferred to a water bath at 37” for a further 15 min. The tubes were then slightly agitated, and the contents of each distributed into four 35mm petri dishes containing 2 ml of EM with 5% fetal calf serum. The medium was changed after 20 hr of incubation at 37”. staining. Immunofluorescent Cells grown on glass coverslips were fixed in an acetone-methanol mixture (7:3) at -20” for 10 min. Anti-polyoma virus capsid (V)-antigen serum was prepared as described previously (Fogel et al., 1967); nonspecific staining of the fluorescein-conjugated antiserum was eliminated as described (Fogel, 1972). The anti-V-antigen serum, after conjugation with fluorescein and purification, stained V-antigen at a dilution of 1:16 when tested on PV-infected mouse embryo cells. Staining for the PV nuclear tumor (T)-antigen and preparation of anti-T serum was done was previously described (Fogel et al., 1967). The proportions of fluorescent cells that stained for V-antigen were calculated by counting a total of about 2 x lo4 cells in 100 microscopic fields on two coverslips. Treatment with vated temperature
mitomycin
C and ele-
(40”). Replicate cultures of each tested line were treated with mitomycin C obtained from the Nutritional Biochemical Corporation, Cleveland, OH) at concentrations of 0.25, 0.5 and 1.O pg/ml of medium for 1 hr at 37”. Four lines which were found inducible either for V-antigen or for both V-antigen and infectious virus at 37” were also subjected to heat treatment at 40 f 0.5” as follows. The cultures were treated with
FOGEL
mitomycin C for 1 hr as indicated. The medium was then changed to one not containing mitomycin C, and the cultures were kept at 37” for an additional 3 hr. A portion from each line was then transferred to an incubator at 40” for 24 hr. The remaining cultures were kept continuously at 37”. Control cultures, untreated with mitomycin C were incubated under identical conditions. The cultures were assayed for V-antigen 48 hr after the addition of mitomycin C and for infectious virus after 96 hr. RESULTS
Induction of PV-Antigen and Virus Synthesis in PV-Transformed Lines We investigated 14 PV-transformed lines of rat origin as well as four hamster and six mouse lines. Induction of PV or V-antigen synthesis was detected in only four lines of rat origin (Table 1). Two of the inducible lines (RPA and RPB) exhibited a different response to mitomycin C when the cultures were subsequently incubated at 40” than when they were kept continuously at 37” (Table 2). The proportions of V-antigen-containing cells in the RPA and RPB cultures preincubated at 40” is 50- to go-fold higher than in corresponding cultures kept at 37”. Infectious virus was not detected in the RPA line at either 37 or 40”; the RPB line, however, produced similar low yields of virus at both temperatures. From the data in Table 2, it is evident that even in the unheated RPB cultures a part of the Vantigen-containing cells do not produce infectious virus. The number of virusyielding cells, determined by the infective-centre assay, was about 5 x lo-’ in RPB cultures incubated at both temperatures. Since l/5,000 cells produce V-antigen in the unheated cultures, l/100 V-antigen-containing cells in these cultures produce infectious virus. In the heated cultures, the ratio of cells containing V-antigen to the cells producing virus increased by a factor of 120. In the control cultures not exposed to mitomycin C, whether incubated at 37 or 40”, neither V-antigen nor infectious virus was detected.
POLYOMA
VIRUS
CAPSID
ANTIGEN
TABLE
449
INDUCTION
1
POLYOMA VIRUS (PV)-TRANSFORMED LINES ASSAYED FOR PV INDUCTION BY MITOMYCIN C Cell species Rat Type of culture infected Infected with PV strain Number of PV-transformed linesb Number of lines inducible for PV or V-antigen
Hamster Primary RLP” 8
Secondary SP” IL110 3 3 0
1
Mouse
Secondary IL11 RLP 2 2
3
0
0
a SP, small plaque; IL11 and RLP are large plaque strains. b In the transformed lines all cells contained PV-T (tumor) antigen. Neither infectious virus was detected in these lines untreated with mitomycin C. TABLE
SP 2
Secondary IL11 2
0
RLP 2
0
0
viral capsid (V) antigen nor
2
PV-TRANSFORMED RAT CELL LINES INDUCIBLE BY MITOMYCIN C AT 37 AND 40” C” Transformed by PV strain
Cell line gbW72 V-antigenb
PFU/ 1 x 106 cells’
Plaqueforming cells/ 1 x 106 cells?
% Cells with V-antiger?
IL11
RPA RPB RP-78 RL
0.01 0.02 0.04
