- Email: [email protected]
Polyoma virus-transformed rat cell lines inducible for viral capsid antigen synthesis
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 <0.005
0 50 8 x lo3 5 x 102
Plaquefye;$ 1 x 106 cell@
At 37” RLP
PFU/ 1 x 10” cells’
At 40” 0 2 NT NT
0.5 1.2 0.02 <0.005
0 35 5 x 103 2 x 102
0 1 NT’ NT
“The cultures were treated with 1.0 rg/ml mitomycin C for 1 hr. Neither V-antigen nor infectious virus was detected in untreated control cultures irrespective of whether they were incubated at 37 or 40”. *Percentages of cells that stained for V-antigen were determined 48 hr after treatment with mitomycin C. Values were calculated from counts of about 2 x 10’ cells in 100 microscopic fields on two coverslips. c The cultures were assayed for infectious virus 96 hr after addition of mitomycin C. * The cultures assayed for infective-centre formation were treated with 1.0 pg/ml of mitomycin C for 1 hr at 37”. After a change to medium not containing mitomycin C, the cultures were kept at 37” for 3 hr and for an additional 24 hr at either 37 or 40”. They were then plated on normal mouse embryo monolayers as indicated in Materials and Methods. The data represent mean values from three experiments. e NT. Not tested.
Single cell clones were isolated from the RPA and RPB lines in the presence of PV antiserum (Fogel and Sachs, 1969) after 19 cell passages of the parental lines. Twelve clones from the RPA and 16 from the RPB line were tested for PV-induction. None of these clones produced either V-antigen or virus spontaneously at 37 or 40”. Following treatment with mitomycin C, V-antigen was detected in six RPA clones and in 14 RPB clones. All these except one (clone 5) produced V-antigen at higher cell frequencies at 40”. In these experiments, the proportion of V-antigen-containing cells
increased 60-65-fold in the RPA clones and 20-120-fold in the RPB clones. Data on the inducibility of representative clones are shown in Table 3. Infectious virus was not detected in the RPA clones. while the RPB clones, except for clone 5, yielded small quantities of virus (12-90 PFU/lOG cells) at both temperatures (Table 3). PV isolated from the RPB line and its progeny clones was of the large plaque strain as was the one by which the rat cultuhs were transformed. In contrast to the lines described above, the two remaining inducible lines (RL and
450
MARIAN
RP-78) exhibited a similar pattern of inducibility by mitomycin C at both temperatures (Table 2); the proportion of Vantigen-containing cells in the RP-78 cultures was similar at 37 and 40” and produced about 20 PFIJAJ-antigen-containing cell. In the mitomycin C-treated cultures of the RL line, low yields of PV were obtained consistently at both temperatures, though no V-antigen was detected in 2 x 10” cells examined (Table 2). When 1 x lo5 of the RL cells were treated with mitomycin C (1 @g/ml) and co-cultivated with mouse embryo secondary cultures for 8 days, V-antigen appeared in these cultures. In the control cultures of both lines, untreated with mitomycin C, neither V-antigen nor virus was detected. Out of 20 clones isolated from the RL line, 60% produced plaques following treatment with mitomycin C. The virus yields in these clones were of a similar order of magnitude as in the parental line. On the other hand, only one out of 20 RP-78 clones could be induced for virus synthesis by mitomycin C. Virus isolated from the RL and RP-78 lines and from the derivative clones was of the large plaque strain. Induction of PVSynthesis in Heterokaryon Cultures of RPA, RPB and Mouse Embryo Cells
If the lack of virus maturation in cells induced for V-antigen synthesis is due to nonpermissiveness of the cells, fusion of such cells with mouse embryo cells might allow complete virus production. The following experiment was performed with two clones from the RPB line and one RPA clone. Cultures of each clone treated with mitomycin C and corresponding untreated cultures were incubated at 40”. The cells of these cultures were then mixed with normal mouse embryo cells in the presence of Sendai virus and cultured as described in Materials and Methods. After 40 hr of incubation, the proportion of mononucleated cells and of cells with two or more nuclei that stained for V-antigen was determined. We also evaluated the proportion of infective centres. In the heterokaryon cultures with mitomycin C-treated
FOGEL
RPA and RPB cells, about 8-13% of V-antigen-containing cells were multinucleated (Table 4). No infective centres were produced by the cells from the RPA heterokaryon cultures. The number of infective centres produced by the cells from the RPB heterokaryon cultures and RPB control cells, identically treated but cultured without mouse cells, was similar (Tables 2 and 4). Neither V-antigen nor virus was detected in heterokaryon cultures with RPA or RPB cells not treated with mitomycin C. Additive Effect of Elevated Temperature and Mitomycin C on PVZnduction
It has been shown that 5-bromodeoxyuridine greatly enhanced the inducibility of LPT cells for PV synthesis by fluorescent light (Fogel, 1973) and by ultraviolet and X irradiation (Fogel, 1972). In the present study, we tested whether elevated temperature (40”) may exert an additive effect on PV induction in the highly inducible LPT cells. Previous data indicated that optimal doses of various PV-inducing agents, including mitomycin C, are very close to those that either abolish the cell capacity for virus synthesis or cause rapid cell death (Fogel, 1972). The highest rates of cell induction with mitomycin C were obtained previously at concentrations of 0.5 and 1.0 &ml of medium. The data in Table 5 indicate that in cultures treated with 0.5 &ml of mitomycin C and incubated at 40”, the proportion of V-antigen-containing cells and virus yields are much lower than in corresponding cultures kept continuously at 37”; conversely, with lower doses of mitomycin C (0.05-0.2 &ml), the proportion of V-antigen-containing cells and virus yields in the heat-treated cultures are increased about two- to six-fold (Table 5). Untreated LPT cells incubated at 37” produce PV spontaneously at a frequency of about 1 x 1O-4 cells (Fogel and Sachs, 1969). The data in Table 5 indicate that in untreated LPT cultures incubated at 40” the number of V-antigen-containing cells and PFU produced are not significantly higher than at 37”. It is evident from these results that elevated temperature enhances PV induction by mitomycin C in the LPT
POLYOMA
VIRUS
CAPSID
ANTIGEN
TABLE
3
SYNTHESISOF PV CAPSID (V)-ANTIGEN AND INFECTIOUSVIRUS IN MITOMYCIN AT 31 AND 40”” Original line
Clone number
451
INDUCTION
% Cells with V-antigen”
PFU/l x lo6 cells’
C-TREATED
RPA AND RPB CLONES
% Cells with V-antigenb
RPB
162 168 2 5 19 30 46
0.01 0.02 0.02 0.09 0.05 0.01 0.03
c
lOa
At 40”
At 37” RPA
PFIJtll,x
0 0 40 0 50 80 12
0.6 1.3 1.8 0.06 1.0 0.6 3.6
0 0 25 0 90 60 20
‘Cultures of each clone were treated with 0.25, 0.5 and 1.0 &ml of mitomycin C. Various clones exhibited a different sensitivity to the drug; optimal induction in a given clone was obtained with one of these concentrations of mitomycin C. The highest values of V-antigen-containing cells and the corresponding numbers of PFU/l x lo6 cells obtained with the optimal concentration of mitomycin C in each particular clone are given. Neither V-antigen nor infectious virus was detected in untreated control cultures kept at either 37 or 40”. b Percentages of cells that stained for V-antigen were determined 48 hr after treatment with mitomycin C. Values were calculated from counts of about 2 x 10’ cells in 100 microscopic fields on two coverslips. c The cultures were assayed for infectious virus 96 hr after addition of the drug.
cells; heat treatment alone does not appear to affect PV induction.
of noninducible cells in the cell populations may account for the fact that the proportion of inducible progeny clones from the DISCUSSION different lines described in the present study varied between 5 and about 80%. Out of 14 polyoma virus (PV)-transOf particular interest are two of the formed lines of rat origin established in the course of this study, three proved inducible presently described inducible lines. Line for PV, and one could be induced only for RPA and 50% of its progeny clones are V-antigen synthesis. None of four hamster inducible for viral capsid (V)-antigen synlines and of six lines of mouse origin thesis by mitomycin C at a cell frequency transformed by PV could be induced. The up to 0.02% at 37”. Incubation of these previously described large plaque-transcultures for 24 hr at 40” increased the cell formed (LPT) inducible line was also of rat induction frequency to 1.3%. However, no origin (Fogel and Sachs, 1969). PV-transinfectious virus could be rescued in the formed lines in which infectious virus could RPA cultures. The RPB line and most be rescued have been rarely observed clones of this line, when treated with mi(Fogel and Sachs, 1969; Vogt, 1970; Saito tomycin C, yield virus titers up to 90 et al., 1970). Apparently, one reason is that PFU/lOG cells irrespective of whether the PV-transformed cells may convert from cultures were incubated at 37 or 40”; inducible to noninducible in the course of whereas the proportion of cells that synthecontinuous culture (Fogel and Sachs, size V-antigen in the cultures incubated at 1970). We described a highly inducible the higher temperature was up to 120-fold LPT clone which yielded 99%-inducible higher than in the cultures incubated at subclones in the course of the first 3 37”. Since the proportion of the clone cells months of culture (Fogel, 1972)_After con- synthesizing V-antigen at 37” does not tinuous culture of the parental clone for 8 exceed 2 x 10e3, theoretically only one out months, 32% of progeny clones proved of about five V-antigen-containing cells noninducible (Fogel, unpublished results). might produce one virus PFU; in fact, it Such conversion and preferential selection was shown by the infective-centre assay
452
MARIAN
FOGEL
that only about l/5 x lo5 RPB cells pro- cells produce infectious virus at 37”. In the duce infectious virus, that is, in these cultures incubated at 40”, the proportion of cells containing V-antigen to cells produccultures l/100-250 V-antigen-containing ing virus increased up to 120-fold. The lack of virus maturation in the RPA cells and in TABLE 4 a great majority of the induced RPB cells PV-SYNTHESIS INDUCTION IN RPA AND RPB CLONES IN may be due to the facts that both the viral HETEROKARYON CULTURES WITH MOUSE EMBRYO genome persisting in these cells is defective CELLS” and the host cells lack a product indispenOriginal Clone % V-antigenPlaquesable for virus maturation. This missing lines containing cells number f”,‘e;;Y factor could probably be supplied to the MultiMono1 x 100 system by fusion of the transformed cells nucleated nucleated cells with permissive to PV mouse embryo cells. cells cells The results of such experiments show, 0.1 0.9 0 RPA 168 however, that virus maturation in V-anti0.14 8 RPB 2 1.6 gen-containing heterokaryons with RPA 6 46 0.18 1.2 cells does not occur at all and does not occur significantly more frequently in het“The clone cells were treated with 1.0 @g/ml of erokaryon cultures with RPB cells than in mitomycin C for 1 hr at 37”. After a change to cultures of RPB cells alone. Thus the lack medium not containing mitomycin C, the cultures of virus maturation seems, rather, to be were kept at 37” for 3 hr and at 40” for an additional 24 hr. The cells were then removed by trypsin and related to a deficiency of the viral genome processed for heterokaryon cultures as described in persisting in these cells. Induction of VMaterials and Methods. The heterokaryon cultures antigen in SV40-transformed human and were assayed for V-antigen and infective-centre normal mouse hybrid cells from which formation after 40 hr of incubation at 37”. In the plaque-forming virus could not be isolated control heterokaryon cultures with clone cells not has been recently described (Croce et al., treated with mitomycin C, neither V-antigen nor 1974). Since plaque-forming virus cannot plaques were detected. About 2 x 10’ cells were be isolated after fusion of these lines with counted, and 1 x lo6 cells from each type of culture the permissive cells the authors assume as were examined for infective-centre formation. TABLE
5
INDUCTION OF PV ANTIGEN AND VIRUS SYNTHESIS BY MITOMYCIN C IN LPT CELL CULTURES AT 37 AND 40” Cell line
Mitomycin WmU
C
% Cells with V-antigena
PFU/l x lo8 cellsb
% Cells with V-antigen”
At 40”
At 37” LPT
16’
16-1
None 0.05 0.2 0.5 None 0.05 0.2 0.5 None 0.05 0.2 0.5
0.01 0.3 1.3 9.0 0.02 0.2 1.2 14.0 0.05 0.3 4.0 26.0
PFU/l x 10s cellsb
3x 5x 3 x 1X 5x 5x 2x 2x 2x 1x 5x 2x
103 10’ 105 108 103 10’ 105 IO’ 10’ 105 105 106
0.02 1.8 6.0 2.5 0.03 0.7 5.6 4.5 0.1 1.6 8.5 3.0
5x 2x 1x 5x 8x 3x 1x 2x 2x 6x 2x 6x
108 lo6 106 10s lOa 105 10’ 106 10’ lo6 10’ IO”
‘Percentages of cells that stained for V-antigen were determined 48 hr after treatment with mitomycin C. Values were calculated from counts of about 2 x 10’ cells in 100 microscopic fields on two coverslips. *The cultures were assayed for infectious virus 96 hr after addition of the drug. The data represent mean values from three experiments. c Clones 16 and 16-l are first- and second-generation clones, respectively, of the original inducible LPT line.
POLYOMA
VIRUS
CAPSID
most plausible that these lines contain a defective viral genome (Croce et al ., 1974). In contrast to the SV40-transformed cell line (Margalith et al., 1970), the RPA and RPB cells do not synthesize V-antigen when incubated at the elevated temperature without treatment with mitomycin C. The results with the SV40- and the PVtransformed lines are, however, hardly comparable, since the former were incubated at 45” at which temperature the PV-transformed cells do not survive. The mechanism involved in phage induction in lysogenic bacteria is yet unclear (Signer, 1968, Borek and Ryan, 1973). Even less understood is this mechanism in the case of virus induction in transformed eukaryotic cells. Our previous studies suggested that in LPT cells which are highly inducible for PV synthesis, selective damage of the cellular DNA is involved in virus induction (Fogel, 1972, 1973). Since elevated temperature accelerates DNA degradation by gamma irradiation in bacteria (Achey and Pollard, 1967), it was plausible to enquire whether heat might also enhance PV induction by mitomycin C. The results showed that elevated temperature did indeed enhance PV induction by mitomycin C in the highly inducible LPT cell cultures. No such effect was observed with the RL and RP-78 lines, probably due to the fact that these lines in any event exhibit a very low rate of inducibility so that significant differences could not be observed. Since enhanced induction obtained in the LPT cells by combined heating at 40” and mitomycin C treatment can also be achieved by raising the concentration of mitomycin C at 37”, it seems that both these factors cause virus induction by a common mechanism (Fogel, 1972, 1973). In contrast to the RPA and RPB lines, in the LPT cells, 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. In the RPA and RPB cells, enhancement of induction could be achieved only by elevated temperature that increased the proportion of cells containing V-antigen in the mitomycin-
ANTIGEN
INDUCTION
453
treated cultures up to 120-fold, while the virus yields were either not at all or not significantly affected. The results thus indicate that in the RPA and RPB lines, a temperature-sensitive factor is involved in the V-antigen induction. Whether this factor is of a viral or a cellular nature remains to be determined. ACKNOWLEDGMENTS The author thanks Professor L. Sachs for reading the manuscript and for his generous help that made this work possible. The technical assistance of Mrs. Nora Pilly and Mr. Eliahou Nava is gratefully acknowledged. REFERENCES ACHEY, PH. M., and POLLARD, E. C. (1967) Studies on the radiation-induced breakdown of deoxyribonucleic acid in Escherichia coli 15T-L-. Radiat. Res. 31,47-62. AXELROD, D., BOLTON E. T., and HABEL, K. (1964) Polyoma viral genetic material in a virus-free polyoma induced tumor. Science 146, 1466-1468. BENJAMIN, T. L. (1966) Virus-specific RNA in cells productively infected or transformed by polyoma virus. J. Mol. Biol. 16, 359-373. BOREK, E., and RYAN, A. (1973) Lysogenic induction. Progr. Nucl. Acid Res. Mol. Biol. 13, 249-300. CROCE, C. M., HUEBNER, K., GIRARDI, A. J., and KOPROWSKI, H. (1974) Rescue of defective SV4O from mouse-human hybrid cells containing human chromosome 7. Virology 60, 276-281. DEFENDI, V., EPHRUSSI, B., and KOPROWSKI, H. (1964). Expression of polyoma-induced cellular antigen(s) in hybrid cells. Nature (London) 203, 495-496. FOGEL, M. (1972) Induction of virus synthesis in polyoma transformed cells by DNA antimetabolites and by irradiation after treatment with 5-bromodeoxyuridine. Virology 49, 12-22. FOGEL, M. (1973) Induction of polyoma virus synthesis by fluorescent (visible) light in polyoma-transformed cells pretreated with 5-bromodeoxyuridine. Nature New Biol. 241, 182-184. FOGEL, M., GILDEN, R., and DEFENDI, V. (1967) Polyoma virus-induced “complement-fixing antigen” in tumors and infected cells as detected by immunofluorescence. Proc. Sot. Exp. Biol. Med. 124,1047-1052. FOGEL, M., and SACHS, L. (1969) The activation of virus synthesis in polyoma transformed cells. Virology 37,327-334. FOGEL, M., and SACHS, L. (1970) Induction of virus synthesis in polyoma transformed cells by ultraviolet light and mitomycin C. Virology 40, 174-177. HABEL, K. (1965) Specific complement-fixing antigens in polyoma tumors and transformed cells. Virol0g.y
454
MARIAN
25, 55-61. MACPHERSON, I., and MONTAGNIER, L. (1964) Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291-294. MANOR, H., FOGEL, M., and SACHS L. (1973) Integration of viral into chromosomal deoxyribonucleic acid in an inducible line of polyoma transformed cells. Virology 53, 174-185. MARGALITH, M., MARGALITH, E., NASIALSKI, T., and GOLDBLUM N. (1970) Induction of simian virus 40 antigen in BSC, transformed cells. J. Viral. 5, 305-308. MEDINA, D., and SACHS, L. (1961) Cell-virus interactions with the polyoma virus. III. The induction of cell transformation and malignancy in uitro. Bit. J. Cancer 15,885-904. SAITO, M., TAGUCHI, F., HASEGAWA, K., YOSHIDA, Y., and NAGAKI, D. (1970) Activation of polyoma virus in polyoma tumor cells. Jap. J. Microbial. 14, 512-515. SIGNER, E. R. (1968) Lysogeny: The integration prob-
FOGEL lem. Annu. Rev. Microbial. 22, 451-488. SJ~GREN, H. O., HELLSTROM, I., and KLEIN, G. (1961) Transplantation of polyoma virus-induced tumors in mice. Cancer Res. 21, 329-337. VOGT, M. (1970) Induction of virus multiplication in 3T3 cells transformed by a therm0 sensitive mutant of polyoma virus. I. Isolation and characterization of Ts-a-3T3 cells. J. Mol. Biol. 47, 307-316. WATKINS, J. F., and DULBECCO, R. (1967) Production of SV40 virus in heterokaryons of transformed and susceptible cells. Proc. Nut. Acad. Sci. USA 58, 1396-1403. WESTPHAL, H., and DULBECCO, R. (1968) Viral DNA in polyoma and SV40 transformed cell lines. hoc. Nat. Acad. Sci. USA 59, 1158-1165. WINOCOUR, E. (1963) Purification of polyoma virus. Virology 19, 158-168. WINOCOUR, E., and SACHS, L. (1960) Cell-virus interactions with the polyoma virus. I. Studies on the lytic interaction in the mouse embryo system. Virology 11, 699-721.
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 <0.005
0 50 8 x lo3 5 x 102
Plaquefye;$ 1 x 106 cell@
At 37” RLP
PFU/ 1 x 10” cells’
At 40” 0 2 NT NT
0.5 1.2 0.02 <0.005
0 35 5 x 103 2 x 102
0 1 NT’ NT
“The cultures were treated with 1.0 rg/ml mitomycin C for 1 hr. Neither V-antigen nor infectious virus was detected in untreated control cultures irrespective of whether they were incubated at 37 or 40”. *Percentages of cells that stained for V-antigen were determined 48 hr after treatment with mitomycin C. Values were calculated from counts of about 2 x 10’ cells in 100 microscopic fields on two coverslips. c The cultures were assayed for infectious virus 96 hr after addition of mitomycin C. * The cultures assayed for infective-centre formation were treated with 1.0 pg/ml of mitomycin C for 1 hr at 37”. After a change to medium not containing mitomycin C, the cultures were kept at 37” for 3 hr and for an additional 24 hr at either 37 or 40”. They were then plated on normal mouse embryo monolayers as indicated in Materials and Methods. The data represent mean values from three experiments. e NT. Not tested.
Single cell clones were isolated from the RPA and RPB lines in the presence of PV antiserum (Fogel and Sachs, 1969) after 19 cell passages of the parental lines. Twelve clones from the RPA and 16 from the RPB line were tested for PV-induction. None of these clones produced either V-antigen or virus spontaneously at 37 or 40”. Following treatment with mitomycin C, V-antigen was detected in six RPA clones and in 14 RPB clones. All these except one (clone 5) produced V-antigen at higher cell frequencies at 40”. In these experiments, the proportion of V-antigen-containing cells
increased 60-65-fold in the RPA clones and 20-120-fold in the RPB clones. Data on the inducibility of representative clones are shown in Table 3. Infectious virus was not detected in the RPA clones. while the RPB clones, except for clone 5, yielded small quantities of virus (12-90 PFU/lOG cells) at both temperatures (Table 3). PV isolated from the RPB line and its progeny clones was of the large plaque strain as was the one by which the rat cultuhs were transformed. In contrast to the lines described above, the two remaining inducible lines (RL and
450
MARIAN
RP-78) exhibited a similar pattern of inducibility by mitomycin C at both temperatures (Table 2); the proportion of Vantigen-containing cells in the RP-78 cultures was similar at 37 and 40” and produced about 20 PFIJAJ-antigen-containing cell. In the mitomycin C-treated cultures of the RL line, low yields of PV were obtained consistently at both temperatures, though no V-antigen was detected in 2 x 10” cells examined (Table 2). When 1 x lo5 of the RL cells were treated with mitomycin C (1 @g/ml) and co-cultivated with mouse embryo secondary cultures for 8 days, V-antigen appeared in these cultures. In the control cultures of both lines, untreated with mitomycin C, neither V-antigen nor virus was detected. Out of 20 clones isolated from the RL line, 60% produced plaques following treatment with mitomycin C. The virus yields in these clones were of a similar order of magnitude as in the parental line. On the other hand, only one out of 20 RP-78 clones could be induced for virus synthesis by mitomycin C. Virus isolated from the RL and RP-78 lines and from the derivative clones was of the large plaque strain. Induction of PVSynthesis in Heterokaryon Cultures of RPA, RPB and Mouse Embryo Cells
If the lack of virus maturation in cells induced for V-antigen synthesis is due to nonpermissiveness of the cells, fusion of such cells with mouse embryo cells might allow complete virus production. The following experiment was performed with two clones from the RPB line and one RPA clone. Cultures of each clone treated with mitomycin C and corresponding untreated cultures were incubated at 40”. The cells of these cultures were then mixed with normal mouse embryo cells in the presence of Sendai virus and cultured as described in Materials and Methods. After 40 hr of incubation, the proportion of mononucleated cells and of cells with two or more nuclei that stained for V-antigen was determined. We also evaluated the proportion of infective centres. In the heterokaryon cultures with mitomycin C-treated
FOGEL
RPA and RPB cells, about 8-13% of V-antigen-containing cells were multinucleated (Table 4). No infective centres were produced by the cells from the RPA heterokaryon cultures. The number of infective centres produced by the cells from the RPB heterokaryon cultures and RPB control cells, identically treated but cultured without mouse cells, was similar (Tables 2 and 4). Neither V-antigen nor virus was detected in heterokaryon cultures with RPA or RPB cells not treated with mitomycin C. Additive Effect of Elevated Temperature and Mitomycin C on PVZnduction
It has been shown that 5-bromodeoxyuridine greatly enhanced the inducibility of LPT cells for PV synthesis by fluorescent light (Fogel, 1973) and by ultraviolet and X irradiation (Fogel, 1972). In the present study, we tested whether elevated temperature (40”) may exert an additive effect on PV induction in the highly inducible LPT cells. Previous data indicated that optimal doses of various PV-inducing agents, including mitomycin C, are very close to those that either abolish the cell capacity for virus synthesis or cause rapid cell death (Fogel, 1972). The highest rates of cell induction with mitomycin C were obtained previously at concentrations of 0.5 and 1.0 &ml of medium. The data in Table 5 indicate that in cultures treated with 0.5 &ml of mitomycin C and incubated at 40”, the proportion of V-antigen-containing cells and virus yields are much lower than in corresponding cultures kept continuously at 37”; conversely, with lower doses of mitomycin C (0.05-0.2 &ml), the proportion of V-antigen-containing cells and virus yields in the heat-treated cultures are increased about two- to six-fold (Table 5). Untreated LPT cells incubated at 37” produce PV spontaneously at a frequency of about 1 x 1O-4 cells (Fogel and Sachs, 1969). The data in Table 5 indicate that in untreated LPT cultures incubated at 40” the number of V-antigen-containing cells and PFU produced are not significantly higher than at 37”. It is evident from these results that elevated temperature enhances PV induction by mitomycin C in the LPT
POLYOMA
VIRUS
CAPSID
ANTIGEN
TABLE
3
SYNTHESISOF PV CAPSID (V)-ANTIGEN AND INFECTIOUSVIRUS IN MITOMYCIN AT 31 AND 40”” Original line
Clone number
451
INDUCTION
% Cells with V-antigen”
PFU/l x lo6 cells’
C-TREATED
RPA AND RPB CLONES
% Cells with V-antigenb
RPB
162 168 2 5 19 30 46
0.01 0.02 0.02 0.09 0.05 0.01 0.03
c
lOa
At 40”
At 37” RPA
PFIJtll,x
0 0 40 0 50 80 12
0.6 1.3 1.8 0.06 1.0 0.6 3.6
0 0 25 0 90 60 20
‘Cultures of each clone were treated with 0.25, 0.5 and 1.0 &ml of mitomycin C. Various clones exhibited a different sensitivity to the drug; optimal induction in a given clone was obtained with one of these concentrations of mitomycin C. The highest values of V-antigen-containing cells and the corresponding numbers of PFU/l x lo6 cells obtained with the optimal concentration of mitomycin C in each particular clone are given. Neither V-antigen nor infectious virus was detected in untreated control cultures kept at either 37 or 40”. b Percentages of cells that stained for V-antigen were determined 48 hr after treatment with mitomycin C. Values were calculated from counts of about 2 x 10’ cells in 100 microscopic fields on two coverslips. c The cultures were assayed for infectious virus 96 hr after addition of the drug.
cells; heat treatment alone does not appear to affect PV induction.
of noninducible cells in the cell populations may account for the fact that the proportion of inducible progeny clones from the DISCUSSION different lines described in the present study varied between 5 and about 80%. Out of 14 polyoma virus (PV)-transOf particular interest are two of the formed lines of rat origin established in the course of this study, three proved inducible presently described inducible lines. Line for PV, and one could be induced only for RPA and 50% of its progeny clones are V-antigen synthesis. None of four hamster inducible for viral capsid (V)-antigen synlines and of six lines of mouse origin thesis by mitomycin C at a cell frequency transformed by PV could be induced. The up to 0.02% at 37”. Incubation of these previously described large plaque-transcultures for 24 hr at 40” increased the cell formed (LPT) inducible line was also of rat induction frequency to 1.3%. However, no origin (Fogel and Sachs, 1969). PV-transinfectious virus could be rescued in the formed lines in which infectious virus could RPA cultures. The RPB line and most be rescued have been rarely observed clones of this line, when treated with mi(Fogel and Sachs, 1969; Vogt, 1970; Saito tomycin C, yield virus titers up to 90 et al., 1970). Apparently, one reason is that PFU/lOG cells irrespective of whether the PV-transformed cells may convert from cultures were incubated at 37 or 40”; inducible to noninducible in the course of whereas the proportion of cells that synthecontinuous culture (Fogel and Sachs, size V-antigen in the cultures incubated at 1970). We described a highly inducible the higher temperature was up to 120-fold LPT clone which yielded 99%-inducible higher than in the cultures incubated at subclones in the course of the first 3 37”. Since the proportion of the clone cells months of culture (Fogel, 1972)_After con- synthesizing V-antigen at 37” does not tinuous culture of the parental clone for 8 exceed 2 x 10e3, theoretically only one out months, 32% of progeny clones proved of about five V-antigen-containing cells noninducible (Fogel, unpublished results). might produce one virus PFU; in fact, it Such conversion and preferential selection was shown by the infective-centre assay
452
MARIAN
FOGEL
that only about l/5 x lo5 RPB cells pro- cells produce infectious virus at 37”. In the duce infectious virus, that is, in these cultures incubated at 40”, the proportion of cells containing V-antigen to cells produccultures l/100-250 V-antigen-containing ing virus increased up to 120-fold. The lack of virus maturation in the RPA cells and in TABLE 4 a great majority of the induced RPB cells PV-SYNTHESIS INDUCTION IN RPA AND RPB CLONES IN may be due to the facts that both the viral HETEROKARYON CULTURES WITH MOUSE EMBRYO genome persisting in these cells is defective CELLS” and the host cells lack a product indispenOriginal Clone % V-antigenPlaquesable for virus maturation. This missing lines containing cells number f”,‘e;;Y factor could probably be supplied to the MultiMono1 x 100 system by fusion of the transformed cells nucleated nucleated cells with permissive to PV mouse embryo cells. cells cells The results of such experiments show, 0.1 0.9 0 RPA 168 however, that virus maturation in V-anti0.14 8 RPB 2 1.6 gen-containing heterokaryons with RPA 6 46 0.18 1.2 cells does not occur at all and does not occur significantly more frequently in het“The clone cells were treated with 1.0 @g/ml of erokaryon cultures with RPB cells than in mitomycin C for 1 hr at 37”. After a change to cultures of RPB cells alone. Thus the lack medium not containing mitomycin C, the cultures of virus maturation seems, rather, to be were kept at 37” for 3 hr and at 40” for an additional 24 hr. The cells were then removed by trypsin and related to a deficiency of the viral genome processed for heterokaryon cultures as described in persisting in these cells. Induction of VMaterials and Methods. The heterokaryon cultures antigen in SV40-transformed human and were assayed for V-antigen and infective-centre normal mouse hybrid cells from which formation after 40 hr of incubation at 37”. In the plaque-forming virus could not be isolated control heterokaryon cultures with clone cells not has been recently described (Croce et al., treated with mitomycin C, neither V-antigen nor 1974). Since plaque-forming virus cannot plaques were detected. About 2 x 10’ cells were be isolated after fusion of these lines with counted, and 1 x lo6 cells from each type of culture the permissive cells the authors assume as were examined for infective-centre formation. TABLE
5
INDUCTION OF PV ANTIGEN AND VIRUS SYNTHESIS BY MITOMYCIN C IN LPT CELL CULTURES AT 37 AND 40” Cell line
Mitomycin WmU
C
% Cells with V-antigena
PFU/l x lo8 cellsb
% Cells with V-antigen”
At 40”
At 37” LPT
16’
16-1
None 0.05 0.2 0.5 None 0.05 0.2 0.5 None 0.05 0.2 0.5
0.01 0.3 1.3 9.0 0.02 0.2 1.2 14.0 0.05 0.3 4.0 26.0
PFU/l x 10s cellsb
3x 5x 3 x 1X 5x 5x 2x 2x 2x 1x 5x 2x
103 10’ 105 108 103 10’ 105 IO’ 10’ 105 105 106
0.02 1.8 6.0 2.5 0.03 0.7 5.6 4.5 0.1 1.6 8.5 3.0
5x 2x 1x 5x 8x 3x 1x 2x 2x 6x 2x 6x
108 lo6 106 10s lOa 105 10’ 106 10’ lo6 10’ IO”
‘Percentages of cells that stained for V-antigen were determined 48 hr after treatment with mitomycin C. Values were calculated from counts of about 2 x 10’ cells in 100 microscopic fields on two coverslips. *The cultures were assayed for infectious virus 96 hr after addition of the drug. The data represent mean values from three experiments. c Clones 16 and 16-l are first- and second-generation clones, respectively, of the original inducible LPT line.
POLYOMA
VIRUS
CAPSID
most plausible that these lines contain a defective viral genome (Croce et al ., 1974). In contrast to the SV40-transformed cell line (Margalith et al., 1970), the RPA and RPB cells do not synthesize V-antigen when incubated at the elevated temperature without treatment with mitomycin C. The results with the SV40- and the PVtransformed lines are, however, hardly comparable, since the former were incubated at 45” at which temperature the PV-transformed cells do not survive. The mechanism involved in phage induction in lysogenic bacteria is yet unclear (Signer, 1968, Borek and Ryan, 1973). Even less understood is this mechanism in the case of virus induction in transformed eukaryotic cells. Our previous studies suggested that in LPT cells which are highly inducible for PV synthesis, selective damage of the cellular DNA is involved in virus induction (Fogel, 1972, 1973). Since elevated temperature accelerates DNA degradation by gamma irradiation in bacteria (Achey and Pollard, 1967), it was plausible to enquire whether heat might also enhance PV induction by mitomycin C. The results showed that elevated temperature did indeed enhance PV induction by mitomycin C in the highly inducible LPT cell cultures. No such effect was observed with the RL and RP-78 lines, probably due to the fact that these lines in any event exhibit a very low rate of inducibility so that significant differences could not be observed. Since enhanced induction obtained in the LPT cells by combined heating at 40” and mitomycin C treatment can also be achieved by raising the concentration of mitomycin C at 37”, it seems that both these factors cause virus induction by a common mechanism (Fogel, 1972, 1973). In contrast to the RPA and RPB lines, in the LPT cells, 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. In the RPA and RPB cells, enhancement of induction could be achieved only by elevated temperature that increased the proportion of cells containing V-antigen in the mitomycin-
ANTIGEN
INDUCTION
453
treated cultures up to 120-fold, while the virus yields were either not at all or not significantly affected. The results thus indicate that in the RPA and RPB lines, a temperature-sensitive factor is involved in the V-antigen induction. Whether this factor is of a viral or a cellular nature remains to be determined. ACKNOWLEDGMENTS The author thanks Professor L. Sachs for reading the manuscript and for his generous help that made this work possible. The technical assistance of Mrs. Nora Pilly and Mr. Eliahou Nava is gratefully acknowledged. REFERENCES ACHEY, PH. M., and POLLARD, E. C. (1967) Studies on the radiation-induced breakdown of deoxyribonucleic acid in Escherichia coli 15T-L-. Radiat. Res. 31,47-62. AXELROD, D., BOLTON E. T., and HABEL, K. (1964) Polyoma viral genetic material in a virus-free polyoma induced tumor. Science 146, 1466-1468. BENJAMIN, T. L. (1966) Virus-specific RNA in cells productively infected or transformed by polyoma virus. J. Mol. Biol. 16, 359-373. BOREK, E., and RYAN, A. (1973) Lysogenic induction. Progr. Nucl. Acid Res. Mol. Biol. 13, 249-300. CROCE, C. M., HUEBNER, K., GIRARDI, A. J., and KOPROWSKI, H. (1974) Rescue of defective SV4O from mouse-human hybrid cells containing human chromosome 7. Virology 60, 276-281. DEFENDI, V., EPHRUSSI, B., and KOPROWSKI, H. (1964). Expression of polyoma-induced cellular antigen(s) in hybrid cells. Nature (London) 203, 495-496. FOGEL, M. (1972) Induction of virus synthesis in polyoma transformed cells by DNA antimetabolites and by irradiation after treatment with 5-bromodeoxyuridine. Virology 49, 12-22. FOGEL, M. (1973) Induction of polyoma virus synthesis by fluorescent (visible) light in polyoma-transformed cells pretreated with 5-bromodeoxyuridine. Nature New Biol. 241, 182-184. FOGEL, M., GILDEN, R., and DEFENDI, V. (1967) Polyoma virus-induced “complement-fixing antigen” in tumors and infected cells as detected by immunofluorescence. Proc. Sot. Exp. Biol. Med. 124,1047-1052. FOGEL, M., and SACHS, L. (1969) The activation of virus synthesis in polyoma transformed cells. Virology 37,327-334. FOGEL, M., and SACHS, L. (1970) Induction of virus synthesis in polyoma transformed cells by ultraviolet light and mitomycin C. Virology 40, 174-177. HABEL, K. (1965) Specific complement-fixing antigens in polyoma tumors and transformed cells. Virol0g.y
454
MARIAN
25, 55-61. MACPHERSON, I., and MONTAGNIER, L. (1964) Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291-294. MANOR, H., FOGEL, M., and SACHS L. (1973) Integration of viral into chromosomal deoxyribonucleic acid in an inducible line of polyoma transformed cells. Virology 53, 174-185. MARGALITH, M., MARGALITH, E., NASIALSKI, T., and GOLDBLUM N. (1970) Induction of simian virus 40 antigen in BSC, transformed cells. J. Viral. 5, 305-308. MEDINA, D., and SACHS, L. (1961) Cell-virus interactions with the polyoma virus. III. The induction of cell transformation and malignancy in uitro. Bit. J. Cancer 15,885-904. SAITO, M., TAGUCHI, F., HASEGAWA, K., YOSHIDA, Y., and NAGAKI, D. (1970) Activation of polyoma virus in polyoma tumor cells. Jap. J. Microbial. 14, 512-515. SIGNER, E. R. (1968) Lysogeny: The integration prob-
FOGEL lem. Annu. Rev. Microbial. 22, 451-488. SJ~GREN, H. O., HELLSTROM, I., and KLEIN, G. (1961) Transplantation of polyoma virus-induced tumors in mice. Cancer Res. 21, 329-337. VOGT, M. (1970) Induction of virus multiplication in 3T3 cells transformed by a therm0 sensitive mutant of polyoma virus. I. Isolation and characterization of Ts-a-3T3 cells. J. Mol. Biol. 47, 307-316. WATKINS, J. F., and DULBECCO, R. (1967) Production of SV40 virus in heterokaryons of transformed and susceptible cells. Proc. Nut. Acad. Sci. USA 58, 1396-1403. WESTPHAL, H., and DULBECCO, R. (1968) Viral DNA in polyoma and SV40 transformed cell lines. hoc. Nat. Acad. Sci. USA 59, 1158-1165. WINOCOUR, E. (1963) Purification of polyoma virus. Virology 19, 158-168. WINOCOUR, E., and SACHS, L. (1960) Cell-virus interactions with the polyoma virus. I. Studies on the lytic interaction in the mouse embryo system. Virology 11, 699-721.