Infection and transformation of mouse cells by human adenovirus type 2

147, 126-141

VIROLOGY

Infection

(1985)

and Transformation

of Mouse Cells by Human Adenovirus

MARCIA Department

qf Molecular

L. ZUCKER’ Biology,

Received

April

Princetcm

AND

S. J. FLINT’ Princetm,

University,

26, 1985; accepted

Type 2

July

New

FJwsey

OXSQ

2h, 19X.5

The susceptibilities of C57Bl/&I mouse embryo fibroblasts (MEF) and baby mouse kidney (BMK) cells to infection or transformation by adenovirus type 2 have been compared to those of rat embryo fibroblasts (REF). Both MEF and BMK cells were some lo-fold less permissive to replication of the virus than were REF cells, even though similar fractions of all three cell types, a maximum of 50-60%,produced viral tumor and structural antigens. This observation suggests that a very late step in adenovirus production, such as assembly or maturation, occurs much less efficiently in mouse cells than it does in rat cells. No significant differences in the frequencies of transformation, as assayed by the appearance of foci of morphologically transformed cells, were observed following transfection of adenovirus type 2 or type 5 DNA into the three cell types. However, it proved extremely difficult to establish permanent lines of adenovirus-transformed mouse cells: only 2 of more than 100 attempts were successful, compared to a success rate of close to 100% with adenovirus type 2-transformed REF or SV40-transformed MEF or BMK cells. The two lines of type 2 adenovirus-transformed MEF that were established have been shown to retain and express viral genetic information. 0 1985 Academic Press, Inc.

INTRODUCTION

Transformation by the subgroup C adenoviruses, such as types 2 and 5 (Ad2 and Ad5), has been intensively studied using primary and secondary rat embryo fibroblasts, as well as established lines of rat cells (see Tooze, 1980; Flint, 1984, for reviews). Rat cells are semipermissive for adenovirus growth, producing considerably lower yields of virus than fully permissive human cells (Gallimore, 1974). Hamster cells have also been transformed by subgroup C adenoviruses following inactivation of replication functions by uv irradiation (Lewis et ah, 1974; Lewis, 1977) or the introduction of mutations (Williams, 1973), steps necessary because hamster cells support efficient adenovirus reproduction (Takahashi, 1972; Williams, 1973). Transformation of mouse cells by a subgroup C adenovirus has, by contrast,

’ Present address: Department of Pharmacology, Yale Medical School, New Haven, Conn. 06510. ‘To whom correspondence should be addressed. 0042-6822/85 Copyright All riyhts

$3.00

b 1385 hy Academic Press. Inc. of reproduction in any form reserved

126

been reported only once and quite recently (Sarnow et al., 1982). Cells of murine origin have been very widely used in studies of transformation by other DNA-containing viruses and retroviruses, as well as by chemical and physical agents (see Tooze, 1980; Weiss ef al., 1982; Heidelberger et al, 1983), and inbred strains of laboratory mice provide the mammalian system whose genetics are best understood. Thus, the dearth of studies of adenovirus transformation of murine cells is surprising. During the course of experiments to establish lines of subgroup C adenovirustransformed primary or secondary mouse cells, we observed some unexpected differences in the susceptibility of rat and mouse cells to transformation. Here we report the results of these experiments, as well as of an examination of the permissivity of C57B1/6J mouse cells to adenovirus replication, undertaken in light of previous suggestions that there exists a direct relationship between the permissiveness of rodent cells and their susceptibility to transformation (Gallimore, 1974; McDougall et al., 1975; Jochemsen et al., 1982).

Ad2

MATERIALS

AND

TRANSFORMATION

METHODS

Cells and virus. Primary mouse embryos were taken from C57B1/6J mice lo-12 days into gestation. Rat embryos were taken after 16 days of gestation from SpragueDawley rats. Seven-day-old C57B1/6J mice were the source of baby mouse kidney cells (BMK). All tissues were removed under sterile conditions and finely minced in Dulbecco’s modified essential medium (DMEM). After gentle pelleting, the pieces were resuspended in 5 ml trypsin-EDTA (0.25% trypsin, 0.02% EDTA, 0.9% NaCl) and 5 ml collagenase solution [0.75% collagenase (Sigma type lA), 5% bovine serum albumin, in phosphate-buffered saline (PBS)] and the suspension was gently rocked at 37” for 1 hr to disperse the cells. Cells were then pelleted gently and washed with sterile saline before plating in 75-mm tissue culture flasks in DMEM supplemented with 2 mMglutamine and 10% fetal calf serum. All transformed cell lines were carried in DMEM supplemented with 5% fetal calf serum, 5% calf serum, and 2 mM glutamine. Adenovirus types 2 and 5 were propagated in HeLa cells in suspension culture as described previously (Flint et al., 1975). Physical particle production was determined by banding 1 ml of the cell lysate in a 5-ml continuous CsCl gradient of density range 1.2-1.4 g/mm3. Concentrations of infectious virus were determined by plaque assay on HeLa cell monolayers, according to the method of Williams (1970). Viral and plasmid DNA. Adenoviral DNA was obtained from purified virions by the method of Pettersson and Sambrook (1973). Plasmids comprising pBR322 DNA into which SV40 DNA or Ad2 EcoRI fragment A had been inserted were obtained from R. Kucherlapati and G. Chinnadurai, respectively, and were propagated in Escheria coli C600 or HBlOl using standard methods (Maniatis et ah, 1982). Adenovirus DNA fragments used in transfection were isolated from full-length viral DNA (HpaI fragments E and C) or the cloned EcoRI A fragment (Sal1 B and BamHI B). Following digestion with the appropriate restriction endonuclease(s), DNA fragments were separated by elec-

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trophoresis in 1% agarose gels cast in 0.04 M Tris-HCl, pH 8.3, containing 0.05 M sodium acetate and 1 mM EDTA. The gels were stained and the fragments of interest were excised. The DNA was eluted electrophoretically and subsequently purified by chromatography or Elutip-d (Schleicher & Schuell) columns. In some experiments, gel slices were dissolved in saturated sodium iodide and the DNA was recovered by chromatography on hydroxylapatite columns, followed by dialysis. In either case, the DNA was precipitated at -20” following the addition of 2 vol of ethanol and 0.2 M sodium acetate. Transfection of DNA into mammalian cells. Transfections were performed as described by Graham and van der Eb (1973), with some modifications. Briefly, cell cultures [mouse and rat embryo fibroblasts and baby mouse kidney cells (MEF), (REF), and (BMK)] were seeded onto 60mm tissue culture dishes at l-2 X lo5 cells per plate 24 hr before transfection. Onehalf hour prior to transfection, O-5 pg of transforming DNA was mixed with carrier DNA to a total concentration of 20 pg/ml in 0.02 M HEPES buffer, pH 7.05, containing 0.17 M NaCl, 5 mM KCl, 1 mM Na2HP04, and 5.5 mM dextrose. Calcium chloride was added to a final concentration of 125 mM The solution was mixed vigorously and allowed to incubate at room temperature for 30 min, when a fine precipitate formed. The medium was then removed from the cells and replaced by 0.5 ml of the calcium phosphate-DNA mixture. After 30 min at room temperature, 5 ml medium was added to each plate, and the plates were placed at 37” in a 5% COr! atmosphere. Five hours after transfection the cells were subjected to a glycerol boost (Frost and Williams 1978). The medium was removed and the plates were washed with sterile saline solution (0.83% NaCl). One milliliter of 25 mM Tris-HCl, pH 7.4, containing 0.14 MNaCl, 5 mMKCl,O.7 mM Na2HP04, 5.6 mM dextrose, 10 mM MgClz, and 20% (v/v) glycerol was applied for 1 min. The plates were again washed with saline solution and 5 ml of growth medium was added per plate. Two days after transfection, the DMEM medium was removed

128

ZUCKER

from those cultures to be subjected to selection by growth in medium containing low calcium concentrations and replaced by SMEM (0.1 mM Ca2+, (GIBCO) supplemented with 10% fetal calf serum and 2 mM glutamine. Colonies appeared in adenovirus DNA-transfected cultures 3 to 4 weeks after transfection and were picked or stained with Wright’s stain (Sigma, 0.2% in methanol) 2 weeks after their initial appearance. Transformation frequencies were expressed as the number of foci per microgram full-length viral genome equivalent. Cultivation of foci. Clearly visible foci were well separated from neighboring foci and were picked for the establishment of clonal cell populations. Dishes containing such foci were washed twice with saline solution and a stainless-steel cloning cylinder whose bottom was coated with sterile silicone grease was placed over the focus to be picked. One or two drops of FC solution (25 mM Tris-HCl, pH 7.3, containing 0.6 mM EDTA, 0.001% phenol red, 0.14 M NaCl, and 5 mM KCl) or 0.05% trypsin was applied to the cells through the cylinder and the cells were washed from the plate. FC solution dislodges cells from their solid support more gently than does trypsin (R. Kucherlapati, personal comunication) and was used in many of the mouse cell studies. Each focus was transferred to an individual well of a 96-well tissue culture plate which contained 0.15 ml of DMEM supplemented with 10% fetal calf serum, 2 mMglutamine, and 10% spent medium. When confluent, each cell line was sequentially transferred to one well of a 24-well tissue culture dish, a 60-mm tissue culture plate, and finally a loo-mm tissue culture plate. After the cultures reached confluence in the loo-mm plate, the serum concentration was changed to 5% fetal calf serum plus 5% calf serum. Primary rodent cells do not divide under these reduced serum conditions (M.L.Z., unpublished observation). Dot blots of mammalian cell DNA. Dot blots were performed as described by Brandsma and Miller (1980), with some modifications. One loo-mm tissue culture dish of each cell line to be tested was scraped, the cells were resuspended in

AND

FLINT

sterile saline solution, counted in a Coulter Counter, and 5 X lo4 cells of each type in 5 yl saline solution was spotted onto a nitrocellulose filter. Each filter was then placed cell side up on Whatman 3MM paper saturated with 0.5 MNaOH for 5 min, twice on 1 M Tris-HCl, pH 6.8, containing 0.6 M NaCl for 1 min, and once on 0.5 M TrisHCl, pH 7.4, containing 1.5 M NaCl for 5 min. Filters were air-dried for 20 min, floated onto 95% ethanol, and air-dried for 5 min. They were then washed twice in chloroform, air-dried for 15 min, rinsed in 0.3 M NaCl, and allowed to air-dry completely. After baking in a vacuum oven at 80” for 18 hr, the filters were prehybridized overnight at 68” in 0.06 M Tris-HCl, pH 7.4, containing 10X Denhardt’s solution, 0.2% SDS, 10 mM sodium pyrophosphate, 100 pug/ml denatured salmon sperm DNA, 20 pg/ml poly(A), and 1 M NaCl. Adenovirus DNA was 32P-labeled by nick translation (Rigby et al., 1977), denatured in 0.3 M NaOH at room temperature for 15 min, neutralized with acetic acid, and applied to the filters in the same solution as during prehybridization except that the concentrations of salmon sperm DNA and poly(A) were reduced to 25 pg/ml and 10 pg/ml, respectively. Hybridization was for 16 hr at 68’. Filters were washed at 68” in fresh hybridization mix for 2-5 hr, overnight in 2X SSPE and 0.1% SDS, 2 hr in fresh 2X SSPE and 0.1% SDS, and finally in 2X SSPE without SDS. They were then dried and exposed to Kodak XAR film with an Ilford Lightening-Plus intensifying screen. Indirect immunc)$uorescence. Cells were grown on 12-mm diameter coverslips until they reached 50-70% confluence. They were then fixed in 10% Formalin (3.7% formaldehyde) in PBS for 10 min. The coverslips were placed in staining racks and prepared by soaking as follows: 1 min in PBS; 3 min in 50% acetone in PBS; 5 min in 100% acetone; 3 min in 50% acetone in PBS; 1 min in PBS. The acetone solutions were chilled to -20” prior to use. The coverslips were then placed cell side up on filter paper saturated with PBS in loo-mm dishes and stored at -80” for up to 2 weeks before staining. Twenty microliters of an appropriate dilution of antiserum (1:lOO for

Ad2

TRANSFORMATION

HT14b antiserum, 1:lO for anti-structural protein antiserum) in PBS was applied to each coverslip and incubated at 37” for 1 hr. Coverslips were then washed individually by dipping three times in each of three beakers of PBS, blotting the coverslip between each wash. The coverslips were placed back on the filter paper and 20 ~1 of a 1:32 dilution in PBS of fluorescein-labeled anti-hamster (HT14B) antiserum or antirabbit (anti-structural protein) antiserum was applied to each and incubated at 37” for 1 hr. The coverslips were again washed in three beakers of PBS with the addition of a final rinse in distilled water and mounted cell-side down on glass microscope slides in 90% glycerol, 10% PBS with 1 mg/ml p-phenylenediamine to inhibit fluorescent bleaching (Johnson and Nogueira-Araujo, 1981). The cells were photographed under mercury illumination. HT14B antiserum was the generous gift of Dr. J. Williams, Mellon Institute, Pittsburgh. To prepare anti-structural protein antiserum, 200 pg protein of disrupted, purified adenovirions in complete Freund’s adjuvant was injected intradermally into a young New Zealand white rabbit. From 6 weeks after this initial injection, booster injections were given every 2 weeks. The rabbit was bled monthly from the ear, and cell-free serum was prepared and stored at -70”. RESULTS

Permissiveness of Rat and Mouse Cells for Adenovirus 2 Replication The production of infectious virus particles, appearance of cytopathic effect and synthesis of early and late viral proteins were compared following Ad2 infection of MEF, BMK, primary Sprague-Dawley REF, or HeLa cells, the permissive cell line in which all virus stocks used in these experiments were propagated. All mouse cells were taken from C57B1/6J mice, as described under Materials and Methods. The various cell types were infected at 20 PFU/ cell and dishes of infected cells were harvested after various periods of incubation at 37”. The yield of infectious virus was

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determined by plaque assay on HeLa cell monolayers after lysis of the cells by repeated cycles of freezing and thawing, with the results shown in Table 1. The yield of virus per HeLa cell increased as a function of time following infection to very high levels by 40 hr after infection, as expected (see Tooze, 1980). Compared to such fully permissive cells, rat embryo fibroblasts displayed a severly impaired ability to support production of Ad2 (Table l), in agreement with previous observations (Gallimore, 1974; Williams et al, 1975). However, the yield of virus recovered from either type of mouse cell was reduced at least a further order of magnitude (Table 1). Thus, these C57B1/6J mouse cells are significantly less permissive for completion of the adenovirus replication cycle than rat embryo fibroblasts. The differences in time required to achieve maximum virus production shown in Table 1, 3 days after infection in MEF, compared to 4 days in BMK or REF, has been reproducibly observed, but is of unknown significance. The cytopathic effects (cpe) of adenovirus infection of the 4 cell types were next compared. Figure 1 illustrates typical changes seen in AdZ-infected HeLa and rodent cell cultures. The former displayed a progressive loss of sharp cellular boundaries, loss of attached cells, and increasingly dense nuclei containing inclusions (panels 2a to 2c of Fig. 1). Uninfected rodent cells were extremely large, with virtually transparent cytoplasm (Fig. la). Following Ad2 infection such cells appeared smaller, i.e., more rounded, and displayed gradually more opaque cytoplasm. Using these criteria, the fraction of AdZ-infected human, rat, or mouse cells showing cytopathic changes was scored during microscopic examination of cells stained after various periods of infection. In all three types of rodent cells examined, obvious cpe increased from a low level 2 days after infection to a maximal value, 50-60% of the cells, by 5 days after infection (Table 1, column 4). Cytopathic changes developed much more quickly in fully permissive HeLa cells and were evident in at least 80% of the cells observed by the end of the infectious cycle: this represents a minimum value, because by these

130

ZUCKER

AND TABLE

PRODUCFIVE

INFECTION

FLINT 1

BY ADENOVIRUS

TYPE

2

% Cells Cell line

Time

(hr)

MEF

24 48 72 96 120

REF

24 48 72 96 120

BMK

24 48 72 96 120

HeLa

14 24 40 44d

PFU/cell” 0.05 0.67 3.70 0.17 0.16 0 11.1 28.0 42.5 19.4 0 0.17 0.19 0.42 0.29 30 1300 2.4 X lo4 3.2 X lo4

positive

for

cpeb

T Ags

Structural

0 O-5% lo-15% 20% 50%

0” 10-15 25 30 50

0’ 10-15 30 30 50

0 O-5% 10% lo-15% 50-60%

10 10 10 20 50-60

0’ 10 10 20 50-60

0 5-10% lo-15% 40% 60%

10 10-15 20 40 60

0’ 10-15 20 40 60

1% 30% 80% 60%

10 20 70 50

15 45 85-90 40

Ags

a Average of three independent experiments. ‘Morphological changes characteristic of infected cells, as illustrated in Fig. 1. ‘Less than 10% of the culture showed a small quantity of nuclear flecks. d Many cells in the 44-hr HeLa cell culture had lysed or detached from the dish.

late times after infection (40 and 44 hr) infected HeLa cells had begun to disintergrate and/or detach from the substratum. The high percentage of AdB-infected rodent cells showing recognizable morphological alterations was interesting in light of the very low yields of virus obtained from them: at least in human cells, an important inducer of cpe has been identified as the penton base (Pettersson and Hoglund, 1968), a viral late protein. To obtain an independent, and more direct assessment, of viral gene expression, synthesis of viral early and late antigens was examined by indirect immunofluorescence. Production of early antigens was monitored using an anti-tumor antigen serum from hamsters bearing tumors induced by the Ad5-transformed hamster cell line HT14B (Williams, 1973). Such cells express only the EIA and EIB regions of the viral

genome (Flint et al, 1976) and HT14B serum has been shown to immunoprecipitate the well characterized EIA and EIB proteins, as well as polypeptides of apparent molecular mass 25,11, and 10 kDa (Ross et al. 1980). The patterns of indirect immunofluorescence observed in infected mouse, rat or human cells exposed to HT14B serum are shown in Fig. 2, panels 1A to 3A: in all cell types, positive cells displayed very strong nuclear fluorescence, as well as less intense cytoplasmic staining, results consistent with previous reports of the locations of the EIA and EIB polypeptides (Feldman and Nevins, 1983; Yee et ah, 1983; Rowe et a& 1983; Spindler et al, 1984). In all three types of rodent cell the fraction of cells synthesizing these adenoviral Tantigens increased gradually to some 50% during the 5-day examination period and at each time point was very similar to the

Ad2

1

TRANSFORMATION

OF

MOUSE

131

CELLS

a

FIG. 1. Cytopathic changes in Ad2-infected HeLa, rat, or mouse cells. Cells were infected with 20 PFU/cell Ad2 and stained and photographed after various periods of infection as described under Materials and Methods. Panel 1: BMK cells, uninfected (a) or at 4 days after infection (b). Panel 2: HeLa cells, uninfected (a) or at 24 hr (b) and 40 hr (c) after infection.

fraction of infected cells displaying cytopathic effects (Table 1, columns 4 and 5). Production of viral late proteins was examined using an anti-structural protein serum prepared as described under Materials and Methods. Immunoprecipitation of extracts of Ad2-infected HeLa cells labeled with [?S]methionine from 16 to 18 hr after infection established that this antiserum contained antibodies which recognized polypeptides II, III, IV, V, and VII, the major structural proteins of the virion (data not shown). Both rodent and human cells infected with Ad2 showed intense nuclear fluorescence and flecked cytoplasmic staining when this antiserum was used in indirect immunofluorescence (Fig. 2, panels 1B to 3B). Moreover, the fraction of cells in the three rodent populations expressing viral structural polypeptides was very similar, after each period of infection, to that positive for synthesis of the early EIA and EIB proteins (Table 1, columns 5 and

6), reaching a maximum of 50 to 60% 5 days after infection. Although cultures of both rat and mouse cells infected by Ad2 eventually displayed similar fractions of cells positive for production of viral proteins, higher proportions of the mouse cell cultures were positive at an earlier time after infection: thus, 20-40% of infected MEF or BMK cells made viral proteins, as judged by immunofluorescence, at ‘72or 96 hr after infection, compared to lo-20% of infected rat embryo fibroblasts (Table 1).

Transformation

of Rat and Mouse Cells by

Ad2 DNA Before undertaking transformation of the various types of rodent cell by adenovirus DNA or DNA fragments, we compared their susceptibility to DNA-mediated transformation using SV40 DNA introduced by the calcium phosphate coprecipitation methods of Graham and van

132

3

FIG. 2. Synthesis of viral early and late proteins in AdB-infected HeLa, rat, or mouse cells. Cells were infected with 20 PFU/cell Ad2 and prepared for indirect immunofluorescence using either HTl4B serum (la, 2a, and 3a) or antistructural antigen serum (lb, 2b, and 3b) or preimmune rabbit serum (lc and 2c) as described under Materials and Methods. Panels 1, 2, and 3 show BMK cells 3 days after infection, REF 3 days after infection, and HeLa cells 40 hr after infection, respectively.

der Eb (1973). The three rodent cell types tested exhibited indentical patterns of DNA concentration-dependent efficiencies of transformation (Fig. 3A). The decrease in transformation frequency in response to increasing quantities of SV40 DNA observed was similar to that reported previously (Graham et al., 1975; Abrahams et al., 1975). The maximal transformation frequencies, attained at the lowest SV40 DNA concentration tested in these experiments, was also similar for MEF, BMK, and REF recipient cells (Fig. 3A) indicating that these three cell types are unlikely to be markedly different with respect to their abilities to take up exogenous DNA or dis-

play the transformed phenotype, appearance of foci of morphologically altered cells, assayed in these experiments. Cultures of MEF, REF, or BMK cells were next transfected with Ad2 DNA at varying concentrations. In most cases, cells were transferred to medium containing low concentrations of Ca2+ ions 2 days after addition of DNA to facilitate detection of foci of transformed cells (Freeman et ah, 1966). In some experiments, such a selection procedure for adenovirus-transformed cells was omitted, such that foci of transformed cells appeared against a background of normal cells. In all experiments, foci of transformed cells were clearly vis-

Ad2

40r

_

A. SV40

2

TRANSFORMATION

OF

DNA

----__ 0.5 6.

Ad

1.0

2 DNA

ii

1.0 pglml. Viral

2.0

3.0

DNA

FIG. 3. Transformation of MEF, BMK, or REF by SV40 or Ad2 DNA. Cultures of MEF, REF, or BMK cells were transfected with the concentrations of viral DNA indicated in the figure in the presence of carrier mouse cell DNA. Transformed foci were counted 3 weeks after transfer into selective medium as described under Materials and Methods. In both panels, the frequencies of transformation of MEF, REF, and BMK cells are represented by 0, A, and 0, respectively.

ible 3 to 4 weeks after transfection in those cultures which received adenoviral DNA. No foci were observed in culture which received only carrier salmon sperm or mouse cell DNA (Table 2). The morphology of cells present in such foci is compared to that of their normal parents in Fig. 4: transformed cells derived from all three cell types were very clearly distinct from their parents, being more compact and refractile. Moreover, transformed cells, by contrast to their normal parents, tended to overgrow one another to form disorganized arrays (compare, for example, panels c and d of Fig. 4). Typical results obtained when transformation frequency was measured as a funtion of Ad2 DNA concentration are shown in Fig. 3B. Maximal frequencies, 16 to 28 foci/pg DNA were obtained at the lowest concentration of DNA tested and no striking differences in susceptibility to trans-

MOUSE

CELLS

133

formation could be discerned among the three cell types. Essentially identical results were obtained when Ad5 DNA replaced Ad2 DNA (Table 2). The efficiencies of transformation observed in these experiments, as well as the morphology and growth properties of the transformed cells, are similar to those reported previously for subgroup C adenovirus DNA transformation of rat cells (Graham and van der Eb, 1973; Graham et cd., 1975; van der Eb et al, 1979; Schrier et ab, 1979, for example). The transformation of mouse embryo fibroblasts by subgenomic fragments of Ad2 DNA which include all or parts of the transforming region was also examined, with the results summarized in Table 2: two fragments of Ad2 DNA which contained both the EIA and EIB transcriptional units, BumHI fragment B and SaZI fragment B, as well as &a1 fragment E, which spans only the EIA transcriptional unit, transformed mouse embryo fibroblasts with efficiencies similar to those seen with the intact Ad2 genome. However, HpaI fragment C, containing the EIB but not the EIA region, failed to induce the appearance of transformed foci, a result in agreement with the observation that EIB products are not sufficient to transform primary or established rat cells, even when produced at high levels under the control of the SV40 early transcriptional control region and enhancer (van den Elsen et cd., 1983). In summary, the quite large number of transformations we have performed failed to detect any significant differences in the susceptibilities of mouse and rat cells to adenovirus DNA-mediated transformation, despite their different degrees of permissiveness for virus growth. One striking difference between rat and mouse transformation did, however, emerge when attempts were made to establish cell lines from foci of transformed cells. As summarized in Table 2, lines of transformed rat cells were readily established from foci arising in cultures which had been transfected with Ad2 DNA. By contrast, it proved to be extremely difficult to establish transformed cell lines from foci appearing in cultures of either type of mouse cell: foci of transformed MEF or

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AND TABLE

TRANSFORMATION

DNA sv40 sv40 Ad2

Ad5 Ad2 BamHI-B (O-29.71) Ad2 SalI-B’ (O-27.35) Ad2 HpaI-E (O-4.36) Ad2 HpaI-C (4.36-25.79) Salmon sperm Mouse

2

OF RAT AND MOUSE

Cell line MEF BMK MEF (5 expts) (2 non-sel.) REF (3 expts) (1 non-sel.) BMK (4 expts) (2 non-sel.) MEF (2 expts) MEF (3 expts)d

FLINT

CELLS BY ADENOVIRUS Maximum” No. foci/pg genome equivalent 33.0 38.5 16.4

DNA Cell lines established/ foci picked 6/X 7/8 Z/43 *

21.4

1203’

27.5

O/25

MEFf MEFB MEF

20.0 19.8 33.7 15.1 37.8 20.6

O/22 O/18 o/12 O/6 o/10 o/7

MEF

0

NA’

MEF MEF

Oh 0”

NA NA

’ From the experiments shown in Fig. 3B and others like them. *Two cell lines established from transformed foci which appeared in the absence of selection for growth in low concentration of Ca2+ ions. ‘Six cell lines were established from foci which appeared in the presence of selection for growth in low concentrations of Ca” ions, six from foci which appeared in the absence of such selection. d Two experiments employed salmon sperm DNA as carrier, one employed C57B1/6J mouse DNA as carrier. eSoII-B, isolated from cloned EcoRI-A (O-55.9%) DNA, contains 200 base pairs of pBR322 DNA. f Salmon sperm DNA used as carrier. g C57B1/6J mouse DNA used as a carrier. h Carrier (240 pg) at 20 pg/ml was transfected into 24 plates of MEF. ’ Carrier (520 pg) at 20 wg/ml was transfected into 52 plates of MEF. ’ NA = not applicable.

BMK could, in the great majority of cases, be picked and grown for no more than two passages before they died. Batch culturing of the cells from dishes which contained up to twenty foci of morphologically transformed cells was no more successful, for the cells died within six passages of transfection. This failure cannot be ascribed to some property of the mouse cells themselves, for cell lines were readily established from foci of SV40 transformed MEF or BMK cells (Table 2). The two successful attempts with AdB-induced foci, of 143 trials, were with colonies from plates of MEF cells which had been transfected with

0.5 or 1.0 pg/ml. Ad2 DNA in the presence of C5’7B1/6J mouse carrier DNA and which were not subsequently selected for growth in medium containing low concentrations of calcium ions. The foci from which these two transformed cell lines, termed MA251 and MA252, were established could be distinguished from normal mouse cells on the basis of their altered morphology and ability to overgrow one another. MA251 and MA252 cells obviously also differ from their parents, MEF, by the criterion of continuous growth in culture. They also grow well under conditions which do not favor growth of MEF, for example, in medium containing

Ad2

TRANSFORMATION

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135

FIG. 4. Morphology of MEF, REF, and BMK cells transformed by Ad2 DNA. Rat embryo fibroblasts (panels a and b), mouse embryo flbroblasts (c and d) and baby mouse kidney cells (e and f) were stained with Wright’s stain and photographed under phase contrast illumination. Parental cells are shown in panels a, c, and e and portions of transformed foci derived from them in panels b, d, and f.

5% calf serum or in medium containing low concentrations of calcium ions, and exhibit somewhat shortened doubling times in medium containing 10% fetal calf serum, 29 or 28 hr for MA251 and MA252, respectively, compared to 35 hr for MEF. Surprisingly, however, neither MA251 or MA252 cells induced tumors in athymic or syngenic neonatal mice, when other transformed rodent cell lines tested under the same conditons induced tumors with up to 100% efficiency.

Presence and Expression of Viral Genetic Infmation in Ad2-Transformed Rat and Mouse Cell Lines As the transformation frequencies listed in Table 2 were determined solely on the basis of formation of foci of morphologically transformed cells, we felt that it was important to establish that adenovirus genetic information was indeed retained and expressed in transformed cells. Moreover, it could be argued that the two exceptional

136

ZUCKER

lines of transformed mouse cells discussed in the previous paragraph arose as the result of spontaneous transformation events which differed from the incomplete transformation induced by Ad2 DNA in the great majority of eases. A dot blot hybridization assay was therefore used to search for the presence of viral DNA in the two lines of transformed mouse cells MA251 and MA252, as well as in several lines of transformed REF established during the course of these experiments. The results obtained when the DNA released from 5 X lo4 cells of various types was hybridized to nick-translated Ad2 DNA and of reconstruction experiments are shown in Fig. 5. No hybridization was found to the DNA of untransformed cells such as REF or BMK, but all transformed cell lines examined did hybridize to Ad2 DNA, with intensities consistent with the presence of about 1 (for example, MA252) to more than 10 (RA113, for example) copies of the Ad2 genome per cell. The ability of the lines of transformed mouse cells to express adenoviral genetic information was examined by indirect immunofluorescence using the HT14B hamster tumor serum discussed previously. Cells of the MA251 and MA252 lines and MEF were grown on coverslips and either stained (Fig. 6, 2a-2c) or treated sequentially with HT14B serum and fluoresceinconjugated rabbit anti-hamster serum (Fig. 6, la-lc). By contrast to normal MEF, both MA251 and MA252 cells exhibited intense nuclear and less strong cytoplasmic fluorescence (Fig. 6). Thus, both cell lines must express the EIA and/or EIB transcriptional units, for their products are the only viral mRNA and protein species made in HT14B cells (Flint et al., 1976; Ross et ah, 1980). DISCUSSION

The results presented here indicate that mouse cells permit some replication of Ad2, a member of subgroup C (see Tooze, 1980), although they have been reported to be completely nonpermissive for the subgroup A Ad12 (Levinthal et aZ., 1966). A similar difference in the reproduction of subgroup

AND

FLINT

a

RAll RA52

RA12

RA13 RA22

RA21

RA23

RAlll

RA113

RA112

RA53

BMK

RA51

REF

MA252

MA251

293

FIG. 5. Presence of Ad2 DNA sequences in Ad2transformed rat and mouse cells. Dot blots of total DNA of AdZ-transformed REF (RA lines) and MEF (MA251 and MA252) were prepared and hybridized to nick-translated Ad2 DNA as described under Materials and Methods. A typical result is shown in panel b, and the location of the various DNA on the filter in panel a. Untransformed REF and BMK cells and cells of the 293 line of Ad5-transformed human cells (Graham et nL, 1975) were included as negative and positive controls, respectively. In the reconstruction experiment shown in panel c, hybridized in parallel with, and to the same probe as, the filter shown in panel b, the amounts of Ad2 DNA corresponding to the numbers of copies shown were mixed with 5 X lo4 uninfected MEF cells prior to transfer to the filter. Several spots were made for each quantity of Ad2 DNA to examine reproducibility and those of similar intensity in each panel used to make a rough estimate of the concentration of viral DNA in the transformed cell lines.

C and A human adenoviruses occurs in hamster cells (Doerfler, 1969; Strohl, 1969a, 1969b; Takahashi, 1972; Williams, 1973). However, hamster cells are far more permissive to Ad2 replication than mouse cells (at least those derived from C5’7B1/6J mice) yielding respectable bursts of infectious virus, 100 to up to 1000 PFU/cell (Takahashi, 1972; Williams, 1973), compared to the maximum yields of 0.4 to 4 PFU/cell we recovered from mouse embryonic fibroblasts or baby mouse kidney cells. In the one previous study of subgroup C adenovirus replication in mouse cells, which also

FIG. 6. Expression of viral early antigens in Adz-transformed mouse cell lines. Indirect immunofluorescence with HT14B serum was performed as described under Materials and Methods. Cultures of MEF (la, 2a), MA251 (lb, 2b), and MA252 (lc, 2c) cells stained with Wright’s stain (panel 1) or after preparation for indirect immunofluorescence (panel 2) are shown.

used cells derived from C57B1/6J mice, but a line established on a 3T3 schedule, Younghusband et al. (1979) reported a similar time course of infection and substantially reduced yields of virus compared to those from other types of rodent cells. However, the maximum yield observed in those studies was even lower, 0.1 PFU/cell (Younghusband et aZ., 1979). As similar multiplicities of infection and cells of the same mouse strain were used in our work, it is possible that this difference reflects different properties of primary and established mouse cells. The expression of viral structural antigens, the products of late transcriptional units whose expression depends upon replication of viral DNA (Flanagan and Ginsberg, 1962; Feldman and Rapp, 1966; Wigand and Schmeider, 1972; Thomas and Mathews, 1980), in 50% of infected mouse cells (Table 1) indicates that the major block to efficient production of infectious virus cannot be a failure to replicate viral DNA. Indeed, Younghusband et al. (1979) have demonstrated directly that mouse

cells support quite efficient replication of Ad5 DNA. It therefore appears that mouse cells can support most steps in the adenovirus productive cycle, synthesis of viral early proteins, replication of viral DNA, and synthesis of late proteins (Table 1; Younghusband et al., 1979) and in this respect behave much like rat embryo fibroblasts (Table 1; Gallimore, 1974). Nevertheless, infected mouse cells produce only extremely low levels of infectious virus (Table 1). Whether the failure to synthesize infectious virus is the result of limiting quantities of viral DNA, failure of infected mouse cells to produce sufficient quantities of late proteins or an inappropriate cellular milieu has not been determined, although the work of Younghusband et al. (1979) tends to argue against the first of these possibilities. Despite the fact that C57B1/6J mouse embryonic or baby kidney cells were considerably more restrictive to subgroup C adenovirus replication than rat embryo fibroblasts, they were found to be as susceptible to transformation, assayed as for-

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mation of foci of morphologically transformed cells (Table 2). While it proved extremely difficult to establish cell lines from such transformed mouse cell foci, it seems certain that their appearance is dependent upon the introduction of adenoviral genetic information: no foci of spontaneous transformants were found in many trials with carrier salmon sperm DNA or C57B1/6J mouse DNA (Table 2). We therefore conclude that the events of adenovirus transformation which lead to the appearance of foci of transformed cells with altered morphology and growth properties occur with similar frequencies in rat and mouse cells. The strikingly different behavior of cells obtained from foci of transformed mouse or rat cells during attempts to establish cell lines does, however, indicate that all transformation events cannot be identical in these two types of rodent cells. Indeed, our failure to establish lines of transformed mouse cells on a routine basis is a surprising result, not least because lines of adenovirus-transformed C3H baby mouse kidney cells appear to have been obtained readily by Sarnow et al. (1982). This failure cannot be attributed to the procedure we employed, for lines of adenovirustransformed rat cells were established with ease in parallel experiments (Table 2). Nor can the small number of lines of Ad2transformed mouse cells recovered reflect some intrinsic resistance to establishment of this type of mouse cell: several lines were established following introduction of SV40 DNA into either MEF or BMK cells (Table 2). It therefore appears that the vast majority of foci observed after transfection of Ad2 DNA sequences into these mouse cell types contain cells which are morphologically altered but not immortalized. Surprising as this observation might seem, it does have precedent in the adenovirus system: primary rat embryonic fibroblasts receiving only the EIA transcriptional unit develop foci of morphologically transformed cells, but such foci are difficult to clone and maintain indefinitely in culture (Graham et al., 1977; van der Eb et al., 1979; Houweling et ah, 1980). In this context, it is noteworthy that all but two foci cloned

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following introduction of Ad2 or Ad5 DNA, or transforming fragments, into the cells of the CCL63 line (derived from C57B1/6J) were established as cell lines and all such lines examined (21) contained viral DNA sequences (M.L.Z., unpublished observations). Foci derived from rat embryo fibroblasts which exhibit altered morphology and growth properties, but possess only a limited potential for proliferation in culture, have also been obtained following introduction of the mouse p53 gene with an acitvated E.J6 Hat-ras gene (Eliyahau et al., 1984). A number of mechanisms which might account for the failure of subgroup C adenovirus DNA to immortalize primary mouse cells can be envisioned. It is, for example, possible that mouse cells, at least those of C57B1/6J origin, do not permit efficient expression of EIB gene products, or synthesis of the form (for example, phosphorylated; Levinson and Levine, 1977; Lassam et al., 1979; Branton et al., 1981) found in human and rat cells: products of this transcriptional unit appear to be essential for complete transformation of rat cells (Graham et ah, 1978; Jones and Shenk, 1979a; van der Eb et al., 1979; Houweling et ab, 1980; Ho et ah, 1982). The ability of cloned Ad2 EIA DNA to induce focus formation in MEF cells at frequencies comparable to those obtained with DNA fragments containing both the EIA and EIB transcriptional units (Table 2) might support this interpretation. The lack of tumorigenicity of MA251 and MA252 cells would also be consistent with this notion, for EIB products have been shown to determine the tumorigenicity displayed by adenovirus-transformed cells in nude mice (Bernards et al., 1982, 1983; Shiroki et ab, 1982). Almost as plausible is the possibility that the EIA transcriptional unit is aberrantly expressed in C57B1/6J mouse cells, such that the 289R protein essential to productive growth (Berk et al., 1979; Jones and Shenk, 1979b; Ricciardi et al., 1981) is made normally, but the 243R protein is not. The latter is not essential for virus replication under most circumstances, but does play an important role in transformation of rat cells (Monte11 et aZ., 1984; Haley et

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aL, 1984; Winberg and Shenk, 1984; A. J. Berk, personal communication). Clearly, it will be necessary to examine in some detail viral gene expression in the two lines of transformed C57B1/6J mouse cells established during the course of this work and in partially transformed foci to begin to distinguish among such possibilites. REFERENCES ABRAHAMS, P. J., MULDER, C., VAN DE VOORDE, A., WARNAAR, S. O., and VAN DER EB, A. J. (1975). Transformation of primary rat kidney cells by fragments of simian virus 40 DNA. J. ViroL 16,818823. BERK, A. J., LEE, F., HARRISON, T., WILLIAMS, J. F., and SHARP, P. A. (1979). Pre-early adenovirus 5 gene products regulate synthesis of viral early messenger RNA. Cell 17,935-944. BERNARDS, R., HOUWELING, A., SCHRIER, P. J., Bos, J. L., and VAN DER EB, A. J. (1982). Characterization of cells transformed by Ad5/Ad12 hybrid early region 1 plasmids. Virology 120,422-432. BERNARDS, K., SCHRIER, P. I., Bos, L., and VAN DER EB, A. J. (1983). Roles of adenovirus types 5 and 12 early region 1B tumor antigens in oncogenic transformation. I/irolog~ 127,45-53. BRANDSMA, J., and MILLER, G. (1980). Nucleic acid spot hybridization: Rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA. Proc. NatL Acd Sci. USA 77,6851-6855. BRANTON, P. E., LASSAM, N. J., DOWNEY, J. F., YEE, S-P., GRAHAM, F. L., MAK, S., and BAYLEY, S. T. (1981). Protein kinase activity immunoprecipitated from adenovirus-infected cells by sera from tumorbearing animals. J. ViroL 37, 601-608. DOERFLER, W. (1969). Non-productive infection of baby hamster kidney cells with adenovirus type 12. Virology 38,587~606. ELIYAHAU, D., RAZ, A., GRUSS, P., GIVOL, D., and OREN, M. (1984). Participation of the p53 cellular tumor antigen in transformation of normal embryonic cells. Nature (kmdwn) 312,646~649. FELDMAN, L. A., and RAPP, F. (1966). Inhibition of adenovirus replication by 1-B-D-arabinofuranosylaytosine. Proe. Sot. Exp. BioL Med 122,243-247. FELDMAN, L. T., and NEVINS, J. R. (1983). Localization of the adenovirus EIA-a protein, a positive-acting transcriptional factor, in infected cells. MoL Cell, BioL 3,829-838. FLANAGAN, J. F., and GINSBERG, H. S. (1962). Synthesis of virus-specific polymers in adenovirus-infected cells: Effect of 5-fluordeoxyuridine. J. Em Med 116, 141-157. FLINT, S. J. (1984). Cellular transformation by adenoviruses. PharmacoL Thw. 26,59-88.

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