VIROLOGY 68, 41-57 (1975)

Analysis

of Transformed

Cl 3 isolated

Cell Variants

as Survivors

of BHK21

of Adenovirus

Type 5 Infections A. MEAGER,’ National

Institute

for Medical

R. NAIRN,

AND

Research, Mill

R. C. HUGHES

Hill, London NW7 IAA, England

Accepted June 2, 1975 Transformed cell variants of the baby hamster kidney cell line, BHK21 C13, selected as survivors after sequential lytic infections with adenovirus type 5 (Ad5) and carrying Ad5-linked tumour-specific antigens, have been examined for several properties that are usually assumed to be characteristic of virally transformed cells. These include the ability of clones to grow in 0.55 fo eta1 bovine serum (FBS) and in agar suspension, sialic acid content and agglutinability induced by concanavalin A. Lactoperoxidase-catalysed cell surface labeling has also been used to compare the cell variants with normal BHK21 Cl3 cells and a clone of BHK21 Cl3 cells transformed by polyoma virus. Considerable variation of cellular properties was observed among the several variant clones examined. Clones isolated from survivors of Ad5 infection grown in medium containing 2.1 mM calcium were large polyploid cells exhibiting density-dependent inhibition of growth, yet able to grow in 0.5’? FBS and agar suspension. However, clones of small round cells. morphologically resembling adenovirus transformants of hamster cells. isolated from survivors of Ad5 infection, grown in medium containing 0.3 mM calcium lacked density-dependent inhibition of growth but were unable to grow in 0.5% FBS and agar suspension. All but one of the clones tested exhibited increased agglutinability with concanavalin A. Marked variation was observed in the expression of an iodinatable, large external glycoprotein (LETS glycoprotein) at the cell surface of the variant cells. It was concluded that the properties we have examined can vary independently among different clones of variant cells. None of these properties exactly parallels the differences observed in growth characteristics of the isolated variant cells. INTRODUCTION

understood, however. One approach to this problem has been the deliberate selection of clones from virally transformed cells that display a more normal phenotype, the so-called flat revertants (Pollack et al., 1968; Culp and Black, 1972; Ozanne, 1973). This can be done by selective killing of cells that grow under conditions in which normal nontransformed cells do not proliferate, for example, at high cell densities, or in the presence of certain lectins such as concanavalin A that selectively either agglutinate or somehow kill cells showing the “classical” transformed phenotype. In this paper we describe a selection system in which several “resistant” variant

Transformation of many cultured fibroblastic cell lines, such as baby hamster kidney cells (BHK21 C13) (Stoker and MacPherson, 1964), with certain viruses, for example, polyoma virus or simian virus 40 (SV40), results in marked alterations in the morphology and growth characteristics of the cells (Burger, 1971). These changes are usually considered to be closely associated with the presence of viral genes incorporated into cellular DNA. The contribution made by the viral genes to maintenance of the transformed state is not well ’ Beit Memorial

Fellow. 41

Copyright@ 1975by Academic Press. Inc. All rights of reproduction in any form reserved.

42

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cell clones of BHK21 Cl3 have been obtained after sequential lytic infections with Ad5. The morphology and growth properties of certain of the variant cell lines resemble a phenotype typical of BHK21 Cl3 transformed by oncogenic adenoviruses, specifically adenovirus type 12 (Ad12). Interestingly, several other of the stable variant cell lines appear to exhibit a more normal morphology and growth pattern, although these cells are also polyploid with regard to chromosome numbers. All of these cell lines have been tentatively classified as harbouring adenovirus genes since tumour-specific antigens associated with Ad5 transformation of secondary hamster embryo cells (Williams, 1973) are detected by immunofluorescence techniques. We describe the segregation among the Ad5-transformed variant BHK21 Cl3 cell lines of several cellular parameters that are assumed in other systems to reflect the state of viral transformation compared to the normal phenotype. MATERIALS

AND

METHODS

Cell cultures. Cell lines and clones were grown in Glasgow-modified minimal essential medium (GMEM) (Flow Laboratories Ltd.) or BHK-Eagle’s medium, supplemented with 10% (v/v) foetal bovine serum (FBS), 10% (v/v) tryptose phosphate broth (TPB), 0.2%’ (w/v) sodium bicarbonate, penicillin (0.1 megaunit/litre) and streptomycin (0.1 g/litre). Polyomaand SV40-transformed BHK21 Cl3 cell lines were kindly donated by Dr. I. MacPherson (Imperial Cancer Research Fund Laboratories, Lincoln’s Inn Fields, London). Cells were transferred every 3-4 days at an inoculation density of about 1 x lo3 cells/ cm2 in Falcon plastic bottles. All cell lines were routinely tested for mycoplasma by the method of Fogh and Fogh (1964) and were negative throughout the course of this study. Growth of cells at the colonial level for determination of plating efficiencies was effected in Ham’s FlO medium (Flow Laboratories Ltd.) supplemented with 10% (v/v) FBS, 10% TPB, 0.2% (w/v) sodium bicarbonate, penicillin (0.1 megaunit/litre) and streptomycin (0.1 g/litre). This me-

AND HUGHES

dium supported colonial growth better than GMEM. No morphological changes were observed microscopically when cells were transferred from GMEM to Ham’s FlO. Viruses and infectivity estimations. Wild-type Ad5, a gift from Dr. W. C. Russell, was propagated in KB cells. Infectivities of Ad5 preparations were measured by plaque formation in HeLa cells (Williams, 1970) at 32”. Ad5 was purified by the method of Russell et al. (1967) with the exception that preformed glycerol-caesium chloride gradients [40% (v/v) glycerol buffered with O.OlMTris-HCl, pH 7.4, was mixed with 42% (w/w) caesium chloride, p = 1.42 g/cm3, to form gradients with increasing glycerol concentration from bottom to top] were used in the final centrifugation steps (Barzilai et al., 1972). Selection of Ad5 cells. Sublines of BHK21 C13, Al (Fig. 1A) and Fl, were cloned in this laboratory and grown routinely in medium containing 2.1 and 0.3 mA4 calcium, respectively. These sublines, which maintained the pseudodiploid chromosome number of 44, were mutagenised at 32” in monolayer cultures (2-5 x lo5 cells per 60-mm plastic petri dish) using 0.5 pg of methyl-N-nitro-N-nitrosoguanidine (MNNG)/ml of medium. The surviving cells, representing about 28% of the initial population, were grown at 32” for 4 days to establish mutant phenotypes (Chu and Malling, 1968; Orkin and Littlefield, 1971), and then transferred to 38.5”. After a further 48 hr, the cells were infected with Ad5 (60 plaque-forming units [PFU]/ml) in medium containing no FBS for 2.5 hr. Virus fluids were then removed and the cells were incubated in growth medium for 60 hr. Most of the cells rounded up and detached completely from the monolayer and were removed. Surviving cells, obtained at a frequency of about 1Om6,were grown out in fresh medium to large cell numbers, and the selection procedure, without further mutagenesis, was repeated twice more with Ad5 at 120 and 1000 PFU/cell. After the three successive lytic infections, colonies of “resistant” cells, one or two colonies per dish, were obtained. One resistant colony derived from the ~ Al

AD5-TRANSFORMED

subline was taken for further study. Another resistant colony was derived from the -Fl subline. The cells from these colonies were grown up to high cell densities to establish cell lines (-Al/3 and -F1/3 in Figs. 2A and B, respectively). Subclones were then picked from these and any subsequently derived isolates as described below. Cloning was achieved by seeding low numbers of cells (100-500) into 60-mm culture dishes containing complete medium. When macroscopic colonies had formed, well-separated colonies were placed under glass clylinders (4-mm internal diameter), the cells removed by brief trypsination and separately transferred to new culture vessels containing growth medium. Chromosom.e analysis. Cells for chromosome analysis were incubated with colchitine (0.04 pg/ml, final concentration) for 2 hr at 38.5”, removed by trypsinisation, swollen in hypotonic sodium citrate-potassium chloride solution, and fixed with Carnoy’s solution [67% (v/v) ethanol -33% (v/v) glacial acetic acid]. These were then spread on damp glass slides and air dried. The dried cells were stained with Giemsa in phosphate buffer, pH 6.8. Viral antigens. Assays for the presence of viral antigens were done by indirect immunofluorescence. For tests concerning Tantigen expression, cells were grown on glass coverslips and fixed in acetone. Cells were reacted with the primary antiserum, hamster T-antiserum (hamster Tantiserum, YAH23, was a generous gift from Dr. W. C. Russell) for 1 hr at room temperature or 37”, rinsed, and then stained with the secondary antiserum, fluorescein-conjugated rabbit anti-hamster IgG(Progressive Laboratories Inc.) for 30 min at room temperature. Hamster Tantiserum, (YAH23) and fluorescein-conjugated rabbit anti-hamster IgG were used in PBS at dilutions of l/sand l/3 respectively. After rinsing, the coverslips were mounted in 90% (v/v) glycerol-10% (v/v) phosphate-buffered saline (PBS) and examined at 4000x magnification under dark field ultraviolet illumination (Zeiss). Hexon production in infected cells was detected

BHK

CELLS

43

by immunofluorescence with rabbit or guinea pig anti-Ad5 hexon (gifts of Dr. W. C. Russell and Dr. V. Mautner) and the appropriate fluorescein-conjugated yglobulins in the manner described for T antigen. Growth in medium containing agar. Efficiencies of plating of cell lines in soft agar were determined by colony formation in 0.33% (v/v) agar-Ham’s FlO medium containing 10% (v/v) FBS at 38.5”, according to Strohl et al. (1970). Agglutination assays. Cultured cells were removed from bottles by incubation in 0.02% (w/v) disodium EDTA in Ca*+and Mg*+-free PBS at room temperature. The suspended cells were washed three or four times with Ca*+/Mg’+-free PBS and diluted to give approximately 2-4 x lo6 cells/ml. Agglutination assays were carried out as described by Inbar and Sachs (1969). The highest concanavalin A concentration was 500 pg/ml. The agglutination of cells was estimated after 30-min incubation at room temperature by direct microscopic examination. Agglutination was scored as ++++, +++, +++ and 0 when 90, 60, 30, 10 and 0% of the total number of cells were present as cell aggregates. The agglutination endpoint was deemed + and is expressed as the reciprocal of the dilution of concanavalin A. Lactoperoxidase-catalysed iodination. This was performed on confluent cell monolayers growing on 50- or 60-mm Falcon plastic tissue culture vessels. Cells were washed four times with complete PBS and labeled in the same buffer containing 5 mM glucose, 20 pg/ml of lactoperoxidase (Sigma Chemical Corp.), 0.2 U/ml of glucose oxidase (Sigma Chemical Corp., type V) and 250 PCi of carrier-free sodium [‘251]iodide (Radiochemical Centre, Amersham). Incubation was for 15 min at room temperature. The reaction was stopped by addition of phosphate-buffered iodide and the monolayers were washed four or five times with the same buffer. The cells were scraped off the plates into complete PBS and washed several times by centrifugation. For analysis the cell pellets were dissolved by heating to 90” for 5 min in electrophoresis spacer buffer (Tris -glycine,

44

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pH 6.7) containing 1% sodium dodecyl sulphate (SDS) and 1% 2-mercaptoethanol. Polyac~lamide-gel electrophoresis. This was performed in buffers containing SDS in vertical slabs as described by Maize1 (1971). The gel slabs were stained with Coomassie brilliant blue R250, dried down and the radioactivity detected by autoradiography on Kodak X-ray film (Kodak Ltd., London). Certain gels were sliced into 2-mm slices and counted directly in a Wallac Autogamma counter. The approximate molecular weights of polypeptide subunits were determined by reference to the mobilities of adenovirus type 5 structural proteins of well-established molecular weight (Russell and Skehel, 1972). Sialic acid assays. Confluent monolayer cultures were washed three times with complete PBS and twice with Ca”+- and Mg2+-free PBS. The cells were harvested from the surface of the growth vessels by scraping with a rubber policeman into 0.02%, (w/v) disodium EDTA in Ca**Mg*+-free PBS and washed extensively by centrifugation in complete PBS. The washed cell pellets were suspended in complete PBS (5.0 ml) and aliquots (0.5 ml) removed for cell counts. The remaining solutions (4.5 ml) were centrifuged again and the cell pellets suspended in 0.05 M H,SO, (0.5 ml). After heating at 80” for 1 hr to release sialic acid, the suspensions were cooled and stored at -70” before analysis. Aliquots of 0.1 ml and 0.25 ml, respectively, were taken for determinations of protein by the Lowry assay and of sialic acid by the method of Amino‘ff (1961) using half the volumes of all reagents. Absorbanties were measured at 549 and 532 nm, and the differences between these values were used for sialic acid estimations in order to eliminate nonspecific chromogens absorbing at 532 nm, as recommended by Warren (1959). RESULTS

Isolation of Ad5 Cells and Preliminag, Characterisation The cell variant selection method used initially was not specifically designed for the isolation of Ad5 transformants. The

AND HLIGHES

primary aim was to use Ad5 infection to select for cells resistant to killing by the virus. In this way we had hoped to isolate variants blocked in virus adsorption on a temperature-sensitive basis and defective in the cell surface receptor for Ad5. This aim has not been realised, and the cell lines described in this paper do not fall into this category. The cell line designated BHK21 Cl3 Al/3 (Fig. 2A) was composed of morphologically heterogeneous cells (Fig. lB), some of which were multinucleated. Serial passage of BHK21 Cl3 A1/3, which in the early period after isolation was extremely difficult since many cells died and those remaining grew very slowly, eventually gave rise (Fig. 2A) to a faster-growing, more stable heteroploid cell line (Fig. 1C). These cells, BHK21 Cl3 A1/3x (Fig. 2A), were retested at this point for resistance to Ad5 killing by infecting with virus at 200 PFU/cell. It was found that cells had lost most, but not all, of their apparent initial resistance giving survivors at the rate of 3 x 10m3. One such stable clone of survivors, BHK21 Cl3 A1/3xP (Fig. 2A), has a more epithelioid morphology (Fig. 3B) and chromosome numbers in the subtriploid range [medium number (MN) of chromosomes = 631 (Fig. 4). BHK21 Cl3 A1/3xP cells have a frequent occurrence of spontaneously endoreduplicating chromosomes, ca. 6Q’ (Fig. 4). It was possible to clone a large cell having endoreduplicated chromosomes and establish a second line (Fig. 2A). BHK21 Cl3 A1/3xD (Fig. 3C). Growth of this line at the colonial level in Ham’s FlO medium revealed a surprising degree of morphological heterogeneity, and further sublines were established such as BHK21 Cl3 Al/3xDl and BHK21 Cl3 A1/3xD2 (Fig. 2A) and BHK21 Cl3 A1/3xD6 (Fig. 3D). All cell lines exhibited limited density-dependent inhibition of growth and parallel orientation at mass culture levels. Generation times, cell densities at confluence and absolute efficiencies of plating of all derived cell lines are detailed in Table 1. The chromosome numbers of BHK21 Cl3 A1/3xD, -A1/3xD2 and -A1/3xD6 were unstable, reducing from subsexaploid in early passage to subtetraploid at high

ADS-TRANSFORMED

passage (Fig. 4). BHK21 Cl3 A1/3xD finally after 70 passages at 38.5” returned to the “stable” subtriploid chromosome set of the cell line -A1/3xP from which it originated. Cell size was reduced as chromosomes were shed. All variant cell lines exhibited gross aneuploidy in their chromosome sets. Since several reports (Freeman et al., 1965, 1966, 1967a, b) indicate that cells resulting from adenovirus-induced tumours or adenovirus transformation in. vitro are sensitive to calcium, attempts to select variant cells using lytic virus infections were made (Fig. 2B) in medium containing a reduced level of calcium (0.3 mM). A colony designated BHK21 Cl3 F1/3 (Fig. 2B) grew out to give predominantly fast-growing fibroblasts that tended to outgrow other morphologically distinct cells (Fig. 5A). However, the small round cells (Fig. 5A) were isolated by shaking them off the monolayer into the medium since they adhered less well to the plastic dishes than the fibroblasts, and then cloning the floating cells in medium with reduced calcium. The small round cells, e.g., BHK21 Cl3 F1/3A4 (Figs. 2B and 5B) grew out to high densities (Table 1) to give the typical piled-up appearance of transformed cells lacking density-dependent inhibition of growth. However, it was observed that all small round cell lines always gave rise on cultivation to a small number of flat fibroblastic and epithelioid cells. Some of these resembled morphologically BHK21 Cl3 A1/3xP and its derivatives. The spontaneous reversion rate of the small round cells to this form was 10~2-10 m3 Preliminary chromosome analysis of the small round cells has indicated that the median number of chromosomes is 44 or 45. Chromosome analysis of the “revertant” that can be obtained from the small round cells and that exhibits a marked increase in density-dependent inhibition of growth has not yet been carried out. Characteristics of Variant Ad5 Cells Serum requirements and growth in agar suspension. Compared to wild-type cells, all of the flat polyploid-type cell lines (Fig. 2A) showed less serum dependence but

BHK

CELLS

45

greater efficiency of plating in agar suspension cultures and were similar in these respects to a polyoma-transformed BHK21 Cl3 cell line (Table 1) (see also Burk, 1966; MacPherson, 1970). The small round cells, -F1/3Al-4 (Fig. 2B), while showing perhaps a lessened serum dependence, plated at much lower efficiencies in either liquid medium or in agar suspension (Table 1) and were sharply differentiated from the polyoma-transformed cell line as well as the other variant cell lines shown in Fig. 2A. Although a surprising finding, rather similar results were reported by Strohl et al. (1970) for AdlS-transformed BHK21 Cl3 cells which morphologically resemble our -F1/3Al-4 cell clones. Synthesis of viral products. None of the variant cell lines produce detectable infectious Ad5 when extracts prepared from 10’ cells were assayed by plaque formation as described in Materials and Methods. Further indirect immunofluorescence tests with specific antisera were performed to detect the production of viral structural proteins in the variant cell lines. These tests were uniformly negative using antiAd5-hexon sera. However, variant cell lines could be infected with Ad5 and subsequently produced virus (Table 1). Direct tests for antigens putatively linked to Ad5 transformation were next carried out. Synthesis of a tumour-specific antigen (T antigen) is characteristic of adenovirus-transformed cells (Schlesinger, 1969). We obtained from Dr. W. C. Russell of this Institute, an antiserum, YAH23, that had been produced by inoculation of Ad5-transformed secondary hamster embryo fibroblasts (HT5 H14a, originally obtained from Dr. J. F. Williams, MRC Virology Unit, Glasgow, Scotland) into a randomly bred, adult male Syrian hamster from which serum was collected when a tumour had appeared. The production of T antigen in our putatively Ad5-transformed BHK21 Cl3 cells was examined by indirect immunofluorescence using this specific antiserum. All3xP and its derivatives as well as --F1/3Al-4 showed specific fluorescence when tested with the antiserum. In the majority of cases, this fluorescence was nuclear or located on the nuclear membrane and took the form of nuclear blobs or

46

MEAGER,

FIG. 1. Phase contrast photomicrographs BHK21

Cl3 Al/&

(stable);

NAIHN

AND HUGHES

of (A), BHK21

Cl3 Al:

(B), BHK21

Cl3 Al/3

(unstable);

(Cl.

x 180.

rods. Sometimes blobs of fluorescence were observed in the cytoplasm as well as the nucleus. This type of fluorescence is identical to that observed when the original HT5 H14a cells, kindly donated by Dr. W. C. Russell, were tested with the same T-

antiserum. Williams et al. (1974) have ialso reported virus-specific antigenic mate rial to be located in the cytoplasm and nucl eus of two established Ad5-transformed rat cell lines. A similar, but weaker, positive react Zion

ADS-TRANSFORMED

BHK

CELLS

FIG. 1 (continued). BHKZI Cl3 Al Ad5 Ad5 after each inlectlcn some “normal” Ad5 fibroblastic survivors were observed i BHKEI Cl3 Al13 (unstable) BHKZI Cl3 Ali3x (stable) Ad5 -f BHKZI iI3

I other clones not tested

AII3xP

BHKZI Cl3 AII3xD BHKZI ;I3 A113xDI Figure 28

BHKZI Cl3 All3xD6

other cionks not tested

BHKEI Cl3 FI Ad5 Ad5 Ad5

BHKLI & Fl/3AI-4 (small round cells1

/

BHKPI 613 All3xD2

after each infection some “normal” flbroblastic survivors were observed

3 BHKLI $13 Fl13 (unstable) I bizarre, heteroploid cells

/ fast growing fibroblasts

“flat reiertants”

FIG. 2. Schematic outline of the selection procedure used to isolate variant cells when (A), BHK21 Cl3 Al cells were infected with Ad5 in medium containing 2.1 mM calcium; (B), BHK21 Cl3 Fl cells were infected krith Ad5 in medium containing 0.3 mM calcium.

with our cell variants as well as with the Ad5-transformed cell line, HT5 H14a, was obtained using pooled sera derived from adult male Syrian hamsters carrying tumours induced by an inoculation ( lo6 cells)

of late-passage-A1/3xD. Induction of tumours. As mentioned above, subcutaneous inoculations of lo6 late-passage-A1/3xD cells into adult male Syrian hamsters were found to cause tu-

48

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AND HllGHES

AD5-TRANSFORMED

BHK

49

CELLS BHK21 Cl3 Al

BHKZl Cl3 A1/3xP

flHK+ 5

Cl3 Al/3.D passage

BHKZI Cl3 A1/3.D 28’” passage s,po

FIG. 4. Histograms of chromosomes per cell against number of cells. For each BHK21 Cl3 line, 100 metaphase preparations were counted. The diploid number (2N) of the hamster is 44. The median number of chromosomes for each line is indicated M. Similar reductions in M for BHK21 Cl3 A1/3xD2 and BHK21 Cl3 A1/3xD6 were obtained.

moms at the site of injection (four out of four animals challenged), usually within 21 days. The wild-type cell lines used in this study produced no tumours at this very high dosage (in four animals). Dispersal of the excised tumours with trypsin and growth of the tumour cells in medium containing 10% (v/v) FBS yielded a cell line, -A1/3xDT, with a subtriploid chromosome set, ability to grow in medium containing 0.5% (v/v) FBS and elevated concanavalin A agglutinability (Table 2). Morphologically, -A1/3xDT was similar to but with the parental line, -A1/3xD, larger numbers of rounded cells. Two sub-A1/3xDl and clones of -mA1/3xD, - A1/3xD6, and the small round cell clone, -F1/3A4, caused tumours (one to four out of four animals) at comparable dosages. Sialic acid contents. Several studies have shown that the total sialic acid content of some cell lines transformed by oncogenic DNA viruses decreases after

transformation (Ohta et al., 1968; Burger, 1971; Culp and Black, 1972). Subsequently, Culp and Black (1972) have shown that concanavalin A-selected revert ants of SV40-transformed mouse 3T3 fibroblast cell lines contain a high content of sialic acid comparable to the nontransformed parent cells. Our results are given in Table 2 showing no significant differences in sialic acid contents between the wild-type -Al and the cell lines in the flat polyploid class (range, 2.6-3.4 pg of sialic acid/mg of protein). However, the small round cell lines, -F1/3Al-4, do have decreased sialic acid content (Table 2; range, 1.6-2.1 ~g of sialic acid/mg of protein). Nevertheless, the validity of this parameter to indicate the transformed or revertant state is clearly questionable, at least for BHK21 Cl3 cells. Thus no significant difference was seen between the sialic acid contents of normal BHK21 Cl3 Al cells and SV40-transformed BHK21 Cl3 cells.

50

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AND HI.(:HES

‘I’AHI,E cHAK.\rTE:KI~I I(‘5 Ok Cl’ll.l-I‘\ Cell line”

Morpho lorrical de&ription”

Mean generation ” time” (hr) 8- 10 8

BHKZI C 13 Al BHKPl Cl3 Polyoma transformed

F F

BHK21 BHK”1 BHKZl BHK21

Cl3 Cl3 Cl:1 Cl3

SR SR SR SK

28 35 N.D.” 20-24

BHK21 BHK21 BHK21 BHK’l BHK21

Cl:IAl/&P Cl3 A1/3xD Cl3 Al/:&D1 Cl3 Al/:&D2 Cl3 A1/3xD6

F + E F+E F F E

11 16 16 16 16

BHK21 Cl3 Al/:lxD’I’

F +E

12

F1/3Al Fl/JA:! Fl/:lA:~ Fl/:jAl

Density of confluent monolayer’ (cells/cm’) 1.3

1 I’F: \I)

Efficiency El’l’iciency Growth in Reaction of olatina 01 ulatinr medium to hamster in agar’ containing Tantiin’liquid 0, ,5’7 serum medium