Transformation of rat cells by DNA of human adenovirus 5

VIROLOGY

64, 536-539 (1973)

Transformation

of Rat Cells F. L. GRAHAM

Laboratory

for Physiological

Chemistry,

by DNA

of Human

A. J. VAN

AND

State University The Netherlands

dccepted

DER

of Leiden,

Adenovirus

5

EB

Wassenaarseweg

62, Leiden,

May 19, 1973

Primary rat embryo and baby rat kidney cells have been transformed by human adenovirus 5 DNA. Transforming activity was resistant to heating (1 hr at 56”C), and to pronase, but was sensitive to DNase. The efficiency of transformation was approximately 1 transformed focus/rg DNA.

We have recently described a new technique (subsequently referred to as the calcium technique) for assaying infectivity of viral DNA (1). This technique proved much more sensitive and reproducible for the assay of infectious adenovirus DNA than the DEAE-dextran technique. Attempts to induce in vitro transformation with adenovirus DNA using the DEAE-dextran technique have not met with success (F.L. G. and A.J. v.d. E., unpublished; Nicholson and McAllister, personal communication) although simian adenovirus 7 (SA 7) DNA has been shown to be oncogenic in new born hamsters (2). In this report we describe the use of the calcium technique to transform rat embryo and rat kidney cells with human adenovirus type 5 (Ad 5) DNA. Although a member of the subgroup of human adenoviruses which are nononcogenic in new born hamsters (subgroup C), Ad 5 can morphologically transform rat cells in vitro (3,4). These cells provided a suitable test system for transforming capacity of Ad 5 DNA. Primary cultures of rat embryo or baby rat kidney cells were prepared by trypsin dispersion of s term Wistar rat embryos or of kidneys from 6-7 days old Wistar rats, respectively. Cells were cultured in 60 mm Falcon Petri dishes in Eagle’s minimum essential medium supplemented with 10% calf serum (MEM + 10% CS) in a humidified atmosphere containing 570 CO2 and were used at subconfluency, usually within 2-3 days from the 536 Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

time of seeding. Exposure of the cells to Ad 5 DNA was carried out following the procedure developed for assaying infectious DNA (I) : viral DNA [prepared as previously described (5)l was diluted in isotonic saline, (HeBS: 8.0 g/liter NaCl, 0.37 g/liter BCl, 0.125 g/liter NazHP04.2Hz0, 1.0 g/liter N-2-Hydroxyethyldextrose, 5.0 g/liter piperazine-N’-2-ethanesulfonic acid, final pH 7.05) and mixed with 2.5 M CaC12 to a final Ca2+ concentration of 125 mM. After 15-20 min at room temperature the mixture was inoculated onto cell monolayers, usually in aliquots of O.Tj ml. After 20 min at room temperature each dish received 5 ml of Eagle’s basal medium supplemented with 2y0 heated horse serum (BME + 2% HS). ’ If the initial DNA inoculum was less than 0.5 ml, the BME was supplemented with additional CaCh to bring the final Ca2+ concentration (comprising Ca2+ from the inoculum plus that from the medium) to approximately 15 mM. After 4-5 hr at 37°C the BME was removed and the dishes received 5 ml MEM + 10% CS. After 3-4 days the medium was changed to low calcium medium (6) (MEM for suspension cultures + 0.1 mM CaCh + 10% CS) and this was replaced every 3-4 days thereafter. Approximately 2-3 wk after exposure of rat embryo cells to Ad 5 DNA, foci of small, densely packed, round or cuboidal cells were observed over a background of normal fibroblasts. These foci, which were similar to those observed in cultures treated with intact

RAT

CELL

TRANSFORMATION

Ad 5 virus, increased in size and number during continued incubation. No such foci were seen in untreated controls or in cultures exposed to DNA without CaC% . For most, experiments, baby rat kidney cells were used because these cells remained more contact-inhibited than embryo cells and gradually disappeared during prolonged incubation in low calcium medium, leaving the foci of transformed cells clearly visible. In rat kidney cultures, foci could often be observed within as little as lo-12 days after exposure to DNA and after 3 wk many colonies were as large as 2-3 mm. As in the case of rat embryo cells, these foci contained small1 round, densely packed cells charact’eristic of adenovirus transformation (E’ig. 1). Cells derived from a rat embryo culture transformed by Ad 5 DNA have been tested for the presence of tumor antigen. Approximately 2 X 10’ cells were SUSpended in 2 ml of PBS and freeze-thawed three times. This extract was used as antigen and tested against 24 units of antibody. The serum, which was kindly provided by Dr. Raymond Gilden (Flow Laboratories, Rockville, llaryland), was obtained from

FI( ::. 1. Part, of a transformed I)NA + CxCl:! 22 days previously. stain.

537

adenovirus %-SV40 hybrid tumor-bearing hamsters and contains antibodies which are reactive with T antigens of the C-group of adenoviruses. By employing this serum in the CF test an antigen titer of y$z was found in an extract of the transformed cells. The complementation test was kindly carried out by Dr. J. van der Noordaa (Laboratorium voor Gezondheidsleer, University of Amsterdam). From morphological and immunological evidence it is thus clear that the transformation which we have observed in DNA treated cultures is adenovirus specific. That this transforming activity is due to DNA is indicated by t’he data presented in Table 1. Pronase treatment and heating to 56°C had little effect, but’ the activit’y was complctcly abolished by DNase. In addition, no transformed foci were observed when cells were exposed to DNA without CaClz . From these results we can certainly exclude the possibility that the t’ransforming activity was due to virus which had survived the DNA purification procedure. The possibility that transformation might require some protein or proteins in addition to DNA cannot be

colony resulting from exposure of primary rat kidney cells to Ad 5 Three normal cells can hc seen to the right of the photograph. (;irnlsa

538

GRAHAM

AND

VAN

TABLE EFFECT OF VARIOUS TREATMENTS Treatment

EB

1

ON TRANSFORMING

ACTIVITY

OF An 5 DNAa

Number of transformed

of DNAb

20°C 56°C 37°C + 50 rg/ml pronase@ 37°C + 6 mM MgClt 37°C + 6 mM MgClt + 20 pg/ml DNase No CaClz added

DER

foci per culture

Expt lc

Expt 2c

2, 2, 1, 1 1, 2, 1, 1

1, 3, 6, 2

Expt 9 7, 5, 0, 5,

1, 0, 1, 1

1, 0, 1, 1 ND

ND, ND

0, 0, 0, 0 0, 0, 0, 0

0, 0, 0, 0 0, 0, 0, 0

0, 2, 3, 3,

0, 0, 1, 4,

4 1 5

0, 0, 0, 0 ND

a Primary rat kidney cells were exposed to Ad 5 DNA + 125 mM CaClz for 20 min at room temperature followed by BME + 2y0 HS for 4-5 hr at 37°C. The cells were then incubated in fresh liquid medium (first MEM + 10% CS for 34 days then low calcium medium, with twice weekly changes) until they were stained with Giemsa and transformed foci counted at 16-22 days. b DNA was incubated under appropriate conditions for 1 hr before addition of CaCl?. c Each dish received 10 rg/ml Ad 5 DNA, 0.5 ml/dish. d Each dish received 12 pg/ml DNA, 0.2 ml/dish, followed by BME + 2yo KS supplemented with 10 mM extra CaC12. e After addition of CaC12, 10% horse serum was added to the DNA to inhibit the pronase. f Not done.

ruled out so rigorously, especially in view of the fact that as little as one active Ad 5 DNA molecule in 3 X 10”’ could account for the observed efliciency of transformation. However, there appears to be little evidence to suggest such a requirement and the most likely hypothesis at present is that pure Ad 5 DNA has the capacity to transform cells in vitro. As illustrated in Fig. 2 the dose response for production of foci was similar to that obtained for infectivity. The number of foci appeared to increase nonlinearly with DNA concentration to a maximum at 10 pg/ml, then decreased. This type of response has been repeatedly observed for both infectivity and transforming activity with a maximum occurring at lo-12 pg/ml. The efficiency of transformation is remarkably high in terms of foci per infective unit. In the experiment illustrated in Fig. 2, focus formation occurred with a frequency only three- to fivefold lower than that of plaque formation. This is in marked contrast to the situation with intact virus where the ratio of infectivity to transforming activity is 106107 (4). When calculated in terms of the amount of input DNA, however, transformation might be considered rather inefficient:

PLaques

Foci

,

5

0 Ad5

DNA

--.--1

10 concentration

15 Lug/ml)

FIG. 2. Dose response for infectivity (A) and transforming activity (0) of Ad 5 DNA. For the assay of infectivity, KB cell monolayers were exposed to various concentrations of DNA, 0.3 ml/dish. Each point represents the mean of two dishes. For transformation, rat kidney cells were exposed to 0.5 ml DNA/dish, 3 dishes/point, and stained with Giemsa after 22 days.

approximately one transformant per 3 X lOlo Ad 5 genome equivalents. The apparently nonlinear relationship between numbers of foci and the DNA concentration should not be taken as evidence that trans-

RAT

CELL

.-,:<9

TRANSFORMATION

formation occurs as the result of the action of more than one molecule of viral DNA. The same nonlinear relationship arises for infectivity and has been shown to be consistent with the hypothesis that one DNA molecule is sufficient for infection (1) and thus presumably also for transformation. A higher transforming activity for DNA compared to int,act virus (expressed as the ratio of focus forming units to plaque forming units) has also been observed for polyoma (7) and SV40 DNA (8). One explanat’ion which has been suggested (7) is that pure viral DNA might be damaged in some way, either during extraction or during the process of uptake into recipient cells. Since infectivity appears to be more sensitive to damage to the viral DNA than is transforming activity (9, lo), a relative increase in transforming activity might bc expected. In agreement with this hypothesis, we have found that Ad 5 DNA, which is linear and relatively large, and should therefore bc much more sensitive to damage due to shearing or nucleases than the DNA of polyoma or SV40, shows a much great’er increase in the ratio of transforming activity to infectivity than that observed for polyoma or SV40 DNA. Furthermore, we have found t’hat by mechanically shearing Ad 5 DNA to fragments with a sedimentation coefficient of approximately 22 S: infectivity could be greatly reduced while transforming activity appeared not to be significantly aff ectcd (unpublished observations). Thus our observations are consistent with the hypothesis that the relatively high ratio of transforming activit,y to infectivity for viral DNA is the result of transformation by defective molecules which have lost infectivity. However, in contradiction to this hypothesis it has been found that cells transformed by SV40 DNA contained nondefective SV40 genomes, since infectious virus could be rescued from each of many DNA transformed lines tested (11). Thus in t’hat case, transformation was evidently not induced by defect,ivc DNA molecules and t’he explanation for the relatively high

transforming &iciency of DN;\ must lie elsewhere, at least for SV40. The results described in t’his communication indicate t,hat the combination of the calcium technique for infecting cells with viral DNA and the rat kidncy cell transformation assay affords a particularly suitable system for the study of transformation by adcnovirus DNA. Foci can usually be observed within 2-R wk and the eticiency of transformation, though not high, appears to be reasonably reproducible. Current research is concerned with the fragment#ation and fractionation of Ad 5 DNL4 by various means and the assay of DNA fract,ions for transforming activity. ACKNOWLEI)GMENTS We carrying Dr. R. serum. Grant

wish to t.hank I)r. J. van der Noordaa for out the complement fixation test and V. Gilden for providing the anti T-antigen This work was supported by Eru-atom No. 102-72-l BIAN. REFERENCES

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