Identification of adenovirus-type 12 gene products involved in transformation and oncogenesis

VIROLOGY

122,15-28 (1982)

Identification of Adenovirus-type 12 Gene Products Involved in Transformation and Oncogenesis H. JOCHEMSEN, G. S. G. DANIELS, J. J. L. HERTOGHS, P. I. SCHRIER, P. J. VAN DEN ELSEN, AND A. J. VAN DER EB’ for Medical Biochemistry, S&G.s Laboratories, Wassaaarseweg 72, 2533 AL Leiden, The Netherlunds

Laboratory

Received September 24, 1981;accepted May 10, 1982 Primary baby rat kidney cells were transfected with Ad12 DNA fragments EcoRI C (left-terminal 16%) and HirudIII G (left-terminal 7.2%), and the resulting transformed cells were established as cell lines. Injection of newborn hamsters with purified Ad12 DNA resulted in the induction of tumors in 2 out of 59 animals. A number of the in vitro transformed cells and a cell line derived from a tumor were found to contain DNA sequences homologous to the fragments used for transfection or tumor induction, and to express virus-specific RNA and T antigens. The cell lines were also studied with respect to their tumorigenicity in nude mice or hamsters. It was found that cells transformed by Ad12 EcoRI C, or by intact DNA or virus, were tumorigenic whereas cells transformed by fragment Hind111 G were unable to induce tumors. To correlate this result with the presence or absence of viral gene products, virus-specific T antigens were identified by immunoprecipitation. From lytically infected cells major proteins of 60,41, 19,14.5, and 13.5 kD were precipitated. Cells transformed by fragment EcoRI C or intact viral DNA contained proteins of 60,41, and 19 kD and of 50 and 36 kD. Hind111 G-transformed cells contained proteins of 41 and 19 kD. Studies of the T antigens in two-dimensional gels and by tryptic peptide analysis have indicated that two virus-specific 60-kD proteins are expressed in AdlZ-infected cells. The major protein probably represents the singlestranded DNA-binding protein encoded by region EII (EII-60 kD), while the minor protein represents a region EI-specific T antigen (EI-60 kD) encoded by region EIb. The 41-kD protein is encoded by region EIa and 19 kD by EIb. Our results suggest that expression of the EI-6OkD protein is required for oncogenicity in nude mice, but not for morphological transformation.

kidney cells transformed by different parts of region EI exhibited different transformed phenotypes, suggesting that the various proteins encoded by the transforming region have discrete functions in this process. Essentially similar results were obtained with weakly oncogenic adenovirus types 3 and 7 (Sekikawa et cd, 1978; Dijkema et cd., 1979). Preliminary data of Graham et al. (1974) had indicated that the transforming activity of the oncogenic adenovirus type 12 is also localized in a left-terminal fragment of its DNA (EcoRI C, the left-most 16%). This was confirmed subsequently by other investigators, using the established rat cell line 3Y1, who also showed that the left-most 7.2 and 4.5% of Ad12 contains

INTRODUCTION

The transforming activity of the human nononcogenic adenovirus types 2 and 5 (Ad2 and Ad5) has been shown to be located in early region 1 (EI), which stretches from about 1.5 to 11.5% of the viral genome (Graham et al, 1974; Sambrook et aL, 1974; Van der Eb et aL, 1979). Transfection studies with restriction endonuclease fragments have demonstrated that it is possible to transform cells not only with DNA fragments comprising the entire region EI, but also with parts of this region. Further studies showed that primary rat ‘To whom dressed.

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0042-6622/82/130015-14$02.00/O Copyright All rights

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16

JOCHEMSEN

transforming activity for these cells (Yano et aL, 1977; Shiroki et aL, 197’7,1979). Established cell lines may have the possible disadvantage for viral transformation studies, however, that the cells already have undergone a transformation step which may obscure certain virus-induced effects. In order to obtain more. information on the role of the Ad12 EI proteins in transformation, we have transformed primary cultures of baby rat kidney cells with the Ad12 DNA fragments EcoRI C and Hind111 G (left-most 7.2%). It will be shown that these two types of transformed cells differ in certain properties, most interestingly in their oncogenic potential, and that this property is correlated with the presence of the 60-kD major T antigen. MATERIALS

AND METHODS

Cells, virus, and viral DNA. Primary cultures of baby rat kidney (BRK) cells were prepared from kidneys of 6- to 7-day-old WAG RIJ or BN-BI rats and were grown in Eagle’s minimum essential medium (MEM) + 8% newborn calf serum (NCS). All transformed cell lines and tumor cell lines were propagated in the same medium. Human adenovirus type 12 (Ad12, strain Huie) was propagated in monolayer cultures of KB or human embryonic kidney (HEK) cells. The purification of the virus and the isolation of viral DNA was performed according to Van der Eb et al. (1969). Preparation of restriction endow&ease fragments of Ad12 DNA. Ad12 DNA was digested with EcoRI (Boehringer) or with Hind111 and the DNA fragments were separated by electrophoresis in 1 or 1.4% agarose slab gels. The fragments were recovered from gel slices as described by Mulder et al. (1974). Assay of transfomning activity. The transformation assays with viral DNA or DNA fragments were carried out with primary cultures of baby rat kidney cells, using the calcium technique as described by Graham and Van der Eb (1973), followed by treatment with a 25% DMSO solution to enhance transformation effi-

ET AL.

ciency (Stowe and Wilkie, 1976; Arrand, 1978). Analysis of renaturation kinetics. To identify the viral DNA sequences present in the transformed cells, the kinetics of renaturation of denatured 32P-labeled viral DNA fragments were analyzed in the presence of unlabeled DNA from transformed or untransformed cells (Gallimore et al, 1974; Van der Eb and Houweling, 1977). Analysis of viral RNA in transformed cells. Poly(A)-containing RNA obtained by oligo(dT)-cellulose selection was electrophoresed in agarose gels containing methylmercury hydroxide according to Bailey and Davidson (1976). After electrophoresis the RNAs were transferred to nitrocellulose paper as described by Thomas (1980) and viral RNA was identified by hybridization with q-labeled Ad12 fragment EcoRI C according to Wahl et al. (1979). Test for tumorigenicity. Cells (1.5 X 10’) in PBS were injected subcutaneously into nude mice (6-12 weeks old, homozygous BALB/c.nu males), or lo6 cells into 2- to 7-day-old hamsters. Cell labeling. KB cells, grown in monolayer culture, were infected with 20-50 PFU of AdlB/cell and were further cultured in MEM + 2% horse serum for lo12 hr after infection (hpi). The cells were washed with PBS and were further incubated in 5 ml of MEM without methionine, 2% NCS and 100-200 &i [35S]methionine (New England Nuclear) per g-cm dish for 2 hr. Transformed and tumor cells were grown to 70-90% confluency and were labeled for 2-8 hr as described above. Extracts of the cells were prepared as described by Schrier et al. (1979). Antisera Ad12 anti-T sera were obtained from hamsters bearing tumors induced by Ad12 virions or by AdlZ-transformed cells, or from tumor-bearing rats. Tumors were induced by inoculating 2- to 7-day-old animals subcutaneously with 2 X 10’ PFU of purified virus in 0.1 ml PBS or with lo6 cells in 0.1 ml PBS. The sera from hamsters carrying virus-induced tumors were kindly provided by Dr. J. D. Verlinde. Immuwprecipitation and electrophoresti. Immunoprecipitation reactions were

IDENTIFICATION

17

OF Ad12 GENE PRODUCTS TABLE 1

TRANSFORMINGACTNITY OF A~12 DNA AND DNA FRAGMENTS

Ad12 DNA fragment Intact DNA Exp. 1 Exp. 2b EcoRI C Exp. 1 Exp. 26 EcoRI (A + B + D + E) Hind III G Exp. 1 Exp. 2b Hind111 (A + E + H) Hind111 (B + C + D) Hind111 (F + I)

Number of dishes

Average number foci/pg genome equivalent

8

9

2

2

0.2 0.75

pg DNA/5 cm dish (genome equivalent)

8

0.1

10 8

0.5 0

10 12 12 12 12

0.05

0.1 0 0 0

DSubconfluent cultures of primary rat kidney cells were infected with Ad12 DNA or specific DNA fragments using the calcium technique. Dense foci of morphologically transformed cells were counted 4-7 weeks after transfection. * Cells treated with DMSO after transfection. ’ Mixture of purified fragments.

carried out as described by Schrier et al. baby rat kidney (BRK) cells. The results (1979). Analysis of T antigens in two-di- are summarized in Table 1. It can be seen mensional gels [isoelectric focusing fol- that colonies of transformed cells were lowed by polyacrylamide gel electropho- obtained with intact DNA, and with the left-terminal fragments EcoRI C and resis] was carried out according to O’Farrell Hind111 G. The transforming activity was (1977). very low as compared to the transforming Trypticfingerprinting. Two-dimensional tryptic peptide analysis was performed as efficiency obtained with Ad5 DNA (3-5 foci/pg DNA; Van der Eb et al, 1977) while described by Bos et al. (1981). the activity of fragment Hind111 G was One-dimensional analysis of the tryptic peptides on a chromobeads-P column was even lower. To investigate whether Ad12 DNA is performed according to Simmons et al. (1977). The pyridine concentration ranged able to induce tumors directly, newborn from 0.1 to 2.0 M. Fractions of 2 ml were hamsters were injected with intact DNA or DNA fragments. Tumors were found in collected and the pH and the radioactivity only 2 out of 59 hamsters; one injected in each fraction was determined. with intact DNA and a second with fragment EcoRI C. This EcoRI C preparation RESULTS appeared to be contaminated with small Transfomzation of Prima7-y BRK CelLsand amounts of other EcoRI fragments, exTumor Induction in Hamsters with plaining why the resulting tumor conAd12 DNA and DNA Jkgments tained other Ad12 DNA sequences, in adAd12 DNA was cleaved with EcoRI or dition to fragment EcoRI C (see below). HindIII. The cleavage maps of Ad12 DNA with these restriction endonucleases have Viral DNA and RNA Sequences in Transbeen presented elsewhere (Jochemsen et formed Cells aL, 1980). The transforming activity of the To investigate whether the cells transresulting fragments and of the uncleaved DNA was tested in primary cultures of formed by specific fragments of Ad12 DNA

18

JOCHEMSEN ET AL. TABLE 2 A~12 DNA SEQUENCESPRESENTIN A NUMBEROFTRANSFORMEDCELL LINES Concentration of DNA fragments” (copies/diploid quantity of cell DNA)

“P-labeled DNA fragment

Map coordinates (%)

EcoRI C D B E F A Hind111 G I F (A + E + H)

12 RI C-RKW d (cl)

12 Hind111 G-RKwe (cl)

0.9 0 0 0.8 0 0

-f -

13.1 11.4 9.7

-f

-

0.9 0 0

1.3

-

0

12 DNA HTb

12 RI-HT”

O-16 16-28 28-55 55-62 62-64 64-100

0.9 0.8 0.9 0

12.0 1.8 0.8 2.1 0

o-7 7-11 11-18 66-78 and 91-100

0.9 0.7 0.5 0.4

-f

-

-f

-

“The concentration of Ad12 DNA fragments present in the transformed cells was calculated as described by Gallimore et al. (1974). *A cell line derived from a hamster tumor induced by Ad12 DNA. ‘A cell line derived from a hamster tumor induced by partially purified Ad12 EcoRI C fragment. d A cell line obtained by transforming primary baby rat kidney (BRK) cells with Ad12 EcoRI C fragment. w denotes that cells are derived from WAG-RIJ rats. eA cell line obtained by transforming BRK cells with Ad12 Hind111G fragment.

‘Not determined.

contained the viral DNA sequences that were used in the transfection experiments, DNA isolated from representative cell lines was subjected to C,,t analysis. The results are shown in Table 2. The tumor induced in a hamster by Ad12 DNA (12 DNA-HT) contained about 1 copy of EcoRI C per diploid amount of cell DNA and 0.40.9 copies of several other fragments. The hamster tumor induced by EcoRI C (12RI HT) contained about 12 copies of fragment C and l-2 copies of other parts of the genome. The presence of DNA sequences other than fragment C can be explained by a contamination of EcoRI C with several other fragments (see previous paragraph). The EcoRI C- and Hind111 Gtransformed BRK cells contained 1 copy of the transforming fragment, while the

transformed cell lines were tested for the presence of virus-specific RNA by Northern blot analysis: the hamster tumor line 12RI-HT3 (derived from 12RI-HT), and a rat line transformed by fragment Hind111 G. Figure 1 shows that the 12RI-HT3 line contained the expected region EI-specific RNAs of approximately 1.3 kb (EIa) and 2.4 kb (EIb), as well as a 3.3 kb species (a read-through product of EIa into EIb; Sawada et aL, 1980; Bos et aL, 1981). The Hind111 G-transformed line contained only RNA of about 1.3 kb (EIa and probably a truncated EIb RNA). The lytically infected cells in addition contained several other RNAs, including transcripts for protein IX (0.9 kb) and most likely protein IVa2 (1.85 kb).

former

Properties of the Transformed Cells

cell

line

in addition

contained

EcoRI E. To investigate whether the transforming DNA segments were expressed, two

The cell morphology and the growth pattern of DNA fragment-transformed

IDENTIFICATION

cells vary somewhat from one line to the other. EcoRI C-transformed cells generally show an epithelial or irregular morphology and a disorganized growth pattern while Hind111 G-transformed cells are epitheloid or polygonal but generally grown in a more regular pattern (data not shown). In order to investigate the subcellular distribution of virus-specific T antigen, cultures of transformed cells were fixed and stained according to the indirect immunofluorescence technique. EcoRI Ctransformed cells were found to contain fluorescent granules and irregular spots predominantly in the nucleus or perinuclear area (see Fig. 2a), whereas Hind111 G-transformed cells showed a (much) weaker fluorescence usually in the form of small fluorescent granules in the nucleus (Fig. 2b). Both categories of fragmenttransformed cells, including the transplantable tumor 12RI-HT3, have a low plating efficiency (O.Ol-0.3%) in media containing 0.33% agarose (results not shown). Tumorigenicity

19

OF Ad12 GENE PRODUCTS

of the Transfomned Cells

A number of the AdlB-transformed cell lines were tested for their ability to induce tumors in nude mice or hamsters. The results are presented in Table 3. The Ad12 EcoRI C-induced hamster tumor cells were tumorigenic in hamsters and nude mice (entries 1, 2). The cell line 12RI-HT3, derived from a primary tumor transplant of the 12RI-HT tumor, had retained its tumorigenicity in both types of animals (entries 3,4). Rat cells transformed by intact DNA or by the EcoRI C fragment were tumorigenic in 100% of the nude mice tested (entries 5-9). The latency period of the tumors varied considerably: from 6 days up to more than 80 days. In contrast, cells transformed by the Ad12 Hind111 G fragment were not tumorigenic, even after two injections of 1.5 X lo7 cells (entries 10-15). This result suggests that the DNA region mapping between 7 and 11% encodes functions that play a role in oncogenicity. Chromosome analysis of a tumor induced in a nude mouse by 12RI C-RK

-4.

.84

kb

.n kb

.s8 kb

123456 FIG. 1. Detection of virus-specific RNA in extracts of KB cells lytically infected with Ad12 (16 and 40 hpi; lanes 1 and 2, respectively), of a hamster tumor line 12RI-HTa (lane 3) and of cells transformed by the Hind111 G fragment (clone 1) (lane 4, poly(A)+ RNA, lane 5, total RNA). Cytoplasmic RNA was fractionated by electrophoresis in 1.2% agarose slab gels containing 5 mM methylmercury hydroxide, and was transferred to nitrocellulose paper. Viral RNA was identified by hybridization with “P-labeled Ad12 DNA fragment EcoRI C. Lane 6: Ad12 DNA size markers of 0.98, 1.72, and 4.34 kb.

(clone 2) cells showed that the tumor cells indeed were of rat cell origin (B. Bakker and P. Pearson, personal communication). Identijcation of Virus-Specific Proteins in Adl2-In$ected KB Cells and in Adl2Transfn-med Cells In order to identify AdlZ-specific T antigens in lytically infected cells ?S-labeled extracts of AdlZ-infected KB cells were incubated with sera from Ad12 tumorbearing hamsters or tumor-bearing rats (anti-T sera). Figure 3 shows an analysis of proteins precipitated from cells harvested 10 hpi. It can be seen that all sera

20

JOCHEMSEN

ET AL.

FIG. 2. T antigen staining patterns of the cell line derived from a hamster tumor induced by Ad12 DNA fragment EcoRI C (a) and of a rat cell line transformed by Ad12 DNA fragment Hind111 G (b).

(

2

3

.

5

8

7

8

9

10

FIG. 3. SDS-gel electrophoresis of proteins immunoprecipitated from &dl2-infected KB cells harvested 10 hpi, with serum from an Ad12 tumor-bearing rat (lane 2) or with ser$ from individual Ad12 tumor-bearing hamsters (lane 3-9). Lane 10: Control precipitation with nonimmune hamster serum (n.i.). Lane 1: Proteins synthesized with poly(A)+ RNA selected by hybridization to Ad12 DNA (see Jochemsen et al, 1930).

precipitated a 13.5-kD protein and that most sera precipitated a 60-kD protein and proteins of 45-50 kD. Only one serum (the rat serum, No. 5) clearly precipitated proteins of 41 and 38 kD (lane 2). A 14.5-kD protein is found with some of the sera only. The proteins synthesized in vitro with early RNA selected by Ad12 DNA are shown for comparison (lane 1; Jochemsen et cd, 1980). Figure 4 shows that the rat anti-T serum (No. 5) precipitated similar proteins from a tumor cell line (12RI-HT3) except that the 13.5- and 14.5-kD proteins were absent and proteins of 19 and 19.5 kD were present. Only a few of the hamster sera precipitated the 19-kD protein. One serum (No. 27) did not appear to contain antibodies against Adl2-specific T antigens. A comparison of Figs. 3 and 4 shows that some of the anti-T sera did not precipitate a 60-kD protein from lytically infected cells, although the same sera did precipitate a 60-kD protein from the tumor cells. To investigate the possibility that two different 60-kD proteins exist, two-dimensional gels were prepared with proteins immunoprecipitated from lytitally infected and transformed cells (Figs.

IDENTIFICATION

21

OF Ad12 GENE PRODUCTS TABLE 3

TUMORIGENICITYOFA~12 DNA FRAGMENT-TRANSFORMED CELLS Transformed cell line”

Origin of cell

Recipient animal*

Number of animals with tumor/ Number of animals injected

Latency period (days)

1. 2. 3. 4.

Hamster Hamster Hamster Hamster

Hamster Nude mouse Hamster Nude mouse

5/6 2/2 M/22 2/2

30-60 80 14-28 8

Rat Rat Rat Rat Rat

Nude Nude Nude Nude Nude

mouse mouse mouse mouse mouse

2/2 2/2 l/2 3/3 6/6

80, 84 6, 9 12 35-50 12-17

Rat Rat Rat Rat Rat Rat

Nude Nude Nude Nude Nude Nude

moused moused moused moused mouse mouse

O/6 o/2 o/2 o/2 o/2 o/2

12RI-HT 12RI-HT 12RI-HT3 12RI-HT3

5. 12DNA-RKBN (cl) 6. 12RI C-RKW (cl) ‘7. 12RI C-RKW (clAc1) 8. 12RI C-RKBN (cl) 9. 12RI C-RKBN (~2) 10. 11. 12. 13. 14. 15.

12HinIII 12HinIII 12HinIII 12HinIII 12HinIII 12HinIII

G-RKW (cl) G-RKW (~2) G-RKW (c3) F-RKW (~4) G-RKW (~6) G-RKBN (cl)

-

a 12RI-HT is a cell line derived from a hamster tumor induced by Ad12 EcoRI C, 12RI-HT3 is derived from a primary tumor-transplant of 12RI-HT (entries l-4). 12DNA-RK and 12RI C-RK denote baby rat kidney cell lines transformed by Ad12 DNA and Ad12 fragment EcoRI C, respectively. 12HinIII G-RK indicates cell lines transformed by Ad12 fragment Hind111 G. Superscripts W and BN indicate inbred rat strains WAG RIJ and Brown Norway, respectively. Cl denotes clone number 1, etc. bHamsters were injected subcutaneously 2-6 days after birth with lo6 cells. Nude mice were injected subcutaneously with 1.5 X 10’ cells. ’ clAc1 is subclone 1 derived from a colony of clone 1 growing in soft agar medium. d Five weeks after the first injection, a second injection of 1.5 X lo7 cells was given.

5a and b, respectively). The 60-kD protein from lytically infected cells has an isoelectric point of 7.5 (Fig. 5, spot l), while the 60-kD protein from the EcoRI C fragment-transformed line runs at pH 4.3 (Fig. 5b, spot 1). The latter protein is only very weakly present in infected cells (Fig. 5a, spot 6). We conclude that Ad12 encodes two different 60-kD proteins: an acidic one encoded by early region I and a neutral protein probably encoded by early region II, and representing the Ad12 DNA binding protein (see Discussion and Jochemsen et al, 1980). The 13.5- and 14.5-kD proteins in lytically infected cells probably represent region EIV-encoded proteins (Jochemsen et aL, 1980). An analysis of the T-antigens present in BRK cells transformed in vitro with fragment EcoRI C (O-16%) or with fragment Hind111 G (O-7.2%) is shown in Fig.

6. As expected the EcoRI C-transformed clones contained essentially the same T antigen species as the 12RI-HT3 line (lanes 2 and 4). In one line (12 RI C RKBN, clone 1; Fig. 6, lane 2) the large T-antigen is smaller than usual, about 56 kD rather than 60 kD. BRK cells transformed by fragment Hind111 G lacked the 60-kD protein, but both lines tested do contain the 41-kD protein (Fig. 6, lanes ‘7 and 9). The 19-kD protein was found in one. cell line (lane 9). It is not known whether the other line does not express this protein or whether its concentration is below the detection level. Peptide Anal&s of Ad12 T Antigens and Proteins Synthesized in Vitro To investigate the relationship between a number of the immunoprecipitated T-

22

JOCHEMSEN

ET AL.

serwn ni

5

97

0

E

F

G

N

1

3

n

92

sl

90

km

H-f4

14

13

FIG. 4. SDS-gel electrophoresis of proteins immunoprecipitated from the 12 RI-HT3 cell line (derived from an Ad12 EcoRI C-induced hamster tumor) with sera from individual Ad12 tumorbearing hamsters (lanes 3-l@, serum from an Ad12 tumor-bearing rat (lane 2), or nonimmune hamster serum (lane 1).

b FIG. 5. Autoradiographs of Ad12 T antigens in two-dimensional gels. (a) Proteins immunoprecipitated with rat anti-T serum from AdlB-infected KB cells (12 hpi). (b) Proteins immunoprecipitated with hamster anti-T serum from an Ad12 EcoRI C-transformed BRK cell line.

IDENTIFICATION

OF Ad12 GENE PRODUCTS

23

FIG. 6. SDS-gel electrophoresis of proteins immunoprecipitated from two different BRK cell lines transformed by Ad12 fragment EcoRI C (lanes 2-5) and from 2 BRK cell lines transformed by Ad12 fragment Hind111 G (lanes 7-10). Precipitations were carried out with serum from an Ad12 tumorbearing rat (lanes 2,4,7, and 9) or with nonimmune rat serum (lanes 3,5, 8, and 10). Lanes 1 and 6 show proteins precipitated from Adl2-infected KB cells (12 hpi).

tides of these proteins were compared by antigens tryptic digests of the proteins two-dimensional mapping on cellulose thin were prepared and analyzed. Figure layer plates (Fig. 8). The mixing experi7 shows ion-exchange chromatography of [SS]methionine-containing tryptic pep- ment (Fig. 8b) shows that the two methionine-containing peptides A and B of the tides of a number of proteins synthesized either in viva or in vitro. The 41-kD protein 19-kD protein (Fig. 8~) are distinct from immunoprecipitated from lytically in- the methionine-containing peptides of the fected cells and the 41-kD protein synthe60-kD protein (Fig. 8a). The 36- and 50-kD sized in vitro with mRNA selected against proteins precipitated by hamster anti-T Ad12 DNA are basically similar (Fig. 7a). sera from RI-HT3 cells (see Fig. 4) have A protein of 33 kD, which represents one tryptic peptides in common with EI- 60kD of a series of proteins that usually appear and therefore might be degradation prodbetween 23 and 41 kD and are encoded by ucts of this 60-kD T-antigen (results not early region Ia (Jochemsen et al, 1980), shown). appears to be related to the 41-kD protein (Fig. 7~). Figure 7b shows that the 13.5-kD DISCUSSION protein immunoprecipitated from lytically infected cells is identical to the 13.5-kD The results presented in this paper show protein synthesized in vitro. This protein that the capacity of adenovirus 12 to seems to be unrelated to the 41-kD protein transform primary baby rat kidney cells and is probably encoded by early region in vitro resides in the left-terminal part IV (Jochemsen et CL&1980). A comparison of the viral DNA. This result is in agreeof one-dimensional peptide maps of the 60- ment with earlier findings showing that and the 19&D T antigens which map in left-terminal fragments of Ad12 DNA early region Ib (Jochemsen et d, 1980) was were able to transform the rat cell line not conclusive. Therefore, the tryptic pep- 3Yl into oncogenic cells (Shiroki et cd,

24

JOCHEMSEN ET AL.

a Ad12

L1k

","O

-

"llrn

-*-

FIG. ‘7. Elution profiles of [?Sjmethionine-containing tryptic peptides fractionated on a chromobeads-P column. The peptides were eluted by a pH gradient as indicated in the figure. (a) 41kD protein synthesized in vitro with region EI-specific RNA from AdlB-infected KB cells, 16 hpi. (X - - - X), and 41&D protein immunoprecipitated from AdlBinfected KB cells 12 hpi (0 0). (b) 13.5-kD protein synthesized in vitro with AdlB-specific RNA from infected KB cells (16 hpi) (x---X) and 13.5kD protein immunoprecipitated from AdlP-infected KB cells (12 hpi) (0 0). (c) 33-kD protein synthesized in vitro with region EI-specific RNA from Adl2-infected KB cells (16 hpi).

IDENTIFICATION

OF Ad12 GENE PRODUCTS

25

C Ad 12 33k wtm

1979), and with results obtained with Ad5, Ad3, and Ad7 DNA fragments (Graham et aL, 1974; Van der Eb et aL, 19’77;Sekikawa et uL, 1978; Dijkema et aL, 1979). In this study we have used primary cultures of baby rat kidneys rather than a cell line because the latter cells already have undergone a “transformation” event (the immortalization step) which may obscure the effects of the viral-transforming genes. Analysis of some of the transformed lines has shown that the cells contained region EI-specific DNA sequences and that this DNA is transcribed into poly(A)+ RNA. The immunoprecipitation studies showed that from lytically infected cells major proteins of 60, 41, 19, 14.5, and 13.5 kD were precipitated. A considerable heterogeneity was observed in the specificity of individual sera. The 41-kD proteins was precipitated only with rat anti-T sera and the 19&D protein was precipitated only with a small proportion of anti-T sera tested. From AdlZ-transformed cells major T antigens of 60, 41, and 19 kD were precipitated. In Hind111 G-transformed cells a 41-kD protein could be clearly detected, while the 19-kD protein was found

in low concentrations only. When the combined results presented in this paper are considered in relation to the mapping data on early proteins (Jochemsen et aL, 1980) the following conclusions can be drawn. In AdlZ-infected KB cells a major 60-kD virus-specific protein is synthesized which maps in region EII. It probably represents the single-stranded DNA-binding protein. Apparently, this protein is expressed in many tumor cells since a large proportion of the anti-T sera precipitate this protein from lytically infected cells (see Fig. 3). Expression of other early regions than EI in AdlZ-transformed cells has been reported previously (Schirm and Doerfler, 1981), which also explains the presence of antibodies reacting with the EIV-specific 13.5- and 14.5-kD proteins. In addition to the EII-specific protein, a second 60-kD protein exists which is synthesized in low concentration early in lytic infection (Fig. 5a, spot 6) but is present in higher amounts in transformed cells (Fig. 5b, spot 1). This protein is probably encoded by region EIb since it is present in cells transformed by the EcoRI C fragment but absent in cells transformed by Hind111 G. The 41-kD pro-

26

JOCHEMSEN ET AL.

60k

a

60k+19k

FIG. 8. Two-dimensional analysis of [YSlmethionine-containing tryptic peptides of proteins precipitated from 12RI-HT3 cells with a hamster anti-T serum: (a) 60-kD protein; (b) a mixture of 60- and 19-kD proteins; (c) 19-kD protein.

tein, expressed in AdlZ-infected KB cells and in all transformed and tumor cells maps in region EIa. At least some of the proteins with molecular weights between 23,000 and 39,000 are related to the 41-kD protein as was shown for the in vitro synthesized 33-kD protein (Fig. 7~). A 19-kD protein was identified in infected and transformed cells. This protein presumably maps between nucleotides 1541 and 2030 of the Ad12 genome (Bos et cd, 1981).

An analogous protein is also found at a comparable position in region EIb of Ad5 (Schrier et cd, 1979; Bos et cd, 1981). Our result, showing that the Ad12 19-kD protein does not share tryptic peptides with the 60-kD protein (Fig. 8), is in agreement with the nucleotide sequence data which indicate that the two proteins are encoded by two different, but partially overlapping, reading frames. A comparison of the proteins identified

b

IDENTIFICATION

OF Ad12 GENE PRODUCTS

in this study in AdlB-infected and transformed cells with the proteins precipitated by Chinnadurai et al. (1977) and Wold et al. (1979) from AdlB-infected KB cells suggest that our proteins of 60,33-41,19,14.5, and 13.5 kD correspond to the proteins of these authors of 60-65,40-46,16.5,12, and 10.5 kD, respectively. The suggestion by Wold et al. (1979) that the 13.5-kD protein (their 10.5 kD) would be encoded by region EI and that the 41-kD (their 46 kD) protein would overlap with the 60-kD (their 60-65 kD) protein is not consistent with our results. An interesting result is that all Hind111 G-transformed cell lines tested were not tumorigenic in nude mice, whereas cell lines containing the entire early region I or more of the Ad12 genome were all found to induce tumors in these animals. Since the Hind111 G-transformed cells lack the 60-kD protein of region EI, which is present in all other types of AdlZ-transformed cells, this result suggests that the 60-kD protein is involved in oncogenicity but is not required for the conversion of a primary cell to a morphologically transformed cell line. An alternative possibility is that the lack of oncogenicity is due to the low concentration of the 19-kD protein. ACKNOWLEDGMENTS The authors gratefully acknowledge the expert technical assistance of Mrs. J. J. L. Hertoghs and thank Dr. J. Maat and Mrs. H. S. C. Lupker-Wille for providing endo R * HindIII. This work was supported by grants from the Netherlands Organization for the Advancement of Pure Research (ZWO) through the Foundation of Fundamental Medical Research (FUNGO). REFERENCES ARRAND, J. E. (1978). Mapping of adenovirus type 5 temperature-sensitive mutations by marker rescue in enhanced double DNA infection. J. Gen Viral 41, 573-586. BAILEY, J. M., and DAVIDSON,N. (1976). Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biodwm 70, 75-85. Bos, J. L., POLDER, L. J., BERNARD& R., SCHRIER, P. I., VAN DEN ELSEN, P. J., VAN DER EB, A. J., and VAN ORMONDT,H. (1981). The 2.2 kb mRNA of the Elb region of human adenovirus types 12 and 5 directs the synthesis of two major tumor antigens from different AUG triolets. CeU 27. 121-131.

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O’FARRELL,P. H. (1977). High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1142. SAMBROOK,J., BOTCHAN,F. M., GALLIMORE, P. H., OZANNE,B., PETTERSSON,U., WIUIAMS, J. F., and SHARP,P. A. (1974). Viral DNA sequences in cells transformed by Simian virus 40, adenovirus type 2 and adenovirus type 5. Cold Spri?zg Harbor Symp. C&ant. Biol. 39, 615-632. SAWADA, Y., and FUJINAGA, K. (1980). Mapping of adenovirus 12 mRNAs transcribed from the transforming region. J. Viral 36, 639-651. SCHIRM,S., and DOERFLER,W. (1981). Expression of viral DNA in adenovirus type 12-transformed ceils, in tumor cells and in revertants. J. Vid 39, 694702. SCHRIER,P. I., VAN DEN ELSEN, P. J., HERTOGHS, J. J. L., and VAN DER EB, A. J. (1979). Characterization of tumor antigens in cells transformed b-r

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