Adenovirus Type 12 DNA Firmly Associates with ... - Journal of Virology

JOURNAL OF VIROLOGY, Oct. 1997, p. 7923–7932 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 71, No. 10

Adenovirus Type 12 DNA Firmly Associates with Mammalian Chromosomes Early after Virus Infection or after DNA Transfer by the Addition of DNA to the Cell Culture Medium ¨ RG SCHRO ¨ ER, IRMGARD HO ¨ LKER, JO

AND

WALTER DOERFLER*

Institute of Genetics, University of Cologne, Cologne, Germany Received 17 June 1996/Accepted 23 May 1997

Human adenovirus type 12 (Ad12) infects human cells productively and leads to viral replication, whereas infection of hamster cells remains abortive, with total blocks in viral DNA replication and late viral gene transcription. The intranuclear fate of Ad12 DNA in productively infected human cells and in abortively infected hamster cells was monitored by using the fluorescent in situ hybridization (FISH) technique. Human HeLa cells, primary human umbilical cord fibroblasts, hamster BHK21 cells, primary embryonal hamster cells, and the Ad12-transformed T637 hamster cell line were studied. As early as 2 h after infection, extensive association of Ad12 DNA with metaphase chromosomes was demonstrated by FISH in all of these cells. Chromosomal association continued until late (24 to 28 h) after infection, when about 100% of the human cell nuclei and 70 to 80% of the hamster cell nuclei showed distinct FISH signals. This chromosomal association of Ad12 DNA in infected cells seemed to be rather firm, since it proved to be resistant to mechanically stretching the chromosomes and to different types of chemical treatment. Moreover, laser scan microscopy of mechanically stretched chromosomes from Ad12-infected HeLa cells and from the Ad12-transformed T637 cell line, with about 20 copies of Ad12 DNA provably integrated, revealed identical FISH patterns. Therefore, it was likely that even in infected cells the chromosomal association of Ad12 DNA was very similar to the integrated state. Late in productively infected cells, large nuclear areas were taken over by viral DNA replication, as visualized by FISH in interphase nuclei. Chromosomal association at many sites was frequently limited to one chromatid, but signals in adjacent positions on both chromatids were also seen. Upon the long-term cultivation and passage of abortively infected BHK21 cells for 96 h after infection, a gradual decrease of viral DNA association with chromosomes was observed. Integration of Ad12 DNA in hamster cells early after infection was previously documented, and recombination between viral and cellular DNAs in human cells was also shown. The FISH data on extensive chromosomal association of Ad12 DNA suggest a means to study the pathway of Ad12 DNA from early steps in viral infection via chromosomal interactions to integration events. In a different approach, Ad12 DNA, Ad12 DNA with the terminal protein covalently linked to its ends (Ad12 DNA-TP), or Ad2 DNA was simply added to the culture medium of HeLa or BHK21 cells. Precipitation or selection procedures were avoided. Depending on the experimental conditions, up to 25 to 30% of the interphase nuclei of HeLa cells and 9 to 19% of the interphase nuclei of BHK21 cells showed positive FISH signals at 24 h after the addition of DNA. Viral DNA also became associated in some cases with both chromatids. The uptake of Ad12 DNA-TP appeared to be 10 to 20 times more efficient than that of Ad12 DNA completely freed of proteins. Control bacteriophage lambda, M13, or plasmid DNA could not be detected in the nuclei under these conditions.

Integration is not sequence specific, although in a few tumor cell lines, Ad12 DNA integration seems to be stable at selective chromosomal sites (20). The mechanism of Ad12 DNA integrative recombination has been partly reconstructed in a cellfree system by using highly purified proteins from hamster nuclear extracts (9, 14, 29). In productively infected human cells, integration of Ad12 DNA is difficult to document, since the cells do not survive infection. Recombinants between Ad12 DNA and cellular DNA do, however, occur (2, 25). When adenovirus DNA is added to the medium of cells growing in culture, the DNA reaches the nucleus and becomes linked to cellular DNA (11). Uptake of adenovirus DNA is markedly enhanced when the viral DNA has been previously complexed with protamine (32). We have now investigated these challenging problems by applying the cytogenetic FISH technique, which is both more sensitive than previously available methods and more powerful for the intranuclear localization of foreign DNA at the singlecell level. We demonstrate that Ad12 DNA can become exten-

Viral oncogenesis by human adenovirus type 12 (Ad12) has been demonstrated in newborn hamsters (30). The adenovirus system has been used to study uptake and integration of foreign DNA into the chromosomal DNA of mammalian cells in culture (for recent reviews, see references 5 and 9). In abortively Ad12-infected hamster BHK21 cells, Ad12 DNA integration can be found early after infection (3). Upon continued cell replication, this integration seems to be unstable in the majority of the cells (8). Association of Ad12 DNA with hamster chromosomes has previously been shown by in situ hybridization techniques less sensitive than the now available fluorescent in situ hybridization (FISH) method (34, 35). Chromosomal integration patterns of viral DNA in clonal lines of Ad12-transformed hamster cells and in Ad12-induced hamster tumor cells have been investigated in detail (6, 16, 21, 26, 28).

* Corresponding author. Mailing address: Institute of Genetics, University of Cologne, Weyertal 121, D-50931 Ko ¨ln, Germany. Phone: 49-221-470-2386. Fax: 49-221-470-5163. 7923

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TABLE 1. Summary of data on association of Ad12 DNA with metaphase chromosomes of human or hamster cellsa Ad12 DNA associationb with metaphase chromosomes at the following time and MOI (PFU/cell): Cells

HeLa HUCF BHK21 T637 EHC a b

2 hpi

6 hpi

16 hpi

24 hpi

0.1

1

10

100

1,000

0.1

1

10

100

1,000

0.1

1

10

100

1,000

0.1

1

10

100

1,000

2 2 2 2 2

2 2 2 2 2

2 2 2 2 2

1 1 2 2 2

1 1 1 1 1

2 2 2 2 2

2 2 2 2 2

2 2 2 2 2

1 1 1 1 1

1 1 1 1 1

2 2 2 2 2

2 2 2 2 2

1 1 2 2 2

1 1 1 1 1

1 1 1 1 1

2 2 2 2 2

2 2 2 2 2

1 1 1 1 1

1 1 1 1 1

1 1 1 1 1

Cells were infected with Ad12 at the indicated multiplicities of infection (MOI). Cell types are described in Materials and Methods. 1, Ad12 DNA associated with metaphase chromosomes as determined by FISH; 2, no association.

sively and firmly associated with chromosomes in human and hamster cells early after infection and after the addition of foreign DNA to the cell culture medium. Ad12 DNA with the terminal protein still covalently attached (Ad12 DNA-TP) (23) is more efficiently taken up by mammalian cells.

carries the luciferase gene under control of the simian virus 40 early promoter (33). Laser scan microscopy. A Zeiss LSM4 microscope and Zeiss software were used for laser scan microscopy.

MATERIALS AND METHODS

Extensive chromosomal association of Ad12 DNA early after viral infection of human or hamster cells. We have previously analyzed the fate of Ad12 DNA in hamster and human cells by molecular biology methods (3, 8, 25). We have now extended these investigations by using the FISH method to elucidate early steps in the stable interaction of the incoming foreign (Ad12) DNA with the cellular nucleus and its chromosomes. This technique is sensitive enough to detect even single copies of fragments of an Ad12 genome per cell and to localize this foreign DNA molecule precisely in the nucleus and on the chromosomes at the level of single infected cells. The experimental plan and the major results of this study are summarized in Tables 1 and 2. The human cell line HeLa, primary HUCF, the baby hamster kidney cell line BHK21, primary EHC, or the BHK21-derived, Ad12-transformed cell line T637 with ;20 integrated Ad12 genomes per cell was infected with Ad12 (Table 1). Multiplicities of infection increased from 0.1 to 103 PFU per cell on a dish with approximately 6 3 106 to 7 3 106 cells. Metaphase chromosomes and interphase nuclei were prepared and analyzed by FISH with biotinylated Ad12 DNA as the hybridization probe and FITCtagged avidin for fluorescent probe detection at 2, 6, 16, and 24 h postinfection (hpi). Starting at 2 hpi, in some experiments at 90 min pi, Ad12-specific signals could be detected on metaphase chromosomes in all cell types when high multiplicities of infection (103 PFU per cell) were used, with BHK21 cells exhibiting weak signals under these conditions. At late times after infection, the metaphase chromosomes or interphase nu-

Cells and virus. Human HeLa cells (American Type Culture Collection [ATCC], CCL2), human KB cells (ATCC, CCL17), hamster BHK21 cells (ATCC, CCL10), primary embryonal hamster cells, and the Ad12-transformed hamster cell line T637 (27) were propagated in culture as described previously (26). Primary human umbilical cord fibroblasts (HUCF), a gift from Sabine Schwemmle, Ulm, Germany, and embryonic hamster cells (EHC) were grown in Dulbecco modified medium (1) containing 10% fetal calf serum. Human Ad12 was obtained from the ATCC (VR863), propagated in KB or HeLa cells, and CsCl purified by standard procedures (4). Infection or mock infection of cells with Ad12 and addition of Ad12 DNA to the culture medium. Multiplicities of infection were calculated on the basis of the empirically determined relation that 1 ml of CsCl-purified Ad12 with an optical density at 260 nm of 1 equals about 1010 PFU (4). Virus inocula were diluted in phosphate-buffered saline (PBS) (7) and added (0 h) to about half-confluent cells on monolayers. In mock infection experiments, PBS devoid of virus was added. In DNA addition experiments, Ad12 or Ad2 DNA was directly administered to the cell culture in 1 ml of serum-free medium per 6-cm-diameter dish, as described previously (11). In some experiments, Ad12 DNA-TP was prepared as described previously (23) and also added to HeLa or BHK21 cells. In other experiments, bacteriophage lambda DNA, M13 DNA, or plasmid pGL2 DNA (33) was used. The number of DNA-positive interphase nuclei in various DNA transfection experiments was determined by FISH and UV light microscopy. FISH technique. The FISH technique (17) with the modifications used in our laboratory was described previously (13). Briefly, cells were arrested in metaphase with colchicine (100 ng/ml), treated with 75 mM KCl for about 20 min at 37°C, and fixed in methanol-acetic acid (3:1) at 4°C. Spread chromosomes and interphase nuclei were treated with 90°C-preheated RNase A (100 mg/ml) in 23 SSC (13 SSC is 0.15 M NaCl plus 0.015 M sodium citrate) prior to hybridization. DNA probes were labeled with biotin-16-29-dUTP by nick translation (22). The DNA fragment length was assessed to be 200 to 500 bp. Chromosomal DNA was hybridized to the 100°C-denatured probe (50 to 100 ng) in 50% formamide–23 SSC–10% dextran sulfate–0.1% sodium dodecyl sulfate (SDS) at 37°C under a sealed coverslip. After the washing procedure, samples were preincubated for 30 min in blocking reagent (3% milk powder in 43 SSC) and then reacted for 30 min at 37°C with avidin-fluorescein isothiocyanate (avidin-FITC) diluted 1:400. The washing and the reactions with biotinylated antibodies against avidin diluted 1:40 and with avidin-FITC were repeated. The chromosomal DNA was counterstained in propidium iodide (1 mg/ml). Color was preserved with 1,4-diazobicyclo[2.2.2]-octane (23.3 mg/ml). For further details, see reference 13. Stretched-chromosome preparations. Chromosomes were mechanically stretched by low-speed centrifugation by a published method (12). In brief, HeLa cells growing in culture and infected 6 h previously with 103 PFU of Ad12 per cell were washed and resuspended in hypotonic buffer (10 mM HEPES [pH 7.3], 30 mM glycerol, 1.0 mM CaCl2) at a concentration of 103 to 104 cells/ml. After 10 min of hypotonic treatment, 0.5 ml of this cell suspension was centrifuged on a glass slide in a Shandon Cytospin centrifuge at 800 rpm for 4 min. The chromosome preparations were fixed in methanol at 220°C for 30 min. Subsequently, FISH reactions were performed as described above. DNA preparations. Human Ad2 and Ad12 DNAs were purified as described previously (4). Ad12 DNA-TP was prepared from purified Ad12 on sucrose gradients (5 to 20%) in TE (0.01 M Tris-HCl, 1 mM EDTA, pH 7.5) containing 4 M guanidinium hydrochloride according to published methods (23). DNAs from bacteriophage l and M13mp18 (19) and from plasmid pGL2 were prepared by standard techniques or were obtained commercially. The pGL2 plasmid

RESULTS AND DISCUSSION

TABLE 2. Summary of data on association of Ad12 DNA with metaphase chromosomes of human or hamster cellsa Ad12 DNA association with metaphase chromosomes at: Method

Cells 2 hpt

6 hpt

16 hpt

24 hpt

DNA added

BHK21 HeLa

1 1

1 1

1 1

1 1

Ca21-phosphate

BHK21 HeLa

1 1

1 1

1 1

1 1

a Cells were transfected with 10 mg of Ad12 DNA plus 5 mg of plasmid DNA. The DNA was either added to the medium or transfected by the Ca21-phosphate precipitation method (10). hpt, hours posttransfection.

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clei of 100% of the HeLa cells (16 hpi) and 70 to 80% of the BHK21 cells (24 hpi) exhibited Ad12-specific signals even at multiplicities of infection of about 10 PFU per cell. The other cell types yielded similar results. In the productively infected human cells, the association of viral DNA was most extensive, required lower multiplicities of infection, and increased with time of infection at low multiplicities of infection. The latter finding suggested that newly synthesized viral DNA was also bound to metaphase chromosomes. The early association of Ad12 DNA with chromosomes long before the start of Ad12 DNA replication in human cells at 12 to 14 hpi, and even in nonpermissive hamster cells with an absolute block for Ad12 DNA replication (4, 15), implied that viral DNA replication was not required for the chromosomal association of Ad12 DNA. Numerous control experiments were performed to rule out unspecific hybridization. The photographs in Fig. 1 show results with the two human cell types studied. There were no or only a few background signals on the chromosomes or in the interphase nuclei of uninfected cells (Fig. 1a and e). Ad12 DNA was associated with human metaphase chromosomes at 2 (Fig. 1b), 6 (Fig. 1c and g), and 24 (Fig. 1d) hpi. At the high multiplicities of infection used, and particularly at late times postinfection, interphase nuclei showed extensive, brightly stained factories of Ad12 DNA replication. Under the same conditions, staining was frequently observed in regions on the glass slide that lay between the chromosomes. This finding is likely caused by the large amounts of Ad12 DNA accumulating in the nuclei of cells at late times after infection at high multiplicities of infection (Fig. 1c, d, and g). This DNA was also liberated and spread during metaphase preparation. It was shown previously by equilibrium sedimentation in an analytical ultracentrifuge that late after infection, up to about 50% of the intranuclear DNA was viral DNA (4). Association versus integration. The presence of true Ad12 DNA integration in productively infected human cells was difficult to establish by either biophysical (25) or cytogenetic (Fig. 1) methods, since the cells did not survive infection. The interpretation that Ad12 DNA and human DNA could become covalently linked was documented previously by the occurrence of symmetric Ad12-human DNA recombinants (2). Chromosomal association of Ad12 DNA in productively infected human cells had been shown earlier, with some apparent preference for association with human chromosome 1 both early (24) and late (18) after infection. Ad12 DNA association with chromosomes and interphase nuclei was also observed in BHK21 and T637 hamster cells at 2 (Fig. 2b and f), 6 (Fig. 2c and g), and 24 (Fig. 2d and h) hpi. Again, signals were not apparent with chromosomes and interphase nuclei from uninfected cells (Fig. 2a and e). The signal in uninfected T637 cells (Fig. 2e) was due to the ;20 copies of integrated Ad12 DNA (16, 26, 28) at a single chromosomal site (5, 13, 16). Uninfected and Ad12-infected T637 cells provided a particularly stringent control for the absence of background hybridization. The data in Fig. 2e to h demonstrated signals due to the integrated Ad12 DNA. Upon Ad12 infection, the integrated Ad12 DNA signals were still apparent on both chromatids of one chromosome, and infecting Ad12 DNA became associated with many additional hamster chromosomes. Chromosomal association of Ad12 DNA in this nonpermissive system was an early event (Fig. 2b, c, f, and g) which progressed and increased with time after infection (Fig. 2d and h; Tables 1 and 2). These findings are in agreement with results published earlier in which linkage of Ad12 DNA to cellular DNA was demonstrated by equilibrium sedimentation in neutral and alkaline CsCl density gradients, by shear experiments,

CHROMOSOMAL ASSOCIATION OF Ad12 DNA

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by DNA-DNA hybridization, and by measurements of reassociation kinetics (3, 8). On the chromosomes of both human cells (Fig. 1) and hamster cells (Fig. 2) infected with Ad12, Ad12-specific signals were frequently observed on only one of the chromatids. This observation supports the notion of chromosomal association and/or unstable integration for many of the hybridization signals. In some instances (arrowheads in Fig. 2), however, signals were detected on both chromatids. In the Ad12-infected nonpermissive BHK21 or T637 hamster cells, hybridization signals with Ad12 DNA in areas in between chromosomes were not observed upon Ad12 infection (Fig. 2b to d and f to h). These data attested to the absence of large amounts of free viral DNA in this abortive system. Stability of the chromosomal association of Ad12 DNA under different experimental conditions. Investigations on the mode and mechanism of Ad12 DNA association with the host cell chromosomes were initiated. In contrast to the previous experiments (Fig. 1 and 2), confluently grown cultures of HeLa cells or BHK21 cells were infected with 103 PFU of Ad12 virus per cell. In either cell line, the chromosomal association of Ad12 DNA at 6 hpi was as extensive as in actively dividing cells (Fig. 1 and 2 and data not shown). Stimulated primary human lymphocytes in culture were infected with 100 PFU of Ad12 virus per cell. At 2, 6, 24, and 48 hpi, Ad12 DNA was found in association with the human chromosomes but by no means as extensively as in the HeLa cell line. After the fixation of interphase or metaphase spreads on the glass slides, the association of Ad12 DNA with the chromosomes of actively replicating HeLa cells in culture was challenged by treating the preparations with 0.25% trypsin or pepsin for up to 10 min at 37°C, with different detergents at 37°C (20% Nonidet P-40 for 30 min [Fig. 3d], 10% deoxycholate for 30 min, 10% SDS for up to 30 min [Fig. 3c], or 20% SDS for 30 min), with 8 M urea or 8 M guanidinium hydrochloride (Fig. 3e) at 37°C for 30 min, with 70% formamide in 23 SSC at 80°C for 5 min (Fig. 3b), or with 0.5 M NaOH at 37°C for 30 min or 0.25 M NaOH at 37°C for 5 min (Fig. 3b). None of these procedures seemed to decrease chromosomal association of Ad12 DNA. This association hence appeared to be very stable and probably due to a rather firm fixation of the viral DNA inside the chromatin structure. It was conceivable that resistance of chromosomal Ad12 DNA to rather stringent chemical procedures could be partly explained by the fixation of chromosomal preparations prior to challenge. Chromosomal association of Ad12 DNA after Ad12 infection can also be demonstrated on stretched-chromosome preparations. The high degree of stability of Ad12 DNA association with the chromosomes of infected cells could be further documented by mechanically stretching the chromosomes by lowspeed centrifugation (12). HeLa cells were infected with 103 PFU of Ad12 per cell, 6-hpi metaphases were prepared, and the chromosomes were mechanically stretched by low-speed centrifugation as described in Materials and Methods. Subsequently, Ad12 DNA sequences on the stretched chromosomes were identified by FISH (Fig. 4a to c). The data unequivocally demonstrate that the high degree of chromosomal association of Ad12 DNA in productively infected cells resists mechanical shear and is stable after the application of both physical and chemical factors designed to disrupt a less specific and firm linkage of Ad12 to the chromosomes. The patterns of chromosomal association of Ad12 DNA in productively infected HeLa cells (Fig. 4a to c) as documented on mechanically stretched chromosomes were compared by FISH and laser scan microscopy to those in the Ad12-transformed hamster cell line T637 (Fig. 4d to f). In this cell line

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FIG. 1. Association of Ad12 DNA with metaphase chromosomes in cultured human HeLa cells (a to d) or in primary HUCF (e to h) after mock infection with PBS (lack of association) (a and e) or 2 (b and f), 6 (c and g), or 24 (d and h) h after Ad12 infection (103 PFU/cell). Ad12 DNA molecules were detected by the FISH method. Magnification, 31,250.

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FIG. 2. Association of Ad12 DNA with metaphase chromosomes in cultured hamster BHK21 cells (a to d) or in the Ad12-transformed BHK21 cell line T637 (e to h). Cells were mock infected with PBS (no association) (a and e) or Ad12 infected (103 PFU/cell) and analyzed at 2 (b and f), 6 (c and g), or 24 (d and h) hpi. The arrowheads designate symmetric Ad12 DNA association with both chromatids, which is indicative of genomic integration in Ad12-infected cells (b to d). In the Ad12-transformed T637 cells, the strong symmetric signal was due to about 20 copies of integrated Ad12 DNA (e to h). Magnification, 31,250.

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FIG. 3. Stability of the association of Ad12 DNA with the chromosomes of infected HeLa cells. (a) Mock-infected cells (no association); (b to e) infected cells after treatment with 0.25 N NaOH for 5 min (b), 10% SDS for 10 min (c), 20% Nonidet P-40 for 30 min (d), or 8 M guanidinium hydrochloride for 30 min (e) under the conditions described in the text. HeLa cells were infected with 103 PFU of Ad12 per cell, chromosomes were prepared at 6 hpi, the FISH reaction was performed, and stability of hybridization was challenged as described above. Magnification, 31,190.

about 20 to 22 copies of Ad12 DNA are covalently integrated into hamster cell DNA (16, 26). The patterns in the Ad12infected HeLa cells (Fig. 4a to c) and the Ad12-transformed T637 cells (Fig. 4d to f) look identical. This finding further supports the notion that the chromosomal association of Ad12

DNA in infected cells is very similar, and perhaps identical, to a truly integrated state. Long-term studies on chromosomal association of Ad12 DNA. Previous biophysical studies using the technique of reassociation kinetics (31) indicated that at late times after in-

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FIG. 4. Laser scan microscopy. Mechanically stretched chromosomes still retain the associated Ad12 DNA in Ad12-infected HeLa cells (a to c) or in Ad12transformed T637 hamster cells with about 20 integrated Ad12 genomes (d to f). Experimental details are described in the text.

fection and with continuous replication of the abortively Ad12infected BHK21 cells, a major portion of the Ad12 DNA was eventually lost from the cells (8). This loss could have been due to a selective growth disadvantage of cells because of damage

due to massive integration of foreign DNA or to an inherent instability of the majority of the chromosome-associated or integrated Ad12 DNA molecules or both. Stable integration of foreign DNA and survival of the cells were probably rare

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J. VIROL. TABLE 3. Summary of gentle Ad12 DNA transfer to mammalian cells by addition of Ad12 DNA or Ad12 DNA-TP to the culture medium

Cells

HeLa

DNA

Ad12

Ad12 DNA-TP

FIG. 5. Percentages of interphase nuclei of Ad12-infected BHK21 cells at different times postinfection with 103 PFU/cell. The FISH method was used to evaluate the number of Ad12 DNA-positive interphase nuclei. Error bars indicate standard deviations.

BHK21

Ad12

Ad12 DNA-TP

events, although transient association and integration could have occurred at many different sites on the chromosomes of infected cells (Fig. 1 and 2). We have determined the kinetics of the association of Ad12 DNA with BHK21 hamster cells after infection with 103 PFU/ cell by the cytogenetic FISH method. The fraction of interphase nuclei with positive Ad12 signals was determined. The data indicated a gradual decline in the number of positive cells (Fig. 5) and paralleled rather precisely the earlier results adduced with the technique of reassociation kinetics (8). Extensive association of Ad12 DNA introduced with infectious virus into BHK21 cells thus was a transient phenomenon. In a few cells, association or integration was found to be stable. Chromosomal association of Ad12 DNA after addition of Ad12 DNA or of Ad12 DNA-TP to the cell culture medium or after Ca21 phosphate transfection. Tables 2 and 3 summarize the results of experiments performed with Ad12 DNA that was introduced into HeLa or BHK21 cells by transfer methods independent of viral infection. The addition of Ad12 DNA to the maintenance medium without any further manipulations (11) proved to be the most gentle method and facilitated chromosomal association of the applied Ad12 DNA (Fig. 6a and c) or Ad12 DNA-TP (Fig. 6b) in HeLa cells (Fig. 6a and b) or in BHK21 cells (Fig. 6c and d), frequently with both chromatids, at 6 (Fig. 6c) or 16 (Fig. 6a and b) hpt. Ad12 DNA was detectable in interphase nuclei up to at least 24 h (Fig. 6d) after DNA application. Addition of Ad12 or of Ad12 DNA-TP proved to be a gentle and efficient transfer method for both HeLa and BHK21 cells in that up to 19% (BHK21 cells) or 30% (HeLa cells) of the exposed cells contained Ad12 DNApositive interphase nuclei at 24 h after DNA transfer (Table 3). Transfer and nuclear uptake were about 10 to 20 times more efficient when Ad12 DNA-TP instead of naked Ad12 DNA was used in the transfer experiments (Table 3). Chromosomal association was observed with either method (Fig. 6a and b). We conclude that Ad12 DNA and, even more efficiently, Ad12 DNA-TP can be gently introduced into mammalian cell nuclei and readily associates with chromosomes when the DNA is simply added to the cell culture medium. Ad2 DNA can also be transferred in this way. However, when 10 mg of bacteriophage l DNA, M13 DNA or plasmid pGL2 DNA was added to the medium of HeLa or BHK21 cells in 60-mm-

Amt of DNA (mg)a

0.5 1 5 10 0.05 0.1 0.5 1 0.5 1 5 10 0.05 0.1 0.5 1

% Ad12 DNA-positive nucleib at: 2 hptc

24 hpt

,1 3 18 22

,1 1 16 25

,1 2 20 26

,1 1 22 30

,1 ,1 7 11

,1 1 7 9

,1 2 11 15

,1 ,1 13 19

a

Amount of DNA added to the medium per 6-cm-diameter culture dish. Percentage of each 500 interphase nuclei Ad12 DNA positive by FISH and UV light microscopy. c hpt, hours after DNA transfer. b

diameter dishes, the DNA could not be detected by FISH in interphase nuclei between 2 and 24 h after DNA transfer. The standard Ca21-phosphate precipitation method (10) also led to chromosomal association of the foreign DNA (data not shown) but proved to be less advantageous for cell survival and continuous growth. Conclusions. As documented by the detection of Ad12 DNA on one chromosome of the revertant cell line TR12 with only about one copy of Ad12 DNA integrated (16), the FISH method allows detection of even a single copy of Ad12 DNA on a mammalian chromosome. We have applied this technique to monitor the fate of Ad12 DNA in human and hamster cells after infection, after Ca21-phosphate transfection, or after gentle transfer by the addition of Ad12 DNA or Ad12 DNA-TP to the cell culture medium. The extensive association of Ad12 DNA with chromosomes both in productively infected human cells and in abortively infected hamster cells suggests a pathway for viral DNA integration in which association and integration events at the single-cell level play an important role. The chromosomal association of Ad12 DNA has proved to be resistant to mechanical stretching of the chromosomes (Fig. 4) and to different DNA-denaturing reagents (Fig. 3). The patterns of chromosomal association upon laser scan microscopy are practically indistinguishable in Ad12-infected HeLa cells and in Ad12-transformed hamster T637 cells (Fig. 4), which carry 20 to 22 copies of integrated Ad12 DNA (16, 26). The cytogenetic data presented here corroborate earlier studies with both hamster and human cells in which biophysical and hybridization methods have been used. The FISH results make it likely, although they cannot prove, that a large part of the Ad12 DNA associated with chromosomes is in a physical state akin to integration. Since the introduction of foreign DNA into mammalian cells is a frequently applied regimen in

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FIG. 6. Transfer of Ad12 DNA into HeLa (a and b) or BHK21 (c and d) cells by different transfection protocols and association of Ad12 DNA with metaphase chromosomes (a to c) or an interphase nucleus (d). (a and b) Ad12 DNA (10 mg) (a) or Ad12 DNA-TP (1 mg) (b) was simply added (11) to the medium of HeLa cells. Metaphase chromosomes were analyzed by FISH with Ad12 DNA as the hybridization probe, as described in the text. (c and d) Association of Ad12 DNA with metaphase chromosomes (c) or an interphase nucleus (d) after the addition of 10 mg of Ad12 DNA plus 5 mg of plasmid DNA to the medium (11) of BHK21 cells growing in monolayer culture. FISH analyses were performed 16 (a and b), 6 (c), or 24 (d) h after the addition of Ad12 DNA. Magnification, 31,250.

molecular genetics and in gene therapy, it will be very interesting to monitor the intranuclear fate of adenovirus DNA as a model, quantitatively and at the single-cell level. The data presented in Fig. 6 confirm previously reported results which have shown that the addition of adenovirus DNA to the cell culture medium without harsh artificial manipulations leads to the uptake and chromosomal DNA association of adenovirus DNA (11), particularly when adenovirus DNA is complexed with protamine (32) or has remained covalently linked to the terminal protein (Table 3; Fig. 6b). Transfection of Ad12 DNA by the Ca21-phosphate precipitation method (10) produced intranuclear signals of viral DNA but interfered with cellular replication in many cells. One of the questions at issue is that of the site specificity of chromosomal association or of integration of Ad12 DNA. In the experiments designed in this study, we could monitor only Ad12 DNA association with chromosomes after infection, transfection, or gentle DNA transfer. Analyses of many metaphase spreads with the FISH technique have not provided evidence for specificities in the association with specific chromosomal sites. However, on the basis of the available data, specific site associations cannot be excluded. It will be necessary to pursue this question in a long-term follow-up of infected or transfected cells at late times after infection or after DNA transfer. In many Ad12-induced tumors (13a) or Ad12transformed cell lines or Ad12-induced tumor cell lines (5, 6, 16), we have not found specific sites of Ad12 DNA integration, although in a few tumor cell lines, selective sites of Ad12 DNA

integration have been observed (20). The transfer of Ad12 DNA-TP, which has the terminal protein still covalently attached, may be a method mimicking viral infection more closely. This method will be investigated for its applicability in gene therapy. With that protocol, the problem of site specificity of Ad12 DNA chromosomal association will be interesting to monitor. ACKNOWLEDGMENTS J.S. thanks T. Haaf and D. C. Ward, Yale University, for introducing him to the technique of extended chromosome preparations. We thank Udo Ringeisen for graphic work and Petra Bo ¨hm for expert editorial contributions. This research was supported by the Deutsche Forschungsgemeinschaft through SFB205 and through Schwerpunkt “Virulenzfaktoren und Wirtstropismus animaler Viren” and by the Center for Molecular Medicine Cologne-BMBF, Bonn, Germany. REFERENCES 1. Bablanian, R., H. J. Eggers, and I. Tamm. 1965. Studies on the mechanism of poliovirus-induced cell damage. I. The relation between poliovirus-induced metabolic and morphological alterations. Virology 26:100–113. 2. Deuring, R., G. Klotz, and W. Doerfler. 1981. An unusual symmetric recombinant between adenovirus type 12 DNA and human cell DNA. Proc. Natl. Acad. Sci. USA 78:3142–3146. 3. Doerfler, W. 1968. The fate of the DNA of adenovirus type 12 in baby hamster kidney cells. Proc. Natl. Acad. Sci. USA 60:636–643. 4. Doerfler, W. 1969. Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology 38:587–606. 5. Doerfler, W. 1995. The insertion of foreign DNA into mammalian genomes

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