Patterns of integration of viral DNA in adenovirus type 2-transformed hamster cells

J. Mol.

R%ol. (1981)

147, 227-246

Patterns Adenovirus

of Integration of Viral DNA in Type 2-transformed Hamster Cells

LILY

VARUIM~N

AND

Institute ~~niversity

(Received

7 July

DOERFLER

of Genetics

of Cologne.

1980, and

WALTER

in

Cologne,

Germany

revised form

10 Decemlw

1980)

The patterns of integration of viral DNA in five lines of adenovirus type 2transformed hamster cells have been investigated. Cell lines HE1 to HE5 were obtained by in vitro transformation of hamster embryo cells by ultraviolet lightinactivated Ad2t. In all lines, segments in the central parts of the viral genome are missing. The lines HEl, HE2, HE3, HE4 and HE5 contain 2 to 4, 2 to 4, 6 to 10, about 10, and 2 to 3 genome fragment equivalents per cell, respectively. The patterns of integration in lines HE2 and HE3 are identical ; however, the viral genome has been amplified in these cell lines to different extents. This result provides evidence for the post-integrational amplification of inserted viral

ganomes. integrated

It is also conceivable that line HE2 may have undergone losses of Ad2 genomes. The persisting Ad2 genomes in lines HE2 and HE3 have

deletions fragments

in parts of the EcoRI are linked to cellular

genome

have been inverted

F and

D fragments.

The

remainders

of these

DNA. The termini of the segments of the viral and linked to each other. This linkage could have

occurred via a circular intermediate in integration or via tandemly integrated viral genomes with subsequent deletion events. The linkage of the termini of viral DNA might be mediated by short sequences of cellular DNA. In line HE5, approximately 40% of the Ad2 genome is deleted, and the truncated segments, again comprising the terminal Ad2 DNA fragments, have been fused. The termini of the viral DNA are linked to cellular DNA. In lines HE1 and HE4 complex deletion and fusion events have altered the inserted Ad2 genomes.

1. Introduction Tnvestigations

integration are related to the general problems both of of malignant transformation of mammalian cells. The results of detailed analyses of integration patterns in a number of adenovirustransformed hamster and rat cell lines, as well as of simian virus 40 (SV40)transformed cell lines suggested that in established virus-transformed cell lines viral DNA is integrated at many different sites of cellular DNA (Botchan et al., 1976; Ketner & Kelly, 1976; Sutter et al., 1978; Galloway et ul., 1979; Sambrook et al., 1979; Visser et al., 1979: Doerfler et al., 1979; Stabel et al., 1980; Eick et al.. 1980). In genetically homogeneous populations of FR3T3 mouse cells transformed insertion

on viral

of foreign

t Abbreviations 002%2836/81/100227-20

DNA

DNA

used: Ad2, $02.00/O

and

adenovirus

type

2: Ad12. 227

adenovirus C

1981

type Academic

12: Ad5, Press

adenovirus Inc.

(London)

type

5. Lt,d

“2X

I>. VARI)IMON

ASI)

b.

I)OEKII‘LEK

by SV40 under special conditions, more specific patterns of integration have brc~n observed (Rassoulzadegan et al., 1979). At present, the question of the specificity of integration sites cannot yet be answered definitely. The st.ructurr of only a few of these sites has been studied (Sambrook et al.. 1979). The mechanisms of recombination between the virus and host grnomes have not been elucidated. Certain features of these mechanisms may be reconstructed from the characteristics of integration patterns. The finding that adenovirus type 2 DNA in rat cells (Sambrook et al., 1980) and adenovirus type 12 DNA in hamster cells (Groneberg et al., 1977; Doerfler et al., 1979; Stabel et al.! 1980) is frequently linked to highly repetitive cellular sequences, is compatible with the following model. Initially, one or a few molecules of viral DNA are linked to cellular DNA and, subsequently, the entire block of viral and cellular DXA sequences is amplified. lt is of course conceivable, albeit less likely, that multiple copies of viral DNA are integrated at identical or very similar sites in cellular Dh’A sequences. There is evidence that in some of the AdlZt-transformed hamster cell lines short sequences at the termini of the viral DKA molecule are amplified disproportionately (Stabrl et al., 1980). (‘omparing some of the characteristics of viral DNA integration of Ad2 DNA in rat cells to that of Ad12 DNA in hamster and rat cells, it is striking that almost invariably parts of the Ad2 genome have been deleted (Sambrook et al., 1974: Visser et al., 1979), whereas the Ad12 DNA molecules persist nearly intact and remain, at least in part, colinear with virion DNA in the hamster and rat cell lines investigated (Fanning & Doerfler, 1976; Green et al., 1976; Groneberg et al., 1977; Sutter et al., 1978; Doerfler et al., 1979: Ibelgaufts et al.. 1980; Stabel et al., 1980). One possible explanation for this apparent discrepancy lies in the fact that in hamster cells, which are fully permissive for Ad2 (Doerfler, 1969), and in rat cells, which are semi-permissive (Gallimore, 1974), there is strong selection against the persistence of the intact viral genome, since the virus would multiply and kill the infected cells. Ad12 DNA, on the other hand, cannot replicate in hamster cells, and thus persistence of the intact viral genomc would not entail disadvantages for the survival of the infected cells. Among the surviving cells, a small proportion would develop the characteristics of transformed cells, and these cells have been selected. In the present paper we have determined in detail the patterns of integration of Ad2 DNA in five lines of Ad2-transformed hamster cells HEl, HE2, HE3, HE4, and HE5 (Johansson et al.., 1978: Cook & Lewis, 1979). These patterns of integration are characterized by varying degrees of deletions of the central portions of the Ad2 genome in the five different lines. In two of the cell lines investigated, the termini of the persisting viral genomes are inverted relative to each other and with respect to the arrangement of Ad2 DNA in the virion. This finding suggests, but does not prove, the notion that a circular DNA intermediate of Ad2 DR’A might have played a role in the integration event. The viral DNA termini could still be separated by cellular DNA sequences. Little information is available as yet on the expression of the integrated viral genes in the five transformed hamster lines. By immunoprecipitation and gel $ See footnote

on p. 227

VIRAL

DNA

IN

Ad2-TRANSFORMED

HAMSTER

CELLS

229

Johansson et al. (1978) have determined some of the AdB-specific proteins synthesized in these lines. Furthermore, it has been demonstrated that certain segments of the integrated Ad2 genomes are extensively methylated (Vardimon et al., 1980). In cell line HEl, the 72K DNA-binding protein is expressed, and the segment of viral DNA coding for this protein is not extensively elrctrophoresis,

methylated. In lines HE2 and HE3, on the other hand, the same DNA segment is strongly methylated, and these lines do not synthesize t,he Ad2-specific 72K pfot,ci n .

2. Materials

and Methods

(a) Cells and eircrs The AdZ-transformed hamster cell lines HEl, HE2, HE3, HE4, and HE5 were obtained by infecting LSH primary hamster embryo cells with u.v.-irradiated Ad2 (Cook & Lewis, 1979). The Ad2 HEl, Ad2 HE2, and Ad2 HE3 lines were established from cultures infected with 4 plaque-forming units/cell, and the Ad2 HE4 and Ad2 HE5 lines were established from cultures infected with 35 plaque-forming units/cell (Johansson et al., 1978). It cannot be excluded that lines HE2 and HE3 could have been derived from the same original transformed cell (A. M. Lewis, personal communication). These cells have been characterized with respect to the persistence of the Ad2 genome and the expression of Ads-specific proteins (Johansson et al., 1978). We have obtained these lines from Drs L. Philipson and U. Pettersson, Uppsala, Sweden. In our laboratory, the cell lines were propagated in Eagle’s medium supplemented with 10% (v/v) fetal bovine serum (Seromed, Munich. Germany). Human Ad2 was grown in suspension cultures of human KB cells as previously described (Doerfler, 1969). The methods of virus purification were outlined elsewhere (Vardimon B Doerfler, 1980).

(b) Viral

DNA

and

viral

05.4

fragments

Ad2 DNA was extracted from purified virions as detailed earlier (Doer-Her et al., 1972), and the Ad2 DNA fragments A to D generated by the BarnHI, and fragments A to F generated by the EcoRI restriction endonucleases were prepared by gel electrophoreses on cylindrical, 2.5 cm diam. 0.5% (w/v) agarose gels as described by Stabel et al. (1980).

(c) Isolation

and

puri$cation

of cellular

DS.4

The DNA from AdS-transformed hamster cells was extracted by the sodium dodecyl sulfate/Pronase B/phenol procedure. The total nucleic acid extracted was further purified by RNAase treatment at low and high salt concentrations followed by treatment with proteinase K. Subsequently, the DNA was sedimented to equilibrium in CsCl density gradients. Details of these procedures have been published (Sutter et al., 1978).

(d) Nick-translation of Ad.2 DX.4 and of pur
The transformed hamster ~11 UN.4 was cleaved with the restriction endonucleases &oRI 1 HamHI, BgIII, or Kp”I (Boehringcr, Mannheim. (irrmany) or jointly with thr ondonucleasrs EcoRI and HamHI The fragments generated were separated IJ~ electrophorrsis on (~50, or I +~~ agarose slab gels and transferred to nitrocellulose fi1tc.r paper (BA85 : Schleicher-Schuell) using the technique of Southern (1976) as descrih~~d &cwhere (Doerflrr et a/., 1979). Subsequent,ly. t,he DX.1 was hybridized to “2P-labeled Ad2 DNA or specific restriction endonuclease fragments of Ad2 DSA. Hybridizat,ion conditions were used as outlined bp Waht rf a/. (1979). (f) Quantitatiorr

of viral DATA frown transformed

sequences cells

in the DA’.4

Autoradiograms were scanned in a Vitatron Electronic photometer, which was capable of integrating intensities of individual bands. Different autoradiograms of varying lengths of exposure were tested for linearity of autoradiographic intensities using a reconstitution series of viral DNA fragments in which I, 2,4,6, and 10 genome equivalents of Ad2 DNA per cell were electrophoresed and were hybridized to Ad2 DNA, which was ‘*P-labeled by nicktranslation. The intensities measured over each of these fragment bands in the reconstitution series were then used to quantitate the amount of each corresponding Ad2 DNA fragment in the DNA extracted from transformed cells (Table 1). The cellular DNA was cut with the BarnHI or EcoRI restriction endonuclease. The fragments were separated by electrophoresis, transferred to nitrocellulose filters and hybridized with 32P-labeled Ad2 DNA.

3. Results ceklar

(a) Survey of patterns of persistence upon cleavage of DNA with the restriction endonwlease EcoRI or BamHI

The DNAs from cell lines HEl. HE2. HE3, HE4, and HE5 were cleaved with the restriction endonuclease EeoRI or BamHI, the fragments were separated by electrophoresis on O5o/o agarose slab gels and were transferred to nitrocellulose filters by the Southern (1975) blotting technique. Subsequently, Ad2-specific sequences were detected in the mass of cellular DNA by DKA-DNA hybridization by nick-translation. Ad2 using as a probe Ad2 DNA that had been 32P-labeled virion DNA cleaved with the same enzyme was co-electrophoresed in all experiments as an internal marker. Comigration of Ad2-specific fragments in cellular DKA with virion DNA fragments suggests identity of Ad2-specific fragments with the corresponding virion DNA segments but does not prove it. Displacement of virus-specific fragments to regions of a molecular weight higher than that of the marker DNA fragments implies linkage of viral DNA to cellular DNA or unusual linkage of viral DNA segments. Such AdS-specific fragments will be designated as off size bands in the autoradiograms. The results of this analysis are presented in Figure 1, which also exhibits the EcoRI and BamHI physical maps of Ad2 DNA for easy reference. It is apparent that in many instances virion marker DNA fragments comigrate with Ad2-specific fragments : the EcoRI fragment B in lines HE1 , HE2, and HE3, fragment D in line HE4, fragment E in lines HE2, HE3, HE4, and HE5; furthermore, BamHI fragment C in lines HEl, HE2, and HE3, and fragment D in lines HE2, HE3 and

VIRAL

9 4.3

DNA

IN

Ad2

TRASSFORMED

HAMSTER

Kbp

C3*6Kbp

231

A 14-I

Kbp

6

9.6

Kbp

C 6.2

Kbp

D 4.5

Kbp

(

OZ.6

Kbp

E 2-l

Kbp

F 1.7

Kbp

Born HI

ECORI I

CELLS

A

1

B

IF[

D IEI

C 1

B

IDI

C

1

A

FIG. 1. Analysis of Ad2 DN4 sequences persisting in the Ad%transformed hamster cell lines HEI, HEZ, HE3, HE4, and HI%. The DNAs from Ad2-transformed cell lines as indicated or Ad2 virion DNA as marker were cleaved with the restriction endonuclease EeoRI (left part of Figure) or BawlHI (right part of Figure). DNA fragments were separated by electrophoresis on 050, agarose slab gels and transferred to nitrocellulose filters by the Southern (1975) blotting technique. The DNA on the filters was subsequently hybridized with Ad2 DNA, 32P-labeled by nick-translation, a,nd AdZ-specific sequences were visualized by autoradiography. The EcoRI and BamHI restriction maps of Ad2 DNA are shown; the sizes of the EeoRI (left margin) and BarnHI fragments (right margin) of Ad2 DNA in kilobase-pairs (lo3 basepairs) (Kbp) are also indicated.

HE4. Off size bands occur in the DNA from all cell lines investigated and correspond to the bands not explicitly listed. It is also worth mentioning that frequently the terminal virion DNA fragments, i.e. the EcoRI fragments A and C, and the BamHI fragments B and A, are absent from cellular DNA from the corresponding marker positions. In some cases, internal fragments are also missing or displaced (Fig. 1). A more detailed analysis of these findings required the use of highly purified restriction endonuclease fragments of Ad2 DNA as probes in order to locate unequivocally specific segments of viral DNA in the cellular genome. (b) Analysis transformed

hamster

of the persisting Ad2 DNA sequences in cells using specijc Ad2 DNA fragments as probes

The EcoRI fragments A to F and the BamHI fragments A to D of Ad2 DNA were highly purified (as described in Materials and Methods) and used as probes to detect

w

t

-_-

”

HE3

HE2

Ad2

Ad2

HEI

i

2 c,

HE2

HE4

$

HE4

m

HE2 HEI

s z /

cD

2Tu

b

2 3 5 I

2 N

HE3

HE4

HE5

Ad2

Ad2

HEI

HE2

HE3

HE5

HE5

Ad2 Ad2

&

Ad2

Ad2

VIRAL

DNA

IN

AdS-TRANSFORMED

HAMSTER

CELLS

233

specific Ad2 DNA segments in the DNA from the Ad2-transformed hamster lines HEl, HE2, HE3, HE4, and HE5. In Figure 2 (a) to (d) the results of hybridization experiments using the BamHI fragments A to D, respectively, are presented. Data obtained with the EcoRI fragments A to F are shown in Figure 3 (a) to (f). respectively. A summary of the results adduced from all hybridization experiments is presented schematically in Figure 4. The following general features can be derived from these results. (I ) Tn all lines investigated the terminal Ad2 DNA fragments, i.e. the EcoRI fragments A and C and the BamHI fragments A and B. are displaced to off size positions, in some instances to off size positions which correspond to lower molecular weights than the respective terminal fragments of virion DNA. (2) The EcoRI and BamHI fragments described to comigrate with the corresponding virion marker DNA fragments (Fig. 1: section (a), above) do in fact represent sequences from these viral DNA fragments. (3) In several instances, internal fragments are missing from the positions of t’he corresponding marker fragments. Sometimes they are completely absent or they are displaced to positions of higher or lower molecular weight, suggesting that certain parts of the viral genome may have been deleted altogether. (4) In lines HE2, HE3, and HE4, off size bands exhibit homology to both terminal BamHI fragments of Ad2 DNA: in lines HEI, HE2, HE3, and HE4. the terminal EcoRI fragments appear to be somehow linked. (5) In cell lines HE1 and HE4, a complicated combination of homologies to different parts of the Ad2 genome is observed in several off size bands. From these data a complex pattern of persistence of Ad2 genomes emerges. In all lines the persisting Ad2 genomes are incomplete. The elucidation of the precise patterns of integration will require more detailed analyses using additional restriction endonucleases. It is apparent from the schemes shown in Figure 4 that the patterns of persistence in cell lines HE2 and HE3 are identical, as revealed upon cleavage of cellular DNA with the restriction endonucleases KpnI and BgZII (not shown) and EcoRI or BamHI, but differ from all other lines. Moreover. the patterns in lines HEl, HE4, and HE5 are all different from each other. (c) Quantitation

of Ad2 genome fragmrnts

persisting

From the data presented in Figures 2 and 3 and from data not shown, the amounts of Ad2 DNA fragments have been estimated using a reconstitution series FIG. 2. Analysis of viral DNA sequences persisting in AdZ-transformed hamster cells using the BarnHI fragments of the viral genome as hybridization probes, The DNAs from Ad2-transformed hamster lines were cleaved with the BumHI restriction endonuclease. The RumHI fragments A (a), B (b), C (c). and D (d) from Ad2 virion DNA were highly purified, s2P-labeled by nick-translation and used as probes in hybridization experiments. Experimental conditions are described in Materials and Methods or in the legend to Fig. 1. In the left-most tracks, Ad2 DNA cleaved with the restriction endonuclease BarnHI was electronhoresed. After transfer of the DNA to nitrocellulose filters by the Southern (1975) method, the left-most strips containing Ad2 DNA were cut off the main parts of the filters and hybridized to intact sZP-labeled Ad2 DNA. The main parts of the filters, which still contained one track with Ad2 marker DNA, were hybridized to the highly purified fragments A (a), B (b), C ( c ) or D (d) of Ad2 DNA. After washing. the filters were joined precisely to the marker strips and autoradiographed.

HEI

HE2

HE3

HE4

HE5

Ad 2

Ad2

HEI

HE2

HE3

HE4

HE5

Ad2

n, 2 ?? / D

i

I 0

::

2

”

0

--

HEI

HE2

HE3

HE4

HE5

Ad2

Ad2

HEI

HE2

HE3

HE4

HE5

Ad2

CL

30 2

,p N

iL

2

o3

VIRAL

DNA

IN

AdSTRANSFORMED

HAMSTER

CELLS

235

of 2, 4. 6, and 10 genome equivalents of Ad2 DNA as internal standards. The intensities of bands in the autoradiograms shown in Figures 2 and 3 and in autoradiograms not presented were scanned photometrically and compared to the intensities of the corresponding Ad2 reference bands (cf. Materials and Methods). Details of this procedure have been described (Stabel et nl.. 1980). The results of this comparison are summarized in Table 1. Only those viral DNA segments in transformed cell lines could be included in the quantitative analysis which comigrated with known marker DNA fragments. Off size bands were less suitable as they contained other parts of the viral genome or cellular DNA or represented deleted segments (Fig. 4). Cell lines HEl, HE2, HE3. HE4, and HE5 contained 2 to 4. 2 to 4, 6 to 10, about 10, and 2 to 3 genome fragment equivalents per cell. respectively. The values calculated from the relative intensities of different Ad2 DKA fragments yielded consistent results (Table 1). These data are in reasonable agreement with those of Johansson et al. (1978). who used measurements of reassociation kinetics for their determination and reported 2, 3, 4, 7. and 3 Ad2

Ad2 N x

EcoRI-E N

Lo P WW

:,,=I,

w”

EcoRI-F

Ad2

lu

-

w

w

2 u

: a

w” I

(e) FIG. 3. Analysis of viral DNA sequences persisting in A&-transformed hamster cells using the EcoRI fragments of Ad2 DNA as hybridization probes. Experimental conditions were similar to those described in the legend to Fig. 2, except that the cellular DNAs were cleaved with the EcoRI restriction endonuclease and that the EcoRI fragments A to F were used ay probes (parts (a) to (f) of the Figure. respectively).

a -

cu

10

2

Y

D,A-

-3 w I

A,C,F-

m w I

A,C-

‘f w I

m w T

-A,B -WA -C,A

-B,A

II)

&

id I

I

Y

A&A,C --B -D,A

c-

c-

B-

B-

B-

E,F-

E,F-

D-

D-

-D

B-

DJ

-&A

-A

-A

-c

-c

-D

-D

cu

w z

Y

:

-A

i

,A -B

-c

-c -D

cD-

DE-

E-

D,E-

F-

DE=

BumHI

EcoRI

FIG. 4. Schematic summary of hybridization experiments using the purified EcoKI or BarnHI fragments of Ad2 DNA as probes. The DNAs of cell lines HEl, HE2, HE3, HE4, and HE5 were analyzed as described in Materials and Methods. Purified restriction endonuclease fragments were used as hybridization probes in many separate hybridization experiments (for details see the legends to Figs 1,2 and 3). The identities of AdPspecific DNA bands in autoradiograms are indicated by letters in the schemes presented, and the map locations of individual restriction endonuclease fragments are designated in the restriction maps.

genome equivalents respectively.

per

cell for

cell

(d) Detailed analyses

L4d2DNA

in

lines

HEl.

of the integration cell

lines

HE2,

HE3,

patterns

HE4,

and

HE5,

of

HE2 and HE3

The data presented so far and summarized schematically in Figure 4 suggest that the patterns of integration of Ad2 DNA in lines HE2 and HE3 are identical or very similar. The AdS-specific off size bands of high molecular weight observed with the DNA of these lines are homologous to both terminal fragments of Ad2 DNA. The internal RamHI fragments D and C. and the internal EcoRI fragments B and E comigrate with the marker DNA fragments. The EcoRI fragment D and BarnHI fragment A occur in off size positions smaller than the virion marker DNA fragments. The small internal EcoRI fragment F is also in an off size position (cf. Fig. 4).

VIRAL

DNA

Quantitation

IS

Ad&TRANSFORMED

TABLE 1 Ad2 llLVil seqwmes in cell HEI, HEZ, HE3, HE4. and HE5 of

Genome

DNA fragment RcoRI B EcoRI I) ECORI E E’coRI F

HAMSTER

fragment

CELLS

237

lines

equivalents

per cell in line:

HE1

HE2

HE3

HE4

HE5

2-4

24 2-i

ti 4-rjt

-10

1-3

23

Relative intensities of bands on the autoradiograms were compared to intensities of the reconstitution series as described. It was apparent from the schemes presented in Fig. 4 which of the Ad2-specific restriction endonuclease fragments in lines HE1 to HE5 comigrated with the corresponding Ad2 marker DNA fragments. Only those fragments could be used in the quantitation of Ad2 genome fragment equivalents. Many of the off size bands contained double homologies to more than one defined Ad2 DNA fragment : they were not included in the analysis. t Occasionally, off size bands were smaller than the corresponding marker DNA fragments. i.e. these fragments were partly deleted. Hence, quantitations based on the intensities of such bands may produce underestimates.

In order to analyze the patterns of integration in more detail, the cellular DNA was cleaved with the EcoRI and BamHI restriction endonucleasessimultaneously (Fig. 5(a) and (b)) or with the KpnI restriction endonuclease (Fig. 5(c) and (d)). Subsequently, the DNA fragments on Southern (1975) blots were hybridized with specific probes, which were selected on the basis of their strategic locations on the Ad2 map : for this purpose the right terminal EcoRI fragment C and the left terminal BamHI fragment B were used. Upon double-cleavage of HE2 or HE3 DNA with the EcoRI and BamHI restriction endonucleases, both the EcoRI fragment C and the BamHI fragment B hybridize to the same off size fragments, which are of slightly higher molecular weight than the BamHI B marker fragment (Fig. 5(a)). These findings suggest that the two terminal fragments of Ad2 DNA might be linked directly as indicated in the schemein Figure 6 (bottom). When HE2 or HE3 DNA is cleaved with the enzyme KpnI, and when the DNA on Southern (1975) blots is hybridized to the EcoRI C or the BamHI B fragment of Ad2 DNA, again identical patterns arise for lines HE2 and HE3 for each probe (Fig. 5(b)) : the EcoRI C fragment hybridizes to one fragment (not visible in line HE2 in this blot). This fragment probably corresponds to the KpnI F-G fragment concatenate, which would have been generated if the two termini of Ad2 had been linked to each other before, during or after integration (cf. schemesat bottom of Fig. 6). In agreement with this interpretation, the left terminal BumHI B fragment hybridizes to the same concatenate fragment and to the authentic KpnI fragments B and C (Fig. 5(b)), as expected.

(a) Ad2

DNA

ECOF

Barn

or the

D

Born B

[

I

iDI

(a)

1

1

B

of integrated wax vlr~vrd

C

BornHI

EC0 RI

FIG:. 5. Detailed analyses DNA of line HE2 or HE3

B

A

A

Ad2

j~lDlE/

.

sequences . r.

c

--

1

in the

I

E

D

t

IGI

I

Ad%-trmsformed ---

3

Ad2

B

B

C IDI

hamster

1

A

lines

HE2

(b)

C

BUmHI

II/HI

Ken

EcoRI

D

I

I

B

JF/D\E~

HE3

1 E 1

and

I

A

A

(F

c

VIRAL

DNA

IN

Ad%TRANSFORMED

HAMSTER

EcoRI

BumHI

N N 4” ii A-

A,C-

239

CELLS

I2 I A,C-

N c;: I a”

I2 -B,A-B,A

-A -B

Bc-

Et-

-A

-A

-c

-c

-c

-12

-D

--D

B-

DE-

E,F-

F-

D-

E,F D-

-vBumHI

FIG. 6. Summary scheme of patterns of integration of Ad2 DNA in lines HE2 and HE3. For all practical purposes the patterns are identical in both lines. At the bottom, the most likely model of integrated viral DNA is presented.% Designates cellular DNA sequences. The data support the model at the bottom.

From these results, the model in Figure 6 has been deduced in which the termini of virion DNA are linked to each other directly. It cannot be ruled out that this link is mediated by a short stretch of cellular DNA or of rearranged Ad2 DNA sequences. Fragments of the EcoRI D and F segments are linked to cellular DNA. This model is also consistent with the results of the following experiment. The DNAs of Ad2 marker DNA and of HE3 DNA were cleaved with both the EcoRI and BumHI restriction endonucleases, and the DNA fragments on Southern (1975) blots were hybridized with intact 32P-labeled Ad2 DNA as probe. There is one high molecular weight off size fragment which is larger than the BamHI B fragment and represents remnants of the linked BamHI B and A fragments, and one low

“40

I>. \‘I\KI)IJlON

ASI) \v l)OEKFI,El<

molecular weight off size fragmentj which might constitute small parts of the BamHI A fragment linked to cellular DNA (cf. scheme at bottom of Fig. 6). As predicted by the model, the BamHI R, EcoRI C. 1). and F fragments arc‘ missing. and all other possibk fragments (BamHI (‘*, BamHI D. EcoRl K*. EcoRI E) migrate to the expected positions (data not shown). The BamHI (‘* and EcoRI K* fragments were generated by double digestion with the E:coRI and RamHI restriction endonuclrases. It is concluded that the integration patterns of Ad2 DNA in the HE2 and HE3 lines are identical. It is obvious from the data presented in Table 1 that the extents of amplification of Ad2 DN\‘A in cell lines HE2 and HE3 are different or that Ad2 DNA has been lost from line HE2 relative to line HE3. The patterns are characterized by deletions of the persisting Ad2 DNA in parts of the EcoRI F and D fragments. In the integrated state. the originally terminal segments of tld2 DNA are linked to each other either directly or via a short segment of cellular DSA. and cellular DXA is linked to viral sequences at the remnants of the EcoRI D and F fragments (see scheme at botOom of Fig. 6).

(e)

Detailed

analysis

=ld2 DiVil

of integration in

cell

line

patterns

of

HE5

An analysis similar to that described in the preceding section was performed for the DNA from cell line HE5. It is apparent from the data presented in Figures 2 to 4 that none of the off size bands of AdB-specific sequences in line HE5 reveals homologies to both terminal segments of Ad2 virion DNA. Hence, it can be excluded that the two termini of Ad2 DNA are linked to each other in this cell line. The data described in Figures 2 and 3 suggest that a large central segment of Ad2 DNA is missing in cell line HE5. To investigate the extent of this defect more precisely, the BamHI D and EcoRI D fragments of Ad2 DNA were used as hybridization probes. Upon simultaneous cleavage of the HE5 DNA with the restriction endonucleases BamHI and EcoRI, both probes hybridize to the same fragment, which is slightly larger than the EcoRI D fragment (Fig. 7(a)). This fragment with dual homology is generated by fusion of parts of the BamHI D and EcoRI D fragments (Fig. 8, schemes on the bottom). HE5 DNA was also hybridized to the BamHI D or EcoRI D probes after cleavage of cellular DNA with the restriction endonucleases Kpni and after blotting (Fig. 7(b)). Again, both probes appear to anneal to the same AdS-specific fragment, which has a molecular weight close to that of the KpnI A fragment. As can be derived from the location of restriction fragments on the maps shown in Figure 7(b), this band with homologies to both probes probably arises by fusion of the KpnI C fragment to the truncated KpnI A fragment with concomitant deletion of the intervening Ad2 DNA sequences. The same conclusion is derived by similar arguments from an analysis illustrated in Figure 7(c). HE5 DNA was cleaved with the restriction endonuclease BgZII, and the fragments on the blot were hybridized with the same probes as described above. Again, the EcoRI D and BamHI D fragments hybridize to the same fragment, which appears to be of slightly higher molecular weight than the BgZII D fragment (Fig. 7(c)), and this fragment contains the fused central region of

- N z

BornHI

- -. I2 ,” I

2 a

I

H

G

F

E

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Fit;. 5. Detailed arrl~ly~eti of the pattern of integration of Ad2 UN.4 in cell line HEB. (a) The DNA of Ad2 or of cell line HE5 was cleaved simultarwousl,v with the restriction endonucleases EroRI and RwnHI. the fragment8 were separated by 32P-l a bel ed Ad2 DNA (AdY), to the BamHI I) fragment electrophoresis and blotted as described. Different parts of the filter were subsequently hybridized 1.11 (HamHI-D) or the B:roKI I, fragment (BlcoRl-I>). The marker USA fragments were designated as described in the leyend to Fig. 5. an& the E’coICI and RamHI restriction maps are @en at the bottom of each autoradiogram. (b) Experimental conditions were similar to those desrrihnd in (a). however. USA preparations were cleaved with the restrirtion endonucleaae KpnI; its restriction map is also included. (r) The restriction rndor~uc~lraxe RglII was used in this experiment. The HglII restriction map of Ad2 DN.4 is also included.

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FIG. 8. Schematic representation summarizing the essential data of the analyses performed on HE5 DNA. The most likely model describing the integration pattern of Ad2 DNA in this line is presented in the two bottom lines.

integrated Ad2 DNA in line HE5. Thus, the model depicted in Figure 8 is supported by detailed analyses of the persistence patterns using four different restriction endonucleases. Moreover, when HE5 DNA was cut with the &oRI and BarnHI restriction endonucleases simultaneously, blotted and hybridized to intact Ad2 DNA, two bands are revealed which represent the termini of Ad2 DNA linked to cellular DNA : one band is slightly larger than the BamHI B fragment (left Ad2 DNA terminus), the other one nearly comigrates with the BamHI D fragment and may contain the right Ad2 DNA terminus linked to cellular DNA (cf. Fig. 8). There are two additional fragments apparently comigrating with the EcoRI D and E fragments, the former representing the region in which fusion had occurred, the latter the authentic EcoRI E fragment (data not shown).

VIRAL

DNA

IN

Adz-TRANSFORMED

HAMSTER

CELLS

243

It is concluded that in line HE5 a large central portion comprising about 40% of the total Ad2 genome is deleted, that the truncated molecules have been fused and linked to cellular DNA at or near the sites of the original termini of virion DNA. It cannot be excluded that short stretches of cellular DNA are interspersed at the sites where fusion of the right and left truncated portions of Ad2 DNA have occurred. Comparably detailed analyses of the patterns of integration of Ad2 DNA in cell lines HE1 and HE4 have not yielded comprehensive models. The data presented in Figures 1 to 3 and summarized in Figure 4 suggest that complicated deletion and fusion events may have taken place in the process of integration of viral DNA.

4. Discussion (a) Survey

and main

characteristics

of integration

patterns

Detailed integration patterns for five lines of AdB-transformed hamster cells have been determined by the Southern (1975) blotting technique combined with DNA-DNA hybridization experiments using specific fragments of the Ad2 genome (cf. Figs 4, 6 and 8 for summary). It is apparent that the integration patterns in lines HE2 and HE3 are identical, although both lines contain different amounts of viral DNA. The patterns in all the other lines are different from that in HE2 and HE3 and different from one another. Using at least two different restriction endonucleases, the integration patterns reveal that in lines HE2, HE3, and HE4 the two terminal viral DNA fragments are directly linked to each other. Some of the internal fragments of Ad2 DNA comigrate with the corresponding marker DNA fragments. others are displaced to off size positions of higher, occasionally of lower molecular weights than the marker DNA fragments (Fig. 4). Each of the lines investigated contains multiple copies of Ad2 DNA, 2 to 4,2 to 4,6 to 10, about 10, and 2 to 3 genome fragment copies per cell for the lines HEl, HE2, HE3, HE4, and HE5, respectively. In lines HE2 and HE3, a small segment of Ad2 DNA comprising parts of the EcoRI fragments F and D (Fig. 6) have been deleted. Cellular DNA sequenceshave become linked to the remnants of these internal fragments giving rise to off size bands with homologies to these fragments. The terminal EcoRI fragments A and C have somehow been joined together and are found in one high molecular weight off size band. The data obtained upon cleavage with the BamHI restriction endonuclease are also consistent with the model shown in Figure 6. It is worth noting that all of the 2 to 4 and 6 to 10 genome fragment copies of Ad2 DR’A persisting in lines HE2 and HE3, respectively, appear to be integrated at the same sites, probably in repetitive sequencesof cellular DNA. Since all copies of Ad2 Dh’A are arranged in the same mode, it is likely that one molecule of Ad2 DNA has been integrated and has been amplified together with cellular DNA sequences subsequent to insertion. The cell lines HE2 and HE3 contain different copy numbers of Ad2 DNA (Table 1). Perhaps these cell lines represent different stable stagesof amplification of viral DNA. It is also possiblethat in line HE2 some of the integrated Ad2 DNA molecules have been lost. It is a matter of conjecture how the terminal viral DNA segments have become linked to each other. A circular

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intermediate of viral DNA might, have played a role in the process of integration : however. it, is also conceivable Ohat) tandem arrangement and subsequent deletions of Ad2 segments might account for the mode observed, In line HE5, again all 2 to 3 copies of Ad2 DNA seem to be arranged in identical fashions. AA rather large central portion of the Ad2 DXA molecule has been deleted in this line. This deletion comprises segments of the tr:coRT A and D fragments and the entire BeoR fragments R and F (Fig. 8). The remaining portions of EcoR’I fragments A and D have been fused intro one fC’coRI (or RamHI) fragment. It cannot be ruled out that a short sequence of cellular DNA or of rearranged viral D?;A sequences has been interspersed at the site of this junction. The t,erminal segments of Ad2 DIVA have become linked to cellular DNA and assumes off size positions on the blot (Figs 4 and 8). 0 ne of these off size bands consisting of portions of the li:coRI A and D fragments and probably of cellular DNA exhibits a size smaller than the virion E’coRI A fragment (Fig. 8). The results of an analysis using the RamHI restriction endonuclease yield results consistent with this interpretation. The integration patterns of cell lines HEI and HE4 have also been analyzed in detail. The patterns appear very complex, and it has not been possible t’o construct simple models.

(b) Rtahility

of integration,

pattems

It has been reported that the patterns of integrated Ad12 DNA in hamster @utter et al.. 1978) and rat cells (Ibelgaufts et al., 1980) are very stable over long periods of passing cells in tissue culture. Even after passaging cells as tumors in hamsters and after re-explantation of cells, the patterns were unchanged (Doerfler et al., 1979). Cells of the AdB-transformed lines HE3 and HE4 (10’ cells/animal) were injected into newborn hamsters. Subcutaneous tumors arose at the sites of injection two to four weeks later. DNA was extracted from these tumors directly, and the patterns of integration of Ad2 DNA sequences were determined. These patterns were indistinguishable from those observed with the original cell lines HE3 and HE4 (R. Gahlmann. I. Kuhlmann, L. Vardimon & W. Doerfler, unpublished experiments). It is concluded that patterns of integration of Ad2 and Ad12 DNA in hamster and rat cells are very stable even after passaging the transformed cells as tumors in animals. (c) Comparison of integration and Adl2-transformed

patterns in Ad2, cell lines

Ad5,

In all of the Adl2-transformed hamster cell lines (Fanning & Doerfler, 1976: Groneberg et al., 1977; Sutter et al., 1978; Stabel et al., 1980) and the Adl2-induced rat brain tumor cell lines (Ibelgaufts et al., 1980), the entire or nearly the entire viral genome has been found to persist in multiple copies. The Ad12 DNA molecules have been found to be inserted colinearly; the possibility exists that in some molecules, stretches at both termini of the Ad12 genome are amplified or rearranged in an as yet undetermined way (Stabel et al., 1980). In contrast, in many

VIRAL

DNA

IN

Ad%TRANSFORMED

HAMSTER

CELLS

245

Ad2 or Ads-transformed hamster and rat cell lines (Sambrook et al., 1974; Visser et al.. 1979: this study) smaller or larger segments of the persisting Ad2 genome have been deleted, prior to, during or subsequent to the integration event. In the case of cell lines HE1 to HE5, which were established upon transformation of primary hamster cells with ultraviolet-irradiated Ad2 DNA, the viral genome may have been predisposed to deletions. On the other hand, hamster cells are fully permissive for Ad2 virus (Doer&r. 1969) and rat cells are semi-permissive for this virus ((:allimore, 1974). Hence, the chances for cells to become transformed when the int’act Ad2 genome persists are decreased, since virus will start replicating and will eventually destroy these cells. In contradistinction, hamster cells are completely non-permissive for and do not facilitate the replication of Ad12 DXA (Doerfler, 1968,1969 ; Fanning & Doerfler, 1976) and, therefore. the persistence and subsequent integration of the intact Ad12 DNA molecule may not be disadvantageous to cell survival. Other, more complicated reasons for the apparent differences in integration patterns of Ad2 and Ad12 DNX can be envisaged. The before-mentioned differences in permissivity of hamster and rat cells towards Ad2 and Ad12 offer a, plausible, albeit not’ the only possible explanation. (d) The

mechanisms

of viral

DSA

insertion

CITY w/known

Aside from speculations, we cannot offer a well-documented model describing the mechanisms of adenovirus DNA integration. In the following we try to summarize certain general elements pertaining to the patterns of persistence. (1) Frequently, multiple copies of the viral genome persist in Ad2 or Ad12transformed cells at a limited number of sites of cellular DNA. In AdS-transformed cell lines, parts of the viral genome are missing. It cannot be decided whether there are multiple integration events of viral DNA molecules at identical sites or whether one DNA molecule is inserted and subsequently amplified by an unknown mechanism. The latter possibility appears to be the more likely. (2) The integration patterns found in lines HE2 and HE3 suggest, but do not prove, that integration might have occurred via a circular intermediate of Ad2 DNA. Such circular forms of viral DNA have not been found (Doerfler et al., 1972), but could exist transiently and occur in low concentrations in the cells. (3) It cannot be decided at what stage deletions have occurred in the Ad2 DNA molecule. Endonucleases in AdB-infected cells have been described (Reif et al., 197i): their role. if any, in integration has not been elucidated. In this respect, it may also be important that ultraviolet-irradiated Ad2 has been used in the initial transformation experiments.

We thank Dr Andrew M. Lewis, National Institutes of Health, Bethesda, Md, U.S.A. and Uppsala, Sweden for making the Ad% Drs Lennart Philipson and Ulf Pettersson, transformed hamster lines available. Hanna Mansi-Wothke rendered invaluable help by preparing media and by culturing cells. This research was supported by the Deutsche Forschungsgemeinschaft through SFB 74 and by the Ministry of Science and Research of the State of Northrhine-Westfalia (IIB8-6440).

I,. VAKI)I?vlOS

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