Loss of viral genomes from hamster tumor cells and nonrandom alterations in patterns of methylation of integrated adenovirus type 12 DNA.

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JOURNAL OF VIROLOGY,Sept.1983,p.631-636

0022-538X/83/090631-06$02.00/0

Vol. 47, No. 3

Loss

of Viral Genomes from

Hamster Tumor Cells and

Nonrandom Alterations in

Patterns

of Methylation of

Integrated

Adenovirus

Type 12 DNA

INGRID KUHLMANNt ANDWALTERDOERFLER*

Instituteof Genetics, University of Cologne, Cologne, Germany Received 11 March1983/Accepted 31 May 1983

The

insertion

stability and DNA methylation patterns of integratedadenovirus

type 12(Adl2)genomes wereinvestigatedin Adl2-inducedtumors and in tumor

cell lines established from them as afunction of time ofpassage under culture

conditions. Upon subcultivation of cells from some of the tumors, the viral

genomes wereeliminated, apparently inastepwise process with segments of the

left termini ofAdl2DNAs persisting the longest. Morphological variants of these

tumorcells lost all viral DNA and yet retained the oncogenic phenotype. All 13

independently isolated clones fromone revertantlinewere devoid ofAdl2DNA.

Itcouldnotbe ruledoutthat very short sequence elements of viral DNA, such as

promoters orenhancing sequences, could have persisted in these variants. The

extentof viral DNA methylation was minimal inAdl2-induced tumors, although

theviralgenome was notextensively expressed, ifatall.Upon passage in culture,

the levels of viral DNA methylation increased. It was interesting that establish-ment

of

thefinalmethylation pattern of integrated Adl2DNAs required many cell

generations after the fixation of foreign DNA in the host genome. The shift in

methylationwasnonrandom. The latepartsoftheinserted viralgenomes became

methylated moreextensively than did the early gene segments.

The results ofa detailed study of the integra-tion andmethylation patterns of adenovirustype 12(Adl2) DNA in 39Ad12-inducedtumors and

cell lines established from these tumors have been

reported previously

(9, 10). In over 70 different adenovirus-transformed cell lines and Adl2-inducedtumorsor tumor

lines,

thesiteof viral DNA insertion differs from cell lineto cell

line (3). In general, the

patterns

of viral DNA

insertion have proven to be quite stable for a

given

cell line (16). However, morphological variants of Ad12-transformed hamster cells (6) orofAdl2-induced hamstertumorcells (9) have been observed whichappearedmorefibroblastic than the

original

tumorcells. Althoughsome of these variants have been characterized by the

complete

elimination of viral DNA fromthehost

genome(5, 9), these cells retain their oncogenic

phenotype upon injection into hamsters (9).

From thesefindings, it has been concluded that

theoncogenic phenotype is compatible withthe

complete loss of Adl2DNAfrom cells that had

originally

been transformed by Adl2. Presum-ably, in hamster cells, Adl2 infection triggers hereto unknown mechanisms that entail

onco-genic transformation,

and thistransformation is

tPresent address:Institute ofVirology,Universityof

Co-logne, CoCo-logne,Germany.

accompanied by

fixation of

multiple copies

of viralDNA. Atleast inthecells in whichtheviral

genome was

completely lost,

stability

of the

oncogenic phenotype

is not

premised

on the persistence of viral genes

(3, 9).

Conceivably,

the decisive

transforming

events areelicited

by

the

fixation

of viral genes,

expression of

viral

genes, or

both,

but thecontinued

realization

of

transformation

once initiated may be

indepen-dentof viral

functions.

There is nowextensive evidence for the

no-tion that DNA

methylation

in

highly

specific

sequences can play a decisive role in the

long-term shutoff ofgenes in eucaryotes

(for

recent

reviews, seereference4and W.

Doerfler,

Curr. Top. Microbiol.

Immunol.,

inpress). It appears

that theabsence ofDNA

methylation

isa

neces-sary but not sufficient

precondition

for gene

expression to proceed (9, 17). This latter con-cepthadbeensupported

by

the

striking

observa-tion thatthelevels ofAdl2DNA

methylation

in

Adl2-induced tumors were very

low,

if DNA

methylation

could be detected at

all,

at 5'-CCGG-3' and 5'-GCGC-3' sites (10).

However,

viral gene expression not at all detectable in

these tumors or at an

exceedingly

low level is

not commensurate with the low level ofDNA

methylation (10). Upon

explantation

and

contin-ued cultivation ofthesetumor

cells,

the extent

631

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632

NOTESJ.Vo.

0

I

:---EcoRIl C D B

FIG. 1. Integration p~

Ad12-induced hamstert and in cell lines Hllll from these tumors, respi fromtumorsorfromcell

explantationfrom thetul

arations (10 p.g) were cl

ments were separated

agarose gels and blotted and Ad12-specific fragnf bridizationto 32P-labele4

diography. Adl2 DNA genome equivalents [20.

10 p.gofcellular DNA

cells)wasmixed with 10 treated in thesame manr

fragments (A to F) and

(Kbp), as well as theEK also shown.

of DNA methylationi

sequences increases g In the present cor

strate that Adl2 DNA cells inastepwisefast,

the viral DNAs vanis the levels of Adl2 D' lished hamster tumor

nonrandom. The 5'-C termini of Adl2 DNA

ylated. Inthisway, tI

patterns approached I

Ad12-transformed cel

co periods oftime

(8, 15).

Viral gene

expression

in

~~ ~ -~~ Ad12-inducedtumors wasminimal.

~~~L'Ci

~The

induction oftumors

by

Adl2

in newborn

E

~~~~~~hamsters

and theestablishmentof cell lines from

X

~~~~Kbp

these tumors, as well as the methods used to

determinepatternsofviral DNA

integration

and

methylation,

have beendescribedearlier

(9,

10).

A1387

In these

experiments,

the Southern

blotting

B7

technique (13)

and a DNA-DNA

hybridization

procedure (20)

were

used,

employing

nick-trans-*

-C5.4

lated

(11)

3P-labeled Adl2 DNAorcloned viral DNA

fragments (18).

DNA

methylation

of

inte-4"4

-0)4.0

grated viral DNA sequences was detected

by

hybfidization

of _Plabeled Adl2 DNA to blot-ted tumor cell DNA that had

previously

been

-E24 cleaved with the isoschizomeric restriction

en--E24donuclease

pair Hpall

or

MspI (19)

or with

HhaIL

Inthe latter

instance, Hhal-cleaved Adl2

virion

DNA,

which is

unmethylated (7),

was

usedas areference.

From 39

Ad12-induced

tumors, 13 cell lines

were established, and in 8 cases, the cell lines

Fo6 were carried

continuously

over 10or more

pas-sages. In three lines, the patterns of

integration

and copy numbers of viral DNA per cell didnot

change.

In three other

lines,

viral DNA was no

- FA

longer

detectable even after the first passage.

One cell line was not

analyzed

further. In cell

)atterns of viral DNA in the ln 11() nymnrcagswr b

,umors Tl111(1) and Tl11112

linvedHFig.12) only, sminor chaneofsiwee

ob-nd

(1) and H1111(2) established

dsaperved(i.

1);

e.g. some

otHell()

offsiz bands

ectively.

DNAwasextracted

disappgearmed.I

eluvlet

lierHe

)

allt

10

tosag

11

Isatstagesof passage(p)after virlgnm qiaet eels npsae2

mnors

asindicated. DNA prep-

(Fig.

1). In passage 12, 3 to 4 genome

equiva-leaved with EcoRI, the frag- lents

remained,

exhibiting

the same

integration

by

electrophoresis

on 0.5% pattern as in

Adl2

genome

equivalents

persisting

in passage

ients were visualized by hy- 12 of line

Hllll(1)

was estimated from the

d1(11)

Adl2DNAandautora-

intensity

of the internal viral DNA bands as (0.5 ng is equivalent to 20 shown in

Fig.

1

by

a

spectrophotometric

scan-x] percell, correspondingto cdr sdsrbdpeiul 1)

and theequivalent numberof ning procdrasecibdpvoul(1)

I

±g

ofB3(BHK-21)DNAand Both the internal

Adl2

fragments

and the Adl-rier.Theauthentic virion DNA

specific

off-size bandsweredecreasedin

intensi-Itheir sizes in kilobase pairs ty. Thus, it was most

likely

that total Adl2

IcoRI mapofAdl2 DNA, are genomes had been lost

during

passage of the tumorcells. In passage 13 of cell line

Hllll(l),

one to two

copies

of the left terminus ofAdl2

DNA

persisted,

the EcoRI D

fragment

and an

iin

the

integrated

Adl2 off-size

fragment

hybridizing

to bothtermini. In

;,radually

(10). this

experiment,

the

molecularly

cloned termini

mmunication, we demon- of Adl2 DNA, the EcoRI C and A*

fragments

A can be lost from tumor (18), were used as

hybridization probes

(refer-iionwith theleft termini of ence9,cf. maps in

Fig.

1 and

2b).

In DNAfrom

;hing

last. The increase in passage 20 onward, viral DNA could not be INA

methylation

in estab-

detected,

even

though

these cells continued to

cell lines wasfound tobe be

oncogenic

in hamsters(9). The

sensitivity

of

.,CGG-3'

sites at the

right

the blot

hybridization

experiments

wassuch that Ls were

increasingly

meth- onecopyof the left 10% of the genome per cell ie

Adl2

DNA

methylation

would have been detected. In passage 12 of cell

those described earlierfor line Hllll(l), 13 cellular clones were

estab-LI lines cultured over

long

lished. In the DNA of these clones, viral DNA

J. VIROL.

0

'r :I? C114 Cl. a a

C--;=. -,;=. -;:x ;.

x T m I

-- - -- ----Cl

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NOTES 633

a Approximatesize

of DNAfragment

inbase pairs [bp]

4150

2464

Band pattern 0kd12DNA MspI

A-B

1700 1650

1250 1150 1050 940 850

760 710 580 550 504

E.F-

_GE,

H,-J

K.L

M

NO0 P Q-R,S

Ad12DNA,leftterminus

b

Ela Elb

Mspl R

od.

Hpall

bpI

Locatedin

fragment

EcoRIC

EcoRIX FIG. 2. Restriction pattern and partial map ofAdl2 DNAfor the isoschizomeric restriction enzyme pair

EcoRIB

MspI

and

HpaII.

(a)

Adl2

DNA was cleaved with

Eco

RIA*

MspI,

and the

fragments

were

separated by

gel

elec-EcoRI A trophoresis on a 1% agarose gel and blotted to nitrocel-lulose paper. Individualfragments were located on the Adl2 map by hybridizing independent blots with 32p-EcoRI B labeled clonedfragments of Adl2 DNA as indicated.

EcoRIB Theestimated sizes of

MspI

fragments

AtoSarealso

indicated in base pairs (bp). The results have been EcoRI A summarized schematically. (b) Restriction maps of the EcoRIB left(26%) and theright

(40%)

termini of Adl2 DNA Eco RIC aftercleavage with HpaII or MspI. These maps have EcoRIC beenpublished previously(8).

punits

MspI

HpaII bp

90

iMap

units

couldnotbedetectedby Southernblotting,even

whenupto 300pgof cellular DNA wasblotted

on nitrocellulose filters. In these hybridization

experiments, the32P-labeled totalAdl2genome

aswellasclonedfragments,inparticularthe left

terminal EcoRI Cfragment (comprising the El

region of Adl2 DNA) and EcoRI D fragment,

wereusedashybridization probes (formaps, see

Fig. 2).

We conclude that viral DNA can be deleted

apparently in a stepwise manner from

Adl2-induced hamstertumorcells.The morphological

variants that lost all viral DNAcan retain their

oncogenic phenotypes, although they do not

carrythe Elregion of Adl2 DNA. It will bevery

difficulttorigorouslyruleoutthepossibility that

smallelements of viralDNA,e.g., apromoteror

enhancer sequence (2, 21), do persist in the

variant cell line. It shouldbe pointedoutthat the

celllinesestablished fromtumorswereprobably

closeto being clonal, as most tumorsexhibited

rather homogeneous integration patterns of

S B Q A

5soi22 2464 | AQ 4150 1

EcoC Eco D Eco B

10 20 3)0 40

E2b (notepPressed)

A1DE2a E3 E4

Adl12 DNAright terminus ,N

D I C G0

li 1

JU

1

Ro I1s

|X

|11700

l>d

T EcoA ] A

60 70 80

VOL. 47,1983

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634 NOTES

rwtl - rx at l

U- C]-e: 3

*~~~~~~~~~

.m

-N O* t. SI

FIG. 3. Nonrandom changes of methylation

pat-ternsofAdl2DNAinanAdl2-induced hamstertumor

line. DNAwasisolated from theAdl2-inducedtumor

T1111(2) andfrom cell line H1111(2)establishedfrom this tumor. DNA waspurifiedin passage 3(p3)or16

(p16) of this cell line. All procedures used in DNA

isolation have beenreportedelsewhere(10, 15).These DNApreparations (10 ,ug perslot)or0.5 ngof Adl2

virionDNA(equivalentto20genomeequivalentsper

cell), mixed with 10,ugof B3 hamster DNAascarrier, were cleaved withMspIorHpaIIasindicated. Frag-ments were separated by electrophoresis on a 1% agarosegel, blotted (13), andhybridizedtoAdl2 DNA that was32P-labeledby nick translation (11).Letters A

toSdesignate marker DNAfragments.

Adl2 DNA. Moreover, the cell lines described

herewere derived fromclonedcells.

We alsoinvestigatedin detailthe shifts in viral

DNA methylation that occurred upon

cultiva-tion of Ad12-inducedhamstertumor cells. These

shifts fromlow-level DNA methylation to more

extensive levels did not occur randomly but

followed certain patterns. Theincrease in Adl2

DNAmethylation was surveyed by cleaving the

DNA fromtumor cells at various passages with

HpaIIorMspI, by gel electrophoresis and

blot-ting,

and by hybridization to

32P-labeled

Adl2 DNA. The MspI (recognition sequence

5'-CCGG-3')

mapof theleftterminal 26%and the

rightterminal40% ofthe Adl2genome has been

J. VIROL.

establishedpreviously (8). AlthoughMspI sites

in the remaining 34% ofthe viralgenome have notyetbeenmapped,the MspIfragments E, F, K, L, M, and Pwere localized in the EcoRI B

fragment by blotting MspI-cleaved Adl2 DNA

and by hybridization to the cloned EcoRI B

fragment(18). Inaddition, fragmentB harbored

a large number ofvery small MspI fragments.

The schemes inFig. 2aassigned map locations

to MspI fragments A to S (4,150 to about 504

base pairs in length). Figure 2b presents

previ-ously published partialMspI maps (8).

The result presented inFig. 3 actually

demon-strated the increase in DNA methylation in the

integrated Adl2 genomes by comparing the

HpaIlandMspI cleavage patterns of viral DNA

from the tumor T1111(2) with those from cell

lineH1111(2)inpassage3 andpassage16. Itwas apparentthat HpaII bands predominantly from

the (late) EcoRI B fragment and from the right

terminal EcoRI A fragmentstarted disappearing

atlowpassagelevels,whereas theHpaIIbands

7-.

C - I

7n- cnc)

I

-M'sp1.

[image:4.492.87.212.73.361.2]

FK

FIG. 4. Changes in methylation

patterns

in Ad12-induced hamstertumorline H1313.Experimental con-ditions were similar to those described for Fig. 3. T1313 and H1313 p6 refer to DNA from the Ad12-induced tumorT1313 and the cell line H1313 in

pas-sage6, respectively.

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VOL. 47, 1983 NOTES 635

o r - t:

+ + + + + ++++ ++ ++++ ++ + ++ + + + + + + *+ + +++++ ++ +++++ ++ +++++ + ++++ ++ ++++ ++ ++ + + + ++ + +++++ + + + + ++ + + ++++ + ++ + + + + + + ++ + ++ + +++ + +++ + + t0J X W _ I.

-gkJ

- 0-_ _ -6- b_-t-J 0 -o _ _ I._ x C) " <n PZ 0e Pz aF a n (L) v) C., (A 9 (A 0

_z

0 n- I.-0. 0 CL co t= 0. 0

I0

Cs m CD

O0

Ds 0 3 Cs rJ C0. 0. CD Cs 0. U) Cs 0z 0q Cs Os

in theearly regions of the genome, particularly

in region El, remained unaltered. The data

presentedin Fig.3and4demonstratedthat with

increasing passage ofcells inculture the HpaII

bands C, D, E, F,H, I, K, L, M,andprobably

smaller fragments as well started to disappear

frompassage 3 onward.Disappearance of HpaII

bands obviously signified methylation at the corresponding 5'-CCGG-3' sites. At higher pas-sage levels, mainly the HpaII sites in early regions oftheintegrated viralgenomeremained unmethylated. Thus, the pattern of DNA meth-ylation approached thatobserved in Adl2-trans-formed cell lines (15). Similar resultswere

pre-sented for DNA fromtumorT1313andcellline H1313 (Fig. 4). It was interesting to note that establishment of the finalpatternofmethylation required many cellgenerations after fixation of the viral DNA in the host genome. Similar observationswere made with othertumors and

tumorlines and also after cleavage of the DNA

withHhaI (10). In severalof the Ad12-induced

tumorsandtumorlinesa

1,400-base-pair off-size

fragmentwasapparentthatcorrespondedtothe junction between the left terminus of viral DNA and cellular DNA. In some cell lines, the 5'-CCGG-3' sites

flanking

this

fragment

became methylated with continued passage; in other lines, thesame sites remainedunmethylated.

In Table 1, the results ofmany experiments

aresummarized.Ingeneral, the conclusion held thatwith

increasing

passage the late

regions

of

theAdl2genome wereprogressively methylated

at5'-CCGG-3' sites.

A total of 17 Adl2-induced tumors and 3

tumor cell lines were screened for the

occur-rence of Adl2-specific cytoplasmic RNA. By

Northernblotting (1, 12), viralRNA was

detect-edinonlytwo tumorsin smallamounts(datanot

shown). Thus, the low level of viralDNA

meth-ylation

did not correspond to

full-fledged

expression of the inserted viralgenome. On the

contrary, the viral genomesseemed to be

com-pletelyshutoff; not eventhe El region seemed

tobeexpressed. Itwill be

interesting

to

investi-gatewhat factors influence the levels of methyl-ation of inserted Adl2DNA andby what mecha-nisms the

expression

of the viral genome is controlled at

early

and late times after the fixa-tion offoreignDNAin thetumorcells. Obvious-ly, the stringency ofthese control mechanisms

could

change

with timeofpassage under culture

conditions.

I.K.wassupported byafellowship oftheFriedrichEbert

Foundation. This researchwasmadepossible byagrantfrom

the Deutsche Forschungsgemeinschaft through

Sonderfors-chungsbereich74C1.

We thank Hanna Mansi-Wothke for the preparation of media.

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636 NOTES

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