adenovirus oncogenesis: alterations in cellular methylation and transcription patterns − factors...

Gene Funct. Dis. 2001, Vol. 2, 4, 1392150 139

Walter Doerfler Adenovirus oncogenesis: alterations in cellularInstitute of Genetics, University methylation and transcription patterns 2 factors inof Cologne, Köln, Germany

viral oncogenesis?

My laboratory has been interested in the consequences of the insertion of foreign DNAinto established mammalian genomes and has studied this problem in adenovirus type 12(Ad12)-transformed cells or in Ad12-induced hamster tumors. Ad12 is a potent oncogenicagent in newborn Syrian hamsters. Since integrated foreign genomes are frequently denovo methylated, it appears that they might be modified by an ancient defense mechanismagainst foreign DNA. In cells transgenic for the DNA of Ad12 or for the DNA of bacterio-phage λ, changes in cellular methylation and transcription patterns have been observed.Thus, the insertion of foreign DNA can have important functional consequences which arenot limited to the site of foreign DNA insertion. These findings appear to be relevant alsofor viral oncology, tumor biology and for the interpretation of data derived from transgenicorganisms. For most animals, the main portal of entry for foreign DNA is the gastrointesti-nal tract. We have investigated the fate of orally ingested foreign DNA in mice. NakedDNA of bacteriophage M13 or the cloned gene for the green fluorescent protein (GFP) ofAequorea victoria have been used as test molecules. At least transiently, food-ingestedDNA can be traced to different organs and, after transplacental transmission, to fetusesand newborns. There is no evidence for germ line transmission or for the expression oforally administered GFP DNA.

Keywords: mechanisms of viral oncogenesis, integration of foreign DNA, de novo methylationof integrated foreign DNA, alterations of transcription patterns, fate of food-ingested foreign DNA

1 A fresh look at a valuable model

In 1962, Trentin, Yabe, and Taylor [1] reported that the injec-tion of purified human adenovirus type 12 (Ad12) into new-born Syrian hamsters (Mesocricetus auratus) induced tu-mors at the site of injection. This seminal observation hasmotivated a large body of research on the adenovirus sys-tem which, in subsequent decades, received recognition notonly as a tumor virus but, perhaps more importantly, as amajor model system in mammalian molecular biology. Manyfundamental principles in mammalian molecular biologyhave evolved from research on adenoviruses and their inter-actions with host cells. Much of this work has become text-book knowledge and the details have been reviewed inmajor treatises on the molecular biology of adenoviruses[224].

Therefore, the present review will not repeat basic infor-mation on the virology or molecular biology of adenoviruses,

Correspondence: Walter Doerfler, Institut für Genetik, Uni-versität zu Köln, Weyertal 121, D-50931 Köln, Germany.Phone: +49-221-470-2386; Fax: +49-221-470-5163; e-mail:[email protected]

WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001 0173-0835/01/0410-0139 $17.50+.50/0

but will rather concentrate on recent results and new aspectsof adenovirus oncogenesis. Among the oncogenic humanadenoviruses, Ad12 has been most extensively studied. Itsoncogenic capacity is not restricted to the transformation ofcells in culture under somewhat artificial conditions. The vi-rus actually can induce tumors soon after the injection ofAd12 in a high percentage of the animals surviving this treat-ment, and details on the molecular biology of these tumorswill be one of the subjects of this article. The Ad12 genomecomprises 34.125 nucleotide pairs [5], and the genome isorganized in a similar way as the genome of adenovirus type2 (Ad2) which has been most extensively studied. Ad12 be-longs to the subgroup A of human adenoviruses, Ad2 to sub-group C. A comparison of the nucleotide sequences be-tween the genomes of Ad2 and Ad12 reveals sequencesimilarities between 30 to 40% and up to 60 to 70% de-pending on the locations of individual viral DNA segments[5].

Ad12 infects human cells in culture productively. The tem-poral course of viral gene expression is similar to that of Ad2DNA, except that genome replication and late transcriptioncommence later in Ad12-infected cells than in cells pro-ductively infected with Ad2. In Syrian hamster cells, Ad2 also

140 W. Doerfler Gene Funct. Dis. 2001, Vol. 2, 4, 1392150

undergoes a productive infection cycle, although the viralyields seem to be lower than in human cells. In contrast,Syrian hamster cells are completely nonpermissive for theproduction of Ad12 and the replication of its DNA. It appearsplausible that the non-permissivity of Syrian hamster cellsto the infection with this virus is closely related to, if not aprecondition for, the tumor induction in Syrian hamsters byAd12. Ad2, however, fails to lead to tumorigenesis in ani-mals. Ad12 does not kill the abortively infected cells, andpersistence of the Ad12 viral genome in an integrated statein the genomes of Syrian hamster cells is compatible withcell survival, probably depending on the site of viral DNAintegration. Since the abortive response of Syrian hamstercells to Ad12 infection has proved to be an essential precon-dition for the oncogenic potential of this virus in hamstercells, we have studied the details of this abortive mode ofinfection by Ad12.

2 The abortive infection of hamster cellswith Ad12

2.1 Absence of Ad12 DNA replication in Syrianhamster cells

It is not certain by which route Ad12 enters into Syrian ham-ster cells, via a receptor akin to the adenovirus coxackievirusreceptor (CAR) [6, 7] or via an independent pathway. Shortlyafter adding the virus to the cells in culture, Ad12 particlescan be visualized, e.g. by electron microscopy, in the cyto-plasm of Ad12-inoculated BHK21 hamster cells, and viralDNA can be traced to the nuclei of these cells. We do notknow how the efficiencies in adsorption and penetration ofAd12 virions compare between permissive human and non-permissive hamster cells. It appears that Ad12 virions ad-sorb by far more efficiently to the membranes of humanHeLa cells than to those of hamster BHK21 cells (D. Webb,M. Hösel, and W. Doerfler, unpublished experiments). TheAd12 DNA can be detected in the nuclei of inoculatedBHK21 cells starting at 2 h postinfection (h p.i.) by themethod of fluorescent in situ hybridization (FISH) [8] or byanalyses of the intranuclear DNA by equilibrium centrifu-gation in CsCl density gradients [9]. Even with the most sensi-tive methods, Ad12 DNA replication cannot be detected inprimary or BHK21 Syrian hamster cells [10213]. Hence, theblock in Ad12 replication in the nonpermissive Syrian ham-ster cells is caused by deficiencies in early stages of theviral replication cycle and/or in the lack of essential cellularfunctions. We have determined two modes in which the repli-cation of Ad12 DNA can be complemented in Syrian ham-ster BHK21 cells:

(i) The Syrian hamster cell line BHK297-C131 carries the leftterminal 18.7% of the adenovirus type 5 (Ad5) genome inan integrated state and expresses its E1 functions constitu-tively. Upon Ad12 infection of these cells, Ad12 DNA can rep-

licate at a moderate rate which, however, lies below that inpermissive human HeLa cells [12, 14-16]. Thus, the E1 func-tions of Ad5, which, like Ad2, can replicate in BHK21 ham-ster cells, provide the necessary factors to stimulate Ad12DNA replication. Ad12 virions are not produced in this comp-lementing system.

(ii) In Ad12-infected BHK21 hamster cells, the early Ad12functions E1A and preterminal protein (pTP), a product ofthe E2B region of the Ad12 genome, are synthesized in mini-mal amounts. In contrast, the E2A function DBP (DNA bind-ing protein) is transcribed efficiently. When the E1 functionsof Ad12 or of Ad2 or, surprisingly, the pTP function by itselfare overexpressed as transfected plasmids in Ad12-infectedBHK21 cells under the control of the strong early humancytomegalovirus promoter, Ad12 DNA is replicated as unitlength molecules as has been determined by zone velocitysedimentation experiments in alkaline sucrose gradients, fol-lowed by Southern blot identification of the peak fractionsas Ad12 DNA by hybridization [16]. The Ad12 DNA newlysynthesized in this system is linked to pTP. An above-thresh-old level of pTP apparently suffices even in the presence ofonly minimal amounts of the E1A functions of Ad12 DNAto elicit Ad12 DNA replication in the nonpermissive Syrianhamster BHK21 cells. In BHK21 cells which overexpress thetransfected pTP plasmid and are subsequently infected withAd12, late Ad12 transcription is barely, if at all, detectable.Late proteins and Ad12 virions are not synthesized.

2.2 Early, but not late genes of the Ad12 genomeare transcribed in Syrian hamster BHK21cells

The early Ad12 functions E1A, E1B, E2A, E2B, and parts ofthe E3 and E4 genes are transcribed in Ad12-infected Syrianhamster cells, some at very low, possibly sub-thresholdlevels, too low at any rate to allow Ad12 DNA replication.The transcription of none of the viral structural protein genesis, however, detectable. The L1 segment of the Ad12 gen-ome or its single VA (virus associated) RNA is not tran-scribed either [17]. In the complementing BHK297-C131 cellsystem, late viral genes are transcribed; the Ad12 fiber-specific mRNA is polyadenylated, carries the tripartite leadersequence and the authentic fiber sequence, but cannot betranslated in the Syrian hamster cells [12]. We surmise thatin addition to the block in Ad12 DNA replication and latetranscription, there exist additional deficiencies which pro-hibit late Ad12-specific RNAs to be translated into late pro-teins. Hence, Ad12 replication in Syrian BHK21 cells ap-pears to be blocked at multiple steps of the viral replicationcycle. However, it is also conceivable, though not very likely,that the expression of one or of several early Ad12 functionsis so low that all the other viral functions cannot be ex-pressed.

Gene Funct. Dis. 2001, Vol. 2, 4, 1392150 Viral oncogenesis 141

2.3 Cellular factors

Recent analyses suggest that the nuclear factor NFIII is notefficiently transported into the nucleus of Ad12-infectedBHK21 hamster cells, whereas the nuclei of Ad2 pro-ductively infected BHK21 hamster or Ad2 or Ad12 pro-ductively infected human HeLa cells contain sufficientamounts of NFIII. NFI, on the other hand, is present in com-parable amounts both in productively and abortively infectedcell nuclei [15].

The cellular promyelocytic leukemia (PML) protein is mor-phologically redistributed from globular to track-like struc-tures in Ad12 or Ad2 productively infected human HeLa cellsor in productively Ad2-infected BHK21 hamster cells, but notin Ad12 abortively infected hamster cells. Since the PML do-mains are thought to play a role as structural nuclear ele-ments also in adenoviral DNA replication [18], the lack oftheir conversion from globular to track-like in the abortivesystem is consistent with the block in Ad12 DNA replication.However, when this conversion is elicited in the Ad12-in-fected nonpermissive BHK21 hamster cells by transfectionand overexpression of the ORF3 (open reading frame) ofthe Ad12 region E4, Ad12 DNA replication still does not en-sue [15]. Hence, the structural conversion of the PML pro-tein-containing complex in hamster cells by itself does notsuffice to render these cells competent for Ad12 DNA repli-cation.

3 Foreign DNA insertion and itsconsequences for the genome

The long-term genome-wide consequences of the insertionof foreign DNA into an established mammalian genomehave been one of the foci of our research. We set out tostudy the mechanism of tumor induction by the human adeno-virus type 12 (Ad12) in newborn Syrian hamsters (Mesoc-ricetus auratus) [1, 19]. In each tumor cell, multiple copiesof the Ad12 genome are covalently linked to the DNA of thecell. The chromosomal insertion site is identical in all cellsof a given tumor but varies from tumor to tumor. The charac-teristics of the adenoviral integration sites and possiblemechanisms involved in the insertion event have been sum-marized in chapter 6 of this review. The Ad12-induced tu-mors are most likely of clonal origins. Revertants of thetumor cells, which have lost all or a part of the multiple cop-ies of integrated Ad12 DNA, arise occasionally and retainthe oncogenic phenotype even when all viral genomes havebeen lost [20, 21].

The mechanism of Ad12 DNA insertion appears to be verysimilar, if not identical, to that for many other types of foreignDNA which can become integrated into mammalian gen-omes as well. Apparently, it does not matter whether theforeign DNA reaches the nucleus of a cell via virus infectionor by one of the somewhat artificial transfection procedures

which have been developed to transport foreign DNA intonuclei of cells. Mammalian cells and their genomes seem toharbor a highly flexible set of mechanisms to accept andrecombine with foreign DNA. It is a challenging question toconsider the evolutionary significance of the integration re-action, e.g., as a means to accept new genetic informationinto the existing genome. The presently available data onthe human genome have raised the question of whethersome of the present-day human genes might have been de-rived by “horizontal transfer from bacteria at some point inthe vertebrate lineage” [22]. However, the extent and func-tional significance of these additions remain enigmatic andcontroversial.

Foreign DNA integrates, particularly those consisting of viralDNA, frequently comprise between 5 to 30 copies of the viralgenome. This number can add up to a megabase (Mb) offoreign DNA, e.g. for Ad12 or bacteriophage λ DNA whichhas been inserted into the accepting genome. This size of aninsert can probably not be accommodated without causingdisturbances in the stability of the genome. This perturbationmight be transmitted to neighboring DNA sequences, to thechromatin structure and possibly to adjacent chromosomeswhich are in contact with the insertion site of that chromo-some which has been targeted by foreign DNA. There is onlyscanty information about the consequences of foreign DNAinsertion for the recipient genome. We have studied

(i) the de novo methylation of the integrated foreign DNAand

(ii) the alterations in DNA methylation and transcription pat-terns of cellular DNA sequences also at sites remotefrom the site of integration.

Over an evolutionary time span, virus infections and the inte-gration of retroviral genomes or of retrotransposons havecontributed to a considerable extent to the accumulation ofrepetitive DNA sequences in mammalian genomes, as hasbeen very precisely documented, e.g. for the human gen-ome [22]. Viruses have perfected highly specialized mecha-nisms to transport their genomes into the nuclei of host cellsand to fix their genomes there permanently by integrationinto the host genome. On the other hand, the main portalof entry of foreign DNA into mammalian organisms is thegastrointestinal tract and the constant oral uptake of foreignDNA by food ingestion. Since integrated foreign DNA inmammalian and plant cell systems becomes frequently denovo methylated, possibly as a consequence of the activityof an ancient host defense mechanism [23], it has been rea-soned that all organisms must have been frequently ex-posed to foreign DNA since the earliest times of evolution.In our laboratory, this argument has led to an extensiveseries of studies on the fate of food-ingested DNA as themain source of foreign DNA in nature [24227] (see alsochapter 10).


4 General significance of the study onforeign DNA integration

The results derived from basic research are often of generalinterest for work on problems in biomedical research. Theinsertion of foreign DNA into established mammalian gen-omes plays an important role in several areas of experimen-tal biology. The following topics will be considered here:

(i) Oncogenesis. For many years, we have pursued theworking hypothesis that the integration of foreign DNA is a“conditio sine qua non” at least in viral oncogenesis [28].The perturbations in chromatin structure and alterations incellular and viral transcription and methylation patterns inthe wake of foreign DNA insertion are thought to render adecisive contribution to the transition from a normal mam-malian cell to a tumor cell [29, 30]. The results on the fateof food-ingested DNA in the mammalian organism raise thequestion of whether food-ingested DNA plays a role inpathogenesis and/or oncogenesis in mammalian organisms.

(ii) In transgenic organisms, foreign DNA has been insertedby homologous or heterologous recombination into animalor plant genomes. So far, the consequences for the stabilityof the recipient genomes have not been thoroughly investi-gated. The interpretations of the results derived from experi-ments with transgenic organisms may still have to be viewedwith caution, since the activity profiles of an unknown num-ber of genes in the transgenic organism may be altered, dueto the insertion of foreign DNA.

(iii) Similar reservations hold for gene fixation regimens insomatic gene therapy.

5 A different way of looking atadenovirus oncogenesis

A great deal of interesting analytical work has been devotedto investigations on the role of the early adenoviral E1 andE4 gene functions for the mechanism of the transformationof cells in culture by human adenoviruses [for reviews31234]. As already mentioned, our group has concentratedon studying the oncogenic mechanisms after the injection ofAd12 virions into Syrian hamsters. Transformation of cells inculture and actual tumorigenesis by Ad12 in living animalsare most likely two different events, although it is difficult tocompare both processes directly.

In this chapter, I wish to delineate an alternate mechanisminvolved in Ad12 oncogenesis. In Ad12-induced tumors aswell as in Ad12-transformed cells in culture, each tumor ortransformed cell carries multiple copies of the persisting viralgenome in a chromosomally integrated form. The foreigngenomes are usually inserted as a bulk integrate at onechromosomal site thus leading to the addition of one half toone megabase of Ad12 DNA to an established mammalian

genome. We have only begun to investigate in what waythis gross alteration of the genome leads to perturbationsaffecting its structure and function. One key to understandthe functional consequences of massive foreign DNA inser-tion for the cell lies in the alterations that the chromatin struc-ture is exposed to at the immediate site of foreign DNA inte-gration and at locations remote from this site. So far, wehave documented extensive alterations in the methylationand transcription patterns of Ad12-transformed cells in cul-ture as well as in cultured cells which are transgenic for theDNA of bacteriophage lambda [35237]. The insertion oflambda DNA is not known to be associated with the onco-genic transformation of cells. It is likely that the integrationof multiple copies of any foreign DNA can elicit alterationsin the cells’ methylation and transcription patterns. Althoughthe mechanisms that connect genome perturbations due toforeign DNA insertions and alterations in gene transcriptionremain enigmatic, it will be interesting to pursue the possibil-ity that these perturbations could be closely linked to theoncogenic transformation of cells. Except for rare examples,tumorigenesis is difficult to explain by changes in the activityof only one gene. The possibility that tumorigenesis is re-lated to fundamental rearrangements of chromatin and theensuing wide-ranging changes in the transcriptional profilesof the cell has not widely been investigated. The literatureprovides ample evidence for multiple alterations in the ex-pression patterns of many different genes in tumor as com-pared to normal cells. A consistent synopsis will be neededfor individual, strictly defined tumor entities and the changesof all transcriptional patterns in these tumor cells vis-a-visnormal cells. It can be expected that the DNA microarraytechnology will be widely applied to these types of studiesfor many different tumor diseases.

6 A synopsis of the characteristicsof foreign (Ad12, Ad2, λ) DNA integrationinto mammalian genomes

Most of the analyses performed in our laboratory have usedAd12 DNA, in some instances Ad2 or bacteriophage λ DNA.It is likely that many of the observations described in thischapter hold true for any type of foreign DNA which is inte-grated into a recipient mammalian genome. However, sincethe mechanism(s) of insertional recombination in mam-malian cells seem to be very flexible, we do not claim thatthe following catalogue of parameters will be complete or all-inclusive. Overviews on related topics have been publishedpreviously [29, 30, 38].

Adenovirus DNA is integrated into the host genome in trans-formed cells and in Ad12-induced hamster tumor cells [9,19, 28, 39242]. Free Ad12 DNA has never been found inAd12-induced tumor cells. In productively infected humancells, Ad12 DNA associates with the chromosomes startingearly after infection [8]. Ad12 or Ad2 DNA can probably re-


combine with human cellular DNA in this system as well [43,44]. A symmetric recombinant (SYREC1) carries the 2081left terminal Ad12 DNA nucleotides which flank a long palin-drome of human cellular DNA on both termini. This DNAmolecule with the length of Ad12 DNA is packaged into Ad12virions [45, 46], since the terminal Ad12 DNA segment stillcarries the viral packaging signal. The existence of this re-combinant has proven recombination to occur between in-fecting Ad12 DNA and the host genome of the target humancells in the productive system. The nucleotide sequenceanalyses of several integration sites of Ad12 or Ad2 DNA inthe host genome have not revealed a unique or specificcellular target sequence for the insertion of viral DNA [38].Most sites of viral DNA integration in transformed and tumorcells or of viral-cellular DNA recombination products gener-ated in a cell-free system are characterized by perfect se-quence homologies of between 8 and 9 nucleotides or bypatchy homologies of up to 20 nucleotides in length betweenthe cellular and viral recombination partners [41, 47250].

In the process of integration, terminal viral nucleotides be-tween 2 2 174 bp in length have been shown to be deletedin different integration events [41]. At the site of insertion,cellular DNA can also be deleted [51]. At one of the existinginsertion sites, however, not a single cellular nucleotide hasbeen missing [52].

In about 60 different Ad12-induced tumors, each tumor cellin a given tumor carries the Ad12 integrates at one, rarelyat two, chromosomal sites which are identical in one tumor,but differ from tumor to tumor [19], even when more thanone tumor arises in one animal. The tumors, therefore, aremost likely of clonal origin.

In Ad12-induced tumor cells or Ad12-transformed cells, upto 30 copies of viral DNA are integrated, at least some ofthem intact. There is a pearl-like array of Ad12 integrateswhich are, however, separated from each other by cellularor rearranged viral DNA sequences [8, 53].

The cellular preinsertion sites are frequently transcriptionallyactive in cells prior to Ad12 infection and viral DNA inte-gration [54, 55]. Transcriptionally active chromatin may exhi-bit an increased propensity for the recombination with for-eign DNA which has entered into the cell nucleus.

Ad12 DNA integration in general is very stable, even afterthe transfer and continuous propagation of tumor cells in cul-ture for many generations [19, 40]. Occasionally however,the Ad12 integrates can be lost in part or completely [56].The cells devoid of Ad12 DNA, nevertheless, retain theironcogenic potential. This observation has supported theidea of a hit-and-run mechanism of Ad12 oncogenesis inhamsters [20, 21].

Ad12 DNA integrated into the hamster cell genome becomesde novo methylated in specific patterns [40, 57]. Virion DNA

extracted from purified Ad12 particles is not methylated [58].An inverse correlation between DNA methylation and adeno-virus gene activity has been described for the first time inadenovirus-transformed cells [57, 59]. De novo methylationappears to be regionally initiated in paracentrally locatedsegments of the integrated Ad12 genomes [60] and spreadsfrom these distinct regions to other parts of the genome[61, 62].

As a further consequence of Ad12 or other foreign (bacterio-phage λ) DNA integration, cellular DNA methylation andtranscription patterns can be altered, even at sites remotefrom the integration locus [35237, 63]. The loci affected bythese genome perturbations may be related, in an as yetunknown way, to the site of foreign DNA integration. In spiteof robust searches, we have not obtained any evidence forheterogenicities in cellular methylation and transcription pat-terns in subclones of nontransgenic BHK21 cells whichmight have preexisted before foreign DNA integration [36,37].

Most of the parameters described here hold true also forbacteriophage λ DNA which has become integrated intohamster cell DNA after the transfection of cells selected forthe expression of the cotransfected and cointegrated neo-mycin phosphotransferase gene [35, 36]. Apparently, thetype of foreign DNA and the mode of transfer into the recipi-ent cell nucleus do not affect the cellular mechanism(s) re-sponsible for the insertional recombination with foreignDNA.

7 Repetitive DNA sequences might protectthe genome by serving as receptors forforeign DNAs

A major part of all mammalian genomes consists of repeti-tive DNA sequences and of retrotransposon-like retroviralelements the function of which is completely unknown.These sequences are likely evolutionary remnants which,nevertheless, may continue to play an important role in thegenome. With the emphasis in our research on the inte-gration of foreign DNA into established mammalian gen-omes, repetitive DNA sequences are of particular interest.The presence of multiple copies of retroviral sequences inmammalian DNA again underscores the frequency and long-term stability of foreign DNA integration events. Is it advan-tageous for the species if essential genes are hidden in ahuge excess of repetitive elements as a protective strategyas it were? Could the potential damage done by high energyradiation or by the integration of foreign DNA be diverted inthis way to less sensitive segments in the genome? Wouldthe repetitive sequences serve a function as that part of thegenome which could rather innocuously and permanently beinvaded by foreign DNA, such as Ad12 DNA, which wouldthus be less likely to destroy essential cellular functions


upon its integration into genes important for the survival ofthe cell?

8 De novo methylation of integrated foreignDNA

We have investigated the de novo methylation of integratedforeign DNA in two different experimental systems, (i) inte-grated Ad12 genomes, and (ii) the mouse B lymphocytetyrosine kinase (BLK) gene which has been reintegrated byhomologous or by heterologous recombination into themouse genome [64]. Our current, still limited understandingof problems related to de novo methylation, which havebeen investigated in these instances, can be summarizedas follows.

8.1 Integrated Ad12 genomes in hamster tumorcells.

In our first experiments in this project, integrated Ad12 gen-omes in hamster tumor and transformed cells could not becleaved with methylation-sensitive restriction enzymes, likeHpaII or HhaI [40], whereas the HpaII isoschizomer MspIcleaved the inserted Ad12 DNA to the same pattern as Ad12virion DNA which we had shown not to be methylated [58].After this discovery of the de novo methylation of integratedforeign DNA in mammalian genomes, we started an inten-sive series of experiments on the biological functions of DNAmethylation in mammalian cells. A further discovery [57, 59]established, for the first time, inverse correlations betweenthe levels of promoter methylation and promoter activity.Soon afterwards, we documented that the sequence-specificmethylation of viral and other eukaryotic promoters led tothe inactivation of these promoters [65271].

In the following years, we investigated DNA methylation pat-terns in the human genome because we reasoned that DNAmethylation must have biological functions which reach be-yond the long-term shut-down of promoters. This more gen-eral role was thought to be in the establishment of chromatindomains for which patterns of DNA methylation might pre-sent the initial scaffolding directly on the DNA molecule onwhich more complex structures could be built by specificDNA-protein interactions. Therefore, a number of humangenome segments were investigated for their specific DNAmethylation patterns [72281]. Patterns of methylation werehighly specific for different parts of the human genome anddifferent from cell type to cell type, possibly even from cellto cell within one cell type [82]. The patterns of DNA meth-ylation in the human genome were frequently found to behighly conserved from individual to individual [72, 74281].

We have started a program to elucidate the establishmentand mechanisms of de novo methylation by using integratedAd12 genomes as models [60]. Currently, our results can be

summarized as follows: Upon the integration of Ad12 DNAinto the genome of Ad12-induced tumor cells, the de novomethylation appears to be initiated at specific sites in thegenome. The results gleaned from analyses using the bisul-fite protocol of the genomic sequencing technique indicatethat the initiation of de novo methylation is not at a specificnucleotide or a narrowly restricted set of nucleotides. Thepatterns of methylation in the cellular DNA surrounding thesite of foreign DNA integration may have a decisive influ-ence on the patterns of methylation generated in the inte-grated foreign DNA and on the time course of establishingthese patterns. From the site(s) of initiation of de novo meth-ylation, this modification spreads gradually and progress-ively, but not uniformly, across major parts of the integratedAd12 genome. Certain parts of the Ad12 genome, particu-larly those that are actively transcribed in the tumor cell, re-main unmethylated or become only hypomethylated. Furtherwork will be required to understand this mechanism in depth.We surmise that the chromatin-like structures neighboringand across integrated genomes of foreign derivation, like theAd12 genome, might have a decisive influence on the in-itiation and spreading [61, 62] of de novo methylation andmight differ from the bulk of the recipient genome.

8.2 De novo methylation of foreign nonviral DNAthat has been integrated into the mousegenome by homolgous or by heterologousrecombination

We have reinserted the mouse gene for the B lymphocytetyrosine kinase (BLK) into the mouse genome by homolo-gous recombination into its authentic genomic site on one ofthe chromosome 14 alleles [64]. In this case, the previouslyunmethylated BLK gene which had been cloned and propa-gated in a methylation-deficient bacterial host became re-methylated in exactly the same pattern which preexisted onthe unmanipulated mouse alleles. When the BLK genelanded, however, by heterologous recombination some-where at random sites in the mouse genome, different pat-terns of de novo methylation were observed. We submit thateach site in the mouse genome might have specific “memorysignals” for the establishment of a given methylation pattern.These signals could be related to specific chromatin proper-ties and/or secondary DNA structures at a given genomicsite. Furthermore, foreign genes which had been attachedto the BLK gene, like the luciferase gene, under the controlof the weak E2AL promoter of Ad2 or the strong early SV40promoter, seemed to be differently methylated, dependingon promoter strength. Tethering of the gene to a weak pro-moter frequently resulted in hypermethylation, neighborhoodto a strong promoter in hypomethylation or the absence ofde novo methylation. This dependence on promoter strengthdid not hold for all sites in the genome, but was most clearlyobserved, when the construct had been reinserted by hom-ologous recombination [64].


9 Genome-wide perturbations in themammalian genome upon foreign DNAinsertion

We have started to investigate the structural and functionalconsequences of the insertion of foreign DNA into estab-lished mammalian genomes [35237, 63]. The de novomethylation of the integrated DNA and alterations in the pat-terns of DNA methylation in the recipient genomes at thesite of insertion and remote from it have been of particularinterest. By using different methods, including the bisulfiteprotocol of the genomic sequencing technique [83, 84], wehave documented extensive changes in the patterns of DNAmethylation at several cellular sites remote from the loci ofinsertion of the DNA of Ad12, and lesser changes in cellstransgenic for the DNA of bacteriophage λ [35, 36]. Since λDNA is not transcribed in transgenic mammalian cells, alter-ations of methylation patterns subsequent to foreign DNAinsertion are not directly dependent on foreign gene tran-scription. It has been shown earlier that cellular DNA se-quences immediately abutting the integrated Ad12 DNA inhamster tumor cells also exhibit changes in DNA methylation[85]. It is presently unknown by what mechanisms the inser-tion of foreign DNA affects the organization and function ofthe recipient genome. Does the site of foreign gene inte-gration determine where the remote effects occur, and is acritical size of integrated foreign DNA required? We surmisethat the acquisition of many kilobases or even a megabaseof inserted DNA alters the chromatin topology and thus influ-ences the function of specific parts of the genome on chro-mosomes that are in contact with the site of foreign DNAintegration in the interphase nucleus.

A wide scope of cellular DNA segments and genes was ana-lyzed and searched for changes in DNA methylation andtranscription. The technique of methylation-sensitive rep-resentational difference analysis (MS-RDA) was based on asubtractive hybridization protocol after selecting againstDNA segments that were heavily methylated and hence rare-ly cleaved by the methylation-sensitive endonucleaseHpaII. The MS-RDA protocol led to the isolation of severalcellular DNA segments that were indeed more heavily meth-ylated in λ DNA-transgenic hamster cell lines [37, 63].

By applying the suppressive subtractive hybridization tech-nique to cDNA preparations from nontransgenic and Ad12-transformed or λ DNA-transgenic hamster cells, severalcellular genes with altered transcription patterns were clonedfrom Ad12-transformed or λ DNA-transgenic hamster cells.Many of the DNA segments with altered methylation patternswere isolated by a newly developed amplicon subtraction(MS-AS) protocol [63], and cDNA fragments derived fromgenes with altered transcription patterns were identified bytheir nucleotide sequences. In control experiments, no differ-ences in gene expression or DNA methylation patterns weredetectable among individual nontransgenic BHK21 cell

clones. This finding argues against methylation or transcrip-tional mosaicism among the BHK21 cell population investi-gated in this context. These studies have been performedwith clonal lines of BHK21 cells which have proved veryhomogeneous with respect to methylation and transcriptionpatterns [35237]. In this respect, clonal cells in culture pro-vide advantages over mouse embryonal stem (ES) cellswhich apparently present problems of heterogeneity with re-spect to methylation patterns.

In one mouse line transgenic for the DNA of bacteriophageλ, hypermethylation was observed in the imprinted Igf2rgene in DNA from heart muscle. Two mouse lines transgenicfor an adenovirus promoter-indicator gene construct showedhypomethylation in the interleukin 10 (IL10) and Igf2r loci.We conclude that the insertion of foreign DNA into an estab-lished mammalian genome can lead to alterations in cellularDNA methylation and transcription patterns. It is conceivablethat the genes and DNA segments affected by these alter-ations depend on the site(s) of foreign DNA insertion.

10 Persistence of food-ingested foreignDNA in the mammalian organism

When foreign DNA, like that of oncogenic viruses or of con-structs transfected into cells, is permanently fixed in estab-lished mammalian or plant genomes by covalent integration,the transgenic DNA is often de novo methylated in specificpatterns [40, 57, 61, 64, 86-88]. This mechanism of long-term gene silencing has been documented in many eukary-otic systems and has been interpreted as an ancient cellulardefense against the activity of foreign genes [23, 89]. Theexistence of this ubiquitous defense machinery raised thequestion of its evolutionary origin and prompted a search forthe main portal of entry of foreign DNA into organisms. Inmammals, the gastrointestinal tract and the large amountsof food-associated DNA undoubtedly qualify as the mostprominent candidates. In the late 1980s, we have thereforestarted to study the fate of food-ingested foreign DNA in thegastrointestinal tract of mice [24227].

Information on the stability and persistence of macromol-ecules in the intestinal system has previously not been avail-able. On the other hand, traces of DNA have been shown tosurvive for centuries, at least in fragmented form, in eventhe remains of extinct organisms [90, 91]. In previous investi-gations on the persistence of foreign DNA during the gastro-intestinal passage of food, we have orally applied test DNAsas unprotected purified molecules to mice in model experi-ments. The DNA of bacteriophage M13 or a vector plasmidcarrying the cloned gene for the green fluorescent protein(GFP) from Aequorea victoria tethered to different viral pro-moters (HCMV, SV40, or RSV) has been shown to persistup to 24 h in fragmented form in minute amounts in differentparts of the gastrointestinal canal and to gain access to cells


of the intestinal wall, of the Peyer’s patches, to peripheralwhite blood cells, and to cells in spleen and liver. Further-more, food-ingested DNA can transgress the placental bar-rier in pregnant mice. However, only a few cells in the em-bryo or fetus take up the foreign DNA into their nuclei [26].

We have recently chosen a natural scenario and fedsoybean leaves to mice [27]. The distribution of the plant-specific gene for the nucleus-encoded ribulose-1,5-bis-phosphate carboxylase (rubisco) gene has been studied inthe mouse organism. The rubisco gene or fragments of itcan be recovered from 2 h up to 49 h after feeding in theintestine, and up to 121 h in the cecum. In some experi-ments, rubisco gene-specific PCR products have also beenamplified from spleen and liver DNA. There is no evidencefor the expression of orally administered genes, as assessedby the RT-PCR method. Moreover, mice have been continu-ously fed daily with GFP DNA for 8 generations and havebeen examined for the transgenic state by assaying DNAfrom tail tips, occasionally by PCR from internal organs ofthe animals. The results have been uniformly negative andargue against the germline transfer of orally administeredDNA.

Upon the intramuscular injection of GFP DNA, authenticGFP DNA fragments have been amplified by PCR up to 17months post injection in DNA from muscle, up to 24 h postinjection in DNA from organs, like kidney, liver, and spleen,which lay remote from the site of injection. GFP fragmentscan also be retrieved from the intestinal contents up to 6 hpost injection. Apparently, the organism eliminates injectedforeign DNA via the liver-bile-intestinal route [27].

After the oral application of the GFP gene construct underSV40-, RSV-, or HCMV-promoter control, 50 µg daily for 21days, transcription of this foreign gene in various organ sys-tems has not been detected by the sensitive RT-PCRmethod. In contrast, expression of the same construct in theinjected mouse muscle, although not at remote sites, hasbeen documented by UV-microscopy and by RT-PCR afterintramuscular injection. Thus, the construct can be tran-scribed and translated at least in the muscle tissue of micewhere the injected foreign DNA persists in higher concen-trations as compared to remote organs. However, we havenot been able to adduce any evidence for the expression oforally administered foreign (pEGFP-C1) DNA.

As might be expected, there is no indication for the germlinetransmission of orally ingested DNA. The data available ar-gue for transplacental transmission to the fetus to a limitedextent when pregnant animals are fed. The nuclei of singlecells in small clusters have been found positive by the fluo-rescent in situ hybridization (FISH) technique, and the DNA-specific signals have been found exclusively in the nucleiand in rare instances in association with both chromatids infetal cells [26]. Apparently, cells of the germ line are sparedfrom this transmission.

The results on the fate of food-ingested foreign DNA in themammalian organism have been discussed among special-ists concerned about food in general and about geneticallymodified organisms in the food chain in particular. Althoughit will be mandatory to consider this problem case by case,the general public can be reassured by the realization thatall kinds of foreign genes in almost limitless combinationshave been part of the food chain throughout the evolution ofthe species Homo sapiens and other species as well. Formillenia, foreign genes and their breakdown products withhigh recombinatorial capacity have been constant partnersin our gastrointestinal inner milieu and that of other species.

AcknowledgmentsWe thank Petra Böhm and Susanne Scheffler for expert editorial

work. At different times, the research described was supported

by the Deutsche Forschungsgemeinschaft through Sonderfor-

schungsbereich 274, Teilprojekt A1, by the Wilhelm Sander Stif-

tung, München, and by the Fritz Thyssen Stiftung, Köln. Some

of the projects were also aided by the Bundesministerium für

Bildung und Wissenschaft (BMBW) (BEO-0311110), by a collab-

orative grant from the BMBW with Coley Pharmaceutical GmbH,

03-12235-06, the Center for Molecular Medicine Köln, TV13,

and by the Bayerische Staatsministerium für Landesentwick-

lung und Umweltfragen.

received August 17, 2001

accepted September 14, 2001

published online October 31, 2001

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