Proc. Natl. Acad. Sci. USAVol. 89, pp. 11523-11527, December 1992Genetics

DNA rearrangement causes hepatocarcinogenesis inalbumin-plasminogen activator transgenic mice

(genomic instability/hepatic regeneration/mitogenesis/tumor progression)

ERIC P. SANDGREN*, RICHARD D. PALMITERt, JANICE L. HECKELf§, RALPH L. BRINSTER*,AND JAY L. DEGENt*Laboratory of Reproductive Physiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104; tHoward Hughes MedicalInstitute and Department of Biochemistry, University of Washington, Seattle, WA 98195; and *Children's Hospital Research Foundation and Department ofPediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229

Contributed by Ralph L. Brinster, September 2, 1992

ABSTRACT Hepatocyte-directed production of uroki-nase-type plasminogen activator (uPA) in transgenic mice ishepatotoxic. Infrequently, hepatocytes arise that do not expressuPA, due to physical loss of transgene DNA, and these cellsclonally repopulate the entire liver within 3 months of birth.Surprisingly, hepatic tumors appear in these mice b nning at8 months of age despite the fact that uPA is not oncogenic orgenotoxic. Analysis of the transgene locus reveals that tumorsarise only from a particular subclass oftransgene-deficient cellsin which the entire transgene array, and possibly a significantamount of flanking DNA, is deleted. Considering that alltransgene-deficient regenerative nodules undergo extensivereplication but only a subset gives rise to tumors, we proposethat loss of genomic DNA, not mitogenesis per se, is a primarycarcinogenic determinant in this model of hepatocarcinogene-sis.

Carcinogenic agents typically induce a spectrum of alter-ations in target tissues and cells. Common to many of theseagents is the ability to enhance cellular proliferation, eitherdirectly or secondary to tissue injury and cell death (1, 2).Recently, the role of mitogenesis in carcinogenesis has be-come a controversial issue (3, 4). According to the classicalview, cell proliferation has two roles during this process(reviewed in ref. 2). First, it serves to permanently fixmutagen-induced (genotoxic) DNA damage before the targetcell can effect repair. This constitutes the step of tumorinitiation. Second, it facilitates clonal expansion of initiatedcells. There are now suggestions, however, based upon thecarcinogenicity of apparently non-genotoxic compounds,that cancer may result solely from enhanced mitogenesis,which serves to create, fix, and expand initiated cells (1-4).The crux of the argument would appear to rest upon both themagnitude of unrepaired DNA damage that accumulatesduring rapid cell division and the rate of spontaneous cellularinitiation.

Since 1985, many transgenic mouse lineages have beenproduced to model potential genetic mechanisms of cancer(reviewed in refs. 5 and 6). For example, the liver has beensubjected to neoplastic transformation by multiple oncogenesor growth factors (6), many of which can be thought of asgenetic tumor initiators that increase the likelihood of sub-sequent tumorigenesis. In contrast, in one hepatitis B virustransgenic model of hepatocarcinogenesis the transgene isnot an oncogene, nor is it obviously genotoxic (7-10). Albu-min (Alb) promoter/enhancer-directed expression of thehepatitis B virus large envelope polypeptide targets produc-tion of hepatitis B virus surface antigen (HBsAg) to hepato-cytes (7), causing chronic hepatocellular injury and death,

inflammation, and nodular hepatic regeneration, and culmi-nating in the development of hepatocellular carcinomas inmice over 1 year of age (9, 10). Chronic regenerative hyper-plasia undoubtedly contributes to carcinogenesis in thismodel. However, the processes of cell injury, cell death, andinflammation are accompanied by potentially genotoxicevents, such as the generation ofmutagenic free radicals, andthese are likely to be necessary agents of hepatocarcinogen-esis in this model (9, 10).We have described another transgenic model that dis-

played extensive and abnormal hepatic regeneration. Hepa-tocyte-targeted expression of murine urokinase-type plas-minogen activator (uPA) by an Alb-uPA transgene causedhigh-level ectopic production and secretion of uPA by theliver. This resulted in neonatal hemorrhaging within 4 days ofbirth in a large fraction of uPA-expressing founder mice (11),consistent with uPA's fibrinolytic and fibrinogenolytic activ-ities (12). Two transgenic lineages, 1353-8 and 1944-6, wereestablished from surviving founder mice, and, interestingly,only about half of the transgenic offspring in each lineagedeveloped bleeding and died as neonates; the remaindersurvived into adulthood and were used to maintain the lines.Unexpectedly, surviving transgenic mice displayed a gradualreduction of plasma uPA and regained normal clotting func-tion. This process was accompanied by overgrowth of theentire liver by clonal, regenerative hepatic nodules that nolonger expressed the transgene, apparently as the result ofchromosomal rearrangements that affected the transgenearray within nodule progenitor cells (13). Thus, cells relievedfrom the hepatotoxic effects ofuPA preferentially replicatedin response to a deficit of liver function, and replaced injured,transgene-expressing cells within 3 months of birth.We now report that transgene-associated DNA rearrange-

ments lead to the development of hepatocellular carcinomasin every adult mouse in these lineages. All tumors lacktransgene expression and appear to be derived from trans-gene-deficient cells. The tumors develop months after theperiod of intensive hyperplasia but in the absence of obviouscellular injury within the regenerative tissue, suggesting arole for mitogenesis in the process. However, our findingsindicate that regeneration per se is not a sufficient stimulusfor tumor formation in these mice; rather, tumor progenitorcells contain transgene-deleting chromosomal rearrange-ments that may extend into flanking DNA, implying thatspecific DNA rearrangements that function as initiatingevents are a requirement for neoplastic transformation in thismodel.

Abbreviations: Alb, albumin; HBsAg, hepatitis B virus surfaceantigen; uPA, urokinase-type plasminogen activator.§Present address: Division ofPediatric Hematology/Oncology, Chil-dren's Medical Center, Medical College of Virginia, Richmond, VA23298.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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MATERIALS AND METHODS

Transgenic Mice. The two lines of Alb-uPA transgenicmice employed in these studies have been described (11, 13)and have been assigned the following genetic designations:line 1353-8, Tg(Alb-1,Plau)Bril44, and line 1944-6, Tg(Alb-1,Plau)Bril45. The transgene contains a 2.3-kilobase (kb)fragment carrying mouse Alb enhancer and promoter ele-ments fused to a 6.4-kb fragment carrying the mouse uPAgenomic coding sequence. The 3' noncoding region has beenreplaced by the corresponding region of the human growthhormone gene, which provides the polyadenylylation signal.Transgenic mice were identified as described (11).

All mice employed in these studies were exposed to mousehepatitis virus (MHV), which is endemic in our colony. MHVis a coronavirus and thus unrelated to human hepatitis viruses(14). The hepatotropic (polytropic) form ofMHV causes onlya transient wave of cell death and regenerative hyperplasia in2- to 4-week-old immunocompetent mice; after this, theinfection is spontaneously cleared and mice retain protectivelevels of circulating antibody (15). Furthermore, nude micesurvive for many months in the colony and at sacrifice displaylesions in gut that are consistent with infection by theenterotropic form of MHV, which generally is not highlypathogenic in liver (data not shown). Thus, we do not believethat infection significantly influences tumor pathogenesis inthese mice. In support of this hypothesis, we did not observea difference in the pathogenesis ofoncogene-induced hepatictumors in other transgenic lineages in our colony before andafter infection with MHV (16).

Tissue Collection and Analysis. Livers and lungs wereexamined grossly, fixed in 10%1 neutral buffered formalin, andprocessed for microscopic viewing according to standardmethods. Hepatic lesions were classified by using publishedcriteria (17). For flow cytometric determination ofploidy andcell cycle characteristics, liver nuclei were isolated andanalyzed for DNA content as described (16). Plasma alanineaminotransferase activity was measured as described (13).

Nucleic Acid Analysis. mRNA was measured by solutionhybridization (13). Southern blots were prepared after stan-dard gel electrophoresis (0.8% agarose gels) or field-inversiongel electrophoresis (0.6% agarose gels) using a DNASTARpulsed-field inversion module. Genomic DNAs were ex-tracted and digested with EcoRV. Electrophoretically sepa-rated DNA restriction fragments were transferred to Gene-ScreenPlus (DuPont) filters, and the filters were hybridizedto a 32P-labeled 1.3-kb Sst I fragment ofthe murine uPA geneencompassing exon 8 through intron 10 (18).

Calculation of Line 1944-6 Transgene Rearrangement Fre-quency. Livers from three hemizygous 12-day-old line 1944-6mice were fixed and sectioned at 6 ,gm, and every eighth

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FIG. 1. Gross appearance ofliver in young Alb-uPA mice. Liverswere removed from line 1353-8 transgenic mice (Upper) or age-matched nontransgenic controls (Lower) at 19 (Left), 33 (Center), and88 (Right) days of age. The small pale mass at the top of the88-day-old transgenic liver is a regenerative nodule that had twistedaround a stalk and become avascular, rather than a remnant oftransgene-expressing liver.

section was examined for the presence of regenerative nod-ules. From this, the numbers of visible regenerative nodulesper liver were estimated to be 0, 8, and 11 (average, 6.3) inthese mice. The total number ofhepatocytes in two additional12-day-old line 1944-6 mice was estimated as described (13),giving a mean value of 1.4 x 108. This information was usedto calculate the approximate frequency of transgene rear-rangement per cell division, /L, according to the followingformula (modified from ref. 19):

,u = n/Nlog2N,

where n is the mean number of regenerative foci per hemi-zygous liver, and N equals the total number of hepatocytesper liver. We estimate that ju = 1.7 x 10-7, so that there willbe m1 rearranged cell per 6 x 106 cell divisions.

RESULTS

Fig. 1 shows the gross appearance oflivers ofyoung Alb-uPAtransgenic mice during the process of clonal regeneration. Inmice beyond 2 weeks of age, tiny red foci became visible inthe otherwise pale-to-white transgene-expressing liver, andin older mice the foci became nodules that progressivelyenlarged. Eventually, between 2 and 3 months of age, rednodules entirely replaced the original hepatic parenchyma(Fig. 1 Right). At this stage, as in subconfluent red nodules,there was no measurable transgene expression within theregenerated liver (13).The kinetics of red-tissue expansion are illustrated in Fig.

2A, in which estimates of percent regenerated (red) liver areplotted as a function of mouse age. The more rapid regener-

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FIG. 2. Growth and DNA synthesis in Alb-uPA mouse liver. (A) Percent regenerated (red) liver was estimated visually for mice from eachAlb-uPA line at several ages and is plotted as a function of mouse age. Each point represents the mean ± SD of 5-11 estimates. +, Signifcantdifference between lineages (P < 0.05, Student's t test). (B) Percent nuclei in S phase of the cell cycle in regenerated (red) liver was deteminedby flow cytometry and is plotted as a function of percent regenerated liver. Samples were collected from mice between 5 weeks and 4 monthsof age. Each point represents the mean + SD of4-10 measurements of samples collected from at least two individual mice; all transgenic valuesare significantly different from corresponding age-matched nontransgenic (non-TG) control values (P < 0.05, Student's t test).

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FIG. 3. Mortality and morbidity in Alb-uPA adult mice. (A) Thefraction of surviving mice in each lineage is plotted as a function ofage and is compared with survival of genetically similar nontrans-genic (non-TG) control mice in our colony. For line 1944-6, mediansurvival is 13.7 months, n = 26; for line 1353-8, median survival is16.5 months, n = 20; for non-transgenic controls, median survival is20.2 months, n = 19. (B) Gross appearance of nontransgenic (Left)and 13-month-old line 1944-6 transgenic (Right) mouse livers.

ation in line 1353-8 versus line 1944-6 is consistent withestimates of the relative frequency of productive transgenerearrangements (i.e., those that abolish transgene expres-sion) in liver cells of these mice: =1 per 104 cell divisions inline 1353-8 mouse liver (13) compared with 1 per 6 x 106 inline 1944-6 mouse liver. Fig. 2B shows the relationshipbetween the regenerative status of Alb-uPA liver and thefraction ofcells undergoingDNA synthesis, as determined byflow cytometry. The number of cycling cells decreased at thelater stages of regeneration and approached that observed inthe livers ofage-matched nontransgenic controls once regen-eration was complete. Thus, the period of intensive mito-genesis ends with the replacement of white, transgene-ex-pressing liver by red, regenerative nodules.The life-span of transgenic mice in both lineages was

reduced relative to genetically similar nontransgenic controlmice (Fig. 3A). Remarkably, at death, every transgenicmouse displayed one or more large hepatic tumors (Fig. 3B),whereas only 1 of the 19 control mice displayed liver lesions(a 5-mm-diameter hyperplastic nodule).The histopathological appearance of hepatic lesions is

illustrated in Fig. 4, and the frequency ofeach type is plottedas a function of age for both lines of Alb-uPA mice in Fig. 5.The first lesions were visible as early as 6 months of age andconsisted of altered or hyperplastic hepatic foci of either the

FIG. 5. Hepatic lesions in Alb-uPA adult mice. Bars show thefraction of transgenic mice from each lineage at different ages thatdisplayed hepatic tumor nodules >3 mm in diameter (open); alteredor hyperplastic hepatic foci (stippled); adenomas (gray); and hepa-tocellular carcinomas (black), almost always of the trabecular type.Stars indicate the number of ademonas that contained foci ofcarcinoma. n.d., Not determined. Five to 10 mice were examined ateach age.

eosinophilic or clear/vacuolated cell types (Fig. 4A). By 10months, eosinophilic (Fig. 4B) or clear cell nodules werepresent that significantly compressed surrounding untrans-formed liver and lacked microscopic evidence of bile ducts.These were classified as adenomas. At 12 months and be-yond, typical hepatocellular carcinomas were observed inmice from each line (Fig. 4C). Adenomas with focal areas ofcarcinoma also occasionally were observed (Fig. 5). In-creased plasma alanine aminotransferase (ALT) activity, anindicator of hepatocellular injury and death, was associatedwith the appearance ofgrossly visible hepatic masses (Fig. 6).No evidence of metastasis was observed upon gross exam-ination of mouse carcasses, and cells from line 1944-6 tumornodules were unable to grow following subcutaneous trans-plant into syngeneic recipients.We further examined the clonality and genomic stability of

tumors by using flow cytometry to measure DNA content ofisolated tumor nuclei (data not shown). Each of five line1353-8 tumor nodules displayed a discrete aneuploid peak (inone case two peaks) between 2.5N and 3N. Nine of 14 line1944-6 tumor nodules also displayed discrete aneuploid peaksbetween 2.8N and 8.3N. Thus, tumor progression is associ-ated with genomic instability in this model, and in most casesthe tumors appear to be clonal, presumably having arisenfrom a single aneuploid cell.

In line 1353-8, the transgene integration site consists of -5copies of the transgene joined head-to-tail in a tandem array;

FIG. 4. Histopathological le-

sions in livers of line 1944-6 trans-genic mice. (A) Clear or vacuolatedcell altered hepatic focus in a 16-month-old mouse. (B) Eosinophilicadenoma in a 16-month-old mouse.

(C) Trabecular hepatoceliular carci-

noma in an 18-month-old mouse.(Bar = 0.1 mm.)

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FIG. 6. Plasma alanine aminotransferase in A1b-uPA adult mice.Plasma alanine aminotransferase (ALT) was measured in individualmice from each line (Upper, 1944-6; Lower, 1353-8) that carriedaltered or hyperplastic foci only (o) or adenomas or carcinomas (0).Values for several nontransgenic control mice are indicated (Upper)by asterisks.

in line 1944-6, the integration site consists of =10 copiesarranged similarly (13). Two types of chromosomal rear-rangements associated with loss of transgene expressionhave been detected in line 1353-8 regenerative nodules. In thefirst, the size ofthe transgene array is reduced, apparently byhomologous recombination that deletes internal transgenecopies. In the second, the entire transgene array is deleted(13). Southern blot analysis of regenerative-nodule DNAdigested with EcoRV, which does not cut within the trans-gene, was used to identify the frequency of these two classesof rearrangements. In white liver and kidney DNA from line1353-8 mice, a transgene probe hybridized to a 50-kb restric-tion fragment in field-inversion gels, which identified theunrearranged transgene array (data not shown); this bandmigrates above 23 kb in standard agarose gels. In DNA fromregenerative nodules of the first type, the transgene probehybridized to a fragment of 8 kb or a fragment of 17 kb (Fig.7 and ref. 13), indicating partial deletion of transgene DNA.Nodules that had undergone complete transgene deletiondisplayed a marked loss of intensity ofthe unrearranged bandbut lacked entirely the lower molecular weight bands (seeFig. 7, mouse 73-4, nodule C; the reduced-intensity highmolecular weight band present in each regenerative-nodule

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FIG. 7. Southern blot analysis ofEcoRV-digested DNAfim line1353-8 kidney and regenerative hepatic nodules. Mouse identifica-tion numbers are given at the top. Nodules are identified by lettersA-C; K, kidney; Non-TG, liver DNA from nontransgenic mouse.The blot was probed with a labeled DNA fagment complementaryto the mouse uPA gene. Arrowhead at right indicates the EcoRVfragment of the endogenous uPA gene, which serves as a control forDNA loading. (Note that kidney DNA from mouse 104-4 wasoverloaded.) DNA isolated from mouse 113-5 was shown unambig-uously in a second experiment to contain a 17-kb rearrangementband.

FIG. 8. Southern blot analysis ofEcoRV-digested DNA from line1353-8 hepatic tumor nodules. DNAs from kidney, white liver, andrepresentative regenerative nodules serve as controls. Arrowhead atright indicates the EcoRV fragment of the endogenous uPA gene.Note that all tumors lack a low molecular weight transgene band,similar to the regenerative nodule C. The sample of non-neoplasticliverfrom mouse 35-19 displays the 17-kb transgene band, confirmingthe occurrence of class I rearrangement events in this mouse.

lane is likely to represent unrearranged DNA from non-hepatocytes, which comprise 35% of normal liver cells). Of39 regenerative nodules examined, 23 (591%) displayed the17-kb band, 11 (28%) displayed the 8-kb band, 2 (5%)displayed both 17- and 8-kb bands (presumably representingconfluent growth of two separate nodules), and 3 (8%)displayed no low molecular weight transgene bands. Wesimilarly analyzed line 1944-6 regenerative-nodule DNA byfield-inversion gel electrophoresis. White liver and kidneysamples from this line produced a band at -'100 kb, whileeach of 12 regenerative nodules analyzed displayed apparentloss of the entire transgene array (data not shown).Transgene expression was not detected in any of 12 tumors

(4 from line 1353-8 and 8 from line 1944-6) or in surroundingnon-neoplastic liver (data not shown), suggesting that tumorswere derived from cells that had lost the transgene. To identifythe type of transgene-inactivating DNA rearrangement pres-ent in line 1353-8 tumor progenitor cells, tumor DNA wasdigested with EcoRV and analyzed by Southern blot. Allexamined line 1353-8 tumors appeared to be derived fromprogenitors with complete transgene deletions; in total, 9 of 9tumors from three different mice displayed no lower molecularweight transgene band but did show the expected reduction inintensity ofthe high molecular weight, unrearranged transgeneband (Fig. 8 and data not shown). The presence within tumorsof only one type of rearrangement at a much higher thanexpected frequency suggests that alterations inDNA structurethat accompany this particular rearrangement must have acritical role in determining the susceptibility of affected cellsto subsequent neoplastic transformation.

DISCUSSIONIn the Alb-uPA transgenic mouse model, transgene-inducedhepatotoxicity selects for clonal expansion of liver cells thatlack transgene expression due to chromosomal rearrange-ment, and this process in turn cu s in the developmentof liver cancer with 100% penetrance, despite the fact thatuPA does not appear to fit into a class of proteins (e.g.,growth factors, membrane or cytosolic signal transducers, ornuclear transcription factors) whose activities have beenassociated with neoplastic transformation. Several mecha-nisms of DNA rearrangement could abolish transgene ex-pression. The first, intrachromosomal recombination (class

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I), involves deletion ofDNA within the transgene array, mostlikely mediated by homologous recombination between in-dividual transgene copies (13). This class retains a remnant ofthe transgene array and leaves surrounding chromosomalDNA unaltered. Despite the fact that class I rearrangementsaccount for >90% of transgene deletion events in the 1353-8lineage, they have never been observed in tumors, suggestingthat uPA-mediated liver damage and regeneration (mitogen-esis) per se are not tumorigenic in these mice. The remainingmechanisms result in complete loss of transgene sequences,and one or more of these apparently increase the risk forsubsequent transformation. Two mechanisms, mitotic re-combination and gene conversion (class II), share with classI events the maintenance of normal disomic cellular DNAcontent. (Class II events may reduce flanking DNA tohomozygosity, but this should not influence tumor progres-sion in inbred strains of mice.) Therefore, the cellular con-sequences of class I and II rearrangements are essentiallyidentical, and thus neither should lead to cancer. In contrast,two additional mechanisms, partial or complete chromosomedeletion and nondisjunction (class III), are accompanied byloss of chromosomal DNA and result in monosomy of all orpart ofthe affected chromosome. We believe that these latterrearrangements function as tumor-initiating events in ourmodel.

Several mechanisms could explain tumor initiating activityof class III rearrangements. (i) Loss of large regions ofendogenous DNA containing multiple genes could signifi-cantly alter gene dosage (the balance of gene expression)within a cell, which then may act in an epigenetic manner topromote altered cellular growth characteristics (20). (ii) Co-deletion of the transgene and a linked tumor-suppressor genewould put the affected cell at risk for progression to neoplasiafollowing subsequent loss of the second allele, as docu-mented in humans for the retinoblastoma, Wilms tumor,adenomatous polyposis col, and p53 loci (21, 22). (iii) Partialor complete monosomy alone may predispose the cell tofurther chromosomal instability, especially in a rapidly di-viding cell population. Effects of these types are thought tobe associated with multistage carcinogenesis in a variety oftissues (reviewed in refs. 20 and 23-26). Finally, it should beemphasized that, for each mechanism, the rearrangementwould serve as both genotoxic first hit and mitogenic stim-ulus; thus, it would be precisely the initiated cell populationthat was selectively expanded.

Extensive cellular replication during the process of regen-eration almost certainly enhances tumorigenesis. However,the fact that regenerative nodules of each class shouldexperience equivalent mitogenic stimuli suggests that mito-genesis per se is not carcinogenic in this model. There is a12:1 ratio of class I to class II + III regenerative nodules inline 1353-8 livers, but 0 of 9 tumors contained class Irearrangements. Even if the next examined tumor displayedthe class I phenotype, there would remain a >100-folddifference between observed and expected ratios of class I toclass II + III tumor progenitor cells. Thus, we conclude thatmitogenesis alone is at least 2 orders of magnitude lesseffective at inducing tumor formation than mitogenesis cou-pled to complete transgene deletion.Both the Alb-uPA and the HBsAg transgenic mice provide

models of hepatocarcinogenesis induced by non-genotoxictransgenes in which tumor incidence and multiplicity aresimilar (this report and refs. 9 and 10). In the HBsAg model,repeated cycles of hepatocyte injury and death lead tochronic inflammation and nodular hepatic regenerationthroughout the life of the mouse (9, 10). In contrast, in theAlb-uPA model the period of cell injury and death is limited

and persistent inflammation is absent. Therefore, the oppor-tunity for exposure of the surviving cell population to poten-tially genotoxic cellular injury and inflammation is greatlyreduced, and residual exposure should affect all nodulesequally. However, there is a selection for transgene deletionevents in Alb-uPA mice, and we propose that loss of chro-mosomal DNA during transgene deletion constitutes an al-ternative form of tumor-initiating activity in our model. Byconstructing a panel of Alb-uPA transgenic mouse lineages,each with a different chromosomal transgene insertion site,we may be able to take advantage of this mechanism of tumorinitiation to map hepatic tumor-suppressor loci and deter-mine whether loss of DNA by itself is capable of furtherdestabilizing the genome. These mice, together with othertransgenic models of hepatocarcinogenesis, provide a re-markable perspective on the diverse pathways that can leadto cancer.

We thank Diane Allen, Mary Avarbock, Nancy Jensen, andRichard Silbiger for technical assistance and Stephen Barthold,Robert Maronpot, and Tom Van Winkle for helpful discussions. Thiswork was supported by National Institutes of Health grants to R.D.P.(HD09172), R.L.B. (CA38635), and J.L.D. (CA44611).

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