Ploidy and Karyotypic Alterations Associated with Early Events in the Development of Hepatocarcinogenesis in Transgenic Mice

[CANCER RESEARCH 56, 2137-2142. May 1. 1996] Ploidy and Karyotypic Alterations Associated with Early Events in the Development of Hepatocarcinogenesis in Transgenic Mice Harboring c-myc and Transforming Growth Factor a Transgenes Linda M. Sargent, Nancy D. Sanderson, and Snorri S. Thorgeirsson1

Laboratory of Experimental Carcinogenesis, Division of Basic Sciences. National Cancer Institute. Bethesda. Maryland 20892

ABSTRACT localization of altered genes than is possible in other species (17). In addition, numerous transgenic mouse lines have been generated car The cooperation of the c-myc oncogene with the growth factor trans rying specific gene additions or knockouts that develop tissue and forming growth factor (TGF)-a in development of liver tumors in trans- cell-specific tumors. These mice can provide a virtually unlimited genie mice has been demonstrated previously. In this study, we analyzed the ploidy and karyotype of c-myc, TGF-a, parental control, and the supply of tumors of a specific stage from genetically identical animals double transgenic c-myc/TGF-a hepatocytes at 3 weeks of age when the and, therefore, offer an ideal experimental model for genetic dissec liver is histologically normal and at 10 weeks when the c-rnyc/TGF-a liver tion of the process of neoplastic evolution. is dysplastic and contains basophilic foci. Eighty % of the 10-week hepa Genes controlling murine tumor susceptibility have been identified tocytes were aneuploid, and 32% had chromosomal breakage. Statistically in chemically induced carcinogenesis (18-20). Tumor susceptibility significant breakage was observed in six different chromosomes. Breakage loci have been observed on mouse chromosomes 1, 4, 7, and 12. at band A5 and at the border of bands C4/S of chromosome 1 was These regions correspond to human chromosomes 18, Ip, lip, and observed. Fragile sites on chromosome 4 were most frequent in the middle 14q (16). Alterations in 1Ip, Ip, and 14q have been reported in human of the chromosome at bands C2 and C6. Chromosome 6 was fragile at liver tumors (1,2, 7). The tumor susceptibility gene on mouse chro band F2. The region of chromosome 7 at bands B5 and D3 was frequently mosome 7 corresponds to the region of rat chromosome 1 that is broken and involved in translocations. Chromosome 12 was broken at duplicated early in liver carcinogenesis. The correlation between bands Dl and D3. The breakage sites on chromosomes 1, 4, 7, and 12 correspond to sites of tumor susceptibility genes in the mouse. Although alterations in the mouse tumor susceptibility genes and stage of tumor there was no consistent change in copy number, recurrent translocations development has not been established. Due to the prior identification between chromosomes 1,4, 7,12, and 19 were also observed. These studies of murine tumor susceptibility genes and the availability of numerous demonstrate that the development of dysplasia and basophilic foci in the genetic markers for mouse chromosomes, as well as the conserved liver is correlated with aneuploidy and chromosome breakage. The spe genetic linkage between mice and humans, we have chosen to exam cific fragile sites indicate genetic regions that are altered during early ine a time course of genetic changes in transgenic mouse models of stages of hepatocarcinogenesis. Due to the conservation of genetic linkage hepatocarcinogenesis. groups between mice and humans, the identification of genetic alterations The transgenic mouse models used in the present study were in the mouse during hepatocarcinogenesis may provide critical informa generated by targeting the expression of the murine proto-oncogene tion about tumor susceptibility genes that are important in the early c-myc and TGF2-a to the liver (21-23). The TGF-a transgenic ani development of human hepatocellular carcinoma. mals develop tumors by 10 months of age (24). The c-myc transgenic mice demonstrate liver tumors at 12 to 15 months of age (25). The INTRODUCTION double transgenic c-myc/TGF-a mice have normal liver histology at 3 Nonrandom chromosome abnormalities associated with the devel weeks, but by 10 weeks of age, numerous basophilic foci, as well as opment of tumors serve as a guide to the identification of genes that dysplastic cells that invade the blood vessels, are evident in the liver (25).3 Hepatocellular carcinoma is observed as early as 4 months of are altered during carcinogenesis. In human cancer, the combination age in male c-myc/TGF-a mice.3 At 8 months of age, 90% of the of classical and molecular cytogenetics has made it possible to detect specific genetic alterations in several tumor types. Molecular analysis c-myc/TGF-a males have liver tumors. The c-myc and TGF-a parental of hepatocellular carcinoma has identified deletions and rearrange strains have normal liver histology at 3 and 10 weeks of age. We have ments in human chromosomes Ip, Iq, 4q, 5q, 8p, 8q, lip, 13q, 14q, chosen to examine the karyotypic changes in hepatocytes at 3 weeks 16q, and 17p (1-10). No correlation between the stage of tumor when the c-myc/TGF-a transgenic liver is histologically normal and at development and the chromosome rearrangement has been made in 10 weeks of age when dysplasia and basophilic foci are evident. human hepatocarcinogenesis. However, results from a number of Analysis of the karyotype of the c-myc, TGF-a, and the c-myc/TGF-a experimental models have demonstrated that many of the fragile sites transgenic mice shows a correlation between the development of that are observed in the transition from the preneoplastic to the dysplasia and basophilic foci with increased chromosome breakage neoplastic morphology are later observed as stable rearrangements in and aneuploidy. Specific breakages on chromosomes 1, 4, 6, 7, and 12 tumors (11-15). are observed in the early stages of murine hepatocarcinogenesis. The mouse is a very useful model to establish the stage-specific genotypic alterations during carcinogenesis due to the extended homology of conserved genetic linkage groups between humans and MATERIALS AND METHODS mice (16). Also, the extensive analysis of the mouse genome has Construction of Fusion Genes and Generation of Transgenic Mice. The produced numerous genetic markers, allowing for a more specific development of the double c-mvc/TGF-a transgenic mouse model using CD1 X (C57BL/6J x CBA)F, mice was described by Murakami et al. (25). Received 10/30/95; accepted 3/4/96. The TGF-a transgene was constructed by Jhappan et al. (23) in CD1 mice and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 2 The abbreviation used is: TGF. transforming growth factor. ' To whom requests for reprints should be addressed, at National Cancer Institute. 3 E. Santoni-Rugiu, P. Nagy, M. R. Jensen, V. M. Factor, and S. S. Thorgeirsson. Building 37. Room 3C28. 37 Convent Drive MSC4255. Bethesda. MD 20892-4255. Evolution of neoplastic development in the liver of transgenic mice coexpressing c-myc Phone: (301) 496-5688; Fax: (301) 496-0734. and transforming growth factor-a. submitted for publication. 2137

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1996 American Association for Cancer Research. OENOMIC INSTABILITY IN HEPATOCARCINOGENESiS the c-mvc transgene by Murakami el al. (25) in (C57BL/6J X CBA)F, mice. Table 2 Chromosome aberrations in 3-week-old mice The screening for the transgene was performed by Southern blot analysis of tail Values are the percentage of affected cells from ihe analysis of 50 banded metaphase spreads from each of 5 animals in the F, control, the c-mvc, the TGF-a, and the DNA (25). The mice were maintained on 50 mmol ZnCL2 drinking water from c-myc/TGF-a transgenic mice 3 weeks of age to maximize induction of the TGF-a expression. Perfusion and Chromosome Preparation. The livers of c-myc, TGF-a, with c-myc/TGF-a mice and F, control mice CD1 x (C57BL/6J x CBA) were TreatmentControl breaks2.0 examined at 3 and 10 weeks of age. Five animals per group were anesthetized Â±1.0 c-myc 1.0 Â±1.0 <1.0 with avertine, and the livers were perfused with a collagenase solution as c-mvc/TGF-a 10.0 Â±1.0Â° <1.0 described previously (26). A section of the liver was tied off with silk thread TGF-aCells 4.0 Â±2.0Translocation<1.0 <1.0 during the initial wash with HBSS, and the liver piece was placed in formalin " Statistically significant. for future pathological analysis. After collagenase digestion, hepatocytes were separated from the littoral cells by a Percoli (Sigma Chemical Co.) isodensity centrifugation and immediately plated in a 75-cm collagen type I-coated flask were aneuploid. A low level of endoreduplication was present in the (Vitrogen 100; Celtrix Laboratories. Santa Barbara. CA) at a density of c-myc, the TGF-a, and the c-myc/TGF-a hepatocytes. The level of 5 X 10" cells (27) in 15 ml of 10% serum DMEM/F12 medium supplemented endoreduplication was neither elevated by the presence of the two with 18 mM HEPES, 5 mM sodium pyruvate, 1 mM NaHCO,, 1 mg/ml transgenes nor increased with age (Table 3). When the diploid c-myc/ galactose. 30 fAg/ml proline, 100 units/ml penicillin. 100 /^g/ml streptomycin, TGF-a hepatocytes were examined separately, the number of aneu and 5 fig/ml ITS (insulin, transferrin, and selenium; Collaborative Research). ploid cells was significantly lower than the polyploid hepatocytes. The medium was changed 2 h later, and 10 ng/ml murine epidermal growth factor (Life Technologies, Inc.) were added. Forty-four h after plating, colchi- Forty-five Â±5.0% of the diploid hepatocytes had an aneuploid chro cine was added to the medium to a final concentration of 0.05 ng/ml. After an mosome number. additional 2 to 3 h incubation, the hepatocytes were removed from the dish Chromosomal Aberrations in Hepatocytes Isolated from Mice with 0.25% trypsin solution and harvested for chromosome analysis by hypo- at 10 Weeks of Age. At 10 weeks of age, the metaphase spreads tonic treatment (0.075 M KCL) for 9.5 min (28). The hepatocytes were then prepared from the c-myc mice had a level of chromosome damage further treated with 3:1 (v/v) acetic methanol fixative, and slides were prepared comparable to that seen in control hepatocytes. The hepatocytes as described (29). Fifty G-banded metaphase spreads of good morphology isolated from the TGF-a mice had a low but statistically significant were randomly selected from cytogenetic preparations from each mouse. level of chromosome breakage (Table 4; P < 0.05). By contrast, 32% Lesions were mapped to specific bands using the karyotypes and ideograms of of the c-mvc/TGF-a hepatocytes had chromosome damage at 10 Cowell (30). A metaphase spread with a loss or gain of at least four chromo somes was identified as aneuploid. The data were analyzed as the mean weeks of age (Table 4; Fig. 1). Eighteen % of the hepatocytes isolated percentage of cells with aberrations, and results were compared with nonpara- from the c-myc/TGF-a hepatocytes at 10 weeks had translocations metric statistics using the x1. (Fig. 2). When the diploid c-myc/TGF-a cells were analyzed sepa rately, 29% Â±8.0% of the hepatocytes had chromosome breakage and 12% Â±4.0% of the hepatocytes had a translocation. Cells with greater RESULTS than 10 breaks/cell were only observed in the octaploid and tetraploid Chromosome Number and Aberrations in Hepatocytes Isolated populations. Fifty % of the octaploid and 33% of the tetraploid at 3 Weeks of Age. At 3 weeks of age. the hepatocytes isolated from hepatocytes had greater than 10 breaks/cell. the controls, the TGF-a, the c-myc, and the c-myc/TGF-a mice were Specific Chromosome Breakage in 10-Week-Old Mice. The approximately 50% diploid (Table 1). Histologically, the liver was level of breakage was statistically significant in six chromosomes essentially normal. The rate of aneuploidy was not statistically ele (Table 5). An example of typical metaphase spreads with chromosome vated. A small but significant level of endoreduplication was observed breakage and rearrangement is shown in Figs. 1-3. Two sites on in the double transgenic liver cells when compared to the control chromosome 1 showed statistically significant damage (P < 0.05). (Table 1; P < 0.05). The level of chromosome breakage in hepato The band regions A5 and the C4/C5 border of chromosome 1 had the cytes isolated from the c-myc and the TGF-a mice at 3 weeks of age greatest level of breakage (Fig. 4, chromosome /; P < 0.05). Chro was not significantly different from that in control liver cells. The mosome 4 had two fragile regions at C2 and C6 (Fig. 4, chromosome hepatocytes isolated from double transgenic mice at 3 weeks of age 4). Chromosome 6 had breakage at band F2 (Fig. 4, chromosome 6; demonstrated a small elevation in chromosome breakage (Table 2; P < 0.05). Breakage of the band regions B5 and D3 of chromosome P < 0.05), but the karyotype of all transgenic mice was essentially 7 was significantly increased (Fig. 4, chromosome 7; P < 0.05). The normal. last light band of chromosome 12 and band D3, as well as band Dl, Chromosome Number in Hepatocytes Isolated at 10 Weeks of were broken (Fig. 4, chromosome 72; P < 0.05). The centromere of Age. The hepatocytes isolated from mice at 10 weeks were predom chromosome 19 at band A was broken 5% of the time (Fig. 4, inantly tetraploid (Table 3). The TGF-a and the c-myc/TGF-a hepa chromosome 19). This breakage was not statistically significant due to tocytes had a small reduction in the number of diploid hepatocytes a high variability between animals; however, the results indicate a (Table 3). Although only 2% of the control, c-myc, and TGF-a trend. Chromosomes 1 and 12 demonstrated the highest breakage. hepatocytes were aneuploid, 83% of the c-myc/TGF-a hepatocytes Chromosomes 1, 4, 6, 7, 9, 12, and 19 were involved in translocations.

Table I Distribution of chromosome number endoreduplication and aneuploidy in 3-week-old mice Values are the percentage of the mean number of affected cells from studying 50 banded metaphase spreads from each of 5 animals from F, control, c-myc,TGF-a, or c-myc/TGF-a transgenic mice at 3 weeks of age. The perfusion methods, cytogenetic preparation, and transgenic constructs are described in "Materials and Methods." The aneuploidy was determined by the loss or gain of at least four chromosomes per metaphase spread. TreatmentControl Â±2.0 Â±3.0 Â±1.0 c-myc 59.4 Â±6.0 38.6 Â±2.0 2.0 Â±2.0 2 Â±1.0Â° 1.0 Â±1.0 c-mvcHXjF-a 59.6 Â±12.055.8 38.5 Â±12.0 1.9 Â±2.0 4Â± 1.0Â° 1.0 Â±4.0 TGF-aDiploid53.0 Â±7.0Tetraploid46.0 42.1 Â±8.0Octaploid1.0 2.1 Â±2.0Endoreduplication<1.01 Â±1.0Aneuploid<1 1.0 Â±1.0 ' Statistically significant.

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Table 3 Distribution of chromosome number endoreduplication and aneuploidy in 10-week-old mice Values are based on the percentage of affected cells from the examined cells of at least 50 cells from each of 5 animals. Ten-week-old F, control, c-myc, TGF-a, and the c-myc/TGF-a transgenic mice were sacrificed, the livers were perfused, and chromosome slides were prepared as described in "Materials and Methods." The near-diploid hepatocytes had approximately 40 chromosomes, the near-tetraploid hepatocytes had approximately 80 chromosomes, and the near-octaploid hepatocytes had approximately 160 chromosomes. Aneuploidy was defined as the loss or gain of a minimum of four chromosomes.

TreatmentControl Â±8.0 Â±8.0 Â±10.0 Â±1.0 4.0 Â±1.0Â° c-myc 21.1 Â±3.0 68.6 Â±4.0 10.3 Â±6.0 2.0 Â±1.0 TGF-o 9.0 Â±1.0" 77.0 Â±2.0 14.0 Â±1.024.2 3.0 Â±1.0" 2.0 Â±2.083.0 c-myc/TGF-aDiploid17.9 9.1 Â±2.0"Tetraploid56.9 66.7 Â±14.0Octaploid25.2 Â±11.0Endoreduplication<1.0 4.0 Â±2.0Aneuploid2.0 Â±7.0" " Statistically significan

dysplastic pathology of the liver in the 10-week c-myc/TGF-a mice was correlated with a dramatic increase in aneuploidy, chromosome breakage, and translocations. The breakage was not high enough to account for the 80% chromosome loss and gain observed in the c-myc/TGF-a hepatocytes, indicating a possible aberration of the spindle formation or a block in synthesis of DNA. The endoredupli cation is further evidence of a disruption of the spindle or DNA synthesis (31-33). Danielsen et al. (34) observed an increased aneu ploidy and translocations in chemically induced mouse liver nodules. A similar increase in aneuploidy and chromosome damage has also been reported in rat liver with neoplastic nodules (27, 29, 35, 36). The correlation of aneuploidy and chromosome damage with the transition

Fig. 1. Photograph of a metaphase spread from a hepatocyte isolated from a 10-week- old c-mvr/TGF-a transgenic mouse. The metaphase spread demonstrates multiple chro mosome breaks designated by an arrow.

Table 4 Chromosome aberrations in 10-week-old mice Values are expressed as the percentage of affected cells examined of at least 50 cells from each of 5 animals. Ten-week-old F, control, c-myc, TGF-a. and c-myc/TGF-a mice were examined. Aneuploidy was defined as the loss or gain of a minimum of four chromosomes.

withbreaks3.0 withbreaks<1.0<1.03.0 TreatmentControlc-mvcTGF-ac-myc/TGF-aCells

Â±1.04.0 Fig. 2. Photograph of a metaphase spread prepared from hepatocytes isolated from a Â±1.09.0 10-week-old c-myc/TGF-a transgenic mouse. Double arrow, a translocation between Â±3.0Â°32.0 Â±1.029.0 chromosomes 7 and 9. Single arrow, a chromosome ring. Â±9.0"Diploidcells Â±8.0Translocation<1.0<1.0<1.018.0Â±2.0" " Statistically significant when compared to controls. Table 5 Fragile sites in the 10-week-old mice Values are based on the percentage of hepatocytes examined that demonstrated these Most of the translocations were centric fusions (Fig. 2). There was no aberrations. Five animals were sacrificed per group, and at least 50 banded metaphase spreads were analyzed from each animal. The F, control, the c-myc, the TGF-a. and the consistent loss or gain of whole chromosomes at 10 weeks. In contrast c-myc/TGF-a transgenic mouse were sacrificed at 10 weeks of age. and the hepatocyles to c-myc/TGF-a, TGF-a hepatocytes demonstrated only three fragile were perfused and prepared as described in "Materials and Methods." sites. Although the level of chromosome breakage was lower, the TreatmentChromosome fragile sites of the TGF-a hepatocytes are a subset of those observed in the c-myc/TGF-a hepatocytes. The bands A5 and C4 of chromo c-myc/TGF-a14679121915.0no. (A5.C4/C5)8.06.09.09.014.01.0Â°Â±5.0Â° C4/C5)3.0Â±1.0"(A5, some 1 demonstrated elevated breakage, as well as band B5 on (C2,C6)1.0" chromosome 7. (F2)4.0" (B5.D3)1.0" Â±1.0" (B5) (B5)1.0"(D1,D3)5.0 DISCUSSION 4.0 (Al)TGF-a5.0 The histology and euploid karyotype of the 3-week c-myc/TGF-a " Statistically significant. The band region that was most frequently broken is indicated mice indicate a normal liver at this time point. By contrast, the after the percentage of breakage. 2139

from benign to malignant tumors has been reported during multistage carcinogenesis in mouse skin and salivary gland (37, 38). Frequently broken regions of chromosomes are designated as frag ile sites. These regions have been demonstrated to be tissue specific and are G-light bands that are actively transcribed, possibly due to local decondensation of the chromosome (39). Cellular regulatory genes such as oncogenes, suppressor genes, and growth factors are frequently located in G-light bands (39-41). These actively tran scribed regions are often altered during carcinogenesis (15, 40, 41). - The identification of specific fragile regions of the chromosome during the early stages of mouse liver carcinogenesis indicates the *'** regions that may be rearranged in tumors. Specific breakage was observed in the TGF-a and in the c-myc/TGF-a at the midpoint of chromosome 1. A tumor resistance gene in the B6C3F1 mouse near the midpoint of chromosome 1 has been reported by Bennett and il **-n Drinkwater (20). Lee et al. (19) identified a tumor resistance gene on chromosome 1 in the DBA/2J mice. Deletions of the band regions C5 and C4 have also been observed in liver tumors from B6C3F1 mice (42, 43). . Another liver tumor resistance gene has been reported on chromo .* some 4 near the break site observed in the c-myc/TGF-a mice (19). Deletions of chromosome 4 have been observed in transformed liver v VcxV epithelial cells at the D4WSMI locus (1, 44). The fragile region of chromosome 4 is homologous to human chromosome Ip32-41. De letions of human Ip have been reported in human hepatocellular carcinoma, breast, and colon carcinoma (45-48). Mouse chromosome Fig. 3. Photograph of a metaphase spread from a hepatocyte isolated from a 10-week- old c-mvc/TGF-a transgenic mouse. Chromosome 1 has a displaced break. 4 and human chromosome 1 suppress the malignant phenotype in cell fusions of normal and tumor cells (48). The fragile region on mouse 4 also has homology to human chromosome 9. Although loss of

Hu2p Hu18p Hu18p *Hu 1q *Hu1p

Fig. 4. Idiogram of chromosomes 1, 4, 6, 7, 12, *Hu1p and 19. Arrows, band regions that were found to have a statistically significant number of breaks. Hu9p Left, homologous human chromosomes; starred re gions, loss of heterozygosity in human hepatocellu Hu7q lar carcinoma. Idiogram of chromosome /. the band regions at the C4/C5 border and band A5 were found to be broken at a significant rate. Idiogram of chromosome 4. the fragile sites occurred at band region C2 and C6. Idiogram of chromosome 6. band region F2 is indicated by an arrow and was found to 12 be fragile. Idiogram of chromosome 7, the band "HuHq region indicated by arrows at bands B5 and at D3 were found to be fragile sites. Idiogram of chromo Hu9q some 12, bands Dl and D3 are indicated by arrows. Idiogram of chromosome 19, the centromere of the Hu10q chromosome was frequently deleted (highlighted in black). *Hu14q *Hu 14q -

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1996 American Association for Cancer Research. GENOMIC INSTABILITY IN HEPATOCARCINIXÃŒLNKSIS heterozygosity of human chromosome 9 has not been reported in Wang. H. P., and Rogler, C. Deletions in human chromosome arms I Ip and 13q in primary hepatocellular carcinomas. Cytogenet. Cell Genet.. 48: 72-78. 1988. hepatocarcinogenesis, the homologous region of 9 has cell cycle Buetow. K. H.. Murray, J. C., Israel. J. L., London, W. T., Smith, M., Kew, M., regulatory genes that may be important in liver carcinogenesis (48, Blanquet. V.. Brechot. C.. Redeker. A., and Govindarajah. S. Loss of heterozygosity 49). suggests tumor suppressor gene responsible for primary hepatocellular carcinoma. The region of chromosome 7 that is fragile in the c-mvc/TGF-a and Genetics. Â«6:8852-8856. 1989. Tsuda. H., Zhang, W., and Shimasato. Y. AlÃeleloss on chromosome 16 associated the TGF-a mice corresponds to rat Iq41 and human llplS.5 and with progression of human hepatocellular carcinoma. Proc. Nati. Acad. Sci. USA, 87: 11p 13. Rearrangements of rat 1q41 and human 11p 13 and 11p 15 have 6791-6794. 1990. been reported in hepatocellular carcinoma (2, 7, 37, 49-52). The loss Emi, M.. Fujiwara. Y.. Nakajima, T., Tsuchiya, E., Tsuda, H.. Hirohashi. S., Maeda, of human chromosome 1Ip has also been observed in Wilms' tumor Y., Tsuruta. K., Miyaki. M.. and Nakamura. Y. Frequent loss of heterozygosity for loci on chromosome 8p in hepatocellular carcinoma, colorÃ©ela!cancer and lung and hepatoblastoma (51, 52). Garibaldi et al. (18) have recently cancer. Cancer Res., 52: 5368-5372. 1992. Zhang, W.. Hirohashi, S., Tsuda, H.. Shimasato. Y., Yokata, J., Terada, M., and identified a tumor susceptibility gene near the H19 and H-ras gene on Sugimura. T. Frequent loss of heterozygosity on chromosome 16 and 4 in human mouse chromosome 7. Activated H-ras has been observed in mouse hepalocellular carcinoma. Jpn. J. Cancer Res.. 81: 108-111. 1990. liver tumors (53-55); however. Ohgaki4 did not detect a mutated Fujimori. M.. Tokino. T., HiÃ±o.O.. Kitagawa, T.. Imamura. T.. Okamoto. E., Mitsunobu. M.. Ishikawa, T.. Nakagama, H., Harada. H.. Yagura. M.. Matsubara. K., H-ras in liver tumors from the c-mycfTGF-a mice. Loss of the normal and Nakamura. Y. Allelotype of study of primary hepatocellular carcinoma. Cancer H-ras alÃelehas been reported when one copy of H-ras is mutated Res.. SI: 89-93. 1991. Simon. D.. Knowles. B. B., and Weith. A. Abnormalities of chromosome I and loss (56). The maternal copy of HI9 on chromosome 7 is lost in SV40 T of heterozygosity on Ip in primary hepatomas. Oncogene, 6: 765-770, 1991. transgenic mouse liver tumors (57). Microcell fusion with human Walker, G. J.. Hayward, N. K.. Falvery, S., and Cooksley, W. G. E. Loss of somatic chromosome 11 will inhibit the tumor phenotype of a Wilms' tumor heterozygosity in hepatocellular carcinoma. Cancer Res.. 51: 4367-4370, 1991. Fujimoto. Y.. Hampton, L. L., Wirth, P. J., Wang, N. J., Xie. J. P.. and Thorgeirsson, cell line (58). S.S. Alterations of tumor suppressor genes and allelic losses in human hepatocellular The breakage observed in the c-myc/TGF-a mouse on chromosome carcinomas in China. Cancer Res.. 54: 281-285. 1994. Aldaz. M. C.. Gollahon. L. S., and Chen, A. Systematic H-ras amplification in 12 is in the region of the chromosome designated by Garibaldi et al. ovary-independent mammary tumors: correlation with progressively anaplastic phe- (18) to have a tumor resistance gene. Loss of heterozygosity of the notypes. Cancer Res.. 53: 5339-5344, 1993. same band region of mouse chromosome 12 has been reported in 12. Yunis, J. J., and Soreng. A. L. Constitutive fragile sites and cancer. Science (Wash ington DC). 226; 1199-1204. 1984. hepatocellular carcinoma isolated from C57BL/6JBY mice (59). This 13. Le Beau. M. M. Chromosomal fragile sites and cancer-specific breakpoints-a mod region is homologous to human 14q32, a region often deleted in erating viewpoint. Cancer Genet. Cylogenet.. 31: 55-60. 1988. hepatocellular carcinoma (6, 7). Aberrations of mouse chromosome 14. Elder. F. B.. and Robinson. F. B. Rodent common fragile sites: are they conserved? Evidence from mouse and rat. Chromosoma. 97: 459-464. 1989. 19 have been observed in neoplastic nodules (34). Although we 15. Yunis, J. J. Multiple recurrent genomic rearrangements and fragile sites in human observed many centric fusions of chromosome 19, extra copies of cancer. Somat. Cell. Mol. Genet., 13: 397-403, 1987. 16. Searle. A. G., Peter, J.. Lyon, M. F.. Evans, E. P., Edwards. J. H., and Buckle, V. J. chromosomes were not detected at this time point. Chromosome maps of man and mouse. III. Genomics. /: 3-18. 1987. The development of dysplasia and basophilic foci in the double 17. Copeland. N. G.. Jenkins. N. A.. Gilbert. D. J.. and Eppig. J. T. A genetic linkage map transgenic mouse is correlated with dramatic increase in aneuploidy of the mouse, current applications and future prospects. Science (Washington DC), 262. 57-66, 1993. and chromosome breakage. This and many other investigations illus 18. Garibaldi, M.. Manenti, G.. Canaan. F.. Falvella. F. S., Pierotti. M. A., Porta, G. D., trate the importance of aneuploidy in the transition from a preneo- Binelli. G., and Dragani. T. A. Chromosome mapping of murine susceptibility loci to plastic to a neoplastic state. Specific breakage indicates genetic re liver carcinogenesis. Cancer Res., 53: 209-211. 1993. 19. Lee. G-H.. Bennett. M. L.. Carabao, R. A., and Drinkwaler. N. R. Identification gions that are important to the development of hepatocellular of hepatocarcinogen-resistance genes in DBA/2 mice. Genetics, 139: 387-395, carcinoma. The regions on chromosome 1. 4, 7, and 12, in which a 1995. high frequency of breakage occurred, have been observed to have 20. Bennett. M. L.. and Drinkwater. N. R. Localization of the Him gene to the distal region of mouse chromosome one. Proc. Am. Assoc. Cancer Res., 34: 144. 1993. tumor susceptibility genes, as well as displaying loss of heterozygos 21. Derynck, R.. Goeddel, D. U.. Ullrich. A.. Gutterman. J. U., Williams, R. D., ity in mouse liver tumors. The break points on mouse chromosomes Bringman, T. S.. and Berger. W. H. Synthesis of mRNAs for transforming growth factor a and ÃŸand the epidermal growth factor receptor by human tumors. Cancer 1, 4, 7. and 12 correspond to human Iq, Ip, llplS.5. Ilpl3, and Res.. 47: 707-712, 1987. 14q32 (16). Human Iq, Ip, lip, and 14q are also rearranged in human 22. Sandgren. E. P.. Quaife, C. J.. Pinkert, C. A.. Palmiter. R. D., and Brinster, R. L. liver tumors. Although many investigations have determined specific Oncogene-induced liver neoplasia in transgenic mice. Oncogene, 4: 715-724. 1989. 23. Jhappan, C.. Stahle. C., Harkins, R. N.. Fausto, N., Smith, G. H.. and Merlino, G. T. gene alterations in hepatic tumors, this study reports the first identi TGF-or overexpression in transgenic mice induces liver neoplasia and abnormal fication of genetic changes in the early stages of murine hepatocar development of the mammary gland and pancreas. Cell, 61: 1137-1146, 1990. cinogenesis. The alteration of the same genetic linkage groups in 24. Sandgren, E. P., Luetteke, N. C.. Qiu. T. H.. Palmiter, R. D.. Brinster. R. L.. and Lee. D. C. Transforming growth factor a dramatically enhances oncogene induced carci mouse and human liver tumors indicates that these regions of the nogenesis in transgenic mouse pancreas and liver. Mol. Cell Biol., 13: 320-330. genome are critical in the neoplastic process. Due to the highly 1993. conserved genetic linkage groups between humans and mice (16, 17), 25. Murakami, H., Sanderson, N., Nagy, P.. Marino, P., Merlino, G. T.. and Thorgeirsson, S.S. Transgenic mouse model for synergistic effects of nuclear oncogenes and growth further examination of these fragile regions may provide critical factors in tumorigenesis: interaction of c-myc and transforming growth factor a in information about tumor susceptibility genes that are important in the hepatic oncogenesis. Cancer Res., 53: 1719-1723. 1993. 26. Martin. I.. Illett. K. J.. and Ninchin. F. Characterisation of putrescine uptake by early development of human hepatocellular carcinoma. cultured adult mouse hepatocytes. Biochim. Biophys. Acta. 1051: 52-59. 1990. 27. Dragan. Y. P.. Sargent, L.. Xu. Y-H, and Pilot, H. C. The initiation-promotion- progression model of rat hepatocarcinogenesis. Proc. Soc. Exp. Biol. Med.. 202: ACKNOWLEDGMENTS 16-24, 1993. Xu. Y-H., Sattler, G. L., and Pilot, H. C. A method for the comparative study of We thank Dr. Glenn Merlino (NIH. Bethesda. MD) for the generous gift of replicative DNA synthesis in GGT-positive and GGT-negative hepatocyles in primary the TGF-a mice and Dr. John Wiley for helpful suggestions on these results. culture isolated from carcinogen-treated rats. In Vitro, 24: 995-1000. 1988. 29. Sargent. L. M.. Sattler. G. L., Roioff, B., Xu, Y-H, Sattler, C. A.. Meisner. L., and Pitot. H. C. Ploidy and specific karyotypic changes during promotion with phÃ©no barbital. 2.5.2'.5'-tetrachlorobiphenyl. and/or 3.4.3',4'-tetrachlorobiphenyl in rat REFERENCES liver. Cancer Res., 52: 955-962, 1992. 1. Yeh, S-H., Chen, P-J.. Lai. M-Y.. Wang, C-C. and Chen. D-S. Frequent genetic 30. Cowell, J. K. A photographic representation of the variability in the G-banded alterations at the distal region of chromosome Ip in human hepatocellular carcinomas. structure of the chromosomes in the mouse karyotype. Chromosoma. 89: 294-320, Cancer Res.. 54: 4188-4192. 1994. 1984. 31. Paweletz, N.. Ghosh. S., and Vig. B. K. Mitosis and the induction of aneuploidy. Prog. Clin. Biol. Res.. 318: 217-229, 1989. 4 H. Ohgaki. personal communication. 32. Brinkley. B. R.. Tousson. A., and Valdivia. M. M. The kinetochore of mammalian 2141

chromosomes: structure and function in normal mitosis and aneuploidy. Basic Life genicity in human colon carcinoma cells by introduction of normal chromosome 1p36 Sci., 36: 243-267, 1985. region. Oncogene, 8: 2253-2258. 1993. 33. Liang, J. C., and Brinkley. B. R. Chemical probes as possible targets for the induction 48. Jonasson, J., Povey. S.. and Harris. H. The analysis of malignancy by cell fusion. VII. of aneuploidy. Basic Life Sci.. 36: 491-505. 1985. Cytogenetic analysis of hybrids between malignant and diploid cells and of tumours 34. Danielsen. H. E.. Brogger. A., and Reith. A. Specific gain of chromosome 19 in derived from them. J. Cell Sci.. 24: 217-254, 1977. preneoplastic mouse liver cells after diethylnitrosamine treatment. Carcinogenesis 49. Nobori. T.. Miura. K.. Wu. D. J.. Lois. A.. Takabayashi. K.. and Carson. D. A. (Lond.), 12: 177-180, 1988. Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers. 35. Becker. F. F.. Fox, R. A., Klein, L. M.. and Wolman. S. R. Chromosome patterns in Nature (Lond.). 368: 753-756, 1994. rat hepatocytes during /V-2-fluorenylacetamide Carcinogenesis. J. Nati. Cancer Inst.. 50. Nishida, N., Fukuda, Y., Komeda. T.. Kita. R.. Sando. T., Motonobu, F., Amenomori, 46: 1261-1269. 1971. M.. Shibagaki. !.. Nakao. K.. Ikenaga. M., and Ishizaki, K. Amplification and 36. Kerens. C., Gonzalez. M. L.. and Barbason. H. Cytogenelic changes in hepatomas overexpression of the cyclin DI gene in aggressive human hepatocellular carcinoma. from rats treated with chronic exposure to diethylnitrosamine. Cancer Genet. Cyto- Cancer Res., 54: 3107-3110. 1994. genet.. 60: 45-52. 1992. 51. Byme, J. A., and Smith. P. J. The I lpI5.5 ribonucleotide reducÃaseMl subunit locus is not imprinted in Wilms' tumor and hepatoblastoma. Human Genet.. 91: 275-277. 37. Cowell, J. K., and Wigley, C. B. Chromosome changes associated with the progres sion of cell lines from preneoplastic to lumorigenic phenotype during transformation 1993. of mouse salivary gland. J. Nail. Cancer Inst.. 69: 425-433, 1982. 52. Albrecht. S., von Schweinitz. D.. Waha, A.. Kraus. J. A., Vondeimling, A., and 38. Aldaz. C. M.. Conti. C. J.. Klein-Szanto. A. J. P.. and Slaga. T. J. Progressive Pietsch, T. Loss of maternal alÃeleson chromosome arm lip in hepatoblasloma. dysplasia and aneuploidy are hallmarks of mouse skin papillomas: relevance to Cancer Res.. 54: 5041-5044. 1994. malignancy. Proc. Nati. Acad. Sci. USA, 84: 2029-2032. 1987. 53. Weiseman. R. W., Stowere, S. J.. Miller, E. C.. Anderson, M. W., and Miller. J. A. 39. Popescu, N. C. Chromosome fragility and instability in human cancer. Crit. Rev. Activation of the mutations of the c-H-ra.v protooncogene in chemically induced Oncog., J: 121-140, 1994. hepatomas of the male B6C3F1 mouse. Proc. Nati. Acad. Sci. USA, 83: 5825-5829, 40. Hecht. D. F. Fragile sites, cancer chromosome breakpoints and oncogenes all cluster 1986. in light G bands. Cancer Genet. Cytogenel.. 31: 17-20, 1988. 54. Reynolds, S. H., Stowers, S. J.. Patterson, R. M.. and Maronpot. R. R. Activated 41. Glover, T. W., and Stein, C. K. Chromosome breakage and recombination at fragile oncogenes in B6C3F1 mouse liver tumors, implications for risk assessment. Science sites. Am. J. Hum. Genet., 43: 265-273. 1988. (Washington DC). 237: 1309-1316, 1987. 42. Davis, L. M.. Caspary. W. J.. Sakallah, S. A.. Maronpot. R.. Wiseman. R.. Barrett. 55. Lee. G. H. Detection of activated c-H-ra.v oncogene in hepalocellular carcinomas J. C.. Elliott. R.. and Hozier. J. C. Loss of heterozygosity in spontaneous and developing in transgenic mice harboring albumin promoter-regulated simian virus 40 chemically induced tumors of the B6C3F1 mouse. Carcinogenesis (Lond.l, 15: gene. Carcinogenesis (Lond.). //: 1145-1148. 1990. 1637-1645, 1994. 56. Bremner. R.. and Balmain. A. Genetic changes in skin tumor progression: correlation 43. Nishimori. H., Ogawa. K., and Tateno, H. Frequent deletion in chromosome 4 and between presence of a mutant ras gene and loss of heterozygosity on mouse chro duplication of chromosome 15 in liver epithelial cells derived from long term culture mosome 7. Cell. 61: 407-417. 1990. of C3H mouse hepatocytes. Int. J. Cancer, 59: 108-113. 1994. 57. Held, W. A.. Pazik. J., Giancola, J.. O'Brien, G.. Kerns. K., Gobey. M., Mels. R., 44. Miyasaka. K.. Ohtake. K.. Nomura, K.. Kanda. H., Kominami. R.. Miyashita. N.. and Kenny. L.. and Rustum. Y. Genetic analysis of liver tumorigenesis in SV40 T antigen Kitagawa. T. Frequent loss of heterozygosity on chromosome 4 in diethylnitrosamine- transgenic mice implies a role for imprinted genes. Cancer Res.. 54: 6489-6495, induced C3H/MSM mouse hepatocellular carcinomas in culture. Mol. Carcinog.. /3: 1994. 37-43. 1995. 58. Weissman, B. E.. Saxon, P. J., Pasquale, S. R., Jones, G. R., Geiser. A. G., and 45. Genuardi. M., Tsihiara. H.. Anderson, D. E.. and Saunders, G. F. Distal deletion of Standbridge. L. J. Introduction of a normal human chromosome 11 into Wilms' tumor chromosome Ip in ductal carcinoma of the breast. Am. J. Hum. Genet., 45: 73-82, cell line controls its tumorigenic expression. Science (Washington DC). 236: 175- 1989. 180. 1987. 46. Bieche. !.. ChÃ¡mpeme. M. H.. Matifas. F.. and Cropp, C. S. Two distinct regions 59. Davis, L. M.. Caspary. W. J., Sakallah, S. E., Maronpot. R.. Wiseman. R.. Barrett. involved in Ip deletion in human primary breast cancer. Cancer Res., 53: 1990-1994. J. C., Elliott. R.. and Hozier, J. C. Loss of heterozygosity in spontaneous and 1993. chemically induced tumors of the B6C3F1 mouse. Carcinogenesis (Lond.), 15: 47. Tanaka, K., Yanoshita, R., Konishi. M.. and Oshimura. M. Suppression of tumouri- 1637-1645, 1994.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1996 American Association for Cancer Research. Ploidy and Karyotypic Alterations Associated with Early Events in the Development of Hepatocarcinogenesis in Transgenic Mice Harboring c-myc and Transforming Growth Factor α Transgenes

Linda M. Sargent, Nancy D. Sanderson and Snorri S. Thorgeirsson

Cancer Res 1996;56:2137-2142.

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