[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 ho- mology 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 ±1.0 c-myc 1.0 ±1.0 <1.0 with avertine, and the livers were perfused with a collagenase solution as c-mvc/TGF-a 10.0 ±1.0° <1.0 described previously (26). A section of the liver was tied off with silk thread TGF-aCells 4.0 ±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.