Nonrandom cytogenetic alterations in hepatocellular carcinoma from transgenic mice overexpressing c-Myc and transforming growth factor-alpha in the liver.

Identification of specific and primary chromosomal alterations during the course of neoplastic development is an essential part of defining the genetic basis of cancer. We have developed a transgenic mouse model for liver neoplasia in which chromosomal lesions associated with both the initial stages of the neoplastic process and the acquisition of malignancy can be analyzed. Here we analyze chromosomal alterations in 11 hepatocellular carcinomas from the c-myc/TGF-alpha double-transgenic mice by fluorescent in situ hybridization with whole chromosome probes, single-copy genes, and 4'-6-diamidino-2-phenylindole (DAPI-) and G-banded chromosomes and report nonrandom cytogenetic alterations associated with the tumor development. All tumors were aneuploid and exhibited nonrandom structural and numerical alterations. A balanced translocation t(5:6)(G1;F2) was identified by two-color fluorescent in situ hybridization in all tumors, and, using a genomic probe, the c-myc transgene was localized near the breakpoint on derivative chromosome der 6. Partial or complete loss of chromosome 4 was observed in all tumors with nonrandom breakage in band C2. Deletions of chromosome 1 were observed in 80% of the tumors, with the most frequent deletion at the border of bands C4 and C5. An entire copy of chromosome 7 was lost in 80% of the tumors cells. Eighty-five percent of the tumor cells had lost one copy of chromosome 12, and the most common breakpoint on chromosome 12 occurred at band D3 (28%). A copy of chromosome 14 was lost in 72%, and band 14E1 was deleted in 32% of the tumor cells. The X chromosome was lost in the majority of the tumor cells. The most frequent deletion on the X chromosome involved band F1. We have previously shown that breakages of chromosomes 1, 6, 7, and 12 were observed before the appearance of morphologically distinct neoplastic liver lesions in this transgenic mouse model. Thus breakpoints on chromosome 4, 9, 14, and X appear to be later events in this model of liver neoplasia. This is the first study to demonstrate that specific sites of chromosomal breakage observed during a period of chromosomal instability in early stages of carcinogenesis are later involved in stable rearrangements in solid tumors. The identification of the 5;6 translocation in all of the tumors has a special significance, being the first balanced translocation reported in human and mouse hepatocellular carcinoma and having the breakpoint near a tumor susceptibility gene and myc transgene site of integration. Moreover, its early occurrence indicates that this is a primary and relevant alteration to the initiation of the neoplastic process. In addition, the concordance between the breakpoints observed during the early dysplastic stage of hepatocarcinogenesis and the stable deletions of chromosomes 1, 4, 6, 7, 9, and 12 in the tumors provides evidence for preferential site of genetic changes in hepatocarcinogenesis.

Molecular analyses of liver tumors in c-myc transgenic mice and c-myc and TGF-alpha double transgenic mice.

It has been demonstrated that co-expression of c-myc and transforming growth factor alpha (TGF-alpha) as transgenes in the mouse liver results in a tremendous acceleration of neoplastic development in this organ as compared to expression of either transgene alone [Murakami, H., et al. (1993) Cancer Res., 53, 1719-1723]. In order to clarify the roles of transgenes and additional other genetic alterations during hepatocarcinogenesis, we analyzed liver tumors developed in albumin/c-myc transgenic mice and albumin/c-myc and MT-1/TGF-alpha double transgenic mice. High expression of TGF-alpha transgene was found in nine of 14 (64%) liver tumors in double transgenic mice, suggesting that TGF-alpha overexpression confers growth advantage during hepatocarcinogenesis. Only one of 14 (7%) liver tumors in double transgenic mice and none of 13 liver tumors in c-myc transgenic mice showed overexpression of insulin-like growth factor II (IGF-II). This result was in contrast to the report by Takagi et al. [Takagi, H., et al. (1992) Cancer Res., 52, 5171-5177] which showed overexpression of IGF-II in 75% of liver tumors in TGF-alpha transgenic mice and suggested that the presence of c-myc transgenes together with TGF-alpha from an early stage of hepatocarcinogenesis may lead to different carcinogenic pathways which are independent of IGF-II overexpression. Expression of c-myc transgene was found in most of the liver tumors, but at lower levels than non-tumorous parts of the liver in c-myc and double transgenic mice. These results suggest that c-myc transgene expression cooperates with TGF-alpha in the early stages of hepatocarcinogenesis but has growth disadvantage in later stages of hepatocarcinogenesis. There was no evidence of mutational activation of the H-ras gene or mutational inactivation of the p53 gene in any liver tumors developed in c-myc or double transgenic mice.

Ploidy and karyotypic alterations associated with early events in the development of hepatocarcinogenesis in transgenic mice harboring c-myc and transforming growth factor alpha transgenes.

The cooperation of the c-myc oncogene with the growth factor transforming growth factor (TGF)-alpha in development of liver tumors in transgenic mice has been demonstrated previously. In this study, we analyzed the ploidy and karyotype of c-myc, TGF-alpha, parental control, and the double transgenic c-myc/TGF-alpha hepatocytes at 3 weeks of age when the liver is histologically normal and at 10 weeks when the c-myc/TGF-alpha liver is dysplastic and contains basophilic foci. Eighty % of the 10-week hepatocytes were aneuploid, and 32% had chromosomal breakage. Statistically significant breakage was observed in six different chromosomes. Breakage at band A5 and at the border of bands C4/5 of chromosome 1 was observed. Fragile sets on chromosome 4 were most frequent in the middle of the chromosome at bands C2 and C6. Chromosome 6 was fragile at band F2. The region of chromosome 7 at bands B5 and D3 was frequently broken and involved in translocations. Chromosome 12 was broken at bands D1 and D3. The breakage sites on chromosomes 1, 4, 7, and 12 correspond to sites of tumor susceptibility genes in the mouse. Although there was no consistent change in copy number, recurrent translocations between chromosomes 1, 4, 7, 12 and 19 were also observed. These studies demonstrate that the development of dysplasia and basophilic foci in the liver is correlated with aneuploidy and chromosome breakage. The specific fragile sites indicate genetic regions that are altered during early stages of hepatocarcinogenesis. Due to the conservation of genetic linkage groups between mice and humans, the identification of genetic alterations in the mouse during hepatocarcinogenesis may provide critical information about tumor susceptibility genes that are important in the early development of human hepatocellular carcinoma.

Transgenic mouse model for synergistic effects of nuclear oncogenes and growth factors in tumorigenesis: interaction of c-myc and transforming growth factor alpha in hepatic oncogenesis.

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc complementary DNA and mouse metallothionein 1 promoter-human transforming growth factor alpha complementary DNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and transforming growth factor alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and transforming growth factor alpha, and possibly other combinations of nuclear oncogenes and growth factors, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion.

Metabolic activation and genotoxicity of N-hydroxy-2-acetylaminofluorene and N-hydroxyphenacetin derivatives in Reuber (H4-II-E) hepatoma cells.

Derivatives of both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxyphenacetin (N-OH-P) were tested for their ability to cause DNA damage in Reuber (H4-II-E) cells using the alkaline elution technique. Reuber cells are devoid of N-OH-AAF deacylase, N,O-acyltransferase, and sulfotransferase activities. The hydroxamic acids themselves caused very little DNA damage, while N-hydroxy-2-aminofluorine (20 to 100 microM), N-hydroxyphenetidine (20 to 200 microM), and p-nitrosophenetole (10 to 100 microM) all caused dose-dependent damage. The dose-dependent DNA damage caused by N-acetoxy-2-acetylaminofluorene (5 to 25 microM) was completely inhibited by the deacylase inhibitor paraoxon (100 microM). In the presence of both partially purified rabbit liver cytosolic N,O-acyltransferase and guinea pig liver microsomal deacylase, N-OH-AAF was genotoxic. Neither paraoxon nor tRNA had any effect on the DNA damage induced by N-OH-AAF in the presence of N,O-acyltransferase, while paraoxon completely inhibited the damage when N-OH-AAF was incubated in the presence of guinea pig deacylase, and N-OH-P only caused slight DNA damage at higher concentrations of enzyme. In addition, partially purified guinea pig liver deacylase and N-OH-AAF (25 microM) caused 2600 revertants in the Salmonella test system, while only 380 revertants were seen with a 40-fold greater concentration of N-OH-P (1000 microM). The mutagenicity of both N-OH-AAF and N-OH-P was completely inhibited by paraoxon. Thus, it is clear that metabolites of N-OH-AAF formed outside the cell are capable of passing both the cellular and nuclear membranes to cause genotoxicity. Metabolic activation of N-OH-AAF by either the membrane-bound deacylase or the cytosolic N,O-acyltransferase caused genotoxicity via a deacetylation process. Metabolic activation of N-OH-P by guinea pig deacylase caused low levels of DNA damage, whereas activation by N,O-acyltransferase was not sufficient to cause genotoxicity.

Kinetic evidence for the involvement of multiple forms of human liver cytochrome P-450 in the metabolism of acetylaminofluorene.

The kinetics of 2-acetylaminofluorene (AAF) metabolism have been studied in liver microsomes from four human subjects over a substrate range of 0.02-200 microM. The N-hydroxylation of AAF was best described by a single enzyme system with a mean Km of 1.63 microM and a mean Vmax of 61 pmol mg-1 protein min-1. Biphasic kinetics for the 7-hydroxylation of AAF in all four subjects were observed and a two enzyme system best described the data. The mean Km and Vmax for the high affinity, low-capacity enzyme were 0.69 microM and 30 pmol mg-1 protein min-1, respectively, while for the low affinity, high capacity enzyme the mean Km was 75 microM and the mean Vmax was 286 pmol mg-1 protein min-1. In one of the four subjects studied the 5-hydroxylation of AAF was similarly resolved into a two enzyme system. The 1-hydroxylation of AAF in human microsomes was a major reaction and was best described by a single enzyme system. The 3- and 5-hydroxylations of AAF were minor metabolic pathways. Non-classical Michaelis-Menten kinetics were observed for the 9-hydroxylation of AAF and at high substrate concentrations this was the major metabolite formed. These data demonstrate the involvement of at least two forms of human cytochrome P450 in the metabolism of AAF.