Development of a transgenic mouse system for the analysis of stages in liver carcinogenesis using tissue-specific expression of SV40 large T-antigen controlled by regulatory elements of the human alpha-1-antitrypsin gene.

alpha-1-Antitrypsin (AAT) is the major antiprotease in human plasma; it is synthesized primarily in hepatocytes and to a lesser extent in several nonhepatic tissues. Under the control of regulatory elements of the human AAT gene, expression of SV40-large tumor antigen (T-ag) in transgenic mice occurred in the liver, stomach, pancreas, and kidney. Among seven founder transgenic animals, six developed liver carcinoma, four showed gastric neoplasia, and one developed pancreatic carcinoma. In three animals the kidneys showed glomerular or tubular epithelial hyperplasia but no malignancy. A stable transgenic line, 1812, was established. Members of this line reproducibly develop liver tumors by 10 weeks of age but do not exhibit any phenotypic changes in other tissues. Histological changes leading to liver tumor formation occurred with predictable kinetics and could be classified into four distinct stages: (a) embryonal/fetal stage, no recognizable histological changes; (b) newborn to 2 weeks of age, hyperplastic hepatocytes with reduced amounts of cytoplasm but no nuclear alterations; (c) between 3 and 8 weeks of age, diffuse liver cell dysplasia without observable tumor nodules; and (d) 8 weeks of age and thereafter, hepatocellular carcinomas in a background of liver dysplasia. Embryonic and newborn liver tissue showed uniform, high level expression of T-ag in the majority of hepatocytes by immunohistochemistry, whereas the dysplastic and tumoral stages were characterized by considerable variation in both the intensity of T-ag staining and the proportion of T-ag-positive cells. Immunoprecipitation analyses showed that T-ag was complexed with cellular protein p53 in all tumor samples. This study showed that SV40 T-ag expression in the liver resulted in cellular hyperplasia and dysplasia; additional event(s) apparently were required for progression to neoplasia. Those cooperating events occurred with predictable kinetics. This transgenic mouse system displays several similarities with human liver disease and provides a practical model for the study of separate steps in hepatocarcinogenesis.

Constitutive achaete-scute homologue-1 promotes airway dysplasia and lung neuroendocrine tumors in transgenic mice.

The transcription factor achaete-scute homologue-1 (ASH1) is essential for neural differentiation during fetal development and is a cardinal feature of neuroendocrine (NE) tumors such as small cell lung cancer. To explore the potential of ASH1 to promote NE differentiation and tumorigenesis in the lung, we constitutively expressed the factor in nonendocrine airway epithelial cells using transgenic mice. Progressive airway hyperplasia and metaplasia developed beginning at 3 weeks of life. ASH1 potently enhanced the tumorigenic effect of SV40 large T antigen in airway epithelium. These doubly transgenic animals developed massive NE lung tumors, implying that ASH1 may cooperate with defects in p53, pRb, or related pathways in promoting NE lung carcinogenesis.

Hepatitis B virus transactivator X protein is not tumorigenic in transgenic mice.

The hepatitis B virus X protein acts as a transcriptional transactivator in vitro. To elucidate possible biological effects of X protein on liver cells in vivo, we generated four lines of transgenic mice carrying the X gene open reading frame under the control of the human alpha-1-antitrypsin regulatory region. The plasmid construct used to introduce the transgene was shown to encode a 16-kDa X protein with transactivating capability. The expression of X protein was detectable in liver tissue of transgenic animals of three of the lines by immunoblot analysis; levels of expression were highest in the first month after birth of the animals. Over 80 animals from the expressing lines were examined histologically. Most transgenic mice, some of which were observed for up to 2 years, remained normal. However, a few transgenic animals developed mild focal hepatitis, nuclear pleomorphism, focal necrosis, and/or nodular hyperplasia in the liver. Increased mitotic activity of hepatocytes also was observed. We conclude that, at the level of expression achieved in these transgenic mice, the hepatitis B virus transcriptional transactivator X protein alone does not appear to mediate the development of serious liver damage or hepatocellular carcinomas.

Tissue-specific regulation of human alpha 1-antitrypsin gene expression in transgenic mice.

The 5'-flanking sequence of the human alpha 1-antitrypsin (AAT) gene contains multiple cis-regulatory elements, including a distal enhancer and proximal sequences essential for its transcription in cultured hepatoma cells. To understand better the promoter specificity of the AAT gene in vivo, transgenic mice harboring the AAT-SV40 hybrid promoter or the natural AAT promoter fused to a reporter gene (CAT) were generated. Examination of CAT activity in various tissues indicated that the CAT gene was expressed primarily in the liver and also, to a lesser extent, in tissues known to express the AAT gene. In addition, the cis-acting elements of the human AAT gene were utilized to drive the transcription of the SV40 T antigen gene in transgenic mice. Hepatocellular malignancy was found in all founder animals examined, while sporadic occurrence of malignancy was also observed in stomach, pancreas, and kidney. These results verify that the 5'-flanking region of the human AAT gene contains cis-regulatory elements sufficient to confer tissue specificity in vivo.

Tissue- and development-specific expression of the human phenylalanine hydroxylase/chloramphenicol acetyltransferase fusion gene in transgenic mice.

Human phenylalanine hydroxylase (PAH) catalyzes the conversion of L-phenylalanine to L-tyrosine. Deficiency of this enzyme results in phenylketonuria, a common genetic disorder of amino acid metabolism that causes severe mental retardation. In primates, PAH is expressed specifically in the liver, while in rodents PAH activity is also present in kidney, although at a much lower level. A 9-kilobase genomic DNA fragment at the 5' end of the hPAH gene (hPAH) was fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The hPAH/CAT minigene was used to generate multiple transgenic mouse lines. In all expressing lines, CAT activity was detected predominantly in the liver and at much lower levels in the kidney. By immunohistochemical staining, CAT expression was localized to hepatocytes and renal epithelial cells, both of which also express the endogenous mouse PAH enzyme. Furthermore, both the transgene and the endogenous mouse PAH were activated at about the same stage of embryonic development in the mouse liver. These results suggest that the 9-kilobase DNA fragment flanking the 5' end of the human PAH gene contains all the necessary cis-acting elements to direct tissue- and developmental-specific expression in vivo.