Somatic mutations of the beta-catenin gene are frequent in mouse and human hepatocellular carcinomas

Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading to liver cell transformation remain largely unknown. The Wnt-beta-catenin pathway is activated in colon cancers and some melanoma cell lines, but has not yet been investigated in HCC. We have examined the status of the beta-catenin gene in different transgenic mouse lines of HCC obtained with the oncogenes c-myc or H-ras. Fifty percent of the hepatic tumors in these transgenic mice had activating somatic mutations within the beta-catenin gene similar to those found in colon cancers and melanomas. These alterations in the beta-catenin gene (point mutations or deletions) lead to a disregulation of the signaling function of beta-catenin and thus to carcinogenesis. We then analyzed human HCCs and found similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations led to the accumulation of beta-catenin in the nucleus. Thus alterations in the beta-catenin gene frequently are selected for during liver tumorigenesis and suggest that disregulation of the Wnt-beta-catenin pathway is a major event in the development of HCC in humans and mice.     

The Helsinki Heart Study: an 8.5-year safety and mortality follow-up

MT-PVLT-10 transgenic mice express the large T-antigen of polyomavirus under the control of the mouse metallothionein-1 promoter. The males of this transgenic line developed testicular tumor and seminal vesicle engorgement at advanced ages. A novel partial cDNA was identified which hybridized to a 2.6 kilobase mRNA. The expression of this mRNA increased approximately two- to fifteen-fold in immortalized cell lines derived from testicular tumors as compared to similar cell lines derived from pre-adenomatous testes. The in vivo pattern of expression for this cDNA as well as its expression in various primary cultures and established cell lines derived from testis of MT-PVLT-10 mice is presented. Overlapping cDNA clones from liver, testes, and brain cDNA libraries containing the entire coding region for this novel cDNA have been isolated and sequenced. The coding region of this gene comprises 1179 nucleotides and predicts a polypeptide of 393 amino acids (calculated molecular mass 44,318). Motif analysis of the amino acid sequence has revealed that it contains several hydrophobic alpha-helices characteristic of transmembrane proteins.     

Expression of bovine beta-lactoglobulin/human erythropoietin fusion protein in the milk of transgenic mice and rabbits

We have generated several transgenic mouse lines and rabbits expressing efficiently (up to 0.3 mg/ml in mice and up to 0.5 mg/ml in rabbits) human erythropoietin in their milk as bovine beta-lactoglobulin fusion protein. Human erythropoietin cDNA was inserted in frame into exon 5 of the bovine beta-lactoglobulin gene with a linker oligonucleotide encoding the cleavage site for bacterial IgA protease. RNA analysis performed on one lactating transgenic mouse and one transgenic rabbit revealed that the fusion gene was expressed almost exlusively in the mammary gland, although low amounts of transgene-derived RNA were detectable in salivary glands and uterus or in the kidney. The fusion protein was specifically cleaved with IgA protease. The erythropoietin part obtained upon digestion had a lower molecular mass than recombinant erythropoietin produced in Chinese hamster ovary cells. By deglycosylation analysis it was shown that the difference in size was due to a different type of glycosylation. Biological activity of the fusion protein, as determined by growth stimulation of TF-1 erythroleukemia cells, was less than 15% of that of human recombinant erythropoietin. Upon digestion of the fusion protein with IgA protease, biological activity comparable to that of the recombinant erythropoietin was recovered. Transgenic males and virgin females did not show signs of enhanced erythropoiesis, but lactating females expressing the transgene displayed transient increases in their hematocrit values.     

The myeloperoxidase gene proximal enhancer directs hematopoietic-specific expression in transgenic mice

The myeloperoxidase (MPO) gene is expressed specifically in immature myeloid cells. The MPO gene includes a promoter proximal enhancer which is coincident with DNaseI hypersensitive chromatin sites and is specifically active in myeloid cell lines. We developed transgenic murine lines in which 1.3 kb of murine MPO proximal 5' flanking region DNA was linked to a TATAA homology and RNA initiation site derived from the HSV-TK promoter and to a luciferase reporter (MPOTKLUC). In each of six founder lines, high-level luciferase activity was evident in marrow, thymus and spleen. Modest- to high-level luciferase expression was also evident in brain and in the heart in several of the lines, and luciferase activity was at or near background levels in lung, liver, kidney, stomach, colon, bladder, skeletal muscle, skin and small intestine in all of the MPOTKLUC transgenic mice. Within marrow cells, luciferase activity was evident in myeloid (GR-1+), B lymphoid (B220+) and T-lymphoid (CD4+) cells. Additional regulatory regions, thus, may be required to further restrict MPO gene expression to immature myeloid cells.     

Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers

To investigate the in vivo role of hepatocyte growth factor/scatter factor (HGF/SF) in liver function, we generated transgenic mice using a mouse HGF/SF cDNA under the control of the mouse metallothionein gene promoter and 5'/3' flanking sequences. In adult HGF/SF transgenic mice, liver weight as a percentage of total body weight was at least twice that of wild-type mice. Comparison of transgenic and control liver morphology revealed dramatic heterogeneity in the size and appearance of hepatocytes as a distinctive feature of HGF/SF overexpression. Transgenic livers exhibited a significant increase in the number of small hepatocytes with a 2N DNA content, accounting for the observed increase in liver mass. The DNA labeling index of hepatocytes increased 11-fold at 4 weeks of age, when liver enlargement first became apparent, and was still elevated about 5-fold in adult HGF/SF transgenic mice. Moreover, hepatocytes isolated by perfusion of transgenic livers doubled every 2 days in culture, whereas little or no growth was observed with isolated control hepatocytes. The mechanistic basis of hepatocyte proliferation was elucidated as the chronic activation of the c-met proto-oncogene product. Met and substrates such as phosphatidylinositol 3-kinase, Src homology and collagen-like, pp60c-src, focal adhesion kinase p125FAK, and paxillin were associated with tyrosine-phosphorylated complexes in a hepatocyte cell line established from the transgenic liver. This proliferative stimulus triggered the formation of hepatocellular adenomas and/or carcinomas in most transgenic mice > or = 1.5 years of age. Finally, the rate of transgenic mouse liver regeneration was increased 3-fold over control livers following partial hepatectomy.     

Apo B100-containing lipoproteins are secreted by the heart

The apo B gene is expressed in the human heart and in the hearts of human apo B transgenic mice generated with large genomic clones spanning the human apo B gene. [35S]Methionine metabolic labeling experiments demonstrated that apo B100-containing lipoproteins are secreted by human heart tissue and by human apo B transgenic and nontransgenic mouse heart tissue. Density gradient analysis revealed that most of the secreted heart lipoproteins were LDLs, even when the labeling experiments were performed in the presence of tetrahydrolipstatin, an inhibitor of lipoprotein lipase. Western blots with a microsomal triglyceride transfer protein) (MTP)-specific antiserum demonstrated that the microsomes of the heart contain the 97-kD subunit of MTP (the subunit involved in the transfer of lipids and assembly of lipoproteins). Metabolic labeling of mouse heart tissue in the presence of BMS-192951, an MTP inhibitor, abolished lipoprotein secretion by the heart but resulted in the secretion of two apo B proteolytic fragments (80 and 120 kD), which were found in the bottom fraction of the density gradient. These studies reveal that the heart, and not just the liver and intestine, secretes apo B-containing lipoproteins. We speculate that lipoprotein secretion by the heart represents a mechanism for removing excess lipids from the heart.     

The effect of mammary gland expression of human lysozyme on the properties of milk from transgenic mice

Transgenic mice were used as model systems to evaluate the impact of human lysozyme expression in the mammary gland. We previously generated two lines of transgenic mice that express human lysozyme mRNA in the mammary gland under the tissue-specific and developmentally correct control of the bovine gene promoter for alpha s1-casein. Concentrations of human lysozyme protein in milk of transgenic mice varied from .25 to .71 micrograms/microliters of milk. Human lysozyme secreted into mouse milk retained its antimicrobial activity, as determined by a denaturing polyacrylamide gel activity assay. The physical and functional properties of the milk were also altered, because mouse milk containing human lysozyme had a 35% decrease in rennet clotting time, a smaller median micelle size (157 nm vs. 172 nm), and a 2.5- to 3-fold greater gel strength than control milk. From these results, we conclude that the use of transgenic animals producing lysozyme in the milk is feasible and potentially useful to the dairy industry.     

Efficient conditional transgene expression in hepatitis C virus cDNA transgenic mice mediated by the Cre/loxP system

Conditional gene expression has greatly facilitated the examination of the functions of particular gene products. Using the Cre/loxP system, we developed efficient conditional transgene activation of hepatitis C virus (HCV) cDNA (nucleotides 294-3435) in transgenic mice. Efficient recombination was observed in transgenic mouse liver upon intravenous administration of adenovirus that expresses Cre DNA recombinase. After transgene activation, most hepatocytes were stained with anti-core polyclonal antibody, and 21-, 37-, and 64-kDa proteins were detected by Western blot analysis in liver lysates using anti-core, E1, and E2 monoclonal antibodies, respectively. Serum core protein was detected in transgenic mice 7 days after transgene activation with concurrent increases in serum alanine aminotransferase levels. Subsequently, an anti-core antibody response was detected 14 days after infection. Furthermore, a CD4 and CD8 positive cell depletion assay normalized both the serum alanine aminotransferase increases and pathological changes in the liver. These results suggest that HCV proteins are not directly cytopathic and that the host immune response plays a pivotal role in HCV infection. Thus, this HCV cDNA transgenic mouse provides a powerful tool with which to investigate the immune responses and pathogenesis of HCV infection.     

Functional correction of renal defects in a mouse model for ARPKD through expression of the cloned wild-type Tg737 cDNA

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the formation of large collecting tubule and ductular cysts that often result in renal insufficiency within the first decade of life. Understanding the process leading to cyst formation will require the identification and characterization of genes involved in the etiology of this disease. In this regard, we previously described the generation of a mouse model (TgN737Rpw) for ARPKD and the cloning of a candidate gene. Here we show direct involvement of the Tg737 gene in collecting duct cyst formation by expressing the wild-type Tg737 cDNA as a transgene in TgN737Rpw mutants. In contrast to TgN737Rpw mutants, the "rescued" animals survive longer, have normal renal function and normal localization of the EGFr to the basolateral surfaces of collecting duct epithelium.     

Protein oxidative damage in a transgenic mouse model of familial amyotrophic lateral sclerosis

The Gly93-->Ala mutation in the Cu,Zn superoxide dismutase (Cu,Zn-SOD) gene (SOD1) found in some familial amyotrophic lateral sclerosis (FALS) patients has been shown to result in an aberrant increase in hydroxyl radical production by the mutant enzyme that may cause oxidative injury to spinal motor neurons. In the present study, we analyzed the extent of oxidative injury to lumbar and cervical spinal cord proteins in transgenic FALS mice that overexpress the SOD1 mutation [TgN(SOD1-G93A)G1H] in comparison with nontransgenic mice. Total protein oxidation was examined by spectrophotometric measurement of tissue protein carbonyl content by the dinitrophenylhydrazine (DNPH) assay. Four ages were investigated: 30 (pre-motor neuron pathology and clinical disease), 60 (after initiation of pathology, but pre-disease), 100 (approximately 50% loss of motor neurons and function), and 120 (near complete hindlimb paralysis) days. Protein carbonyl content in 30-day-old TgN(SOD1-G93A)G1H mice was twice as high as the level found in age-matched nontransgenic mice. However, at 60 and 100 days of age, the levels were the same. Then, between 100 and 120 days of age, the levels in the TgN(SOD1-G93A)G1H mice increased dramatically (557%) compared with either the nontransgenic mice or transgenic animals that overexpress the wild-type human Cu,Zn-SOD [TgN(SOD1)N29]. The 100-120-day increase in spinal cord protein carbonyl levels was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic separation and western blot immunoassay, which enabled the identification of heavily oxidized individual proteins using a monoclonal antibody against DNPH-derivatized proteins. One of the more heavily oxidized protein bands (14 kDa) was identified by immunoprecipitation as largely Cu,Zn-SOD. Western blot comparison of the extent of Cu,Zn-SOD protein carbonylation revealed that the level in spinal cord samples from 120-day-old TgN(SOD1-G93A)G1H mice was significantly higher than that found in age-matched nontransgenic or TgN(SOD1)N29 mice. These results suggest that the increased hydroxyl radical production associated with the G93A SOD1 mutation and/or lipid peroxidation-derived radical species (peroxyl or alkoxyl) causes extensive protein oxidative injury and that the Cu,Zn-SOD itself is a key target, which may compromise its antioxidant function.     

NK cells from human MHC class I (HLA-B) transgenic mice do not mediate hybrid resistance killing against parental nontransgenic cells

We have investigated the capacity of human MHC class I HLA-B gene products, HLA-B27, -B7 (fully human), and -B7kb (human-mouse hybrid consisting of the alpha1 and alpha2 domains of HLA-B7, and the alpha3 and cytoplasmic domains of mouse H-2Kb), expressed on mouse NK cells during ontogeny to influence NK recognition of otherwise syngeneic mouse target cells. Despite a high level of surface expression of the transgene (comparable to that of endogeneous H-2DbKb molecules), the direct killing of YAC-1 targets, and the killing of P815 targets in a redirected lysis assay, the NK effectors of these transgenic mice could not mediate hybrid resistance-like killing of nontransgenic C57BL/6 target cells either in vitro or in vivo. Splenocytes from B6-B27 mice could be used to generate CTL lines against a B27-binding peptide, implying that T cells restricted by HLA-B27 developed during ontogeny. NK cells from B6-B27 could lyse B6-B27 Con A lymphoblasts pulsed with Db-binding peptide but not B27-binding peptides. Taken together, our results show that these human HLA-B transgene products cannot function as class I MHC "self" elements for mouse NK cells, even when present throughout ontogeny.     

Parameters affecting the use of the lac repressor system in eukaryotic cells and transgenic animals

Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals. A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences. In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction. Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene. In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent. After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary. In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes. Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures. The applicability and challenges of the system for regulation of gene expression are discussed.