Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice.

We previously reported the disruption of the murine gene encoding the transcription factor USF2 and its consequences on glucose-dependent gene regulation in the liver. We report here a peculiar phenotype of Usf2(-/-) mice that progressively develop multivisceral iron overload; plasma iron overcomes transferrin binding capacity, and nontransferrin-bound iron accumulates in various tissues including pancreas and heart. In contrast, the splenic iron content is strikingly lower in knockout animals than in controls. To identify genes that may account for the abnormalities of iron homeostasis in Usf2(-/-) mice, we used suppressive subtractive hybridization between livers from Usf2(-/-) and wild-type mice. We isolated a cDNA encoding a peptide, hepcidin (also referred to as LEAP-1, for liver-expressed antimicrobial peptide), that was very recently purified from human blood ultrafiltrate and from urine as a disulfide-bonded peptide exhibiting antimicrobial activity. Accumulation of iron in the liver has been recently reported to up-regulate hepcidin expression, whereas our data clearly show that a complete defect in hepcidin expression is responsible for progressive tissue iron overload. The striking similarity of the alterations in iron metabolism between HFE knockout mice, a murine model of hereditary hemochromatosis, and the Usf2(-/-) hepcidin-deficient mice suggests that hepcidin may function in the same regulatory pathway as HFE. We propose that hepcidin acts as a signaling molecule that is required in conjunction with HFE to regulate both intestinal iron absorption and iron storage in macrophages.

The consequence of p53 overexpression for liver tumor development and the response of transformed murine hepatocytes to genotoxic agents.

To analyse the effect of p53 on liver tumor development, we generated transgenic mice overexpressing wild-type p53 in the liver and crossed them with transgenic mice in which the expression of the SV40 large T antigen (TAg) induces hepatic tumors. Remarkably, whereas preneoplastic TAg liver exhibited anisocaryosis and anisocytosis, TAg/p53 liver never presented any dysplastic cells. Moreover, whereas expression of p53 did not affect hepatic development, its constitutive expression in tumorigenic livers resulted in a significantly enhanced apoptosis once nodules had appeared. In contrast, p53 overexpression did not modify the elevated proliferation of TAg-transformed hepatocytes and had no effect on hepatocarcinoma progression. In vitro analysis of primary hepatocytes exposed to various genotoxic agents showed that p53 failed to sensitize normal or TAg-transformed hepatocytes to apoptosis, except when high doses of doxorubicin, UV-B and UV-C radiation were used. Our results confirmed that the hepatocyte cell type is very resistant to genotoxic agents and showed that constitutive expression of p53 failed to improve their responsiveness. In addition, our results showed that suppression of dysplastic cells, probably by restoring normal cytokinesis and karyokinesis, and enhancement of apoptosis by means of p53 overexpression were insufficient to counteract or delay the TAg-induced liver tumoral progression. Oncogene (2000) 19, 3498 - 3507

Annexin 1 expression and phosphorylation are upregulated during liver regeneration and transformation in antithrombin III SV40 T large antigen transgenic mice.

We have used a transgenic animal model, which constitutively develops hepatocarcinoma (Antithrombin III SV40 T large Antigen: ASV), to study the involvement of Annexin 1 (ANX1) in liver regeneration and malignant transformation. Primary hepatocytes isolated from normal mice did not express ANX1. In contrast, ANX1 was strongly expressed in hepatocytes of transgenic mice during constitutive development of hepatocarcinoma. In ASV transgenic mice, an elevated ANX1 level preceded the appearance of the tumor, indicating that it could be a good marker in the diagnosis of cancer. One-third hepatectomy in normal mice resulted in stimulation of ANX1 synthesis and phosphorylation. This upregulation correlated with increased synthesis of EGF and consequently with increased phosphorylation of the EGF receptor (EGF-R). Stable transfection of a hepatocyte cell line derived from ASV transgenic mice (mhAT2) with antisense complementary DNA for ANX1 reduced the proliferation rate as well as cytosolic phospholipase A(2) (cPLA(2)) activity. Thus, ANX1 expression and phosphorylation could be a factor implicated in liver regeneration and tumorigenesis, either through modulation of cPLA(2) activity or EGF-R function.

The amino-terminal region of SV40 large T antigen is sufficient to induce hepatic tumours in mice.

The transforming activity of SV40 large T-antigen (Tag) depends on its binding to cellular proteins involved in the control of the cell cycle (p53, pRb, p300..) and on the J-domain region in the amino-terminus. We established transgenic lines expressing wild-type or Tag mutant proteins lacking one of the three transforming domains, to determine the respective contributions of these domains to hepatic tumour formation. Tag mutants with no pRb-binding domain or N-terminal fragment did not cause neoplastic liver abnormalities. The d11137 Tag mutant protein, which inhibits pRb function without affecting p53, induced hepatic tumours. These tumours grew significantly faster than those induced by wild-type Tag. Our results demonstrate different requirements for each of the inactivating functions of SV40 Tag in hepatocyte transformation and show that the loss of p53 function has only a moderate effect on hepatic tumour formation.

Critical stages of tumor growth regulation in transgenic mice harboring a hepatocellular carcinoma revealed by distinct patterns of tumor necrosis factor-alpha and transforming growth factor-beta mRNA production.

There is now good evidence that cytokines contribute to the regulation of tumor growth. The cytokine-driven modulation of tumor growth was investigated during the progression of a hepatocellular carcinoma (HCC) in SV40 large T tumor antigen transgenic mice. In vivo, an increased rate of liver growth correlated with increased transforming growth factor (TGF)-beta 1 mRNA expression, while the greatest amounts of tumor necrosis factor (TNF)-alpha mRNA were detected earlier during tumor development. Conversely, no particular alteration of IL-1 alpha, IL-1 beta, IL-6, IL-2, IL-4 and IFN-gamma mRNA production could be reported. In vitro, hepatocyte-like tumor cell lines established at two stages, either before or after HCC differentiation, were characterized. The early-stage-derived cell line produced TNF-alpha mRNA, but had barely detectable expression of TGF-beta 1 mRNA, while later-stage-derived cell lines showed the reciprocal pattern. All cell lines displayed a lack of sensitivity to TNF-alpha, although some degree of sensitivity to TNF-alpha could be observed in the presence of actinomycin-D or after treatment with IFN-gamma. The early-stage-derived cell line was sensitive to the growth inhibitory effects of TGF-beta 1, but late-stage-derived tumor cell lines displayed a loss of sensitivity to TGF-beta 1 which correlated with the increased expression of TGF-beta 1 mRNA. Altogether, this suggests that tumor cells contribute to the discrete TNF-alpha and TGF-beta 1 expression patterns during HCC progression. This model of HCC could be of valuable interest to assess the impact of various immunotherapeutic strategies on modulation of tumor growth.

Hepatocyte growth factor expressed by a retrovirus-producing cell line enhances retroviral transduction of primary hepatocytes: implications for in vivo gene transfer.

Hepatocyte Growth Factor (HGF) is the more potent mitogen of mature hepatocytes. We have examined the effect of human HGF expression by a recombinant retroviral cell line (MFG-LacZ) on retroviral transduction of primary mouse and human hepatocytes. The HGF in the supernatant of MFG-LacZ cell line was correctly processed and biologically active. Transduction of mouse and human hepatocytes with the supernatant of transfected cells was increased 5-fold, as determined by beta-galactosidase activity. The production of HGF was stable and did not interfere with the viral titers of the producer cells. This study provides evidence that expression of HGF within a retrovirus-producer cell line increases the transduction rate of primary hepatocytes. Since the number of corrected cells is a limiting step for phenotypic correction of liver deficiencies, our approach should improve hepatic gene therapy efficiency. Furthermore this cell line should be useful for in vivo liver gene therapy.

Compensatory apoptosis in response to SV40 large T antigen expression in the liver.

Transgenesis allows the in vivo determination of the effects of oncogene expression in normal tissues. In an attempt to understand the mechanism underlying liver transformation, we have previously created transgenic mice carrying the SV40 early gene sequences, which developed hepatocarcinoma in a reproducible way. In the present study, we show that constant expression of the transgene was directly correlated to an abnormally increased hepatocyte proliferation, even at the adult stage. We further demonstrate in this model that the preneoplastic stage of hepatocarcinoma is characterized by marked ploidy alterations as early as 1 month, including the emergence of aneuploid and hyperpolyploid cells, and the persistence of an important diploid cell population. We show that this elevated proliferation is early and transiently counterbalanced by a mechanism of apoptosis, which maintains liver homeostasis. The disappearance of this programmed cell death response effective during preneoplasia might signal the commitment of the liver to neoplasia.

Fas-dependent apoptosis is impaired by SV40 T-antigen in transgenic liver.

A transgenic mouse model for hepatocarcinoma has been previously produced by targeting SV40 T-antigen expression to the liver. To evaluate the perturbation of cell death occurring during hepatocarcinogenesis, we examined the Fas-induced apoptosis on hepatocytes expressing T-antigen. Whereas anti-Fas antibody induced apoptosis in primary cultured normal hepatocytes, they imparted a weak cytotoxicity on primary cultured hepatocytes expressing T-antigen. This resistance of hepatic Fas-mediated apoptosis appears to result in an enhancement of a protective mechanism involving the protein kinase C signaling pathway rather than in a down-regulation of Fas-antigen expression. We further demonstrated that anti-Fas antibody does not have as efficient a lethal effect in T-antigen transgenic mice as in wild-type mice. The livers of transgenic mice injected with anti-Fas mAbs showed large intact regions with a few scattered apoptotic bodies: these regions strictly corresponded with carcinoma nodules, expressing high level of T-antigen. Our results describe a novel function for SV40 T-antigen which could contribute to viral pathogenesis by protecting infected cells against the host apoptotic defense mechanism.

Efficient retroviral-mediated gene transfer into primary culture of murine and human hepatocytes: expression of the LDL receptor.

The ex vivo approach to hepatic gene therapy involves several steps, which include the isolation and culture of hepatocytes, followed by their transduction with a retrovirus. Subsequently, autologous hepatocytes are transplanted. The number of hepatocytes that can be transduced by retroviruses bearing the therapeutic gene is one of the limiting steps that can impair the success of this strategy. We presently describe an experimental approach that leads to improved transduction efficiency in mouse and human hepatocytes in vitro. By using a recombinant retrovirus bearing the Escherichia coli beta-galactosidase gene, we show that addition of growth factors to the cells, namely human hepatocyte growth factor (HGF), allows marked increase in the transduction efficiency in mouse (up to 80%) and human (40%) hepatocytes. Familial hypercholesterolemia (FH) is due to mutation in the low-density lipoprotein (LDL) receptor gene and results in a deficiency in LDL receptors. Transduction of the human LDL receptor cDNA under the transcriptional control of the L-type pyruvate kinase promoter-activator into mouse hepatocytes led to an elevated tissue-specific expression of the human protein. These results suggest that the ex vivo approach remains a promising alternative for hepatic gene therapy.

Hepatitis B virus X gene product acts as a transactivator in vivo.

It has previously been shown that the hepatitis B virus X gene product, pX, transactivates homologous and heterologous transcriptional regulatory sequences of viruses and various cellular genes in vitro. However, there is no evidence about the reproducibility and the relevance of this phenomenon in vivo. In this study we crossbred transgenic mice expressing the X gene under the control of the human antithrombin III (ATIII) gene regulatory sequences with transgenics carrying either the chloramphenicol acetyl-transferase or the LacZ bacterial reporter genes driven by the HIV1-LTR, which is known to be activated in trans by pX. Expression of pX in the liver stimulates the HIV1-LTR driven expression of both chloramphenicol acetyl-transferase and beta-galactosidase reporter genes in double transgenic mice. No detectable increase in chloramphenicol acetyl-transferase expression was observed in tissues, such as the spleen, brain and heart, that do not express pX. Our results confirm the transactivating properties of pX in vivo for the first time and support the hypothesis that pX might indeed modify gene expression in HBV-infected hepatocytes and influence viral pathogenesis.

Oval cell proliferation in early stages of hepatocarcinogenesis in simian virus 40 large T transgenic mice.

In transgenic mice bearing the Simian Virus 40 large T antigen under the control of the human antithrombin III regulatory sequences, a stepwise progression toward hepatocellular carcinoma is observed. We have used two monoclonal antibodies (A6 and G7) developed against a surface antigen expressed in oval cells from dipin-treated mice, to analyze the emergence of such preneoplastic populations in the livers of antithrombin III Simian Virus 40 T transgenic mice. We show that a unique population of small heterogeneous epithelial cells, which probably corresponds to oval and/or transitional cells according to their morphological features, consistently appears at approximately the 10th week after birth and proliferates thereafter. This oval cell-like population stained positively for A6 and G7 monoclonal antibodies. Furthermore, different subpopulations usually recognized as possible precursors of carcinoma cells including hyperplastic foci and neoplastic nodules as well as carcinoma cells, were also positive for A6 but not G7 monoclonal antibodies. Stimulation of cell proliferation by partial hepatectomy performed at the time of emergence of the oval-like cells resulted in a rapid increase in the number of oval/transitional A6-positive cells. Our findings support the view that a common mechanism may be involved in the development of carcinomas that are induced by chemical carcinogens and in transgenic mice expressing a potent oncogene under the control of a hepatic specific promoter. In addition, our findings demonstrate a specific precursor-product relationship between the appearance of the oval/transitional cells and the development of neoplastic hepatocytes in this transgenic model.

Retroviral infection of primary hepatocytes from normal mice and mice transgenic for SV40 large T antigen.

Cultured adult rodent hepatocytes are extensively used as a model system for gene transfer in vitro. In the present study, we examined the influence differentiation status and growth capacity of the hepatocytes on their infectivity in vitro by a retroviral vector. These parameters were initially studied in primary cultures of rat hepatocytes transduced with an ecotropic retroviral vector containing Escherichia coli beta-galactosidase. However, significant differences observed in the infectivity of hepatocytes from 12-day-old and adult rats led us to also examine hepatocytes from a transgenic mouse strain in which the SV40 large T antigen is fused to the regulatory sequences of the human anti-thrombin III gene. The large T antigen is expressed in the liver and these mice develop hepatoma within 7 months. A comparison of infectivity of hepatocytes from normal and transgenic mice of different ages indicated that in contrast to previous reports, hepatocytes which express differentiated functions during the first week of culture can still be efficiently infected by retroviral vectors. Optimal infection was observed between the second and fourth day of culture and does not appear to be due to transient cell dedifferentiation, but is more likely due to transient mitotic activity of mice cells since the role of growth factors seems crucial for infection. The peak of infection did not appear to correspond to transient cell dedifferentiation. We also found differences of infectivity between hepatocytes from normal and transgenic mice of different ages. Such differences are correlated with differences in in vitro BrdU incorporation, which was used to determine the proportion of dividing hepatocytes. These results indicate that the efficiency of infectivity of hepatocytes by recombinant retrovirus is probably related to their normal proliferative potential and not to some dedifferentiated stage. Hence these findings provide a model for efficient gene transfer in differentiated cells and suggest an approach for studies of liver-specific gene regulation and for somatic gene therapy of metabolic diseases as well.

The hepatitis B virus X gene product transactivates the HIV-LTR in vivo.

It has previously been shown that the hepatitis B virus (HBV) X gene product, HBx, transactivates homologous and heterologous transcriptional regulatory sequences of viruses, including the human immunodeficiency virus type 1 (HIV1) long terminal repeat (LTR), and various cellular genes in vitro. To evaluate the transactivating function of HBx in vivo, we generated transgenic mice carrying the X open reading frame under the control of the human antithrombin III (ATIII) gene regulatory sequences. These mice express the 16 Kd HBx protein in the liver, as demonstrated by immunoprecipitation studies. Crossbreeding of HBx mice with transgenics carrying either the chloramphenicol acetyl transferase (CAT) bacterial or the lacZ reporter gene driven by the HIV1-LTR allowed us to demonstrate, for the first time, the in vivo transactivating function of HBx protein.

Time-course development of differentiated hepatocarcinoma and lung metastasis in transgenic mice.

A precise targeting of the SV40 T early region expression in the liver of transgenic mice was obtained using 700 bp of the antithrombin III regulatory sequences to control oncogene expression. In the strain expressing the highest level of large T antigen (Tag), the incidence of hepatocarcinoma was 100%. The evolution was reproducible and characterized by a marked cytolysis occurring as early as 4 weeks, when no morphological and histological modifications were visible, a preneoplastic state marked by a progression from hyperplasia to proliferative nodules composed of highly differentiated cells exhibiting a high Tag expression, which elicited tumor formation in nude mice and could proliferate in vitro, and hepatocellular carcinoma associated, in 10% of the cases, with lung metastasis. These transgenic mice constituted a useful model for therapeutic assays and fundamental studies on carcinogenesis.

In vivo activation by ultraviolet rays of the human immunodeficiency virus type 1 long terminal repeat.

It has been previously shown in vitro that the human immunodeficiency virus type 1 long terminal repeat (LTR) is activated by ultraviolet irradiation. In order to analyze if a similar effect could occur in vivo, transgenic mice carrying the lacZ gene under the control of the viral LTR were irradiated at 280-300 and 254 nm. These mice spontaneously expressed the transgene in the epidermis and the lens of both adults and embryos. Irradiations caused a significant increase in skin beta-galactosidase activity. This phenomenon might be involved in viral activation and could be of interest in regard to the skin pathology observed during an HIV infection.

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line.

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.

At least seven ribosomal proteins are involved in the control of translational accuracy in a eukaryotic organism.

In the filamentous fungus Podospora anserina, ribosomal proteins of 60 mutants impaired in the control of translational fidelity have been submitted to electrophoretic analysis. The "four corners" system combining four different two-dimensional polyacrylamide gel electrophoretic systems has been used. An altered electrophoretic pattern has been observed for 12 mutants. In mutants su3, su12 and su11 (decreased translational fidelity), proteins S1, S7 and S8, respectively, are altered. For AS mutants (increased translational fidelity), proteins S9, S12 and S19, respectively, are altered in AS9, AS1 and AS6 mutants, and protein S29 is lacking in AS3 mutants. The data suggest that five of these genes (at least) are the structural genes for the relevant proteins (su3:S1, su12:S7, AS1:S12, AS6:S19, AS9:S9), while the AS3 gene may code for a modifying enzyme.

Introns, protein syntheses and aging.

In the fungus Podospora, a correlation has recently been established between the presence of circular DNA molecules arising from the mitochondrial genome (SEN-DNAs) and the senescence syndrome. Here, I propose a hypothesis which accounts for the initial event which leads to the first SEN-DNA. A molecule in the most frequent situation where the SEN-DNA is an intron which might code for a maturase. This hypothesis is based upon several observations made either in Podospora or in the yeast S. cerevisiae. It assumes that mitochondrially synthesized maturases are unspecific nucleases able to work at the level of RNA and DNA molecules. Their specificity for RNA splicing instead of DNA is given by cytoplasmic proteins. Therefore, if the balance between cytoplasmic and mitochondrial protein syntheses is disturbed in favour of the mitochondrial compartment, the maturase would be accumulated and allowed to splice introns from DNA instead of RNA molecules. This hypothesis can account for aging of higher eucaryotic cells by postulating analogous processes in their nuclear compartment.

Relationship between the intracellular level and growth inhibition of a new anthracycline 4'O-tetrahydropyranyl-Adriamycin in Friend leukemia cell variants.

The relationship between the intracellular amount of a new anthracycline derivative, 4'-O-tetrahydropyranyl-adriamycin (THP-ADM) and its cytotoxic activity in Friend leukemia cells (FLC) was investigated. By comparison to adriamycin (ADM), the uptake of THP-ADM is a very rapid process reaching maximal levels within 5 min. Both drugs are accumulated and retained in the nuclear fraction. The two main consequences associated to these different uptake rate are: following short-time cell exposure to comparable drug concentration, the higher cytotoxic effect of THP-ADM correlates to the ease with which it crosses the cell membrane; the intracellular amount of THP-ADM but not of ADM decreases with the cell density. These results emphasize the importance of considering drug uptake kinetics and its relationship to cytotoxicity. Studies comparing uptake and efflux of both drugs in ADM-resistant cells showed that THP-ADM extrusion correlate more to cytotoxicity than that of ADM. The relevance of these in vitro findings to clinical application is considered.