Reduced mammary gland carcinogenesis in transgenic mice expressing a growth hormone antagonist.

Several reports have provided evidence that body size early in life is positively correlated with risk of subsequent breast cancer, but the biological basis for this relationship is unclear. We examined tumour incidence in transgenic mice expressing a growth hormone (GH) antagonist and in non-transgenic littermates following exposure to dimethylbenz[ a ]anthracene (DMBA), a well characterized murine mammary gland carcinogen. The transgenic animals had lower IGF-I levels, were smaller in terms of body size and weight, and exhibited decreased tumour incidence relative to controls. The demonstration that both body size early in life and breast cancer incidence are influenced by experimental perturbation of the GH-IGF-I axis in a transgenic model provides evidence that variability between individuals with respect to these hormones underlies the relationship between body size early in life and breast cancer risk observed in epidemiological studies.

Genetic correction of sickle cell disease: insights using transgenic mouse models.

Sickle cell disease is a hereditary disorder characterized by erythrocyte deformity due to hemoglobin polymerization. We assessed in vivo the potential curative threshold of fetal hemoglobin in the SAD transgenic mouse model of sickle cell disease using mating with mice expressing the human fetal Agamma-globin gene. With increasing levels of HbF, AgammaSAD mice showed considerable improvement in all hematologic parameters, morphopathologic features and life span/survival. We established the direct therapeutic effect of fetal hemoglobin on sickle cell disease and demonstrated correction by increasing fetal hemoglobin to about 9-16% in this mouse model. This in vivo study emphasizes the potential of the SAD mouse models for quantitative analysis of gene therapy approaches.

Altered hematopoiesis in murine sickle cell disease.

We investigated the mechanisms of sickle cell disease (SCD) hematopoietic/erythropoietic defects using bone marrow, spleen, and/or peripheral blood from the transgenic SAD mouse model, which closely reproduces the biochemical and physiological disorders observed in human SCD. First, the erythropoietic lineage late precursors (polychromatophilic normoblasts to the intramedullary reticulocytes) of SAD mouse bone marrow were significantly altered morphologically. These anomalies resulted from high levels of hemoglobin polymers and were associated with increased cell fragmentation occurring during medullary endothelial migration of reticulocytes. Secondly, analysis of bone marrow erythropoiesis in earlier stages showed a marked depletion in SAD erythroid burst-forming units (BFU-E; of approximately 42%) and erythroid colony-forming units (CFU-E; of approximately 23%) progenitors, despite a significant increase in their proliferation, suggesting a compensatory mechanism. In contrast to the bone marrow progenitor depletion, we observed (1) a high mobilization/relocation of BFU-E early progenitors (approximately 4-fold increase) in peripheral blood of SAD mice as well as of colony-forming units-granulocyte-macrophage (CFU-GM) and (2) a 7-fold increase of SAD CFU-E in the spleen. Third, and most importantly, SAD bone marrow multipotent cells (spleen colony-forming units [CFU-S], granulocyte-erythroid-macrophage-megakaryocyte colony-forming units [CFU-GEMM], and Sca(+)Lin(-)) were highly mobilized to the peripheral blood (approximately 4-fold increase), suggesting that peripheral multipotent cells could serve as proliferative and autologous vehicles for gene therapy. Therefore, we conclude the following. (1) The abnormal differentiation and morphology of late nucleated erythroid precursors result in an ineffective sickle erythropoiesis and likely contribute to the pathophysiology of sickle cell disorders; this suggests that transfer of normal or modified SCD bone marrow cells may have a selective advantage in vivo. (2) A hematopoietic compensatory mechanism exists in SAD/SCD pathology and consists of mobilization of multipotent cells from the bone marrow to the peripheral blood and their subsequent uptake into the spleen, an extramedullary hematopoietic site for immediate differentiation. Altogether, these results corroborate the strong potential effectiveness of both autologous and allogeneic bone marrow transplantation for SCD hematopoietic therapy.

C-myc-induced apoptosis in polycystic kidney disease is Bcl-2 and p53 independent.

The SBM mouse is a unique transgenic model of polycystic kidney disease (PKD) induced by the dysregulated expression of c-myc in renal tissue. In situ hybridization analysis demonstrated intense signal for the c-myc transgene overlying tubular cystic epithelium in SBM mice. Renal proliferation index in SBM kidneys was 10-fold increased over nontransgenic controls correlating with the presence of epithelial hyperplasia. The specificity of c-myc for the proliferative potential of epithelial cells was demonstrated by substitution of c-myc with the proto-oncogene c-fos or the transforming growth factor (TGF)-alpha within the same construct. No renal abnormalities were detected in 13 transgenic lines established, indicating that the PKD phenotype is dependent on functions specific to c-myc. We also investigated another well characterized function of c-myc, the regulation of apoptosis through pathways involving p53 and members of the bcl-2 family, which induce and inhibit apoptosis, respectively. The SBM kidney tissues, which overexpress c-myc, displayed a markedly elevated (10-100-fold) apoptotic index. However, no significant difference in bcl-2, bax, or p53 expression was observed in SBM kidney compared with controls. Direct proof that the heightened renal cellular apoptosis in PKD is not occurring through p53 was obtained by successive matings between SBM and p53(-/-) mice. All SBM offspring, irrespective of their p53 genotype, developed PKD with increased renal epithelial apoptotic index. In addition, overexpression of both bcl-2 and c-myc in double transgenic mice (SBB+/SBM+) also produced a similar PKD phenotype with a high apoptotic rate, showing that c-myc can bypass bcl-2 in vivo. Thus, the in vivo c-myc apoptotic pathway in SBM mice occurs through a p53- and bcl-2-independent mechanism. We conclude that the pathogenesis of PKD is c-myc specific and involves a critical imbalance between the opposing processes of cell proliferation and apoptosis.

Limk1 is predominantly expressed in neural tissues and phosphorylates serine, threonine and tyrosine residues in vitro.

We have isolated the murine Limk1 gene, which is a single copy gene located at the distal end of mouse chromosome 5. Limk1 exhibits a 95% homology to the human homologue, LIMK, which contains two LIM domains and a putative protein kinase domain. Although Limk1 and LIMK contain all motifs found in catalytic kinase domains, amino acids previously described to be diagnostic of either serine/threonine- or tyrosine-kinases are not present. It is demonstrated that GST-Limk1-fusion protein can autophosphorylate on serine, tyrosine and threonine residues in vitro and that mutation of residue D460 within the IHRDL motif abolishes kinase activity. Northern blot showed preferential expression of a 3.5 kb message in adult spinal cord and brain. In situ hybridisation confirmed high expression levels in the nervous system, particularly in the spinal cord and the cranial nerve and dorsal root ganglia. Limk1 also contains two tandem LIM-domains. These zinc-finger like domains can mediate protein-protein interactions and have been described in nuclear and cytoskeletal proteins. The combination of LIM- and kinase domains may provide a novel route by which intracellular signalling can be integrated.

Specialization switch in differentiating embryonic rat liver progenitor cells in response to sodium butyrate.

Embryonic (E) 12 rat liver epithelial cells constitute a population of bipotential progenitor cells which can differentiate along the hepatocyte (Hep) or biliary epithelial cell (BEC) lineage in primary culture. In the present study, E12 cells were seeded on fibronectin-coated substratum and exposed to sodium butyrate (SB) for various exposure times, and the emergence of the Hep or BEC phenotype was monitored by following the variations in albumin production and assessing the appearance of the two surface-exposed markers HES6 and BDS7. Continuous exposure to SB resulted into a major reduction in albumin production and, at Day 9 postseeding, few cells coexpressed BDS7 and albumin. When cells were exposed to SB for 5 days and then cultured for an additional 5 days without SB, they massively express BDS7, but very little HES6. Moreover, the reverse sequence, i.e., 5 days without SB followed by 5 days with it, led to the appearance of many cells expressing both HES6 and BDS7. These results indicate that progenitors committed preferentially along the Hep lineage still have the option to switch to BECs, at a transitional stage that we refer to as a "differentiation window."

Cytokeratin 14 expression in rat liver cells in culture and localization in vivo.

Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES6+ cells) and was expressed in some biliary epithelial (BDS7+ cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.

Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, alpha-fetoprotein, albumin, and cell surface-exposed components.

The differentiation patterns of epithelial cells in fetal rat liver were analyzed in situ and in primary culture by indirect immunofluorescence microscopy using polyvalent and monoclonal antibodies directed against cytokeratins with molecular weights of 55,000 (CK55), 52,000 (CK52), and 39,000 (CK39) and against vimentin, albumin, alpha-fetoprotein, and surface-exposed components of bile ductular cells (BDS7) and hepatocytes (HES6). The anti-CK52 antibody, which reacted with biliary ductal cells in the liver of adult rats (Germain et al., Cancer Res., 45:673, 1985; Germain et al., Cancer Res., 48: 368-378, 1988), stained essentially all of the epithelial cells of embryonic day 12 (E12) rat liver. The anti-BDS7 antibody reacted with a few cell foci, which enlarged and became more numerous at later developmental ages. At E12 essentially all of the cells were positive for albumin and alpha-fetoprotein but did not express HES6. In fact HES6 was not detected until E15 in cells with the morphology of immature hepatocytes. By E18 staining with anti-HES6 reached the level of that observed on adult rat hepatocytes. Liver cells isolated from E12 rats were seeded on fibronectin-treated dishes and their response to various combinations of growth- and differentiation-promoting factors was evaluated with respect to their capacity to express either the hepatocytic or the bile ductular phenotype. In medium supplemented with serum, insulin, dexamethasone, and dimethyl sulfoxide, the E12 cells were capable of differentiating in culture to mimic over a 6-day period the sequential phenotypic changes which occur in vivo during normal hepatoontogeny, namely the loss of CK52 and the appearance of HES6. In contrast, the addition of sodium butyrate to the above supplement mixture resulted in the massive expression of BDS7. To further assess the developmental potential of fetal rat liver cells toward the biliary epithelial cell lineage, the in vitro assay was performed using cells isolated from livers of E18 rats and also from 2-day-old (P2) and P14 rats. While a slight expression of BDS7 was induced in cell culture from E18 liver, essentially no expression was observed in cells from postnatal livers. These findings strongly suggest that the emerging hepatic tissue in rat embryo is composed of bipotential progenitor epithelial cells that are capable of differentiating along either the hepatocytic or biliary epithelial cell lineage. These observations constitute a clear demonstration of the plasticity of liver differentiation and also provide a striking example of environmental influences on liver progenitor cell differentiation.