Embryonic versus adult stem cell pluripotency: in liver only fusion matters.
Explore the source record for details and available documents.
Adult↗
Biomedical subjects
Markus Grompe
Publications and source records attributed to Markus Grompe.
AAV serotype 2 vectors preferentially integrate into active genes in mice.
Recombinant adeno-associated virus serotype 2 (rAAV2) is a promising vector for gene therapy because it can achieve long-term stable transgene expression in animals and human subjects after direct administration of vectors into various target tissues. In the liver, although stable transgene expression primarily results from extrachromosomal vector genomes, a series of experiments has shown that vector genomes integrate into host chromosomes in hepatocytes at a low frequency. Despite the low integration efficiency, recent reports of retroviral insertional mutagenesis in mice and two human subjects have raised concerns about the potential for rAAV2-mediated insertional mutagenesis. Here we characterize rAAV2-targeted chromosomal integration sites isolated from selected or non-selected hepatocytes in vector-injected mouse livers. We document frequent chromosomal deletions of up to 2 kb at integration sites (14 of 14 integrations, 100%; most of the deletions were <0.3 kb) and preferred integration into genes (21 of 29 integrations, 72%). In addition, all of the targeted genes analyzed (20 of 20 targeted genes, 100%) were expressed in the liver. This is the first report to our knowledge on host chromosomal effects of rAAV2 integration in animals, and it provides insights into the nature of rAAV2 vector integration into chromosomes in quiescent somatic cells in animals and human subjects.
Animals↗
The Fanconi anaemia/BRCA pathway.
Fanconi anaemia (FA) is a rare genetic cancer-susceptibility syndrome that is characterized by congenital abnormalities, bone-marrow failure and cellular sensitivity to DNA crosslinking agents. Seven FA-associated genes have recently been cloned, and their products were found to interact with well-known DNA-damage-response proteins, including BRCA1, ATM and NBS1. The FA proteins could therefore be involved in the cell-cycle checkpoint and DNA-repair pathways. Recent studies implicate the FA proteins in the process of repairing chromosome defects that occur during homologous recombination, and disruption of the FA genes results in chromosome instability--a common feature of many human cancers.
Animals↗
The role of bone marrow stem cells in liver regeneration.
Hepatic oval cells involved in some forms of liver regeneration express many markers also found on hematopoietic stem cells (HSCs). In addition, multiple independent reports have demonstrated that bone marrow cells can give rise to several hepatic epithelial cell types, including oval cells, hepatocytes, and duct epithelium. These observations have resulted in the hypothesis that bone marrow resident stem cells, specifically HSCs, are an important source for liver epithelial cell replacement, particularly during chronic injury. The function of such stem cells in hepatic injury responses is the topic of this article. Taken together, the published data on the role of bone marrow stem cells in liver damage suggest that they do not play a significant physiological role in the replacement of epithelial cells in any known form of hepatic injury. Fully functional bone marrow-derived hepatocytes exist but are extremely rare and are generated by cell fusion, not stem cell differentiation. Nonetheless, bone marrow-derived cells may play important indirect roles in liver regeneration. First, they may serve as a source for the replacement of endothelial cells. Second, hematopoietic cells, including lymphocytes, neutrophils, macrophages, and platelets, may provide crucial factors required for efficient healing of damaged liver.
Animals↗
Mutations in PHF6 are associated with Börjeson-Forssman-Lehmann syndrome.
Börjeson-Forssman-Lehmann syndrome (BFLS; OMIM 301900) is characterized by moderate to severe mental retardation, epilepsy, hypogonadism, hypometabolism, obesity with marked gynecomastia, swelling of subcutaneous tissue of the face, narrow palpebral fissure and large but not deformed ears. Previously, the gene associated with BFLS was localized to 17 Mb in Xq26-q27 (refs 2-4). We have reduced this interval to roughly 9 Mb containing more than 62 genes. Among these, a novel, widely expressed zinc-finger (plant homeodomain (PHD)-like finger) gene (PHF6) had eight different missense and truncation mutations in seven familial and two sporadic cases of BFLS. Transient transfection studies with PHF6 tagged with green fluorescent protein (GFP) showed diffuse nuclear staining with prominent nucleolar accumulation. Such localization, and the presence of two PHD-like zinc fingers, is suggestive of a role for PHF6 in transcription.
Amino Acid Motifs↗
Gene therapy of Fanconi anemia: preclinical efficacy using lentiviral vectors.
Fanconi anemia (FA) is an inherited cancer susceptibility syndrome caused by mutations in a DNA repair pathway including at least 6 genes (FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts to use retroviral vectors in humans have proven ineffective given the impaired proliferation potential of human FA hematopoietic progenitors (HPCs). In this work, we show that using lentiviral vectors efficient genetic correction can be achieved in quiescent hematopoietic progenitors from Fanca(-/-) and Fancc(-/-) mice. Long-term repopulating HPCs were transduced by a single exposure of unfractionated bone marrow mononuclear cells to lentivectors carrying the normal gene. Notably, no cell purification or cytokine prestimulation was necessary. Resistance to DNA- damaging agents was fully restored by lentiviral transduction, allowing for in vivo selection of the corrected cells with nonablative doses of cyclophosphamide. This study strongly supports the use of lentiviral vectors for FA gene therapy in humans.
Animals↗
BRCA1 interacts directly with the Fanconi anemia protein FANCA.
Fanconi anemia (FA) is a rare autosomal recessive disease characterized by skeletal defects, anemia, chromosomal instability and increased risk of leukemia. At the cellular level FA is characterized by increased sensitivity to agents forming interstrand crosslinks (ICL) in DNA. Six FA genes have been cloned and interactions among individual FANC proteins have been found. The FANCD2 protein co-localizes in nuclear foci with the BRCA1 protein following DNA damage and during S-phase, requiring the FANCA, C, E and G proteins to do so. This finding may reflect a direct role for the BRCA1 protein in double strand break (DSB) repair and interaction with the FANC proteins. Therefore interactions between BRCA1 and the FANC proteins were investigated. Among the known FANC proteins, we find evidence for direct interaction only between the FANCA protein and BRCA1. The evidence rests on three different tests: yeast two-hybrid analysis, coimmunoprecipitation from in vitro synthesis, and coimmunoprecipitation from cell extracts. The amino terminal portion of FANCA and the central part (aa 740-1083) of BRCA1 contain the sites of interaction. The interaction does not depend on DNA damage, thus FANCA and BRCA1 are constitutively interacting. The demonstrated interaction directly connects BRCA1 to the FA pathway of DNA repair.
BRCA1 Protein↗
S-phase-specific interaction of the Fanconi anemia protein, FANCD2, with BRCA1 and RAD51.
Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and defective cell-cycle progression. Six FA genes (corresponding to subtypes A, C, D2, E, F, and G) have been cloned, and the encoded FA proteins interact in a common pathway. DNA damage activates this pathway, leading to monoubiquitination of the downstream FANCD2 protein and targeting to nuclear foci containing BRCA1. In the current study, we demonstrate that FANCD2 also undergoes monoubiquitination during S phase of the cell cycle. Monoubiquitinated FANCD2 colocalizes with BRCA1 and RAD51 in S-phase-specific nuclear foci. Monoubiquitination of FANCD2 is required for normal cell-cycle progression following cellular exposure to mitomycin C. Our data indicate that the monoubiquitination of FANCD2 is highly regulated, and they suggest that FANCD2/BRCA1 complexes and FANCD2/RAD51 complexes participate in an S-phase-specific cellular process, such as DNA repair by homologous recombination.
BRCA1 Protein↗
SV40 large T-antigen disturbs the formation of nuclear DNA-repair foci containing MRE11.
The accumulation of DNA repair proteins at the sites of DNA damage can be visualized in mutagenized cells at the single cell level as discrete nuclear foci by immunofluorescent staining. Formation of nuclear foci in irradiated human fibroblasts, as detected by antibodies directed against the DNA repair protein MRE11, is significantly disturbed by the presence of the viral oncogene, SV40 large T-antigen. The attenuation of foci formation was found in both T-antigen immortalized cells and in cells transiently expressing T-antigen, indicating that it is not attributable to secondary mutations but to T-antigen expression itself. ATM-mediated nibrin phosphorylation was not altered, thus the disturbance of MRE11 foci formation by T-antigen is independent of this event. The decrease in MRE11 foci was particularly pronounced in T-antigen immortalized cells from the Fanconi anaemia complementation group FA-D2. FA-D2 cells produce essentially no MRE11 DNA repair foci after ionizing irradiation and have a significantly increased cellular radiosensitivity at low radiation doses. The gene mutated in FA-D2 cells, FANCD2, codes for a protein which also locates to nuclear foci and may, therefore, be involved in MRE11 foci formation, at least in T-antigen immortalized cells. This finding possibly links Fanconi anaemia proteins to the frequently reported increased sensitivity of Fanconi anaemia cells to transformation by SV40. From a practical stand point these findings are particularly relevant to the many studies on DNA repair which exploit the advantages of SV40 immortalized cell lines. The interference of T-antigen with DNA repair processes, as demonstrated here, should be borne in mind when interpreting such studies.
Antigens, Polyomavirus Transforming↗
Biallelic inactivation of BRCA2 in Fanconi anemia.
Fanconi anemia (FA) is a rare autosomal recessive cancer susceptibility disorder characterized by cellular hypersensitivity to mitomycin C (MMC). Six FA genes have been cloned, but the gene or genes corresponding to FA subtypes B and D1 remain unidentified. Here we show that cell lines derived from FA-B and FA-D1 patients have biallelic mutations in BRCA2 and express truncated BRCA2 proteins. Functional complementation of FA-D1 fibroblasts with wild-type BRCA2 complementary DNA restores MMC resistance. Our results link the six cloned FA genes with BRCA1 and BRCA2 in a common pathway. Germ-line mutation of genes in this pathway may result in cancer risks similar to those observed in families with BRCA1 or BRCA2 mutations.
Alleles↗
Adult versus embryonic stem cells: it's still a tie.
Explore the source record for details and available documents.
Adult↗
In vivo correction of murine tyrosinemia type I by DNA-mediated transposition.
Gene therapy applications of naked DNA constructs for genetic disorders have been limited because of lack of permanent transgene expression. This limitation, however, can be overcome by the Sleeping Beauty (SB) transposable element, which can achieve permanent transgene expression through genomic integration from plasmid DNA. To date, only one example of an in vivo gene therapy application of this system has been reported. In this report, we have further defined the activity of the SB transposon in vivo by analyzing the expression and integration of a fumarylacetoacetate hydrolase (FAH) transposon in FAH-deficient mice. In this model, stably corrected FAH(+) hepatocytes are clonally selected and stable integration events can therefore be quantified and characterized at the molecular level. Herein, we demonstrate that SB-transposon-transfected hepatocytes can support significant repopulation of the liver, resulting in long-lasting correction of the FAH-deficiency phenotype. A single, combined injection of an FAH-expressing transposon plasmid and a transposase expression construct resulted in stable FAH expression in approximately 1% of transfected hepatocytes. The average transposon copy number was determined to be approximately 1/diploid genome and expression was not silenced during serial transplantation. Molecular analysis indicated that high-efficiency DNA-mediated transposition into the mouse genome was strictly dependent on the expression of wild-type transposase.
Animals↗
Kinetics of liver repopulation after bone marrow transplantation.
Recent work has convincingly demonstrated that adult bone marrow contains cells capable of differentiating into liver epithelial cells in vivo. However, the frequency and time course with which fully functional hepatocytes emerge after bone marrow transplantation remained controversial. Here, we used the fumarylacetoacetate hydrolase knockout mouse to determine the kinetics of hepatocyte replacement after complete hematopoietic reconstitution. Single donor-derived hepatocytes were first detected 7 weeks after lethal irradiation and bone marrow transplantation. Liver disease was not required for this transdifferentiation. In the presence of selective pressure the single cells evolved into hepatocyte nodules by 11 weeks after transplantation and resulted in >30% overall liver repopulation by 22 weeks. The frequency with which hepatocytes were produced was between 10(-4) and 10(-6), resulting in only 50 to 500 repopulation events per liver. Hepatic engraftment was not observed without previous hematopoietic reconstitution even in the presence of liver injury. In addition, significant liver repopulation was completely dependent on hepatocyte growth selection. We conclude that hepatocyte replacement by bone marrow cells is a slow and rare event. Significant improvements in the efficiency of this process will be needed before clinical success can be expected.
Animals↗
Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. 1991.
Explore the source record for details and available documents.
Albumins↗
Fanconi anemia group A and C double-mutant mice: functional evidence for a multi-protein Fanconi anemia complex.
OBJECTIVE: Fanconi anemia (FA) is a genetically heterogeneous disorder associated with defects in at least eight genes. The biochemical function(s) of the FA proteins are unknown, but together they define the FA pathway, which is involved in cellular responses to DNA damage and in other cellular processes. It is currently unknown whether all FA proteins are involved in controlling a single function or whether some of the FA proteins have additional roles. The aim of this study was 1) to determine whether the FA group A and group C genes have identical or partially distinct functions, and 2) to have a better model for human FA. MATERIALS AND METHODS: We generated mice with a targeted mutation in fanca and crossed them with fancc disrupted animals. Several phenotypes including sensitivity to DNA cross linkers and ionizing radiation, hematopoietic colony growth, and germ cell loss were analyzed in fanca-/-, fancc-/-, fanca/fancc double -/-, and controls. RESULTS: Fibroblast cells and hematopoietic precursors from fanca/fancc double-mutant mice were not more sensitive to MMC than those of either single mutant. fanca/fancc double mutants had no evidence for an additive phenotype at the cellular or organismal level. CONCLUSIONS: These results support a model where both FANCA and FANCC are part of a multi-protein nuclear FA complex with identical function in cellular responses to DNA damage and germ cell survival.
Animals↗
In vivo administration of interferon gamma does not cause marrow aplasia in mice with a targeted disruption of FANCC.
OBJECTIVE: Hematopoietic cells from patients with Fanconi anemia (FA) and mice carrying a targeted disruption of the gene encoding complementation group C protein (FANCC(-/-)) demonstrate an apoptotic phenotype in response to alkylating agents and cytokines including interferon gamma (IFN-gamma) in vitro. The aim of this study was to explore these apoptosis-inducing effects of IFN-gamma on the bone marrow of FANCC(-/-) mice as a potential strategy to select gene-corrected cells in vivo. Following pharmacokinetic studies to determine if serum concentrations effective in vitro can be achieved in vivo, we injected FANCC(-/-) mice with recombinant murine IFN-gamma. Hematopoietic effects were investigated by serial determinations of blood counts, progenitor colony formation, and marrow cellularity. RESULTS: Serial weekly intraperitoneal administrations of escalating doses of rmIFN-gamma did not affect peripheral blood counts in FANCC(-/-) mice, even after subsequent antibody-mediated fas ligation. Additionally, prolonged exposure after sequential daily administration of recombinant IFN-gamma did not impair progenitor cell clonogenicity in vitro. Pharmacokinetic data confirmed that the failure of IFN-gamma to induce marrow aplasia occurred in spite of peak serum levels greater than 100-fold in excess of those effective in vitro. CONCLUSION: We conclude that in spite of the well-documented in vitro apoptotic tendency of FA-phenotype hematopoietic cells, the in vivo administration of IFN-gamma with and without subsequent fas ligation does not induce bone marrow failure in FANCC(-/-) (129SvJ strain) mice. Additional selective pressure may be necessary to achieve targeted ablation of uncorrected, FA-phenotype, marrow cells.
Anemia, Aplastic↗
FANCD2: a branch-point in DNA damage response?
Explore the source record for details and available documents.
Chromosome Aberrations↗
Attenuation of the formation of DNA-repair foci containing RAD51 in Fanconi anaemia.
The role of the Fanconi anaemia genes in DNA repair was examined by a quantitative analysis of nuclear DNA repair foci in FA primary fibroblasts after ionising irradiation using antibodies directed against RAD51, MRE11 and BRCA1 for visualisation. IR induced foci detected with anti-RAD51, but not those detected with anti-MRE11, are reduced in fibroblasts of all eight FA complementation groups in comparison to control cells. Correction of FA-A, FA-C and FA-G cells by retroviral cDNA transfer specifically corrected the RAD51-foci response but did not affect formation of foci containing BRCA1 or MRE11. Since all FA cells, except FA-D1, lack the monoubiquitinated FANCD2-L protein, this isoform is likely to be involved in the formation of nuclear foci containing RAD51 in diploid FA cells. FA-D1 cells show the same attenuation in RAD51 foci formation, suggesting that the unknown FANCD1 protein is similarly involved in RAD51 foci formation, either independently or as a subsequent step in the FANCD2 pathway. These findings indicate that Fanconi anaemia cells have an impairment in the RAD51-dependent homologous recombination pathway for DNA repair, explaining their chromosomal instability and extreme sensitivity to DNA cross-linking agents.
BRCA1 Protein↗