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Biochemical purification has identified a new component of the Fanconi anemia complex. The protein has ubiquitin ligase activity and may be the long-sought enzyme responsible for monoubiquitination of FANCD2.

Abstract SCI-1 Fanconi anemia (FA) is a genetically heterogeneous inherited disorder characterized by bone marrow failure, birth defects, and cancer susceptibility. At the cellular level, FA features hypersensitivity to DNA cross-linking agents, cell cycle abnormalities, and abnormal responses to cytokines. Therapy for the hematopoietic defects of FA has consisted of supportive care, stem cell transplantation, and administration of androgens such as oxymetholone. Unfortunately, even successfully transplanted FA patients have a very high risk of subsequent solid tumors, especially squamous cell carcinomas of the oropharyngeal and urogenital areas. The pharmacologic treatment of FA has been unchanged for several decades, and for this reason our group has embarked on a systematic search for small-molecule therapeutics that could benefit FA patients. This effort has been made possible by the cloning of 15 FA genes and biochemical insights into the function of FA proteins. Mouse models, zebra fish models, and FA patient cell lines, including induced pluripotent stem cells, are available for screening and validation. The specific goals of small-molecule interventions are to delay or prevent hematopoietic failure and also to delay or prevent carcinogenesis. To date, we have shown that the superoxide dismutase mimetic drug Tempol can significantly delay tumor formation in FA mice. Similarly, resveratrol clearly improved hematopoiesis in the same model. Medium-throughput screens have revealed numerous new molecules that ameliorate the FA phenotypes of cells, mice, and zebrafish. Using genetic approaches we also have identified the p53 pathway as a therapeutic target in FA. These therapeutic opportunities and progress in FA small-molecule screening will be discussed. Disclosures: No relevant conflicts of interest to declare.

Cell fusion plays a well-recognized, physiologic role during development. Bone marrow-derived hematopoietic cells have been shown to fuse with non-hematopoietic cells in a wide variety of tissues. Some organs appear to resolve the changes in ploidy status, generating functional and mitotically-competent events. However, cell fusion exclusively involving hematopoietic cells has not been reported. Indeed, genomic copy number variation (CNV) in highly replicative hematopoietic cells is widely considered a hallmark of malignant transformation. Here we show that cell fusion occurs between cells of the hematopoietic system under injury as well as non-injury conditions. Experiments reveal the acquisition of genetic markers in fusion products, their tractable maintenance during hematopoietic differentiation and long-term persistence after serial transplantation. Fusion events were identified in clonogenic progenitors as well as differentiated myeloid and lymphoid cells. These observations provide a new experimental model for the study of non-pathogenic somatic diversity in the hematopoietic system.

Cells from Fanconi anemia (FA) patients have defective DNA repair and are hypersensitive to DNA crosslinking agents such as mitomycin C (MMC). We examined the possibility that topoisomerase I is involved in the DNA crosslink repair system and is deficient in FA group A cells. FA cells and control cells were exposed to MMC with or without camptothecin (CPT), a topoisomerase I inhibitor. The cells did not show any increased sensitivity to killing by MMC with CPT, suggesting that the topoisomerase I is not involved in MMC-damaged DNA repair. However, FA cells showed increased sensitivity to CPT in comparison to control cells, raising the possibility of altered topoisomerase I in FA cells. Therefore, a mutation analysis was performed on topoisomerase I cDNA from FA cells by using chemical cleavage mismatch scanning and nucleotide sequencing. No mutation was detected from GM1309, a group A FA cell line. A base transition (C to T) at position 241, causing an amino acid change (His to Tyr), was found in GM2061, a FA cell line of unknown complementation group. However, allele-specific oligonucleotide hybridization analysis showed that this is a gene polymorphism. We conclude that FA cells have normal gene structure for topoisomerase I.

Fanconi anemia (FA) is an autosomal recessive disease for which at least four complementation groups exist. Recently the gene that corrects the defect in Fanconi anemia complementation group C cells (FACC) has been cloned. We have previously identified a common mutation in the FACC gene, which accounts for a majority of FA cases in Ashkenazi Jewish individuals. We here describe the use of allele-specific oligonucleotide (ASO) hybridization to determine the frequency of this mutation among additional Jewish FA patients and to determine the carrier frequency in the Jewish population. The common IVS4 + 4A T allele was found on 19/23 (83%) Jewish FA chromosomes, indicating that it is indeed responsible for most cases of FA among Ashkenazi Jews. The carrier frequency was 2/314 for Jewish individuals and the mutant allele was not detected in 130 non-Jewish controls. © 1994 Wiley-Liss, Inc.

The Fanconi anemia D2 protein is vital for cellular resistance to DNA cross-linking. Now, FANCD2 is shown to also be involved in a second, independent function, the arrest of DNA synthesis after ionizing radiation.

Mice lacking all three Rb genes in the liver develop tumors resembling specific subgroups of human hepatocellular carcinomas, and Notch activity appears to suppress the growth and progression of these tumors., Hepatocellular carcinoma (HCC) is the third cancer killer worldwide with >600,000 deaths every year. Although the major risk factors are known, therapeutic options in patients remain limited in part because of our incomplete understanding of the cellular and molecular mechanisms influencing HCC development. Evidence indicates that the retinoblastoma (RB) pathway is functionally inactivated in most cases of HCC by genetic, epigenetic, and/or viral mechanisms. To investigate the functional relevance of this observation, we inactivated the RB pathway in the liver of adult mice by deleting the three members of the Rb (Rb1) gene family: Rb, p107, and p130. Rb family triple knockout mice develop liver tumors with histopathological features and gene expression profiles similar to human HCC. In this mouse model, cancer initiation is associated with the specific expansion of populations of liver stem/progenitor cells, indicating that the RB pathway may prevent HCC development by maintaining the quiescence of adult liver progenitor cells. In addition, we show that during tumor progression, activation of the Notch pathway via E2F transcription factors serves as a negative feedback mechanism to slow HCC growth. The level of Notch activity is also able to predict survival of HCC patients, suggesting novel means to diagnose and treat HCC.

Fanconi anaemia is an autosomal recessive disease for which four known complementation groups exist. Recently, the gene defective in complementation group C (FACC) has been cloned. In order to determine the fraction of Fanconi anaemia caused by FACC mutations, we used reverse transcription PCR and chemical mismatch cleavage (CMC) to examine the FACC cDNA in 17 FA cell lines. 4/17 patients (23.5%) had mutations in this gene. Two Ashkenazi-Jewish individuals were homozygous for an identical splice mutation. Three additional Jewish patients bearing this allele were found upon screening 21 other families. We conclude that a common mutation in FACC accounts for the majority of Fanconi anaemia in Ashkenazi-Jewish families.

Tyrosinemia type I is an autosomal recessive inborn error of metabolism caused by deficiency of the enzyme fumaryl acetoacetate hydrolase (FAH, EC 3.7.1.2.). We have used reverse transcription and the polymerize chain reaction to amplify the peptide coding region of the FAH cDNA from four patients with tyrosinemia type I. Chemical mismatch cleavage analysis and DNA sequencing were utilized to determine mutant alleles in all cases. A French Canadian patient was homozygous for a splice error mutation in the 3′ portion of the gene. A second patient, from a consanguineous pedigree in Iran, had the identical splice alteration. The third patient has a missense mutation, changing valine to glycine in codon 166. And finally two nonsense mutations in codons 357 and 364 were found in the fourth patient. © 1993 Wiley-Liss, Inc.

Abstract 504 The hematopoietic niche supports both normal and malignant hematopoiesis. We have evaluated the functional interactions between leukemic cells and the endothelial cell component of the hematopoietic niche. Highly enriched populations of circulating human acute myeloid leukemia (AML) cells were obtained by therapeutic leukapheresis. These primary AML cells rapidly homed to and associated with pre-existing vascular tubes in vitro. Co-culture of human lung microvascular endothelial cells (HLMVEC) with AML cells both inhibited AML apoptosis by ∼50% (p 50% human AML cells in the bone marrow) we observed leukemic infiltration into the perivascular region of both large and small vessels of the mouse liver, and AML cells were frequently integrated into the vascular endothelium. Confocal microscopy surprisingly revealed the co-expression of human CD45 and the mouse endothelial cell marker CD31. To evaluate these apparently fused leukemia/endothelial cells further, single cell suspensions from the livers of mice engrafted with AML cells were FACS sorted based on the co-expression of human CD33 and mouse CD31. Multiplex PCR analysis of single hCD33+/mCD31+ cells verified the presence of both mouse genes (fah and tspy or D2Mit206) and human genes (flt3 and tty-15). The presence of the patient specific, leukemia-associated Flt3-ITD mutation confirmed that fusion occurs between malignant cells and vascular endothelium. AML and endothelial cell fusion was also confirmed by interphase fluorescent in situ hybridization (FISH) with species specific centromeric probes. In conclusion, the results from this study indicate that the functionally important cross-talk between normal hematopoietic stem/progenitor cells and vascular endothelium extends to malignant hematopoiesis. The fusion of primary AML cells with normal endothelial cells raises the possibility that vascular endothelium may serve as a reservoir for occult leukemia. Disclosures: No relevant conflicts of interest to declare.

Hereditary tyrosinemia type I is caused by deficiency of the enzyme fumarylacetoacetate hydrolase (FAH) (EC 3.7.1.2), the final step in tyrosine degradation. We report here the cloning and sequencing of a full length cDNA coding for murine FAH. This cDNA is highly homologous to the previously cloned human and rat genes.

About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.

The cobalamin C (cbl C) complementation group of combined MMA/HCYS is the most common o f the inborn errors of vitamin B12 metabolism, affecting both methylmalonyl-CoA and homocysteine (Hcys) degradation. We have recently diagnosed 2 patients with this disorder. AH. a 3 week old male, presented with lethargy, decreased oral intake, dehydration and pancytopenia. There had been a preceding upper respiratory infection. Parents were first-cousin Saudi Arabians. VM, a Hispanic female, presented at 19 days of life with lethargy. hyptonicity, dehydration, cough, decreased oral intake, significant thrombocytopenia, and mild pancytopenia. For both, worsening neurologic status was accompanied by seizures, and sepsis was suspected followed by appropriate intervention. Subsequent metabolic work-up revealed MMAIHCYS; fibroblast analysis for AH verified the cbl C complementation subgroup (Dr. D. Rosenblatt, Montreal, Canada; studies in VM are pending, although she likely falls within the same subgroup). Plasma total Hcys remains persistently elevated in AH (26-39 uM, n=8; nl < 10) and was 216 uM for VM (n=l). CSF total Hcys was 35 uM for VM (nl < 0.08). We conclude that I) diagnosis of patients with MMA/HCYS of the cbl C complementation subgroup may be hampered by absence of acute metabolic derangement (acidosis, hyperammonemia, hypoglycemia, ketosis) combined with features (lethargy, obtundation) suggesting sepsis; and 2) significantly increased CSF Hcys may be the underlying cause of altered neurologic status in these patients.

Targeted recombination in murine embryonic stem cells promises to be a powerful tool for introducing specific mutations into target genes to study development in mice and to create animal models of human disease. Gene targeting also holds potential for correcting genetic defects as an approach to human gene therapy. To precisely modify target genes, homologous recombination must proceed with high fidelity. However, several results have suggested that targeted recombination may be highly mutagenic. To test the accuracy of gene targeting we analyzed 44 independent targeted recombinants at the hypoxanthine phosphoribosyltransferase (HPRT) locus in a human fibroblast cell line and in mouse embryonic stem cells. We surveyed 80 kilobases around the sites of recombination by using chemical cleavage of mismatches. Only two mutations were found: a T----G transversion and a thymidine deletion. Thus, gene targeting in mammalian cells can be extremely accurate. These results demonstrate the feasibility of generating precise modifications of mammalian genomes by gene targeting.

Since the cloning of the cDNA for X-linked ornithine transcarbamylase (OTC) in 1984, diagnostic accuracy of OTC deficiency for prenatal and carrier detection has been greatly improved by the use of linkage analysis. However, the use of RFLP-based diagnosis is limited in this and in other new mutation diseases. Here we report both the use of direct mutation detection by new PCR-based techniques and our experience with linkage-based diagnosis in 18 families. We have previously reported the use of chemical mismatch cleavage to detect mutations first in amplified mRNA and then in genomic DNA of patients. This technique has now been utilized for prenatal diagnosis. Primers for specific amplification of OTC exons 1, 3, 5, 9, and 10 have been developed and been employed to map deletions of the OTC gene in two families. These primers also have been used to detect alterations in the TaqI sites found in exons 1, 3, 5, and 9. Four novel mutations of the OTC gene leading to abolition of a TaqI site in the OTC cDNA were discovered. One of these mutations is in exon 1; two lie in exon 3; and one is in exon 9. In addition, we have used the PCR products as probes to identify the exon-specific bands seen on Southern blots and to map the polymorphic BamHI and MspI sites, which are commonly used for linkage analysis. This information will facilitate the interpretation of altered band patterns seen in deletion cases and in cases of point mutations affecting restriction sites. Utilization of the appropriate combination of these molecular techniques permitted accurate diagnostic evaluations in 17 of 18 families.

X-chromosome inactivation results in the cis-limited dosage compensation of genes on one of the pair of X chromosomes in mammalian females. Although most X-linked genes are believed to be subject to inactivation, several are known to be expressed from both active and inactive X chromosomes. Here we describe an X-linked gene with a novel expression pattern--transcripts are detected only from the inactive X chromosome (Xi) and not from the active X chromosome (Xa). This gene, called XIST (for Xi-specific transcripts), is a candidate for a gene either involved in or uniquely influenced by the process of X inactivation.

Gene therapy is defined as the delivery and expression of a functional gene into somatic tissues of patients (or animals) endogenously deficient in this gene. Depending on the natural tissue pattern of expression and the sites most affected by disease processes, different anatomic sites may be the targets for gene transduction. To date, retrovirus-mediated gene transfer into hemopoietic stem cells is the most advanced of gene therapy systems and the most promising in terms of clinical applications in the near future. There are, however, additional efforts under way in our laboratory to develop efficient means of transducing genes into the liver, intestine, skeletal and cardiac muscles.

Endothelial progenitors of hematopoietic origin have a role in promoting recovery from vascular injury. Recently we have shown that following radiation damage, transplanted hematopoietic stem cells (HSCs) give rise to endothelial cell outcomes at the clonal level. This raises the question of whether prospectively identifiable HSC progeny have endothelial cell activity. Using β-galactosidase and EGFP expression as donor markers, we show that lineage-restricted myeloid progenitors can give rise to endothelial cells. Transplanted common myeloid progenitors (CMPs) and granulocyte/macrophage progenitors (GMPs) gave rise to similar numbers of endothelial cells in liver portal vein branches. Donor-derived endothelial cells expressing functional endothelial markers including CD31, von Willebrand factor (vWF) and Tie-2 were clearly distinguished from mature hematopoietic cells by the absence of CD45 and F4/80 expression. Confocal microscopy showed the co-localization of donor and endothelial cell marker expression in individual cells so that endothelial cells could be discriminated from perivascular cells. To evaluate cell fusion as a potential mechanism for the emergence of endothelial cells of hematopoietic origin, HSCs transgenic for EGFP and Tie-2-Cre recombinase were transplanted into ROSA26 reporter (R26R) mice. Activation of β-galactosidase reporter gene expression, indicative of cell fusion with a donor-derived hematopoietic cell, could be detected in hepatocytes but not in endothelial cells. However, due to the tissue-specificity of the Tie-2 promoter, endothelial fusion products in which the donor nucleus had failed to activate the Tie-2-driven Cre recombinase would have also lacked detectable reporter gene expression. To ensure that the absence of β-galactosidase expression in the donor-derived endothelial cells was not due to insufficient nuclear reprogramming, HSCs transgenic for EGFP and R26R were transplanted into Tie-2-Cre recipients. Donor-derived endothelial cells were again negative for both β-galactosidase expression and activity. To determine whether radiation injury is required for the incorporation of donor cells into the endothelium, a parabiosis model was utilized. Both long-term donor-derived endothelial cell outcomes and multilineage hematopoiesis were detected in parabiotic mice. These results demonstrate that CMPs and further lineage-restricted GMPs are capable of differentiating into endothelial cells in the absence of cell fusion. Importantly, radiation injury is not required for endothelial cell engraftment, indicating that circulating progenitors contribute to angiogenesis during steady-state conditions.

The life span of patients with Fanconi Anemia (FA) is severely shortened by bone marrow failure and malignancies and currently, allogeneic bone-marrow transplantation is the only therapeutic option. Despite the successful modeling of FA viral vector gene therapy in the mouse, application of the same approach to humans is complicated by the requirement for prolonged ex vivo manipulation and the scarcity of HSCs in this disease. Here, we utilized the non-viral Sleeping Beauty (SB) transposon system to facilitate stable, gene transfer of the human FANCC cDNA without ex vivo culture. This was achieved either by electroporation of naked DNA into whole bone marrow or by direct injection into the femur cavity. Our studies demonstrate that a) hematopoietic stem cells from Fancc -/- mice can be transduced by plasmid DNA; b) stable integration of plasmid DNA into the genome of hematopoietic stem cells (HSC) is possible; c) animal model was phenotypically corrected; and d) long-term therapeutic benefit could be achieved using this method. Non-viral gene transfer may be a viable alternative for the treatment of hematopoietic diseases.

Recent reports on severe adverse effects induced by retroviral integration into the LMO2 proto-oncogene in humans and the demonstration of preferential AAV serotype 2 (rAAV2) vector integration into genes in our previous study with a limited number of integration sites in mice1, have raised a possible concern about rAAV-mediated insertional mutagenesis. In order to further address this issue, we have expanded our previous study and performed a high-throughput analysis of rAAV2 integration sites isolated from in vivo selected hereditary tyrosinemia type I (HTI) mouse hepatocytes transduced with a human fumaryl acetoacetate hydrolase (FAH)-expressing rAAV2 shuttle vector, AAV-EF1α-hFAH.AOS. Briefly, total liver DNA was isolated from 4 HTI mice that underwent a 7-month in vivo selection of transplanted hepatocytes isolated from the donor HTI mice having received 3.0×1011 vector genomes of AAV-EF1α-hFAH.AOS via the portal vein and undergone an 8-week in vivo selection. The whole proviral rAAV2 vector genome and flanking genomic DNA sequences were isolated as a plasmid from the liver DNA by a plasmid rescue technique as previously described1. All four mice had a different donor HTI mouse, therefore, any integrations found in different mice should be considered as independent integration events. To date, we have characterized 307 independent integration events and found: 1) 191 of 307 (62%) of integrations occurred in genes; 2) there was a strong bias toward integrating into host genomes around transcription start sites, as has been observed in murine leukemia virus integrations (approximately a quarter of the total rAAV2 integrations occurred within ±1 kb from transcription start sites); 3) rAAV integration involved large genomic deletions of over 1 kb in 15% of the cases, suggesting that such large deletions may not be rare events associated with rAAV2 integration. The most striking finding was that among the 307 independent integration events, at least 4 integrations from 3 different mice (1.5 % of total integrations and 2.1 % of the genes targeted by rAAV) were found within a 30-kb region in the mouse ubiquitin C gene, and at least 4 integrations from 4 different mice fell on another sequence stretch of approximately 2.5 kb. Our preliminary analysis by restriction enzyme mapping and sequencing over a thousand base pairs has indicated that the hot spot of 2.5 kb in length likely resides in a proto-oncogene, the Evi-1 gene. It should be noted that murine leukemia found in a retroviral gene marking study was induced by insertional mutagenesis at this locus. Although the oncogenic potential of this gene in the liver is not known, overexpression of this gene can transform not only hematopoietic cells but also other cell types. Thus, the preliminary results in our high-throughput rAAV2 integration site analysis further emphasize the importance of pursuing the study on the elucidation of the mechanisms of rAAV vector integration and its consequences in the host.

Objective: Determine the minimal number of phenylalanine hydroxylase (PAH) positive hepatocytes and minimal level of PAH activity required to correct hyperphenylalaninemia in Pahenu2 mice, a model of human phenylketonuria (PKU).

The life span of patients with Fanconi Anemia (FA) is severely shortened by bone marrow failure and malignancies. Currently, the only efficient therapy is bone-marrow transplantation from a matched sibling donor. Therefore, FA is considered a good candidate disease for hematopoietic gene therapy. Our previous preclinical gene therapy experiments with Fancc knock-out mice have shown that wild-type hematopoietic stem cells (introduced by transplantation or generated by gene transfer) have a strong selective growth advantage in vivo. However, despite the successful modeling of FA gene therapy in the mouse, application of the same approach to humans is complicated by the requirement for ex vivo manipulation and scarcity of HSCs in FA patients. Consequently, the development of new gene transfer methods that support stable integration of therapeutic transgenes in vivo are greatly desired. Here, we have utilized the Sleeping Beauty (SB) transposon system to facilitate stable, transposase-mediated gene transfer of the human FANCC cDNA in vivo from a plasmid-based system. The transposase and FANCC transposon plasmid constructs were co-transfected into bone-marrow cells of Fancc mutant mice by two different methods: 1) via electroporation into enriched hematopoietic cells; 2) via direct injection into the femur cavity. In each case, the presence of the transgene was followed in treated animals by semi-quantitative PCR. Although no transgene was detectable in peripheral blood DNA 2 weeks after injection, 2/4 mice injected into the femur cavity were PCR positive 4 weeks after injection, even in the absence of selective pressure. At this time, a single low dose of cyclophosphamide (CPA) was given to provide a selective advantage for FANCC corrected cells. Four weeks after CPA administration, all the experimental electroporated animals (N=2) and femur injected animals (N=8) were strongly PCR positive in their peripheral blood and have remained stably positive for at least 9 months. Furthermore, upon transplantation of bone marrow from these mice into wild-type lethally irradiated recipients, we observed that 81/110 (74%) of day 12 CFU-S colonies were transgene positive. Similarly, 64/110 (64%) of the 12-day CFU-S colonies were transgene positive from the femur-injected donors, thus confirming that transgene integration had occurred in bona fide stem cells. Collectively, these studies demonstrate that a) hematopoietic stem cells from Fancc mutant mice are proficient in taking up plasmid DNA; b) stable integration of plasmid DNA into the genome of hematopoietic stem cells is possible and c) the efficiency of stable gene transfer using these nonviral approaches compares favorably to that achieved by alternative viral-based methods. We are in the process of analyzing transposon host cellular DNA junctions to determine the mechanism of transgene persistence in each experimental group. The above experiments have been repeated independently with more animals: N=5 for electroporated and N=6 for femur injected. Details of all these results will be discussed.

Reply to “Involvement of oxidative stress in Fanconi's anaemia: from phenotype to FA protein functions”

Alkaptonuria (aku), an inborn error of metabolism caused by the loss of homogentisate 1,2-dioxygenase (HGD), has been described in a mouse model created by ethylnitrosourea mutagenesis but the mutation in these mice has not previously been identified. We used RT-PCR to amplify the Hgd cDNA from Hgd(aku)/Hgd(aku) mice. Two products shorter than the wild-type product were amplified. Restriction mapping and DNA sequencing were then used to identify the Hgd(aku) mouse mutation, found to be a single base change in a splice donor consensus sequence, causing exon skipping and frame-shifted products. This base change allowed us to create a non-radioactive genotyping assay for this allele.

SummaryAdult liver progenitor cells are biliary-like epithelial cells that emerge only under injury conditions in the periportal region of the liver. They exhibit phenotypes of both hepatocytes and bile ducts. However, their origin and their significance to injury repair remain unclear. Here, we used a chimeric lineage tracing system to demonstrate that hepatocytes contribute to the progenitor pool. RNA-sequencing, ultrastructural analysis, and in vitro progenitor assays revealed that hepatocyte-derived progenitors were distinct from their biliary-derived counterparts. In vivo lineage tracing and serial transplantation assays showed that hepatocyte-derived proliferative ducts retained a memory of their origin and differentiated back into hepatocytes upon cessation of injury. Similarly, human hepatocytes in chimeric mice also gave rise to biliary progenitors in vivo. We conclude that human and mouse hepatocytes can undergo reversible ductal metaplasia in response to injury, expand as ducts, and subsequently contribute to restoration of the hepatocyte mass.

Summary Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2−/− mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2−/− mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure., Graphical Abstract, Highlights • OXM treatment causes loss of quiescence and stem cell exhaustion • RNaseq analysis reveals transcriptional changes in key pathways in Fancd2−/− HSPC • OXM suppresses transcription of osteopontin, a known stem cell quiescence promoter • Unlike previous notions, OXM has no effect on telomerase expression or EPO signaling, In this article, Zhang and colleagues show that 18-month-old Fancd2−/− mice recapitulated key human Fanconi anemia phenotypes, including peripheral pancytopenia and macrocytosis. Chronic oxymetholone treatment improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells, but eventually resulted in stem cell exhaustion. RNaseq analysis implicated downregulation of osteopontin as an important mechanism for the drug’s action.

We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or α-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of α, β, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.

Novel cell surface-reactive monoclonal antibodies generated against extrahepatic biliary cells were developed for the isolation and characterization of different cell subsets from normal adult human gallbladder. Eleven antigenically distinct gallbladder subpopulations were isolated by fluorescence-activated cell sorting. They were classified into epithelial, mesenchymal, and pancreatobiliary (PDX1+SOX9+) subsets based on gene expression profiling. These antigenically distinct human gallbladder cell subsets could potentially also reflect different functional properties in regards to bile physiology, cell renewal and plasticity. Three of the novel monoclonal antibodies differentially labeled archival sections of primary carcinoma of human gallbladder relative to normal tissue. The novel monoclonal antibodies described herein enable the identification and characterization of antigenically diverse cell subsets within adult human gallbladder and are putative tumor biomarkers.

The mutations in X chromosome-linked diseases are frequently heterogeneous, and yet only a small fraction can be detected by Southern analysis. We therefore adapted the chemical cleavage method of Cotton et al. [Cotton, R. G. H., Rodrigues, N. R. & Campbell, R. D. (1988) Proc. Natl. Acad. Sci. USA 85, 4397-4401] and the polymerase chain reaction to rapidly scan for point mutations in X chromosome-linked ornithine transcarbamoylase (carbamoyl-phosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) deficiency. This simple heteroduplex mapping method identified different mismatch sites in polymerase chain reaction-amplified liver cDNA from five unrelated ornithine transcarbamoylase-deficient patients. The predicted sequence alteration was confirmed by DNA sequencing in all five patients and indicated a likely disease-causing mutation in four of these patients. In one atypical ornithine transcarbamoylase-deficient patient a sequence alteration compatible with a cDNA polymorphism was found. One family was studied in detail. Female-carrier detection was performed by chemical cleavage of amplified genomic DNA and verified by allele-specific oligonucleotide hybridization. This mutation scanning approach is simple, sensitive, and applicable to many genetic loci.

Steroid sulfatase (STS) and arylsulfatase C ( ARSC ) were studied in fibroblast clones from a 45,X/47,XXX mosaic and from a 69,XXY triploidy with one or two active X chromosomes. The comparison of the 47,XXX with 45,X clones showed an incomplete gene dosage effect (1.8 for STS and 2.0 for ARSC ). This was not the case for the triploid clones with different X-inactivation patterns. These results confirm previous reports on the non-inactivation of the STS gene, and establish X linkage and non-inactivation for the ARSC gene as well.

Mice homozygous for the c14CoS albino deletion die as neonates as a result of liver dysfunction. Previous mapping studies have associated this defect with a 310-kb fragment encoding the hepatocyte-specific developmental regulation locus (alf/hsdr-1). The gene encoding fumarylacetoacetate hydrolase (Fah), a metabolic enzyme that catalyzes the last step of tyrosine catabolism, also maps to the same deletion interval. To test whether the neonatal defects found in the albino deletion mutants are attributable to loss of Fah, and not to another gene mapping to the deletion, we have generated Fah mutant mice by gene targeting in embryonic stem cells. Fah-deficient mice die within 12 hr after birth from hypoglycemia and liver dysfunction. In addition, the same pattern of altered liver mRNA expression found in the albino deletion mutants was also found in affected animals. We conclude that the neonatal lethal and liver dysfunction phenotype of the alf/hsdr-1 deletion is entirely attributable to loss of Fah.

Fanconi anemia (FA) is an autosomal recessive disorder caused by defects in at least eight distinct genes FANCA, B, C, D1, D2, E, F and G. The clinical phenotype of all FA complementation groups is similar and is characterized by progressive bone marrow failure, cancer proneness and typical birth defects. The principal cellular phenotype is hypersensitivity to DNA damage, particularly interstrand DNA crosslinks. The FA proteins constitute a multiprotein pathway whose precise biochemical function(s) remain unknown. Five of the FA proteins (FANCA, C, E, F and G) interact in a nuclear complex upstream of FANCD2. FANCB and FANCD1 have not yet been cloned, but it is likely that FANCB is part of the nuclear complex and that FANCD1 acts downstream of FANCD2. The FA nuclear complex regulates the mono-ubiquitination of FANCD2 in response to DNA damage, resulting in targeting of this protein into nuclear foci. These foci also contain BRCA1 and other DNA damage response proteins. In male meiosis, FANCD2 also co-localizes with BRCA1 at synaptonemal complexes. Together, these data suggest that the FA pathway functions primarily as a DNA damage response system, although its exact role (direct involvement in DNA repair versus indirect, facilitating role) has not yet been defined.

IN mammals, equal dosage of gene products encoded by the X chromosome in male and female cells is achieved by X inactivation. Although X-chromosome inactivation represents the most extensive example known of long range cis gene regulation, the mechanism by which thousands of genes on only one of a pair of identical chromosomes are turned off is poorly understood. We have recently identified a human gene (XIST) exclusively expressed from the inactive X chromosome1. Here we report the isolation and characterization of its murine homologue (Xist) which localizes to the mouse X inactivation centre region and is the first murine gene found to be expressed from the inactive X chromosome. Nucleotide sequence analysis indicates that Xist may be associated with a protein product. The similar map positions and expression patterns for Xist in mouse and man suggest that this gene may have a role in X inactivation.

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.