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The role of reactive oxygen and nitrogen species in the development of Fanconi Anemia, an inherited bone… Hadjur, Suzana 2002

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The Role of Reactive Oxygen and Nitrogen Species in the Development of Fanconi Anemia, an Inherited Bone Marrow Failure Disorder by SUZANA HADJUR B.Sc , The University of Western Ontario (Honors), 1997 A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Biology; Genetics Graduate Program) We accept this thesis as conforming to the required standard: THE UNIVERSITY OF BRITISH C O L U M B I A September 6, 2002 © Suzana Hadjur, 2002 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of £j &oe-hcS 6 r g i d u gyfg- ?r^<^cz^rr\ The University of British Columbia Vancouver, Canada Date <2 QcA-ober 2 0 0 2 _ DE-6 (2/88) A B S T R A C T The objective of this thesis was to investigate the role of endogenous reactive oxygen and nitrogen species in the pathogenesis of Fanconi's Anemia (FA) using Fanconi Anemia Complementation Group C (Fancc) - deficient mice. This objective was examined in two distinct ways. First, through the generation and characterization of a novel mouse model for F A with intrinsic oxidant stress and secondly, through the observation that nitric oxide (NO) may have a role in cytokine - mediated inhibition of hematopoiesis in FA. F A is an autosomal recessive disorder, which primarily affects children and young adults, resulting in morbidity and mortality due to B M failure or acute myelogenous leukemia (AML). Currently, eight complementation groups have been identified and six F A genes have been cloned. Although knockout mice have been generated for each of the genes cloned, they do not exhibit the primary hematopoietic defect of FA. Thus, no spontaneously occurring mouse model for F A exists with which to better define the pathogenesis of this disease. Several lines of evidence have pointed to abnormal regulation of intracellular reactive oxygen species (ROS) in individuals with FA. To investigate the possible role of the Fancc protein in the regulation of an in vivo redox state, mice were generated having combined deficiencies in the genes encoding the cytoplasmic antioxidant, Cu/Zn superoxide dismutase (Sodl) and Fancc. Fancc''Sodl~'~ mice developed hepatic lipid accumulation, peripheral blood bicytopenia, marrow hypocellularity, little to no growth of committed progenitor cells in vitro, and decreased frequencies of long-term progenitors. This novel murine model of F A partially replicates the hematopoietic defect of this disease and may be useful in defining novel therapies. The second theme of this thesis involved the observation that NO may have a role in F A B M failure. Cytokine inhibition of hematopoietic progenitor colony growth from Fancc'' mice was completely rescued in the presence of an iNOS inhibitor, L - N M M A . Fancc'' progenitor cells were hypersensitive to NO generating drugs in vitro while primary macrophages had elevated expression of iNOS and N O production in response to rFNy and IFNy /LPS. To date, no information exists regarding F A and NO and these studies have opened a new avenue of investigation in F A research. i i T A B L E O F C O N T E N T S ABSTRA CT " TABLE OF CONTENTS . List of Tables — v List of Figures . vi List of Abbreviations viii Acknowledgements . ix Dedication ix Publications arising from the work in this thesis x CHAPTER ONE - Introduction 1 1.1 REVIEW OF CURRENT LITERATURE 1 Clinical and Cellular Phenotypes 1 Complementation Analysis and Cloning of the F A Genes 2 F A Molecular Interactions 5 Role of the F A Proteins 7 F A and D N A Repair 7 F A and Redox Regulation 9 Animal Models 11 Bone Marrow Failure in F A 13 1.2 THESIS GOALS 17 CHAPTER TWO - Generation and Analysis of Mice with Combined Deficiencies of the Genes Encoding Fanconi Anemia Complementation Group C (Fancc) and Cu/Zn Superoxide Dismutase (Sodl). 18 2.1 INTRODUCTION 18 2.2 MATERIALS AND METHODS 21 2.3 RESULTS 25 Fancc'Sodl' mice develop zonal microvesicular hepatic steatosis 25 Primary Fancc'~Sodl:'~ liver cell cultures generate increased levels of superoxide 26 Increased expression of Mn SOD and HO-1 in Fancc''Sodl'' livers. 27 Peripheral blood and bone marrow abnormalities of Fancc^'Sodl"7" mice 28 Fancc''Sodl'' total marrow cells fail to show increased apoptosis or chromosomal aberrations 30 In vitro hematopoietic colony growth is severely impaired in Fancc''Sodl'' mice : 30 Primitive progenitor numbers are normal in Fancc''Sodl''' mice 31 Aging study reveals persistant aberrant hematopoiesis in Fancc''Sodl'' mice. 32 2.4 DISCUSSION 33 CHAPTER THREE - Absence of Cu/Zn Superoxide Dismutase (Sodl) Limits Long-Term Progenitor Cell Self-Renewal in Fanconi Anemia Complementation Group C (Fancc)-deficient Mice. 38 3.1 INTRODUCTION 38 3.2 MATERIALS AND METHODS 40 3.3 RESULTS 46 i i i CAFC frequencies are significantly decreased from Fancc''Sodl'' mice and can be partially rescued in hypoxia. 46 Column purified early HPC from Fancc'1'Sodl'1' mice have reduced growth rates and increased apoptosis. 48 HPC can be partially rescued when grown in the presence of hypoxia or antioxidants. 49 Inability to generate cell lines from Fancc''Sodl'' mice. 51 3.4 DISCUSSION 52 CHAPTER FOUR - FancA''' Sodl'1' mice have a less severe phenotype compared to 57 Fancc'Sodl'' Mice . 57 4.1 INTRODUCTION 57 4.2 MATERIALS AND METHODS 59 4.3 RESULTS 60 Histological analysis of FancA'''Sodl'' mice. 60 BM cellularity and body weights are slightly decreased in FancA''Sodl'mice. 60 Colony Forming Assays reveal defective growth from FancA'''Sodl'' committed progenitors. 61 4.4 DISCUSSION . 62 CHAPTER FIVE - A Novel Role for Nitric Oxide in Mediating Cytokine - Induced Growth Inhibition of Fancc -deficient Bone Marrow Cells. 65 5.1 INTRODUCTION 65 5.2 MATERIALS AND METHODS 67 5.3 RESULTS - 71 Cytokine - inhibited colony growth of Fancc'' BM cells is rescued with L-NMMA. 71 Fancc'' BM cells are hypersensitive to NO donors. 72 Apoptosis of IFNy-treated HPC from Fancc'' mice is inhibited by L-NMMA. 73 Fancc'' macrophages have elevated expression of iNOS. 74 Fancc'' macrophages have increased nitrite production. 75 Phosphorylated Statl is increased in Fancc'' macrophages stimulated with IFNy 76 5.4 DISCUSSION 77 CHAPTER SIX - DISCUSSION 81 6.1 Results Summary . 81 6.2 A Role for FA Proteins in Redox Regulation. 83 6.3 A Role for NO in FA BM Aplasia 85 BIBLIOGRAPHY 90 TABLES and FIGURES 104 iv List of Tables Table I. Clinical and Cellular Phenotypes in Fanconi Anemia p. 104 Table II. Fanconi Anemia Complementation Groups p. 105 Table III. Current Mouse Models for Fanconi Anemia p. 108 Table IV. Peripheral Blood Values from 8 - 10 week old mice p. 115 Table V. Total B M Cellularity and absolute number of cell types from 8-10 week old mice p. 117 Table VI. PI/Annexin V FACS Analysis from total B M samples p. 119 Table VII. Reagents used to try to rescue growth of methylcellulose colonies fromFancc'Sodl'' mice p. 121 Table VIII. C A F C Frequencies in 20% and 5% Oxygen p. 124 v List of Figures Fig. 1 Mutations identified in Fanconi anemia senes p. 106 Fig. 2 Current knowledge, of protein interactions and function of FA proteins p. 107 Fig. 3 Major sources of reactive species found within cells p. 109 Fig. 4 Body weights of 8 wk old Fancc''Sodl~'' mice p. 110 Fig. 5 livers Histologic examination reveals zonal hepatic microvesicular steatosis in Fancc'Sodl'' mouse p . l l l Fig. 6 F.lectron microscopy of hepatocytes from Fancc'Sodl'' mice p.112 Fig. 7 Primary hepatocytes from Fancc'Sodl'' mice demonstrate increased superoxide levels. p.113 Fig. 8 Liver specific expression of MnSOD and HO-1 is increased in Fancc'Sodl'' mice p. 114 Fig. 9 Hypocellnlariry and increased fat accumulation in Fancc''Sodl~'~ bone marrows p.116 Fig. 10 FACS analysis of L i n + cells for progenitor markers p.118 Fig. 11 Colony forming assays reveal abnormal progenitor growth in Fancc'Sodl'' mice p. 120 Fig. 12 FACS analysis of T.in" cells from Fancc'Sodl'' marrows after column selection p. 122 Fig. 13 Colony Forming Assavs from 6-8 month old mice p.123 Fig. 14 Representative C A F C colonies at dav 7 in 20% and 5% O. p. 125-6 Fig. 15 Morphological changes in C A F C colonies from Fancc'Sodl'' cultures p. 127 Fig. 16 HPC growth in liquid media reveals abnormal proliferation and increased apoptosis p.128 Fig. 17 Hypoxia partiallv rescues Fancc'Sodl'' HPC growth p. 129 Fig. 18 TTRON partiallv rescues Fancc'Sodl'1' HPC erowth p. 130 Fig. 19 MnTMPyP dose response and partial rescue of HPC proliferation in vitro p.131 Fig. 20 Primary mast cell cultures from Fancc'Sodl'' marrows fail to grow in vitro p.132 Fig. 21 Histologic examination of FancA'Sodl''mouse livers p.133 Fig. 22 Rody weights are significantly decreased in FancA'Sodl''mice p. 134 Fig. 23 Marrow cellularitv is decreased in FancA~'~Sod1''' mice p.135 Fig. 24 Colony forming assays reveal abnormal progenitor growth in FancA'SodV'' mice p.136 Fig. 25 Frp.qiie.nry of R M progenitors from FaneA~'~Sodl'1' mice is abnormal p.137 Fig. 26 Inhibition of Fancc'' colony formation by TFNy is reversed bv L - N M M A p.138 Fig. 27 Rerliir.ed colony formation bv TNFo: and MTP1 a is reversed bv L - N M M A p.139 Fig. 28 Fancc'' R M progenitors show increased sensitivity to NO-seneratins drugs p. 140 Fig. 29 Inhibition of Fancc'' FTPC growth bv TFNy is reversed bv L - N M M A p.141 Fig. 30 Elevated iNOS expression in stimulated Fancc'' peritoneal macrophages p. 142 Fig. 31 Elevated iNOS expression in Fancc'' BM-derived macrophages stimulated with IFNy p. 143 Fig. 32 Increased NO production by Fancc'' macrophages p. 144 Fig. 33 Statl phosphorylation is augmented in IFNy-stimulated Fancc'' macrophages p. 145 Fig. 34 Hif 1 nr. expression is increased in IFNy-stimulated Fancc'' macrophages p. 146 Fig. 35 NO and superoxide anion: Converging Pathwavs to FA? p. 147 vii List of Abbreviations A L T Alanine Amino Transferase A M L Acute Myeloid Leukemia B M Bone Marrow B R C A Breast Cancer C A F C Cobblestone Area Forming Cell C F U Colony Forming Units Cu/Zn SOD Copper/Zinc Superoxide Dismutase DEB Diepoxybutane DSB Double Strand Breaks F A Fanconi Anemia FACS Florescence Actiavted Cell Sorter GSTP1 Glutathione S-Transferase Protein 1 H 2 0 2 Hydrogen Peroxide HO-1 Heme Oxygenase 1 HPC Hematopoietic Progenitor Cell IFNy Interferon - y MJPlcc Macrophage Inflammatory Protein 1 - a M M C Mitomycin C Mn SOD Mangenese Superoxide Dismutase NLS Nuclear Localization Signal NO Nitric Oxide NOS Nitric Oxide Synthase ONOO" Peroxynitrite ORO Oil Red 0 Stain o2- Superoxide anion RED N A D P H Cytochrome P-450 Reductase RNS Reactive Nitrogen Species ROS Reactive Oxygen Species SCF Stem Cell Factor SOD Superoxide Dismutase TNFct Tumor Necrosis Factor - a T U N E L Terminal viii Acknowledgements I would like to extend my most sincere thanks to my supervisor Dr. Frank Jirik, who believed in my abilities as a graduate student and who provided constant encouragement. He gave me the confidence to become a scientist. I thank the entire Jirik Lab, both in Vancouver and in Calgary, especially Jennifer Moody, and all the members of the respective animal units. A very special thank you to Mark Cordy for his endless support, love and positivity. Dedication This thesis is dedicated to my parents. It is because they had the courage to leave their homeland that I was allowed this opportunity. ix Publications arising from the work in this thesis Hadjur S., Ung K. , Wadsworth L. , Dimmick J., Rajcan-Separovic E., Scott R.W., Buchwald M . , F.R. J. (2001) Defective hematopoiesis and hepatic steatosis in mice with combined deficiencies of the genes encoding Fancc and Cu/Zn superoxide dismutase. Blood. 98:1003-1011 Hadjur, S. and Jirik, F.R. (submitted) Role of nitric oxide in cytokine-mediated growth inhibition of Fancc-deficient hematopoietic cells. Hadjur, S. and Jirik, F.R. (in preparation) Absence of Cu/Zn superoxide dismutase (Sodl) limits progenitor cell self-renewal in murine hematopoiesis. Work contained within this thesis not completed by the author • Metaphase chromosome preparation and breakage analysis: Results, page 30 x CHAPTER ONE - Introduction 1.1 REVIEW OF CURRENT LITERATURE Clinical and Cellular Phenotvpes Fanconi Anemia (FA) is an autosomal recessive disorder clinically characterized by progressive pancytopenia, developmental abnormalities of the skeleton, kidneys and heart, hypogonadism (resulting in reduced fertility) and an increased risk of malignancy F A has a wide spectrum of severity ranging from congenital abnormalities and acute myeloid leukemia (AML) in the first decade of life to mild anemia and oral cancer in the fifth decade of life (Sable I). The mean age of onset of bone marrow (BM) aplasia is 8 years and the mean life expectancy of F A patients is 20 years with the major causes of fatality being B M failure (aplastic anemia) or malignancies, primarily A M L and squamous cell carcinomas 2 . The disease has a frequency in the population of 1-5 per million and is found in all ethnic groups, with an estimated heterozygous mutation carrier frequency between 0.3 and 1% 3 . Currently, there is no cure for FA. Pancytopenic F A patients become transfusion dependent and B M transplants are the only way to permanently correct the hematological phenotype. F A was originally classified as a chromosomal instability disorder due to the observation that metaphase spreads from F A patient lymphocytes exhibited spontaneous chromosomal breaks, interchanges and arrest during the G2 phase of the cell cycle 4 . When F A cells are challenged with D N A cross-linking agents such as mitomycin C (MMC), diepoxybutane (DEB) and cisplatin there is a marked increase in chromosomal aberrations, which include the formation of radial and quadrilateral chromosomes as well as increased numbers of chromosomal breaks and interchanges 3 ' 5 ' 6 . This dramatic cross-linker sensitivity of F A cells serves as a tool for disease diagnosis. F A cells are also hypersensitive to elevated oxygen concentrations 1 . Cells grow slowly at elevated oxygen levels (35%) and tend to arrest at G 2 , while at low oxygen concentrations (< 5%), growth is normal and accompanied by decreased chromosomal aberrations 1 7"9. In fact there is indirect evidence that the effect of M M C on F A cells is mediated through oxidant stress 1 0 . Several other complex genetic diseases have been identified which have chromosomal instability and an increased predisposition to cancer. The genes mutated in these diseases are thought to have a role in genome surveillance, otherwise known as 'care-taker' genes. Examples include xeroderma pigmentosa (XP), hereditary non-polyposis colorectal cancer (HNPCC) and ataxia telangiectasia (AT) u . These disorders all involve genes that are required for double strand break repair response (nucleotide excision repair, mismatch repair and A T M , respectively) and thus the inability to maintain genome integrity leads to the accumulation of mutations and eventual malignant transformation of the cell Due to the chromosomal instability and increased cancer risk in F A patients, F A genes were immediately assumed to be involved in the repair or recognition of D N A damage. Complementation Analysis and Cloning of the F A Genes Genetic complementation analysis was employed with cultured F A lymphoblasts to determine the number of genes involved in the disease 1 2" 1 4. This method took advantage of the fact that F A cells are characterized by their high sensitivity to M M C (10-100 times over wildtype controls). A panel of somatic cell hybrids was generated by fusing together lymphoblast cell lines derived from different F A patients and the DEB and M M C sensitivities of each hybrid and parental line was assayed in order to determine the extent of genetic complementation. If the M M C - sensitive phenotype persisted then the mutations occurred in the same F A gene and the complementation group was identical, however if the M M C - sensitive phenotype was corrected, then the two cell lines were from different complementation groups and likely represented different genes 1 5 . The F A N C C cDNA that corresponded to the human F A - C complementation group was cloned using essentially the same functional complementation method 1 4. The FA-C cell line, 2 selected because of its extreme sensitivity to M M C and DEB, was transfected with a cDNA library and the.FA-C cells were selected for the complementing cDNA by repeated exposure to high doses of M M C and following outgrowth of clones, to repeated doses of DEB. The resultant clones represented the potential FANCC gene 1 4 . The plasmid carrying the wildtype cDNA was shown to directly correct the M M C sensitivity of its particular complementation group (FA-C) and not other complementation groups. Subsequently, pathogenic mutations were identified within this gene 1 6 . Clearly, the rarity of F A mutations in the population has been an obstacle in the cloning of the genes. Despite this, complementation studies have identified that F A is genetically heterogeneous, with at least eight F A complementation groups (A, B , C, D l , D2, E, F and G) identified to date (Table II). The genes corresponding to the groups (A) FANCA, (C) FANCC, (D2) FANCDZ (E) FANCE, (F) FANCF and (G) FANCG, have now been cloned using a variety of techniques including positional and complementation cloning and microcell - mediated chromosome transfer strategies 1 4 1 7 " 2 2 . Complementation group D appears to be heterogeneous since the FANCD2 gene has mutations that have been described in only a subset of F A - D patients, suggesting that another gene must be involved, temporarily called FAN CD 1. Fig. II is a cartoon showing each of the six cloned F A genes and pathogenic mutations identified within each gene. Due to founder effects, the prevalence of mutations within a particular F A gene may vary with ethnic background. For example, most South African F A patients belong to group A and deletion mutations in exons 11-17 and 12-31 of the FANCA gene are frequently found in this population 2 3 . Complementation group C is most common among the Ashkenazi-Jewish population 2 4 . The most common mutations in FANCC are the splice-site mutation (IVS4 + 4A —> T) and the frameshift mutation (322delG) and these are found most often in the Ashkenazi-Jewish and in the Dutch populations, respectively. Interestingly, mutations in FANCA account for approximately 80% of all F A patients 2 5 . There are upwards of 100 unique-site pathogenic mutations in the FANCA gene alone and greater then one-third of these mutations are deletions, likely due to the large number of Alu repeats found within the gene. As more 3 mutation information is collected, genotype - phenotype correlations for the F A genes have begun to emerge. F A - C patients are generally considered to have the most severe form of the disease 2 6 . Patients with mutations in FANCC and FANCF show onset of hematological problems at a much earlier age, and have an increased risk of malignancy compared to patients with'mutations in FANCA, which are considered to have an overall milder form of the disease 2 6'. Furthermore, disease severity is also correlated to particular mutations within an F A sub-type. For example, genotype - phenotype analyses of F A - C patients divided these into three distinct groups; a) patients with the IVS4 + 4A —» T mutation, b) patients with at least one exon 14 mutation, and c) patients with at least one exon 1 mutation. The median age of hematological onset was 2.7, 2.4 and 7.6 years respectively for each group and the mean age of survival was 6.7, 7.8 and 14.3 years, respectively 2 6 . Initial attempts to identify the role of the F A proteins within the cell focused on sequence homology and expression studies. Homologous regions within the predicted F A proteins do exist in lower organisms, however these are not significantly similar to functional motifs in non-vertebrate, yeast or bacterial sequences 1 6 . Significant conservation of protein and gene sequence in FANCA, FANCC, FANCG and FANCF is observed within vertebrates (Table II), while FANCD2 is relatively well conserved and appears to have orthologs in lower organisms, including Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana 1 6 ' 2 2 . Unfortunately, the role of the proteins in these organisms is not known. Interestingly, FANCG was found to be identical to XRCC9, a gene that is defective in specific radiation sensitive CHO cell lines 1 9 . The significance of this observation is unknown. Studies have revealed that the mouse orthologs of FANCA and FANCC are ubiquitously expressed at low levels in adult tissues. During embryogenesis, Fancc is expressed in whisker follicles, brain, kidney, lung, gut, stomach and in osteogenic and hematopoietic lineages 2 1 . The expression patterns of Fanca are very similar to Fancc with the major differences being high expression in lymphoid tissue (spleen, thymus and lymph nodes), testis and ovary and no 4 expression in the lung and gut 2 8 , 2 9 . Fancg has similar expression patterns to the Fanca knockout moues, primarily in the spleen, thymus and the testis 3 0 . F A Molecular Interactions Since homology and expression studies were not useful in elucidating the function of the F A proteins, researchers focused on subcellular localization and protein interactions. Cell fractionation and confocal microscopy studies have indicated that F A N C A , F A N C C and F A N C G are nuclear as well as cytoplasmic proteins 3 1 , 3 2 . The majority of the F A N C C protein is cytoplasmic and this is in keeping with the observation that F A N C C is required in the cytoplasm in order to complement the M M C sensitivity of F A - C cells 3 3 . The remainder of the known F A proteins are thought to be nuclear, although some results suggest that a small fraction of F A N C E and F A N C F can also be cytoplasmic 3 4 . Using yeast two-hybrid, co-immunoprecipitation and immunofluorescence studies, a substantial body of evidence has shown that most of the F A proteins interact, directly or indirectly in a common functional pathway. These methods have shown that there are direct interactions between F A N C A and F A N C G and between F A N C C and F A N C E in the cytoplasm of normal cells. Furthermore, all five F A proteins (FANCA, F A N C C , F A N C E , F A N C F and F A N C G ) can 32 35 38 be co-purified out together as a complex from nuclear extracts ' " . Binding affinities vary depending on the F A protein interactions in question. For example, F A N C A binds very strongly to F A N C G while F A N C C binds with lower affinity to F A N C A , and this is strongest in the presence of F A N C G . These observations suggest that in normal cells, the F A proteins interact sequentially in a linear pathway within the cytoplasm forming an initial complex that then translocates into the nucleus where it becomes part of a larger complex. It is important to note that: a) an intact F A complex is found only in the nucleus and not in the cytoplasm; b) complex formation does not occur in response to D N A damage or to the cell cycle 3 9 and; c) the formation of an intact F A complex is disrupted in cells from all F A complementation groups except F A - D 3 9 . 5 A recent study has shown that the F A complex has been found specifically within the nuclear matrix and in condensed chromatin and when F A N C G becomes phosphorylated the complex exits the chromatin sites 4 0 . Both F A N C A and most recently, F A N C G have been identified as phosphoproteins, however, the stimulus for the phosphorylation events, and the kinase(s) remain unidentified. In mutant F A cell lines (except FANCD2), F A N C A is not phosphorylated, suggesting that phosphorylation may be important for the assembly of the F A proteins or for the proper function of the complex 4 1 . The phosphorylation of F A N C A was shown to be regulated by a cytoplasmic serine protein kinase 4 1 , later identified as A k t 4 2 . Once F A N C A becomes phosphorylated, the F A proteins move into the nucleus, presumably via the NLS of F A N C A . A mutation within the phosphorylation domain or in the NLS region of F A N C A prevents nuclear inclusion of the complex and does not correct M M C sensitivity of F A - A cells 4 1 . F A N C B cells contain only cytoplasmic F A N C A protein, suggesting that the F A N C B protein is needed for F A N C A and/or F A complex nuclear localization. FANCD1 cells have an intact F A complex and a monoubiquitinated form of FANCD2, therefore it is believed to act downstream of the FANCD2 protein. In F A complementation groups A , B , C, E, F and G, the formation of the complex is disrupted, thus all proteins are required for the proper assembly and translocation of the nuclear complex. However, in FANCD2 mutant cells, the F A complex is intact suggesting that the FANCD2 protein functions downstream of complex formation and is not required for the assembly or stability of the complex (Fig. 2) 4 3 . Taken together all of these studies support the hypothesis that the F A proteins sense a signal in the cytoplasm and are eventually translocated into the nucleus in order to maintain chromosomal integrity. Several key questions remain, such as what is the stimulus that precipitates F A complex formation, what is the eventual downstream effect of nuclear inclusion and does the F A complex have a direct or indirect role in damage repair? 6 Role of the FA Proteins FA and DNA Repair While it is quite clear that the FA complex forms and translocates into the nucleus in response to a cellular signal, the exact molecular role for the FA proteins is still actively debated. Two major theories exist in FA research: one hypothesis proposes that the FA proteins constitute a DNA damage recognition/repair pathway, whose impairment is manifested by chromosomal instability and increased sensitivity to inter-strand DNA cross linking agents such as MMC, DEB and cisplatins 4 4 . While some FA cell lines (primarily FANCD2) have been shown to have modest sensitivities to ionizing radiation (IR) as well 3 9 , it is worthy to note that FA cells are not particularly sensitive to ultra-violet (UV) or to monofunctional alkylating agents such as ethyl methane sulfonate (EMS) or methyl methane sulfonate (MMS) 4 5 . Based on these sensitivity studies, the primary defect in FA cells involves the removal of DNA inter-strand cross-links. The mechanism for the removal of DNA inter-strand cross-links from mammalian cells is not well understood, however in lower eukaryotes and bacteria these lesions are removed by homologous recombination (HR) or non-homologous end-joining (NHEJ) repair pathways 3 9 . Defects in both of these repair mechanisms have been described for FA cells. HR frequencies and activity were found to be increased in nuclear extracts from primary FA fibroblasts 4 6 , while the fidelity of end-joining of specific DSB was lost in FA cells 4 7 . Mutation analyses done on FA lymphoblasts found hypo-mutability at the HPRT locus, however the spectrum of mutations revealed a significant number of deletions 4 8 . Deletion events represent up to 65% of spontaneous mutations occurring at the HPRT locus in FA lymphoblasts of the D2 complementation group compared to 18% in wildtype cells 4 9 . A heptamer motif exists at the 3' breakpoint of these deletions and suggests that site-directed activity may be involved. These reults suggest that the FA proteins may have a role in the repair of DSB through HR or NHEJ mechanisms. 7 A major step was made in support of a role for F A proteins in D N A repair when the F A N C D 2 gene was cloned 1 1 . Subsequent functional studies showed that the F A complex is required in the nucleus for the ubiquitination of the F A N C D 2 protein, shown to interact with the BRCA1 D N A repair pathway 4 3 . FANCD2 is exclusively nuclear in location and is present in wildtype cells in two isoforms, FANCD2-short and FANCD2-long. The long isoform is monoubiquitnated and this form is absent in cells from F A complementation groups A , B , C. E, F and G 4 3 . Interestingly, these complementation groups do not have an intact F A nuclear complex and therefore, it is assumed that the F A complex has a role, direct or indirect, in either the addition of ubiquitin groups to F A N C D 2 or the recruitment of a ubiquitin ligase to perform this task 3 9 . The ubiquitination of F A N C D 2 occurs in response to D N A inter-strand cross-linkers such as M M C and immunofluorescence studies have shown that FANCD2 can be found at nuclear foci in conjunction with BRCA1 when cells are challenged with these agents 4 3 . Nuclear foci are formed by other D N A repair proteins upon induction of D N A inter-strand croslinks, including B R C A 1 , B R C A 2 and Rad51. Using the novel technique of micro-irradiation of human fibroblast cells and fluorescent markers for various repair proteins, F A N C D 2 has been shown to directly associate with damaged D N A , in particular double strand breaks (DSB) and proteins involved in genetic recombination repair 5 0 . This important study was the first to show the F A proteins interacting with either damaged D N A or other repair proteins in vivo, and that F A N C D 2 does associate with BRCA1 and B L M (components of recombination repair). However, this association is not perfect since nuclear DSB foci can be found without the F A N C D 2 protein present even though B R C A 1 and B L M are there 5 0 , thus BRCA1 and F A N C D 2 do not always co-localize. This study also showed that the localization of F A N C D 2 with DSB does require F A N C A , presumably for the proper ubiquitination of the FANCD2 protein. FA and Redox Regulation The other hypothesis for the role of the F A proteins suggests that F A might result, at least in part, from an abnormal regulation of cellular redox state and/or of the cellular response to oxidative stress. For example, increased production of ROS by F A cells, such as leukocytes and fibroblasts, has also been reported, suggesting that F A proteins might regulate the generation of these species 5 1 ' 5 2 . Furthermore, reduction of M M C in F A cells has been shown to lead to the production of reactive oxygen species (ROS) that indirectly may generate cross-links and other types of oxidative lesions 5 3 . Many studies have presented evidence in defense of this hypothesis. The addition of cellular antioxidants, such as superoxide dismutases (SOD) to the culture medium of F A cells was reported to attenuate chromosomal breakage as well as M M C cytotoxicity 5 4 ' 5 5, an effect also observed in F A cells over-expressing thioredoxin, another cellular antioxidant56. SOD administration has been used in pilot studies as a potential therapeutic agent for F A B M recovery with apparently promising results 5 7 . To further support the redox dysregulation hypothesis, some of the F A proteins have been shown to interact with various non-FA cytoplasmic and nuclear proteins involved in regulating oxidant stress. These protein interactions have also indicated that F A proteins may have numerous additional roles within the cell besides D N A repair, and might conceivably explain the fact that multiple congenital defects can occur in a patient with FA. Specifically, F A N C C is known to interact with the molecular chaperone GRP94 5 8 , N A D P H cytochrome P-450 reductase 5 9 , the zinc-finger F A Z F 6 0 and most recently with the Phase II detoxification enzyme, GSTP1 6 1 . The N A D P H cytochrome P-450 reductase (RED) system is a potential source of endogenous superoxide. The cytochrome P-450 enzymes are a superfamily of heme-containing proteins that are expressed in all tissues and catalyze the oxidation of many endogenous and xenobiotic chemicals 5 2 . P-450 metabolism usually requires two protein components, P-450 and RED. These enzymes are embedded in the microsomal membrane and RED is responsible for shuttling electrons to P-450 from N A D P H 6 2 . Not only were chromosomal breaks in F A cells 9 reduced to background levels by cytochrome P-450 inhibition 6 3 , but evidence of a direct physical interaction between F A N C C and R E D was reported, where F A N C C modulated the activity of R E D 5 9 . These results led to the hypothesis that F A N C C might protect cells from R O S via regulation of R E D activity. Interestingly, the cytochrome P-450 system has also been implicated in association with F A N C G . C Y P 2 E 1 is a member of the P-450 superfamily responsible for the production of R O S , the bioactivation of carcinogens, and the removal of lipid-peroxidation products 6 2 . Constituitively elevated levels of C Y P 2 E 1 protein were found in F A - G lymphoblast lines which was reduced when the cells were complemented with wildtype FANCG. This study also showed that there was a dose dependent increase in oxidized D N A bases in mutant F A - G lines post M M C treatment M . F A N C C also interacts with glutathione S-transferase P l - 1 (GSTP1) , a phase II detoxification enzyme responsible for the reduction of glutathione (GSH) and various xenobiotics 6 1 . G S T P 1 and G S H are required by the cell for the detoxification of exogenous and endogenous electrophilic compounds and in doing so prevent protein oxidation overload. R O S can damage proteins through the oxidation of sulfhydryl groups which results in the formation of disulfide bonds and the eventual inactivation of the protein 6 5 . The study by Cumming et al. revealed that F A N C C prevents the formation of these inactivating disulfide bonds within G S T P 1 . Thus, F A N C C indirectly regulates cellular oxidant stress by maintaining an active form of G S T P 1 , which in turn functions to detoxify xenobiotics and reduce G S H proteins. The overexpression of F A N C C maintains G S T P 1 , and thus indirectly, G S H in an oxidized or 'open' state, preventing apoptosis in hematopoietic cells following growth factor deprivation 6 1 . Surprisingly, F A N C C lacks homology with any conventional disulfide reductases (there is no active - site motif C - X - X -C ) , however other proteins have been described with reductase function that also lack conventional active - site mo t i f s 6 1 . F A N C C may be part of an undiscovered subset of disulfide reductases within the cytoplasm and may have a role in the general regulation of redox sensitive proteins. The lack of F A N C C in F A - C cells may result in the oxidation of an increased number 10 of protective cytoplasmic proteins which in turn cannot function properly to clear oxidative stress, leading to an increased susceptibility to apoptosis. The observation that F A proteins may have potential functions outside of the F A complex is not specific to F A N C C . Yeast two-hybrid analysis using F A N C A as a bait revealed a novel interaction between F A N C A and brm - related gene 1 (BRG1) product66'. BRG1 is a component of the SWI/SNF complex which is involved in chromatin remodelling. BRG1 and F A N C A have been shown to co-localise in the nucleus of the cell, however chromatin re-modelling is normal in F A - A cel ls 6 6 , suggesting that F A N C A may have a role in opening chromatin at specific sites for the transcription of specific genes. The F A N C A protein may also have roles in redox regulation since amino acid residues located within the N-terminal region of F A N C A have been identified that are homologous to heme peroxidases 6 7 . Mutations of highly conserved sites within the putative peroxidase domain of F A N C A abolished the ability of F A N C A (mut) to correct the M M C sensitivity of F A - A cells, however this mutant did not compromise either the stability of the F A N C A protein or the interaction of F A N C A with F A N C G 6 8 . Animal Models Mice have been generated with targeted mutations of Fancc, Fanca, Fancg and Fancdl 69' 7 4 . With the exception of Fancdl, all mouse models have similar phenotypes, demonstrating compromised gametogenesis, and an increase in the number of chromosomal aberrations, both spontaneously and following exposure of cultured embryonic fibroblasts and primary splenocytes to M M C . The targeted strains do not spontaneously display developmental or hematological defects typical of human FA, and do not have a predisposition to malignancy (for a comprehensive list of F A mouse models see Table III). Peripheral blood counts of F A knockout mice remain normal over time and there is no significant difference in committed progenitor growth over time in most knockout mice. Two Fancc - null murine strains have been developed with slightly 11 different targeting mutations, disruption in exon 8 6 9 and disruption in exon 9 of Fancc ™. Both Fancc'' strains undergo progressive hematopoietic failure and eventual death (within 3-8 weeks) when M M C is administered in vivo 7 5 and when challenged with inhibitory cytokines in vitro 16. Interestingly, only the Fancc'' mice generated by Whitney et al. develop age-dependent decreases in the number of committed progenitors from the B M , suggesting that a disruption of exon 9 leads to a more severe phenotype. While Fancg'' mice have the same phenotypes as the Fanca and Fancc null mice, preliminary studies of Fancg'' mice reveal that the cellular sensitivities to M M S , U V and X-rays observed in the C H O cell lines have not been duplicated from embryonic fibroblasts isolated from these mice 7 3 . The phenotype of Fancdl mutant mice is slightly more severe than the phenotype of the remaining F A mouse models. Gonads of both sexes show significantly decreased numbers of germ cells over wildtype, Fanca'' and Fancc'' mice 7 4 . Moreover, embryonic fibroblasts from Fancd2~'' mice have increased sensitivity to cross-linkers as well as a modest sensitivity to IR in vitro that becomes significantly more pronounced in vivo 1A. This difference is in keeping with the fact that the FANCD2 protein product has a role in the cell that seems to be quite different from the other F A proteins. However, despite this difference, even Fancdl'' mice do not demonstrate spontaneous B M aplasia. The lack of an FA- l ike B M failure phenotype in mutant mice is surprising since all murine FA c D N A s are able to fully complement the M M C sensitivity in human lymphoblast cells, suggesting that the proteins are able to function properly in vitro. The reason(s) for this interspecies difference is unknown, but it has limited the utility of the mutant mice as potential models of F A . A stressful insult or an affected modifier gene may be required in order to uncover an FA- l ike hematological defect in these mice. Thus, double mutant mice have been generated to better understand the role of the F A proteins in human B M failure. T o date, three 'double mutant' models have been described in the literature. Since T N F a is known to be elevated in the serum of F A patients and is known to prime the fas pathway, Fancc'' mice were generated to overexpress human T N F a ( F N T mice) and the effect on hematopoiesis was examined 7 7 . F N T mice had 12 normal peripheral C B C and marrow cellularity, and while whole marrow from FNT mice had increased levels of CD95 + cells, myeloid progenitors assayed in methylcellulose cultures were not spontaneously sensitive to the in vivo effects of overexpressed TNFa. Erythroid progenitors from FNT mice did however show decreased growth in methycellulose cultures both spontaneously and upon treatment with fas ligand 1 1 . Mice with targeted mutations in different F A genes have also been generated. Not surprisingly, Fanca/Fancg double mutant mice have a phenotype that is identical to the single knockout mice, reduced fertility with no hematological defects or tumor development 7 8 . The overexpression of human F A N C C has been shown to protect murine hematopoietic progenitor cells (HPC) from Fas-mediated apoptosis 7 9 . Committed progenitors from these mice were resistant to the killing effects of fas in methylcellulose assays and Lin" HPC treated with/as had significantly less apoptosis compared to littermate controls 7 9 . These reults indicated that F A N C C was required for the proper protection of progenitor cells from/as-induced apoptosis. Bone Marrow Failure in F A Despite the fact that the F A genes are expressed in most tissues, the B M is the only organ that consistently fails in F A patients. Thus, there is a preferential defect within the B M compartment of F A patients that leads to aplasia and eventual death. Studies of B M aspirates from F A patients indicate that there is a dramatic reduction (up to a 15-fold decrease) in the frequency of committed hematopoietic progenitors from both the erythroid (BFU-E) and the myeloid (CFU-GM) lineages of F A patients. Occasionally, committed progenitors from' F A patients do not grow in vitro at all, even when cultured in the presence of SCF 8 0 ' 8 1 . This reduction is seen in pancytopenic as well as asymptomatic (non-anemic) F A patients 8 2 ' 8 3 . Interestingly, the colonies enumerated in the above named studies are consistently decreased in 13 size as well as frequency. These results indicate that there is a defect in the proliferation of lineage constricted progenitors. Long - term B M cultures (LTC) derived from F A patients reveal that hematopoiesis is impaired at the multipotent progenitor level as well. Frequencies of early L T C progenitors from F A B M samples in vitro have not been directly determined, rather C F U - G M was determined from the non-adherent nucleated cells in L T C . Two independent studies indicate that C F U - G M from L T C cultures were markedly decreased or null from F A patients 8 0 ' 8 4 and while adherent stromal layers consistently formed from F A B M samples, they took significantly longer to achieve confluency than normal controls 8 0 . These studies indicated that while progenitors exist in the marrow of F A patients, their proliferative potential is affected, or they cannot respond properly to growth factor stimulation. In addition to defective progenitors, it is possible that there is also a defect in the proper production of cytokines from F A cells, although the data has been somewhat conflicting and dependent on the cell type analyzed. F A lymphocyte cell lines revealed a consistent increase in T N F a production 8 5 and decreased production of IL-6 8 6 . When these cell lines were treated with anti-TNFa antibodies or supplemented with IL-6, their M M C sensitivity was corrected. These results are in keeping with the observation that both T N F a and flt3 ligand are elevated in the serum of F A patients 8 7 ' 8 8 . In contrast to the these results, L T C cultures from primary F A B M samples reveal a significant decrease in T N F a and DLip production over a 4 week time period in vitro and no difference in IL-6 production 8 9 . The above data all suggest that loss of F A protein function results in quantitative or qualitative injury to hematopoietic progenitor cells or to the progenitor environment. In keeping with this hypothesis, a specific role for F A N C C in the survival and/or proliferation of HPC has been established in both murine and human cells 9 0 . Committed progenitor cells from Fancc'' animals were found to be less responsive to stem cell factor (SCF) stimulation, suggesting that 14 the lack of Fancc may lead to an inability to respond to growth factor stimulation 9 1 . Furthermore, in addition to initial reports which showed that Fancc'' HPC cells were 75 70 76 / hypersensitive to M M C and to inhibitory cytokines ' , Fancc" mice were also used in competitive repopulation assays to determine the role of Fancc in short and long-term reconstitution of the B M compartment. Competitive repopulation assays involve the co-transplantation of test cells (Fancc'') with congenic competitor (wildtype) cells into irradiated mice and reconstitution of the marrow is measured. These experiments showed that Fancc'' cells had 7-12 fold decreased repopulation ability compared to wildtype controls 9 2 . Despite these results, no hematologic malignancy has ever been observed in Fancc'' mice, perhaps owing to the lack of a sufficient insult to exacerbate the phenotype. The observation that Fancc'' progenitor cells are hypersensitive to low doses of the inhibitory cytokines IFNy, TNFa , and M l P l a 7 0 , 7 6 suggested that these cytokines may be mediating apoptotic responses in F A N C C progenitor cells. In fact, progenitors from F A N C C patients were shown to induce fas and interferon response factor 1 (IRF-1) gene expression at significantly lower doses of JFNy compared to normal controls 9 3 . An increase in CD95 (fas receptor) expression was also found on CD34 + hematopoietic progenitors from Fancc'' mice 7 7 . These apoptotic responses were found to be mediated by caspase 8-dependent activation of caspase 3 family members 9 4 . The suppressive effects of fFNy and T N F a on clonal growth of Fancc'' B M cells in vitro can be augmented with the addition of agonistic anti-fas antibodies, and the reverse is also true, neutralizing anti-fas antibodies abolish the inhibitory effects of these cytokines on clonogenic growth 7 7 ' 9 3 . Similarly, caspase 3 inhibitors can rescue IFNy - mediated committed progenitor growth from Fancc'' B M cells and human F A - C CD34 + cel ls . 9 4 Although F A N C C cells are known to be hypersensitive to the effects of IFNY, paradoxically, the activation of STAT1 in response to IFNY m EBV-transformed F A N C C lymphoblast cell lines appears to be suppressed 9 1 . It was later reported that this STAT1 15 abnormality is dependent on the type of mutation that occurs within the F A N C C gene y 3 . Furthermore, Fancc'' mouse embryonic fibroblasts were found to have increased levels of active RNA-dependent protein kinase (PKR) after treatment with dsRNA and I F N Y . When catalytically inactive forms of the P K R were overexpressed in Fancc'' embryonic fibroblasts, the cells were protected from apoptosis 9 6 . This provided a mechanism through which the apoptotic response could be mediated. 16 1.2 THESIS GOALS The main goals of this thesis were: 1) To generate, through breeding protocols, a novel model of F A that has an in vivo pro-oxidant state. Using this genetic model of F A (Fancc and Sodl double mutant animals), we investigated the role of intrinsically elevated superoxide anion on the pathogenesis of the disease, specifically on hematopoietic failure. 2) To determine the potential role of the Fanca protein in the regulation of cellular redox potential through the generation of Fanca and Sodl double mutant mice. The phenotype of these mice was compared to the Fancc and Sodl double mutants to examine if ROS-mediated F A pathogenesis is a Fancc-specific effect of a more general F A defect. 3) To investigate the potential role of nitric oxide in the response of Fancc'' B M cells to growth - inhibitory cytokines. 17 CHAPTER TWO - Generation and Analysis of Mice with Combined Deficiencies of the Genes Encoding Fanconi Anemia Complementation Group C (Fancc) and Cu/Zn Superoxide Dismutase (Sodl). 2.1 INTRODUCTION A number of hypotheses regarding the nature of the primary defect in F A have been suggested, including the proposal that F A proteins constitute a D N A damage recognition and signaling pathway, whose impairment is manifested by chromosomal instability and increased sensitivity to interstrand D N A cross linking agents 4 4 . While a reduced ability to process D N A cross-links is clearly evident, it has also been proposed that an abnormal reduction of M M C in F A cells leads to the production of reactive species that in turn generate cross-links and other types of oxidative lesions 5 3 . Thus, F A might also result, at least in part, from an abnormal regulation of cell redox state and/or of the cellular response to oxidative stress. In support of this notion, SOD1 addition to the culture medium of F A cells was reported to attenuate chromosomal breakage as well as M M C cytotoxicity 5 4 ' 5 5 , an effect also observed in F A cells over-expressing thioredoxin 5 6 . In keeping with an inability to regulate either production, or the consequences of ROS, some F A cells have been shown to be hypersensitive to oxygen 1 . Thus, cells grew slowly at elevated oxygen levels (e.g. 35%) and tended to arrest at G 2 , while at low oxygen concentrations (e.g. < 5%), growth was normal and accompanied by decreased chromosomal aberrations 7"9. Increased production of ROS by F A cells, such as leukocytes and fibroblasts, has also been reported, suggesting that F A N C C might regulate the generation of these species 5 1 ' 5 2 . Furthermore, a high level of 8-hydroxy-2-deoxyguanine residues have been detected in the D N A of F A cells which is a marker of oxidative D N A damage and is thought to reflect oxidative stress in F A cells 5 2 . A potential endogenous source of superoxide, the N A D P H cytochrome P-450 reductase (RED) system, has also been implicated in FA. Chromosomal breaks in F A cells were reduced by cytochrome P-450 inhibition, and a direct physical interaction between F A N C C and RED has been 18 reported 5 9 ' 6 3 , leading to the hypothesis that F A N C C might protect cells from ROS via regulation of RED activity. Furthermore, a recent paper describes a role for F A N C C in the redox regulation of GSTP1, a Phase II detoxification enzyme 6 1 . The overexpression of F A N C C and GSTP1 results in enhanced survival of a growth factor dependent hematopoietic cell line. Aerobic organisms generate energy through the conversion of molecular oxygen (O2) to H 2 0 and A T P via the process of oxidative phosphorylation. It has been estimated that approximately 2-4% of the oxygen used during mitochondrial respiration results in aberrant production of superoxide anion (02~*), one of several reactive forms of oxygen (ROS) (Fig. 3 for a list of reactive species) 9 1 . ROS, are free radicals associated with the oxygen atom, some of which have strong reactivity with other molecules 9 1 . In addition to respiration, ROS are also generated in the cytosol and extracellular spaces and their presence can be both beneficial as well as harmful to the cell. For example, macrophages use the respiratory burst oxidase to generate 02~* within cellular membranes to kill microorganisms and many ROS are critical components of cellular signal transduction pathways 9 8 , 9 9 . However a balance must exist since excessive production of ROS is also capable of causing cellular damage to protein, lipids and D N A , which can ultimately lead to mutagenesis and cell death 9 1 . In order to maintain an equilibrium and to combat toxic 0 2~\ organisms have evolved three forms of superoxide dismutase as an antioxidant defense, cytosolic Cu/Zn SOD (Sodl), mitochondrial MnSOD (Sod2), and extracellular E C SOD (Sod3) 1 0 ° . SODs are homodimeric metalloenzymes that catalyze the dismutation of 0 2 * to hydrogen peroxide (H 2 0 2 ) and 0 2 1 0 ° . These enzymes are ubiquitously expressed and their importance is highlighted by their appearance in all aerobic organisms. Organisms genetically engineered to be null for one of the SOD enzymes have defined phenotypes. Bacteria and yeast the SOD1 gene have severe growth defects under an aerobic environment 1 0 1 " 1 0 3 while Drosophila have severely shortened lifespans and are very sensitive to paraquat and radiation 1 0 4 1 0 5 . Mice lacking MnSOD die within 2 weeks 19 of birth due to cardiomyopathy, degenerative brain neuron injury and severe mitochondrial j 106,107 damage Sodl constitutes the major portion of total Sod activity within most tissues and is considered to be the primary CV* scavenger within the cytosol. Surprisingly however, mice with a targeted disruption of the gene encoding the cytosolic Cu/Zn SOD (Sodl) exhibit normal growth and development, age at similar rates as wildtype mice and do not have an increase in the expression of secondary antioxidant genes 1 0 8 1 1 0 . However, Sodl'' mice do show a distinctive motor axonopathy 1 0 8 , 1 u , as well as impaired gametogenesis 1 0 9 . The limited spontaneous pathology of Sodl'' mice suggested that while this enzyme might function to modulate superoxide-mediated effects in some tissues under basal conditions, that it was of critical importance during exposures to specific pro-oxidant stimuli. In keeping with this, Sodl'' embryonic fibroblasts exposed to the superoxide-generating herbicide, paraquat, exhibited a pronounced sensitivity compared to both Sodl+/+ and Sod2+/' controls 1 1 2 ' 1 1 3 . Other studies have shown a clear role for dominant mutations of Sodl in familial amyotrophic lateral sclerosis (FALS) 1 1 4 , as well as other neurodegenerative diseases, aging and cancer " 5 . We hypothesized that a lack of Sodl might reveal a role for alterations in redox state with respect to the development of an FA-like syndrome in Fancc deficient mice. To examine this possibility, we generated mice with combined deficiencies of both the Fancc and Sodl genes. Fancc'1'Sodl'' mice developed not only a novel liver pathology, but also demonstrated B M hypocellularity and peripheral blood bicytopenia (RBC and leukocytes) accompanied by a profound reduction of the clonogenic growth of hematopoietic precursors in vitro, that persisted in aged (6-8 month) Fancc''Sodl'' mice. 20 2.2 MATERIALS AND METHODS Generation of Fancc''Sodl'' mice and histological analysis Fancc+/' mice 6 9 were crossed with Sodl+/' mice 1 0 8 until mice that were heterozygous at both loci were obtained (three generations of matings). Locus-specific PCR was used to genotype mice. Brother-sister matings of Fancc+/~ Sodl+/' mice were carried out to produce litters having Fancc''Sodl'' mice. Mice, 8-10 weeks of age, were of a mixed genetic background and thus littermate controls were used in all experiments. Viral antibody-free mice were housed in the Center for Molecular Medicine and Therapeutics barrier facility according to protocols approved by the Animal Care Committee at the University of British Columbia. For light microscopy, tissue samples were either frozen, or fixed in 4% paraformaldehyde solution and embedded in paraffin and bone sections were first de-calcified before processing. For paraffin-embedded sections; hematoxylin and eosin (H&E); periodic acid-Schiff (PAS) with and without diastase; and Masson's trichrome were used. Frozen sections were stained with oil-red-0 (ORO). For electron microscopy, liver blocks were fixed in cold 3% gluteraldehyde and stored at 4 °C. Samples were rinsed twice in Millonig's buffer (pH 7.4) and were subsequently fixed in 1% osmium tetroxide in Palade's solution for 1.5 hours at 4 °C. Samples were stained en bloc for 15 minutes with 2 % aqueous uranyl acetate and then dehydrated before embedding. Sections were examined with a Phillips 400 electron microscope. Blood collection, serum ALT measurement and peripheral blood counts Following avertin overdose, blood was obtained by cardiac puncture, and either allowed to clot at room temperature, and/or added to microtainer tubes, pre-treated with EDTA, for blood counts. Clotted blood was centrifuged at 14,000 rpm for 5 min, serum was removed and frozen at -80 °C. Serum alanine aminotransferase (ALT) levels were determined using a Beckman 21 Synchron CX7. Peripheral blood (PB) counts were performed using a Sysmex 9500 analyzer. RBC, W B C , PLT, hemoglobin (HGB) and mean cell volume (MCV) values were determined. Tissue isolation and BM cell Preparation Mice were sacrificed at 8-10 weeks of age by intraperitoneal (ip) injection of 4% avertin (0.01 ml/g). Samples were harvested from the same region of the liver in all mice. Single-cell suspensions were prepared by pressing samples through a wire mesh into cold serum-free RPMI 1640 (Life Technologies). Cells were then passed through a 40 p nylon filter to remove clumps and debris. Liver cells were pelleted at 1500 rpm, and resuspended in cold RPMI. Total B M cells were collected by flushing femurs from mice with cold Hank's Balanced Salt Solution containing 5% FCS. Cell viability, > 90% in all samples, was determined by trypan blue exclusion. Superoxide quantitation Isolated liver cells were resuspended, in triplicate, at a density of 5.0 x 105/ml in serum-free RPMI and centrifuged at 1500 rpm, before being resuspended in 100 pi Superoxide Assay Medium (Calbiochem, San Diego, CA). Each culture was then placed into a well of an opaque 96-well polystyrene flat-bottomed microtiter plate (VWR Canlab) kept on ice until analysis. Then, 5.0 pi of 4.0 m M luminol solution (Calbiochem, San Diego, CA), diluted in 95 pi of Superoxide Assay Medium, was added simultaneously to all samples. Chemiluminescence was measured at one minute after luminol addition using a M L X Microtiter Plate, Luminometer (Dynex Technologies, Chantilly, V A ) . The average intensity of the triplicates was recorded as Relative Light Units (RLU). Purified SOD (Calbiochem, San Diego, CA) was added to the cells as a specificity control to show that chemiluminescence was due to superoxide anion. 22 Immunoblotting and densitometry Flash-frozen liver samples were lysed in Nonidet P-40 lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 m M Tris (pH 7.5) and 10% glycerol) in the presence of multiple protease inhibitors (Boehringer Mannheim, Indianapolis, IN and B D H , Toronto, Canada). Lysates were centrifuged for 15 min at 14,000 rpm. Liver protein concentration was determined by Bradford-method-based assay. Lysate volume corresponding to 250 pg of total protein was diluted 3:1 with Laemmli sample buffer. Samples were boiled for 5 min prior to electrophoresis. Total cell lysates were separated by SDS-PAGE at 150V and transferred to nitrocellulose paper by electroblotting at 100 V for 1 hr at RT in a solution containing 192 mM glycine, 25 m M Tris and 20% methanol. Filters were blocked overnight at 4 °C in TBST (10 mM Tris (pH8.0), 150 m M NaCl and 0.05% Tween-20) containing 5% BSA. Filters were then incubated for 60 min at room temperature in TBST with 1% B S A with one of the following monoclonal antibodies (StressGen Biotechnologies, Victoria, BC): anti-MnSOD (1/5000), anti-HO-1 (1/2000), or anti-3-tubulin (1/250). After three TBST washes, filters were incubated for 1 h with a horseradish peroxidase-conjugated secondary antibody (Dako Diagnostics, Mississauga, ON). Proteins were detected by chemiluminescence (Amersham, Arlington Heights, EL) using Biomax MR. film (Eastman Kodak, Rochester, NY) . Densitometry was performed using a GS300 reader (Hoefer Scientific Instruments, San Francisco, CA), and results were analyzed using the GS370 1-D Data System, version 2.0 for Macintosh. Flow cytometery A total of 1 x 106 cells was resuspended in 500 pL PBS + 2% FCS (FACS buffer), blocked on ice with 1 pg of anti-FcYRHb (2.4G2, Pharmingen, Mississauga, ON) for 20 min, and then stained with either 0.5 pg anti-CDllb-FITC (for liver samples) or one of the following FITC-conjugated antibodies for 30 min on ice ( B M cells); PGP1; B220; Ly6G (Gr-1); 7-4 (PMN); C D l l b ; CD14 and TER-119; and (primitive populations); Seal, ckit, CD34 (Pharmingen, 23 Mississauga, ON). Cells were washed 3x with FACS Buffer and resuspended in 500 pL of FACS buffer before analysis on a FACSort (Becton Dickinson, Mountain View, CA) flow cytometer equipped with CellQuest software (Becton Dickinson). The viable cells that remained unstained represented hepatocytes, while the CDllb+ population included Kupffer cells and contaminating peripheral blood phagocytes. For BM samples, the percent staining was multiplied by the total cellularity (obtained from one femur) to determine the absolute number of each cell type. Chromosome analysis For Fancc+/+Sodl+/+, Fancc+/'Sodl+/' and Fancc SodY'' marrows, an aliquot of RPMI + 5% FCS containing 1.5 x 106 resuspended BM cells was added to a tube containing 1 ml Trypsin-EDTA (Irvine Scientific) and 0.75 M KC1. The tubes were incubated at 37 °C for 25 min, spun for 10 min at 1000 rpm and the pellet carefully resuspended in Carnoy's fixative (3 parts methanol: 1 part glacial acetic acid). The fixative was changed two more times and the slides made by air-drying. Approximately 10 metaphases per sample were examined for evidence of chromosomal breaks, gaps or detectable rearrangements. Methylcellulose colony forming assays and lineage depletion of total BM cells Whole BM cells were plated in 1.1 ml of 1% methylcellulose media supplemented with 10% FCS, 2 mM L-glutamine, 10"4 M 2-mercaptoethanol and the following recombinant growth factors: for myeloid assays, methylcellulose was supplemented with 1% bovine serum albumin, 10 pg/ml bovine pancreatic insulin, 200 pg/ml human transferrin, 3 units/ml recombinant human erythropoietin, 10 ng/ml recombinant mouse IL-3, 10 ng/ml recombinant human interleukin-6 and 50 ng/ml recombinant mouse stem cell factor (SCF). For Pre-B assays 10 ng/ml recombinant human IL-7 was used (StemCell Technologies, Vancouver, BC). Cells were dispensed using a blunt-ended needle and cultured at a density of 1.7 x 105 and 5.5 x 104 cells per 35 mm dish for 24 Pre-B and Myeloid colonies respectively (each sample done in duplicate). Dishes were incubated for 6 (for Pre-B) or 12 (for myeloid) days at 37 °C, 5 % C 0 2 in air, > 95 % humidity. Colonies (> 20 cells) were counted on a gridded stage using an inverted light microscope. Lineage depleted samples were collected by resuspending the cells at 5.0 x 107 nucleated cells/ml in PBS with 2% FBS, plus 5% rat serum for 15 min at 4 °C. Samples were first incubated with an antibody cocktail (CD5, C D l l b , CD45R, GR1, 7-4 and TER-119) and subsequently with an anti-biotin tetrameric antibody (both antibody cocktails from StemCell Technologies, Vancouver, BC) complex (each step for 15 min at 4 °C), then a magnetic colloid was added for cell separation as recommended (StemCell Technologies, Vancouver, BC). To isolate Lin" populations, the suspension was applied to a primed 0.3 inch magnetic column and washed 3x with PBS containing 2% FBS. The cells in the flow-through were enumerated and trypan blue exclusion used to determine viability (> 95%). Statistical Methods The Student's t test (Microsoft Excel) was used when analyzing the results, p < 0.05 was considered significant. 2.3 RESULTS Fancc'Sodl'' mice develop zonal micro vesicular hepatic steatosis No developmental defects or gross skeletal abnormalities were seen in Fancc'Sodl'' mice. Body weights of Fancc'', Sodl'~, Fancc+'"Sodl+/', Fancc+/+Sodl+/+ and Fancc'Sodl''mice, both male and female, were not statistically different from one another (EigS4). Liver and spleen weights were not increased in any of the mutants as compared to Fancc+/+Sodl+/+ controls. However, necroscopy and histological analysis of Fancc'Sodl'' mice revealed abnormalities of the liver and B M . 25 On inspection, livers of Fancc''Sodl'' mice (n=6) were pale and exhibited a yellow reticular surface pattern. Liver sections were examined by light and electron microscopy, with a typical sample shown in Fig. 5. Liver sections from a, e) Fancc'', b, f) Sodl'', c, g) Fancc*'' Sodl*'' and d, h) Fancc'Sodl''mice were stained with Masson's trichrome (a-d). While periportal (zones 1 and 2) hepatocytes were unremarkable, zone 3 cells were distended by numerous PAS negative (data not shown) cytoplasmic vacuoles that did not displace the nuclei. No inflammatory cell infiltrates were present in the liver, and trichrome stain did not reveal evidence of hepatic fibrosis or increased collagen deposition. Oil red O staining (Fig. 5 e-h) demonstrated microvesicular steatosis (Fig. Ih) in zone 3 hepatocytes of Fancc''Sodl'' mice. Fancc'' mice (Fig. 5e) revealed no increase in lipid staining over controls, while Sodl'' mice (Fig. 5f) revealed modest amounts of oil red O-positive droplets distributed in a non-zonal pattern. Transmission electron microscopy (EM), performed on Fancc*''Sodl*'' (Fig. 6, a,c), and Fancc''Sodl'' (Fig. 6, b,d) liver samples, revealed no morphological abnormalities of Kupffer cells or endothelial sinusoidal cells, and aside from the obvious lipid fdled vacuoles, the structure of hepatocyte smooth endoplasmic reticulum and mitochondria was unremarkable (Fig. 6 c,d). To search for evidence of hepatocyte injury, serum alanine aminotransferase (ALT) and T U N E L assays were employed. A L T levels were as follows: Sodl'' (55.3 ± 8.67), Fancc'' (45.5 ± 2.87), Fancc*'*Sodl*'* (41.3 ± 7.98), Fancc*''Sodl*'' (22.7 ± 4.2) and Fancc''Sodl'' (126.8 ± 52.0). While serum A L T from Fancc''Sodl'' mice was increased (~3-fold) over littermate controls, suggesting that low levels of hepatocyte damage may have been present, this data is not statistically significant (p=0.12). T U N E L assay of liver sections revealed no difference with respect to the rare apoptotic cells seen when Fancc''Sodl'' and littermate mice were compared. Primary Fancc''Sodl'' liver cell cultures generate increased levels of superoxide To determine whether oxidant stress was present in Fancc''Sodl'' livers, we assayed spontaneous superoxide production from primary liver cell cultures. Luminol, which undergoes 26 chemiluminescence when oxidized by superoxide, enabled quantitation of the relative amounts of this species. The average intensity of the samples was recorded as relative light units (RLU), with the R L U values being proportional to the level of superoxide in the samples. Fig. 7 shows the average R L U values for 5 mice per group, each mouse sample assayed in triplicate, taken immediately after luminol addition. In all samples, the luminol signal was ablated when SOD protein was added to the culture medium. Fancc+/+Sodl+/+, Fancc'' and Fancc+/'Sodl+/' controls all had statistically similar R L U values of 0.33, 0.38 and 0.44, respectively. Sodl'' samples showed a marginally elevated R L U of 0.55 that was statistically different from Fancc+/+Sodl+/+ mice (p=0.02). In contrast, there was a 4.8-fold increase in the R L U value obtained from Fancc'' Sodl'' cells (1.62) compared to Fancc+/+Sodl+/+ controls (p=0.0008). While hepatocytes were likely the source of the increased levels of superoxide in Fancc'Sodl'' cells, we are unable to define the potential contribution of Kupffer cell-derived superoxide. FACS analysis demonstrated that the percentage of CD1 l b + cells was similar in all samples. Increased expression of Mn SOD and HO-1 in Fancc'Sodl'' livers. Heme-oxygenase-1 (HO-1) is induced by various cellular stressors, including ROS 1 1 6 . Similarly, MnSOD can also be induced by ROS, including superoxide anion 1 1 7 . Thus, an increased level of MnSOD and/or HO-1 in total liver cell lysates, as assessed by immunoblotting with anti-MnSOD and HO-1 antibodies, would be predicted to accompany the putative pro-oxidant state in Fancc'Sodl'' hepatocytes. Fig. 8 is a collection of representative Westerm blots showing protein levels of HO-1 and MnSOD in liver lysates from A) suppliers control, B) Fancc+/+Sodl+/+, C) Fancc+/'Sodl+/' and D) Fancc'Sodl'' samples. Densitometric analysis of a number of immunoblotting experiments was carried out, with the ratio of protein band intensities for MnSOD or HO-1 normalized according to band intensities following stripping and re-immunoblotting of the same filters with an anti-(3-tubulin Ab. The results indicated that there were increased levels of MnSOD and HO-1 of ~4-fold, and ~10-fold, respectively, in Fancc'Sodl'1' 27 livers, as compared to Fancc+/+Sodl+/+ littermates, consistent with in vivo oxidative stress, however this difference was not significant. Peripheral blood and bone marrow abnormalities of Fancc^Sodl^mice To search for evidence of marrow dysfunction we evaluated peripheral blood (PB) cells from Fancc+/+Sodl+/+, Fancc+'~Sodl+/', Fancc'', Sodl'' and Fancc'Sodl'' mice (both males and females) (Table IV). Significant decreases were observed in the R B C (p=0.005) and W B C (p=0.03) compartments of Fancc'Sodl'"mice, as compared to Fancc+/+Sodl+/+ littermates. W B C values from Fancc'', Sodl'' and Fancc''Sodl'' mice, however, were not significantly different from one another (p=0.18 and p=0.12, respectively). Fancc''Sodl'' differentials (n=4) revealed that the W B C decrease was due to a reduction in both neutrophils and lymphocytes. There was no indication of a granulocyte maturation arrest in any of the samples. PB smears revealed that lymphocytes from Fancc''Sodl'' blood were often larger with more immature nuclear chromatin then either Fancc+/+Sodl+/+ or Fancc+'~Sodl+'~ controls. Red cell M C V was significantly increased (p=1.4 x 10"5), and there were higher numbers of polychromatic erythrocytes in Fancc'' Sodl'' mice compared to controls (data not shown). Fancc'' mice demonstrated a trend towards reduced W B C , however, this decrease was not significant (p=0.07). Platelet counts were normal in Fancc''Sodl'' mice, consistent with the normal megakaryocyte numbers observed in the marrow. Interestingly, 8 wk old, but not older (platelet count 587 ± 12.9) Fancc'' mice showed a significant (p=0.007) decrease in platelet numbers. Furthermore, there were reductions in both R B C (p=0.03) and HGB (p=0.04) in Sodl'' mice, as compared to Fancc+/+Sodl+/+ controls. Peripheral counts were obtained from mice up to the age of 3 months. With age, W B C values from Fancc'' mice (10.5 ± 1.5) increased to Fancc+/+Sodl+/+ levels (10.21 ± 0.86), ceasing to be statistically similar to W B C values from Fancc''Sodl'' mice (5.9 ± 0.86). Thus, in contrast to Fancc''Sodl'' mice, the reductions of W B C and R B C seen in 8-10 wk old Fancc'' mice normalize over time. 28 The marrows of Fancc+/'SodF'~, Fancc'', Sodl'' and Fancc''Sodl'' femora were then assessed (representative examples are shown in Fig. 9). Decreased cellularity was present in Fancc'Sodl'', suggested by increased fat cell numbers, particularly in the long bone metaphyses. In 5/5 Fancc''Sodl'' femurs analyzed, a large increase in the amount of B M fat was present, while in only 1/4 Fancc+/+Sodl+/+ and 1/4 Fancc+/'Sodl+/' controls there was an increase in fat, and when present this was less pronounced than that of Fancc''Sodl'' mice. To quantitate these observations, total B M cell numbers per femur were obtained from Fancc+/+Sodl+/+, Fancc+/' Sodl+/', Fancc'', Sodl'' and Fancc'Sodl'' mice (Tabje-Vj; n is as shown). Cellularity was decreased in Fancc''Sodl'' mice (2.33 x 10 7 ± 0.19) compared to Fancc+/+Sodl+/+ controls (3.98 x 107 ± 0.62) by 58%, however, this was not significant (p=0.06). There was also no statistical difference in total B M cellularity between Fancc+/'Sodl+/', Fancc'' and Sodl'' controls. To determine whether any specific marrow cell type might be differentially affected, flow cytometry was carried out on total B M samples with the following monoclonal antibodies: PGP1, B220, Ly6G, 7/4 (PMN), C D l l b , CD14, and Ter-119. As shown in Table V, the average number of Fancc''Sodl'' cells of each type was decreased by at least 40%, as compared to Fancc+/+Sodl+/+ controls. To investigate whether these reductions were mirrored by reduced numbers of committed (Lin +) progenitors, L in + Sca l + , Lin + ckit + and Lin + CD34 + values were obtained from Fancc+/'Sodl+'r and Fancc'Sodl'' mice (n=3). The average absolute number of these phenotypes for Fancc+/'Sodl+/' mice was 1.8 x 106, 3.6 x 106 and 1.7 x 106, and for Fancc''Sodl'' mice, was 0.9 x 106, 1.26 x 106 and 0.8 x 106, respectively, demonstrating that committed progenitor populations were similarly decreased in Fancc''Sodl'marrows (Fig. 10). There was no evidence of increased extramedullary hematopoiesis, as spleens obtained from two sets of animals, revealed no change in cellularity: Fancc'Sodl'' (9.65 x 107 ± 0.45) and Fancc+'~Sodl+/' (13 x 107 ± 3.85). Furthermore, the average number of Fancc''Sodl'' cells of each type from the spleen was the same when compared to Fancc+/+Sodl+/+ controls. 29 Fancc''Sodl'' total marrow cells fail to show increased apoptosis or chromosomal aberrations Since B M hypocellularity in Fancc''Sodl'' mice might have been due to an increased level of apoptosis, total marrow samples were analyzed by flow cytometry and propidium iodide / annexinV (PI/A) staining. However, this revealed no gross increase in apoptotic cells in Fancc'' Sodl'' mice, as compared to Fancc+'~Sodl+'ccmtrols (Table^i). While suggesting that increased apoptosis might not be the explanation for the B M hypocellularity, the possibility of increased apoptosis within a progenitor subset was not excluded by this procedure. As gross cytogenetic abnormalities would impair hematopoietic cell development, we evaluated metaphase chromosome spreads from Fancc+/+Sodl+/+, Fancc+/'Sodl+'~, and Fancc''Sodl'' B M cells. However, there was no evidence of increased chromosomal aberrations (breaks, gaps or detectable rearrangements) in Fancc''Sodl'' mice (n=2) as compared to control mice (n=3) upon examination of 10 metaphase cells per mouse. In vitro hematopoietic colony growth is severely impaired in Fancc''Sodl'' mice As B M hypocellularity would also result from inadequate growth of hematopoietic progenitors in Fancc''Sodl'' mice, we examined the in vitro clonogenic potential of committed myeloid (CFU-GM) and lymphoid (CFU-pre-B) progenitors from Sodl'', Fancc'1', Fancc+/+Sodl+/+, Fancc+/'Sodl+/' and Fancc''Sodl''mice. Fig. 1 l a represents the average number of progenitors/femur ± S E M from myeloid (dark bars) and pre-B (hatched bars) methylcellulose assays for n=6-8 animals per genotype, with each experiment done in duplicate (for Sodl'' pre-B cultures, n=4). This data clearly shows that the numbers of colonies from myeloid and pre-B progenitors/femur from Fancc''Sodl'' mice (p=0.0002 for both) was severely decreased, as compared to Fancc+/+Sodl+/+ controls. This data indicates that the number of myeloid and pre-B progenitors/femur from Fancc''Sodl'' mice was approximately 75-fold lower than from Fancc+/+Sodl+/+ controls. The number of colonies obtained from Sodl'' and Fancc'' marrows was 30 also significantly reduced (p=0.04 and p=0.01, respectively) for both the myeloid and pre-B assays when compared to Fancc+/+Sodl+/+ and Fancc+/'Sodl+/' controls. In vitro colony forming assays provide additional information about the quality of committed progenitors since both the size of the colonies as well as the frequency of different cell types arising from a myeloid progenitor can be evaluated. For example, most colonies scored from Fancc''Sodl'' mice just met the criteria for colony size (> 20 cells/colony), as compared to colonies from Fancc+/+Sodl+/+ controls which were highly cellular. Furthermore, the colonies described in Fig^ llaj were scored by cell morphology into C F U - G E M M , C F U - G M / G / M and BFU-E groups. Fig. f i b represents the frequency of progenitors/105 B M cells. We found that colonies enumerated from Fancc'Sodl'' samples were mostly erythroid in origin, 57.4 ± 20.44% (p=0.12) with very few C F U - G M / G / M , 8.78 ± 8.0% and C F U - G E M M , 0.46 ± 0.21% colonies being present (p=0.002; p=0.003, respectively). Sodl'', Fancc'1', Fancc+/+Sodl+/+, Fancc+'~Sodl+/' progenitors, on the other hand, all gave rise to the different cell types at similar frequencies. It was important to determine whether we could rescue colony growth from Fancc''Sodl'' progenitors in methylcellulose since this would give us an indication of what was causing the lack of growth. We plated whole B M cells from Fancc+/+ Sodl+/+ and Fancc'Sodl'' mice in the following conditions and scored for colony rescue: a) increased numbers of cells to 2-, 5-, or 10-times the original cell number, b) growth in 5% O2, c) a pan-caspase inhibitor (ZVAD-fmk), d) various antioxidants, e) and increasing concentrations of SCF (Table VII). None of the above tests augmented colony growth. Each rescue experiment represents 3 mice for each of Fancc''Sodl'' and Fancc+/'Sodl+'\ with each experiment performed in duplicate. Primitive progenitor numbers are normal in Fancc'Sodl'' mice Although B M hypocellularity could result from reduced levels of early progenitors, Fancc''Sodl'' marrows did not exhibit a significant reduction in the lineage-negative (Lin) compartment. The absolute number of Lin" cells, as determined by flow cytometry of non-31 fractionated total B M samples, was similar for Fancc+'~Sodl+/' (1.7 x 105 ± 0.3/femur) and Fancc1' Sodl'' mice (1.3 x 105 ± 0.7/femur); n=3 for each genotype. Furthermore, the absolute number of Lin" cells (obtained after L i n + cell depletion-column experiments) from Faracc+/"5W/+/"controls (7.6 x 105 ± 1.52) was similar to that of Fancc''Sodl'' mice (5.2 x 105 ± 1.05), n=6 (values represent cell numbers obtained from both femurs and tibiae per mouse). Flow cytometry of Lin" cells, obtained following L i n + depletion, using monoclonal antibodies against CD34, Seal and c-kit revealed no significant differences in absolute number (Fig. 12). The values below represent experiments using 5 animals per group and are the average absolute number ± S E M . Thus, the absolute number of Lin"Scal+c-kit" and Lin"Scal +c-kit + cells from Fancc''Sodl'' mice was 1.3 x 104 ± 0.3 and 5.1 x 104 ± 2.0, while for Fancc+''Sodl+/'controls were 2.0 x 104 ± 0.9 and 3.4 x 104 ± 0.9, respectively. The similarity is also observed in the Lin"CD34 +Scal" compartment, where both Fancc''Sodl'' mice and Fancc+/'Sodl +/"controls had 13 x 105 ± 0.7 cells. We conclude that the absolute number of cells within the Lin ' compartment of Fancc'Sodl'~ B M is similar to controls and that subpopulations within the Lin" compartment of these mice are also similar to controls, as assessed by immunophenotyping. Aging study reveals persistant aberrant hematopoiesis in Fancc''Sodl'' mice. Cohorts of mice (wildtype controls, Fancc'', Sodl'' and Fancc''Sodl') were aged up to 8 months at which point the mice were sacrificed and committed progenitor growth was analyzed (n = 3-4 per genotype). We hypothesized that the lack of progenitor growth observed in the 8 week old Fancc''Sodl'' mice may persist in this older population and eventually lead to complete B M aplasia. We observed a significant decrease (p=0.03) in B M cellularity/femur of approximately 43% from Fancc''Sodl'' mice (1.6 x 107 ± 0.1) compared to wildtype mice (3.7 x 107 ± 0.4), consistent with previous B M cellularity data from 8 week old mice (see Table V) . To measure the level of apoptosis within the whole B M samples, B M cells were analyzed by flow cytometry for propidium iodide/annexinV (PI/A) staining. We did not detect a significant increase in apoptotic 32 cells (A + /P f ) from Fancc'Sodl'' mice (11.7 ± 0.8 %), as compared to Fancc+/'Sodl+'contro\s (8.9 ± 1 . 0 %). We also assessed clonogenic growth potential from committed B M progenitors from 6-8 month old mice and our results were very similar to those obtained for younger 8 wk old mice. Fig. 13; represents the average number of progenitors/femur ± S E M from myeloid (dark bars) and pre-B (hatched bars) methylcellulose assays for n=3-4 animals per genotype, with each experiment done in duplicate. This data clearly shows that the numbers of colonies from myeloid and pre-B progenitors/femur from Fancc'Sodl''mice (p=0.0003 and p=0.005, respectively) was severely decreased, as compared to Fancc+/+Sodl+/+ controls. The number of progenitors obtained from Sodl'' marrows was also significantly reduced for both the myeloid and pre-B assays (p=0.04 and p=0.03, respectively) when compared to Fancc+/+Sodl+/+ controls, while myeloid and pre-B progenitors from Fancc'' marrows were never significantly decreased compared to wildtype, however this did not reach statistical significance. This data clearly indicates that the B M hypocellularity and lack of committed progenitor growth that was observed from the B M of 8 week old mice persisted in the marrows of older Fancc''Sodl'' mice and suggests that there is a defect in the ability of progenitors to proliferate and populate the marrow in vivo in Fancc'Sodl'' mice. 2.4 DISCUSSION We have shown that mice having combined deficiencies of Fancc and the primary cytosolic superoxide-detoxifying enzyme, Sodl, exhibit two novel phenotypes: fatty liver and an impairment of hematopoietic cell development. Fancc'Sodl''liver pathology was characterized primarily by zone 3 microvesicular steatosis, possibly a manifestation of in vivo superoxide toxicity as evidenced by increased superoxide production. In keeping with superoxide-mediated pathology, we found increases in the oxidative stress-inducible enzymes, MnSOD and HO-1 within Fancc''Sodl'' livers. While microvesicular 33 fatty liver is often encountered as a result of mitochondrial dysfunction, it can also result from impaired egress of lipids from hepatocytes as seen following specific hepatotoxin exposures 1 1 8 . ROS, such as superoxide anion, either alone, or in combination with nitric oxide (NO) yielding peroxynitrite (ONOO), are able to react with a variety of cellular macromolecules, including membrane lipids 1 1 9 ' 1 2 0 . Lipid peroxidation and accumulated hydroxy fatty acids within membranes of the endoplasmic reticulum, for example, can interfere with transport of lipids and/or components of V L D L particles that are responsible for removing lipids from hepatocytes 1 1 8 . Interestingly, and possibly in keeping with superoxide-mediated organelle, and possibly plasma membrane damage, the livers of mice lacking MnSOD (Sod2) also demonstrated microvesicular steatosis and increased levels of serum A L T 1 0 7 . It should also be noted that ROS can function as second messengers, modulating the activities of intracellular signaling molecules and transcription factors l 2 1 . Thus, liver pathology in Fancc''Sodl'' mice might stem from abnormal gene expression patterns secondary to elevated superoxide levels or reduced dismutation of this species into hydrogen peroxide. Increased superoxide production by Fancc''Sodl'1' liver cell cultures was interesting given the reports of elevated ROS generation by F A cells 5 1 ' 5 2 , and the reported protective effects of SOD where extrinsic SOD reduced the high rates of chromosomal breakage in F A cells and also diminished M M C cytotoxicity 5 4 ' 5 5 . Reduced SOD1 levels have been reported in F A erythrocytes 122,123 while there are no definitive reports that implicate SOD1 as a direct modifier of the human F A disease, genetic evidence suggesting that modifier genes may exist in F A is emerging. Clinical analysis of Japanese F A patients carrying a classic 'severe' mutation in FANCC, paradoxically have a mild phenotype 1 2 4 , suggesting that modifyer genes may be involved in determining severity of the clinical phenotype. Unlike the Fancc''Sodl'' cross, hepatic steatosis has not been reported human FA. Assuming that a similar pathogenetic mechanism were involved in FA, this discordance might be explained by the known species-specific differences in C Y P P-450 genes, or in differences in xenobiotic exposures. 34 Since lipid accumulation in hepatocytes can be accompanied by necrosis J Z 5 and inflammation, we searched for evidence of hepatocyte damage, and cell infiltrates. Only the modest elevations of serum A L T were suggestive of hepatocyte damage, and this occurred in the absence of overt necrosis or pathological collagen deposition. Activation of Kupffer cells, which also leads to ROS, NO, as well as pro-inflammatory cytokine production, can injure hepatocytes, and is often accompanied by neutrophil infiltration " 9 . The lack of infiltrates, the normal percentages of C D l l b + cells in Fancc'Sodl'' liver samples, and the zonal liver pathology, however, would suggest a defect intrinsic to the hepatocytes of Fancc''Sodl'' mice. The second phenotype of Fancc''Sodl'' mice was that of marrow hypoplasia, accompanied by a striking impairment of in vitro hematopoietic colony formation. Given the normal levels of primitive precursors (as assessed by immunophenotyping), this was suggestive of a growth and/or survival defect in committed progenitor populations. Similar to Fancc''Sodl'' mice, in vitro colony generation by F A marrows is impaired at both the multipotential and differentiated progenitor levels in vitro 8 0 ' 8 1 , with the mean C F U - G M values for human F A being approximately 15-fold lower than controls 8 0 . In contrast to humans with FANCC mutations, however, Fancc'' mice do not show spontaneous permanent cytopenias or decreased clonogenic potential 6 9 ' 7 0. Like their human counterparts, however, hematopoietic cells from Fancc'' show increased sensitivity to I F N Y , T N F a and M l P - l a , as well as deregulated apoptosis 1 6 , and Fancc'' B M cells exhibit a decrease (7-12 fold) in short-term and long-term multilineage repopulating ability 9 2 . The mild thrombocytopenia in young (8-10 wk), but not older (3 mo) Fancc'' mice had not been reported previously, and is likely attributable to differences in the genetic backgrounds of the mice. This variable may also account for the modest reductions in myeloid and lymphoid colony formation in our Fancc'' mice. Although, unlike FA, platelet counts of young Fancc'Sodl'' mice were normal, it is possible that thrombocytopenia would occur over time if marrow failure is progressive in these mice. Interestingly, increased mean corpuscular volume of Fancc''Sodl'' red cells was 35 analogous to the macrocytosis commonly observed in F A patients 3 , however this morphology has many causes, including liver dysfunction 1 2 6 . The finding of impaired hematopoiesis in Fancc''Sodl''mice is most strongly supported by our functional studies of in vitro growth of committed progenitors. C F U - G E M M , C F U - G M , and CFU-preB from Fancc'Sodl''marrows failed to grow and this observation was also made in older (6-8 month) Fancc''Sodl'' mice as well. Furthermore, despite normal levels of the earliest (lineage-negative) progenitors, there were considerably lower numbers of lineage-positive progenitors/femur in Fancc''Sodl'' mice. In addition, the ability of marrow cells to produce colonies of normal size and containing the usual range of cell types was compromised. In keeping with a markedly reduced proliferative potential (and/or an increased rate of apoptosis) C F U numbers in vitro was not increased by plating more cells (up to 10-fold), adding antioxidants, caspase inhibitors or growth in hypoxia. Thus, poor Fancc''Sodl'' colony formation did not appear to result from the plating of lower progenitor numbers, but instead pointed to a progenitor cell growth or survival defect. Although Fancc''Sodl'' total marrow samples did not reveal evidence of overt apoptosis, increased death restricted to a progenitor subset(s) would readily explain the lack of growth in the Fancc''Sodl'' CFAs. This might be attributable to superoxide-mediated genotoxicity superimposed on a background of reduced D N A repair capacity due to the lack of Fancc. While oxygen-dependent toxicity did not appear to play a role in C F A inhibition (growth in 5% oxygen did not 'rescue' growth), it is possible that committed progenitors have pro-oxidant intracellular environments that result in toxicity even at reduced oxygen tensions, that 5% oxygen was still not sufficient to rescue growth, and that 1 - 0.1% oxygen would be necessary to see such an affect. It is also possible that marrow Fancc''Sodl'' progenitors are damaged by ambient oxygen during harvesting and initial C F A plating procedures. Alternatively, as ROS appear to be required for the normal proliferative response to various growth factors 1 2 7 1 2 8 , it is possible that Sodl deficiency led to loss of a positive growth signal. There is evidence that ROS, such as superoxide and hydrogen peroxide, can act as second 36 messengers for a variety of stimuli, including growth factors 12!*. GM-CSF stimulation, for example, lead to rapid increases in cellular hydrogen peroxide levels, accompanied by elevated levels of tyrosine phosphorylation 1 1 1 . The latter may be due to the transient inhibition of protein-tyrosine phosphatases by this species, an event favoring protein-tyrosine kinase-dependent signaling 1 2 8 . Furthermore, alterations in redox potential impact a wide range of cellular processes 1 2 1 . The balance between ROS and antioxidant systems can thus regulate cellular responses to external stimuli 1 2 1 , 1 2 9 ; for example, interfering with hydrogen peroxide generation attenuated the proliferative response of hematopoietic cells to colony stimulating factors 121. A lack of Sodl would be predicted to inhibit growth factor mediated cell growth by reducing conversion of superoxide into hydrogen peroxide. Thus, hematopoietic progenitors from Fancc''Sodl'' mice might be intrinsically hypo-responsive to growth factor stimulation. Perhaps in keeping with this, we observed a consistent reduction in colony formation in vitro when Sodl'' marrow samples were plated. It is also notable that the abnormalities of Fancc''Sodl'' mice are analogous to those of W/Wv mice that lack normal stem cell factor receptor kinase (c-kit) activity and demonstrate a marked suppression of C F U growth in vitro 1 3 ° . Interestingly, there is evidence that F A N C C is required for normal STAT1 activation following growth factor stimulation 9 1 . This intriguing finding raises the possibility that a 'two hit' signaling abnormality might account for the hematopoietic defect of Fancc''Sodl'' mice: namely, that a decreased level of growth factor-induced ROS (specifically, hydrogen peroxide), together with a defect in Stat 1-mediated signaling, act synergistically to inhibit proliferation and/or survival of hematopoietic progenitors. 37 CHAPTER THREE - Absence of Cu/Zn Superoxide Dismutase (Sodl) Limits Long-Term Progenitor Cell Self-Renewal in Fanconi Anemia Complementation Group C (Fancc)-deficient Mice. 3.1 INTRODUCTION Chapter 2 described a hematopoietic defect in Fancc'Sodl'' mice that manifested itself as peripheral blood bicytopenia, whole B M hypocellularity, and a complete lack of committed progenitor growth in methylcellulose. These results indicate that excess endogenous superoxide anion results in decreased committed progenitor growth from Fancc''Sodl'' mice and suggests a mechanism by which human F A N C C HPC with elevated levels of superoxide anion have abnormal growth. As a result of these observations, we were interested in enumerating a) earlier hematopoietic progenitors in functional assays and b) progenitor growth recovery, to identify i f decreased growth is due to a block in this progenitor compartment, or if the defect lies in an earlier population of progenitors. The cause of human F A B M failure is unknown and previous to our Fancc''Sodl'' strain, spontaneous B M failure has not been observed in any F A mouse models (see Table LTJ for current list of mouse models). This gave us a unique opportunity to examine the nature of bicytopenia in these mice and to generate some hypotheses about the nature of aplasia in FA. One of the possibilities for F A B M failure is that it results from absent or defective hematopoietic precursors. In F A patients (and our Fancc''Sodl'' mice), progenitors do exist, however the growth of C F U -G M , C F U - G E M M and BFU-E is completely absent and the addition of SCF does nothing to correct progenitor outgrowth 8 1 . This effect is seen both in aplastic anemia patients as well as patients that have not yet developed B M failure. Studies measuring the growth of B M cells from F A patients in long-term cultures (LTBMC) have been difficult to interpret. One study found that differentiated myeloid cells were generated in normal numbers and that the cells were able to 38 initiate secondary L T B M C cultures 8 4 . It is important to note that these cultures were grown in 5% oxygen since F A cells are known to have a significant growth advantage in lower oxygen conditions. This study is important because it clearly shows that early hematopoietic progenitors are in fact present in appropriate numbers in F A patients with concurrent B M failure. A second study investigating L T B M C from F A patients (in 20% oxygen conditions) revealed that adherent stromal layers did develop in all F A cases, however, stromal growth rate was slower than that of controls 8 0 , and impaired C F U - G M was observed in all F A patients. C F U - G M was measured from patient B M aspirates as well as cells harvested from the suspension of L T B M C . To our knowledge, there has been no study done that has measured the frequency of long-term culture initiating cells (LTCTC), the earliest hemopoietic progenitor cells, in vitro from F A patients or murine models. The Cobblestone Area Forming Cell (CAFC) assay can be used as an alternative to the traditional L T B M C assays that measure LTC-IC frequencies in vitro. The C A F C assay was developed as a quantitative in vitro assay that allows the direct measurement of the frequency of both short-term (CFU-S) and long-term (MRA) hematopoietic engraftment ability 1 3 1 . The most primitive hematopoietic cells are preferentially located within the adherent layer, and can be seen as colonies. Previous reports indicate that in vivo CFU-S are accurately estimated by day 7 C A F C frequencies while M R A are measured by day 28 C A F C frequencies 1 3 1 1 3 2 . The major goal of this work was two-fold; first to define the hematopoietic progenitor compartment that was defective in Fancc'Sodl'' mice and was responsible for the peripheral bicytopenia and B M hypocellularity observed in these mice. Secondly, to try to rescue this growth defect using various antioxidants and hypoxic conditions in an attempt to identify the nature of decreased growth. Using the C A F C assay, we measured the frequency of the earliest hematopoietic progenitors (LTC-IC) in vitro from Fancc''Sodl'' mice and found that while C A F C did exist in the marrow of these mice, that there was a profound decrease in frequency with time that was only partially rescued with growth in 5% oxygen. Furthermore, lineage depleted, column 39 - purified progenitors were assessed for growth in liquid media and found to have stunted growth potential in vitro as well as a concomitant increase in apoptosis. If one of the roles of the Fancc protein is to regulate oxidant states within the cell, and the loss of the Fancc protein leads to ROS -mediated cellular toxicity, then forcing a pro-oxidant state (through the introduction of modifier genes) onto a Fancc - null background should result in a phenotype reminiscent of FA. If this were successful then we would be able to infer that the B M failure seen in these mice might be a good reflection of the events occurring in human F A patients. It would also be useful in aiding the identification of the causes for B M death, and possibly ways to correct this phenotype. Based on this logic and our results herein, we suggest that B M failure in F A results from the death of the earliest progenitors in the marrow. This may, in part, be due to ROS - mediated (or other reactive species) toxicity, as the specific scavenging of superoxide anion species partially corrects the reduced growth of HPC from Fancc'SodV'' mice. 3.2 MATERIALS AND METHODS BM Cell Preparation and Column Purified Progenitor Cell Isolation Mice were sacrificed at 8 weeks of age by C 0 2 asphyxiation. Total B M cells were collected by flushing femurs with cold a M E M containing 5% FCS. Cell viability, > 90% in all samples, was determined by trypan blue exclusion. Lineage depleted samples were collected by resuspending the cells at 5.0 x 107 nucleated cells/ml in PBS with 2% FBS, plus 5% rat serum for 15 min at 4 °C. Samples were first incubated with an antibody cocktail (CD5, C D l l b , CD45R, GR1, 7-4 and TER-119) and subsequently with an anti-biotin tetrameric antibody (both antibody cocktails from StemCell Technologies, Vancouver, BC) complex (each step for 15 min at 4 °C), then a magnetic colloid was added for cell separation as recommended (StemCell Technologies, Vancouver, BC). To isolate Lin" populations, the suspension was applied to a primed 0.3 inch 40 magnetic column and washed 3x with PBS containing 2% FBS. The cells in the flow-through were enumerated and trypan blue exclusion used to determine viability (> 95%). Column purified progenitors were plated in Iscove's modified Dulbecco's media ( IMDM, Stemcell Technologies) supplemented with 15% FBS (lot # 6250, StemCell Technologies), 50 ng/ml SCF, 10 ng/ml IL3 and 10 ng/ml IL6 and allowed to grow in a humidified chamber in 5% C 0 2 in air (+/- 5% 0 2 in air). Chemicals 4,5-Dihydroxy-l,3-Benzene-Disulfonic Acid (TIRON) was purchased from Sigma Company (Cat no. D 7389) and [Mn(UI)tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride] (MnTMPyP) was purchased from Calbiochem (Cat no. 475872). Clonogenic Assays for committed hematopoietic progenitor cells Whole B M cells were plated in 1.1 ml of 1% mefhylcellulose media supplemented with 10% FCS, 2 m M L-glutamine, 10"4 M 2-mercaptoethanol and the following recombinant growth factors: for myeloid assays, methylcellulose was supplemented with 1% bovine serum albumin, 10 pg/ml bovine pancreatic insulin, 200 pg/ml human transferrin, 3 units/ml recombinant human erythropoietin, 10 ng/ml recombinant mouse IL-3, 10 ng/ml recombinant human interleukin-6 and 50 ng/ml recombinant mouse stem cell factor (SCF). For Pre-B assays 10 ng/ml recombinant human LL-7 was used (StemCell Technologies, Vancouver, BC). Cells were dispensed using a blunt-ended needle and cultured at a density of 1.7 x 105 and 5.5 x 104 cells per 35 mm dish for Pre-B and Myeloid colonies respectively (each sample done in duplicate). Dishes were incubated for 6 (for Pre-B) or 12 (for myeloid) days at 37 °C, 5 % C 0 2 in air, > 95 % humidity. Colonies (> 20 cells) were counted on a gridded stage using an inverted light microscope. 41 Long term Cobblestone Area Forming Cell Assays (CAFC) Primary B M stromal feeder layers were prepared from 4-6 week old female Bl/6 mice. 3.0 x 105 B M cells were plated into 96 well dishes in 0.2 ml Myelocult media (StemCell Technologies, Vancouver BC) supplemented with freshly prepared 10"6 M hydrocortisone hemisuccinate (StemCell Technologies). The cultures were grown until they reached 80% confluency in a 33°C incubator in 5% C 0 2 in air (> 95% humidity). Confluency was achieved by 2-3 weeks of culture with weekly half-media changes. One day before B M overlay, the cultures were irradiated with 15 Gy using a 1 3 7 Cs source that emitted 280 rad/min and the cells were allowed to recover overnight (as per the protocol of StemCell Technologies). There were no C A F C colonies observed in the irradiated cultures without B M overlay up to 6 weeks post-irradiation. Fresh, non-fractionated B M cultures were obtained from the femurs of mice sacrificed by C 0 2 asphyxiation. The marrow was flushed into cold cdvlEM + 5% FBS. The B M cells were diluted in MyeloCult + 10"6 M hydrocortisone starting at a density of 3.0 x 105 c/ 0.2 ml media and five further dilutions were made, each titrated down by half to a final dilution of 937 c/0.2 ml of media. Each dilution from each mouse was repeated 6 times on the same plate and 32 wells were scored from each oxygen concentration (n = 5 mice/genotype). A l l of the media was removed from the irradiated stromal cultures and the B M cells were plated on top of the adherent irradiated stromal cells (StemCell Technologies). Weekly half media changes were done for 4 weeks with fresh MyeloCult + 10"6 M hydrocortisone and scoring of C A F C colonies was done before addition of fresh media. Scoring was done by scanning each well at 400 x magnification using an inverted scope on phase contrast. A well was scored positive if it contained at least one C A F C , which represented a cluster of cells with at least 5 cells grouped together1 3 1. A C A F C was defined as cells that were large and homogenous, not refractive and in the same plane as the stromal layer. The frequency of CAFCs were calculated using the frequency of negative wells at each cell dilution in a Poission-based limiting dilution analysis. 42 Poission statistical software was provided by StemCell Technologies and was used to calculate significant differences. Column Purified Progenitor Growth Curves Column purified progenitors were grown in HVIDM + 15% FBS, 50 ng/ml SCF, 10 ng/ml IL-3 and 10 ng/ml IL-6 in a 37°C incubator with 5% C 0 2 in air (+/- 5% 0 2 ) , > 95 % humidity. Growth was measured using trypan blue exclusion. For studies without drug addition, cells were plated at a density of 2.0 x 105 in 1 ml of media, in triplicate in a 24 well plate (n=5 for each genotype). For TIRON and MnTMPyP studies, chemicals were diluted directly into I M D M media (+ growth factors) at the concentrations indicated and cells were plated at a density of 4.0 x 104 in 0.2 ml of media, in triplicate in 96 well plates (n=3 for each genotype). Flow cytometry for TUNEL Assays of Column Purified Progenitors Cells were collected on day 13 for T U N E L analysis. Samples were washed twice in cold PBS + 1% BSA and were transferred to a U-bottom 96 well plate. Cells were resuspended in 0.1 ml of freshly prepared 4% paraformaldehyde, pH 7.4 and allowed to incubate at room temperature for l h on an orbital shaker. The plate was centrifuged using a flat-bed spinner at 300 g for 10 min to remove fixative. Cells were washed once in cold 0.2 ml/well PBS + 1% B S A and resuspended in 0.1 ml/well permeabilization solution (0.2% Triton X in PBS) for 10 min on ice. Cells were washed twice in cold PBS and resuspended in 25 pl/well of T U N E L reaction mixture, which was made up of 2.5 pi TdT enzyme + 22.5 pi dUTP-FITC labeling solution (Boehringer Mannheim, Cat no. 1684795). Labeling was done for l h in a 37°C incubator in the dark. Cells were washed twice with cold PBS (0.2 ml/wash) and resuspended in a final volume of 0.3 ml in PBS. The samples were collected using a Becton Dickinson FACS machine equipped with Cell Quest Software and the samples were analyzed using F l o J o Software. 43 Primary Mast Cell Cultures The femurs and tibiae from wildtype and Fancc''Sodl'' mice were flushed and cell density was determined by trypan blue staining. The B M cells were grown at a density of 1-2 x 106 c/ml for the first week in a 24-well tissue culture plate in Iscove's media supplemented with 15% FCS (#6250, StemCell Technologies), 10 ng/ml IL-3, 10 ng/ml IL-6, and 50 ng/ml SCF. In the second week, the adherent stromal cells were discarded and the suspension cells were centrifuged, washed and resuspended in new media (the same as above, but without IL-6). The cells were then maintained and expanded at 2.0 x 105 c/ml and were not split until the sixth week in culture at which point the cells began to grow slowly and were homogenous mast cell cultures Murine Embryonic Fibroblast Cultures Female mice were set-up in cages with proven stud males and were checked for vaginal plugs in the morning of each day. Once a plug was found, this was considered day 1 and the pregnancy was timed so that the female is sacrificed on day 14 by CO2 asphyxiation. The uterus was dissected carefully from the female, so as not to puncture the gut, and placed into a sterile petri-dish within a tissue culture hood. The individual embryos were removed from the uterus into 100 mm sterile dishes using freshly cleaned scissors and tweezers sterilized with 70% ethanol between embryos. The embryos were minced as finely as possible using re-flamed scissors and 1.5 ml of warmed trypsin was added to the plate and incubated at 37 °C for 5 minutes to fully break up the embryo. 8.5 ml of warm a - M E M with 20% FCS was added to the plate and transferred to a 50 ml Falcon tube. The petri-dish was washed with the same medium to collect all of the cells and then added to the same tube. The suspension was then passed through a syringe fitted with a 15 gauge and subsequently an 18 gauge needle to finely break up the embryo pieces. The suspension cells were counted with acetic acid and plated at a density of 5.0 x 106 c/100 mm dish (this yields approximately 10 dishes per embryo). The media was changed every 3-4 days until the cells became confluent, which took 1-2 weeks. The confluent cells were transfected by 44 calcium phosphate precipitation. Briefly, on day one, cells were plated at a density of 5-9 x 10° c/60 mm dish in a - M E M + 10% FCS. On day two, the plasmid D N A was prepared using the following components; 0.25 ml C C T (0.25M CaCl 2 , 40 pg/ml salmon sperm carrier D N A , 0.1 x TE, pH 7.05), 5 pg SV40 large T antigen plasmid, 0.5 pg selectable pSV 2neo plasmid and 0.25 ml 2x HBS (280 m M NaCl, 50 m M Hepes, 1.5 m M Na 2 HP0 4 , pH 7.10). The HBS was added dropwise while bubbling air into the CCT/plasmid mixture through a plugged small pasteur pipette at a rate of 1 bubble/sec. After addition of the HBS, the precipitate was left to form for 30-40 minutes. Once the precipitate formed, 0.5 ml was added to the 60 mm dish, swirled and incubated at 37 °C for 16 hours or overnight. On day 3, the media was removed, the monolayer was rinsed and 5 ml of fresh a - M E M + 10% FCS was added to the cells. Five hours later, the cells were trypsinized and split into 6 x 60mm dishes containing 5 ml a - M E M + 10% FCS + 400 pg/ml G418. The media was changed every 3-4 days until the selected clones were grown out and confluent. Bone Marrow Derived Macrophage Cell Lines The B M from two femurs (per mouse) were flushed, pooled together and resuspended into 4.5 mis of D M E M + 10% FCS. To this suspension, 4.5 mis of J2 viral supernatant (3T3 cells stably transfected with retroviruses carrying the v-raf and v-myc oncogenes) and 5 pg/ml protamine sulfate are added and plated onto a 10 cm 2 petri-dish and left overnight. The viral supernatant was first collected from fully confluent viral producer cells (for maximal viral titer) and filter sterilized using a 0.45 micron filter. The following day the adherent cells were discarded and the suspension cells were collected into a fresh Falcon tube and resuspended in 4.5 mis D M E M + 10% FCS, 4.5 mis fresh viral supernatant and fresh 5 pg/ml protamine sulfate. The cells were plated and left overnight for a second time. On the third day, the cells were collected again into a fresh Falcon tube, washed in PBS once and resuspended into 12 mis of D M E M + 10% FCS. 45 The cells were aliquoted into 6 well tissue culture dishes in decreasing concentrations and left to culture for 5-6 weeks. During this time, the media was changed once a week until the sixth week when the culture became confluent and the media began to turn yellow, at this point the cells were terminally differentiated. Cytodex beads were added to the cultures and left for 48 hours at which point the beads were transferred to a new tissue culture dish and the cells were allowed to move off of the beads and adhere to the dish. Once the cultures grew out into confluent culture, the cells were then cloned into base methylcellulose media and frozen in aliquots as polyclonal and clonal cultures. Statistical Methods The Student's t test (Microsoft Excel) was used when analyzing the results other than those obtained for CAFC frequencies, p < 0.05 was considered significant. 3.3 RESULTS CAFC frequencies are significantly decreased from Fancc''Sodl'' mice and can be partially rescued in hypoxia. We were interested in defining the exact progenitor compartment within which the hematopoietic defect occurs. LTC-IC assays could not be used to examine the frequency of the earliest progenitor populations because cells from Fancc''Sodl'' mice did not survive in vitro for the duration of the assay (28 days). Thus, the alternative was to examine the frequency of the earliest progenitor populations on a daily basis from the initial day of culture using the CAFC assay system. The frequency of CAFC/105 BM cells from wildtype, Sodl'', Fancc'' and Fancc'' Sodl'' mice was determined on days 3, 7, 14, 21 and 28 and cultures were grown either in 20% or 5% oxygen. jTable_.''yiTJ represents the CAFC frequencies from 5 mice, each set done with 6 repeats of 6 dilutions of BM cells/mouse. The data clearly shows that progenitors from Fancc'' 46 Sodl'' mice only exist ex vivo for a maximum of two weeks in 20% 0 2 and these C A F C frequencies are significantly decreased in Fancc'Sodl'' mice at every day measured compared to wildtype, Fancc'' and Sodl'' controls in 20% 0 2 . When cultures were grown in 5% 0 2 , there was a partial increase in C A F C frequency in Fancc''Sodl'' mice and C A F C now existed ex vivo for the duration of the assay, albeit at constant declining frequencies. C A F C frequencies from Fancc''Sodl'' mice at 5% 0 2 were still statistically different from wildtype at all time points, however they were no longer different from Sodl'' and Fancc'' controls at days 3, 7 and 14. This data suggests that early progenitor cells do exist in the marrow of Fancc''Sodl'' mice, albeit at lower frequencies then in wildtype marrows. A benefit of using the C A F C assay system to measure progenitor frequencies and proliferation is that qualitative observations of C A F C can be made at the time of scoring. Fig. 14 is a representative collection of pictures of C A F C colonies from wildtype, Sodl'', Fancc'' and Fancc''Sodl'' cultures taken at day 7 in a) 20% and b) 5% 0 2 . The C A F C colonies can be seen as a group of refractive, homogenous cells located under the stromal layer. It is clear from these pictures that there is a difference in the quality of C A F C from Fancc''Sodl'' mice. C A F C colonies from wildtype cultures were always large when they appeared in culture, a result that was also seen in Fancc'' cultures. Thus, the decrease in C A F C frequency seen in wildtype and Fancc'' cultures was due to a normal reduction in C A F C frequency over time and not to a change in the size of the actual colonies. C A F C colonies from Sodl'' B M were consistently smaller then either the wildtype or Fancc'' colonies, but were always significantly larger then C A F C colonies from Fancc''Sodl'' cultures. This result was partially ameliorated by a reduction in oxygen concentration, since Fancc''Sodl'' C A F C grown in 5% O2 were larger (Fig. 14b). Fig. 15; is a collection of C A F C colonies from wildtype (a, b) and Fancc''Sodl'' (c - f) cultures taken at day 7 (a, c, e) and day 10 (b, d, f) in either 20% (a - d) or 5% (e, f) 0 2 on high magnification. These figures show morphological changes in the C A F C colonies from Fancc'' Sodl'' cultures. As we had already observed, wildtype C A F C were always large when they 47 appeared in culture, whether it is an early C A F C (day 7) or a later C A F C (day 10) colony and this observation was identical in 5% 0 2 . Conversely, C A F C progenitors from Fancc''Sodl'' cultures barely constituted a significant colony with only 4 cells being present (Fig. 15 c), on day 7 in 20% oxygen) and the progenitors within a given colony were irregular in volume and shape (Fig. 15 f). It is also clear from Fig. 15 f) that Fancc''Sodl'' C A F C grown in 5% 0 2 were significantly larger and more homogenous then those grown in 20% 0 2 (Fig. 15 d). These results indicate that while early progenitors do exist in the marrow of Fancc''Sodl'' mice, they appear to be exquisitely sensitive to the toxic effects of 20% oxygen, exhibiting abnormal proliferative capacities. Column purified early HPC from Fancc''Sodl'' mice have reduced growth rates and increased apoptosis. To further explain the reduced growth of both the committed progenitors in methylcellulose as well as the earlier C A F C progenitors in long-term B M cultures, as well as to isolate a population of cells that can be assessed biochemically, early hematopoietic progenitors (HPC) were purified by negative selection and grown in the presence of SCF, IL-3 and IL-6. HPC growth was measured by direct cell counts of wildtype, Fancc'', Sodl'' and Fancc''Sodl'' cells on days 3, 6, 9 and 13 post-isolation (Fig. 16a), (n=5 for each genotype, each count done in triplicate). Both Fancc'' and Sodl'' HPC had similarly decreased growth rates compared to wildtype controls, however this difference was not statistically significant at any time point measured. Not surprisingly, Fancc''Sodl'' HPC were found to have significantly reduced growth rates in liquid media in vitro compared to wildtype controls at all time points measured (p < 0.01). We wanted to assess whether the reduced growth of HPC from Fancc''Sodl'' mice was due to increased apoptosis or decreased response to growth factor stimulation. Thus, HPCs were assessed for the presence of apoptotic nuclei on day 13 post-isolation by the F A C S T U N E L assay (Fig. 16b). There was a significant increase in the number of positively staining d-UTP nuclei 48 from Fancc'Sodl'' FfPC (26.7 ± 1.1 %) on day 13 compared to wildtype HPC (12.2 ± 1.5 %) controls (p = 0.001). We hypothesize then that the lack of proliferation seen in B M cells isolated from Fancc'Sodl'' mice and grown in vitro was, at least partially, due to increased apoptosis of progenitor cells. HPC can be partiallv rescued when grown in the presence of hypoxia or antioxidants. Since we hypothesized that the increased apoptosis was a ROS-mediated effect, we next attempted to identify the ROS responsible for this effect. HPCs were thus grown in the presence of the cell permeable superoxide anion scavenger, 4,5-dihydroxy-l,3-benzene-disulfonic acid (TERON); a Sodl mimetic, [Mn(IU)tetrakis (l-mefhyl-4-pyridyl) porphyrin pentachloride] (MnTMPyP), or hypoxia (5% 0 2). Similar to the partial rescue of C A F C colonies grown in 5% 0 2 , Fancc'Sodl'' HPC were partially rescued when the cultures were grown in hypoxic conditions, n=3-4 per genotype, each done in triplicate (Fig. 17). Fancc''Sodl'' HPC grow at 43% of wildtype in 20% 0 2 and this difference is statistically significant (p=0.009) while hypoxia rescues HPC growth to 66% of wildtype and the difference is no longer statistically different (p=0.08). This data, from a different subset of cells, nicely mirrors the observations we accumulated from the C A F C colonies and suggests that the HPC are undergoing excessive apoptosis, likely due to ROS - mediated toxicity. We also attempted to rescue HPC growth by the specific scavenging of superoxide anion, to try to determine whether this species was involved. A dose response of TLRON on HPC cultures grown for 9 days in vitro shows that 0.001 m M TIRON maximizes growth from all genotypes, n=3-4 per genotype, with each count done in triplicate (Fig. 18). At this dose, TIRON increases HPC growth over basal (no drug) conditions in wildtype, Fancc'', Sodl'' and Fancc'Sodl'' cultures by 3.1, 1.81, 1.78 and 1.90 fold, respectively. When Fancc''Sodl'' HPCs are grown in the presence of 0.001 m M TIRON, the average cell number at day 9 (3.61 x 105 ± 0.4) is no 49 longer statistically different from the average cell number of wildtype HPC grown under basal conditions (3.75 x 105 ± 0.3) (p=0.9). An unexpected observation was made when HPC were grown in the presence of 0.001 m M TIRON. We found that growth in TIRON stimulated differentiation of HPC cultures into a larger, highly granular and slightly adherent population as assessed by FACS analysis in all genotypes except for Fancc''Sodl'' cultures. The significance of this observation is unknown however, it suggests that the rescue of HPC growth in the presence of TIRON is not attributable strictly to superoxide scavenging, since wildtype cells have significantly increased growth rates as well. It does suggest however, that HPC from Fancc'' Sodl'' mice are unable to respond normally to growth factors. Similarly, a dose response of MnTMPyP, a SOD mimetic done on day 9 post-isolation, revealed that 5 p M MnTMPyP maximally increased growth of cultured HPC from all genotypes, n=5 for each genotype, each done in duplicate (F^j^a ) . MnTMPyP increases HPC growth over basal no drug conditions in wildtype, Fancc'', Sodl'' and Fancc''Sodl'' cultures by 1.1, 1.32, 1.31 and 1.69 fold, respectively. The average number of HPC from Fancc''Sodl'' cultures counted on day 9 in basal conditions are statistically different from wildtype HPC cell numbers under the same conditions (p=0.02). However, when Fancc''Sodl'' HPCs are grown in the presence of 5 p M MnTMPyP, the average cell number at day 9 (3.0 x 105 ± 0.3) is no longer statistically different from the average cell number of wildtype HPC grown under basal conditions (3.87 x 105 ± 0.5) (p=0.52). HPC from wildtype and Fancc''Sodl'' mice were subsequently cultured in the presence of 5 p M MnTMPyP and counted to determine the rate of growth over 13 days (Fig. 19b). Interestingly, MnTMPyP increased growth of wildtype HPC over wildtype cells grown in basal conditions, however this was not a statistically significant difference. The resultant growth of HPC from Fancc''Sodl'' mice is only partially rescued over 13 days in culture. Unlike the TIRON supplemented cultures, we did not find additional, differentiated cell populations in HPC cultures grown in the presence of MnTMPyP. Taken 50 together, these observations suggest that the specific scavenging of superoxide anion, either with TIRON or MnTMPyP, results in a partial correction of HPC growth which may, in part, be due to ROS. Inability to generate cell lines from Fancc''Sodl'' mice. Since it was very difficult to obtain or grow sufficient numbers of primary BM-derived progenitors from Fancc''Sodl'' mice, and also because we were interested in examining the biochemical events causing death in Fancc''Sodl'' cells, we attempted to generate cell lines from Fancc''Sodl'' mice. Mast cell lines were initially chosen for study because large numbers of these cells can be readily grown from primary B M cultures. Thus, while wildtype controls differentiated successfully into large numbers of homogenous mast cells over the 6 weeks in culture, Fancc''Sodl'' B M cells failed to proliferate and after approximately 3 weeks in culture, the resulting cell population was highly heterogenous, with a combination of small cells and large, very granular cells (Fig.20). The average number of mast cells recovered from wildtype cultures after 5 weeks in vitro was 4.5 x 107 c/ml (represents a 225-fold increase from the original cell number plated), while the average number of cells from Fancc''Sodl'' cultures was 9.3 x 105 c/ml, a 5-fold increase. This result suggests that Fancc''Sodl'' B M cells cannot properly expand and differentiate in vitro, and likely in vivo as well. This result was supported by our previous study which showed that TIRON induced differentiation of HPC in all cultures except for Fancc'' Sodl'' cultures. Murine embryonic fibroblasts (MEFs) were chosen as a good alternative for the generation of a Fancc'Sodl'' cell line. As these cells are not hematopoietic in origin, we hypothesized that the proliferation defect observed in BM-derived cells might not be manifested in fibroblasts. Thus, embryos were harvested from pregnant females on day 14 of a timed pregnancy and after processing all of embryos, they were genotyped. Wildtype, Fancc'', Sodl'' and Fancc''Sodl'' MEFs were retained and left to grow until confluency at which point they were to be transformed. 51 Intriguingly, MEFs from Fancc''Sodl'' embryos ceased proliferating once they were plated and could never be grown to confluency. Thus, out of a total of three Fancc''Sodl'' embryos, none grew to confluency, while all the wildtype and Fancc'' cells became confluent within a week and were successfully transformed. Furthermore, 3 of 5 Sodl'' MEFs were capable of growing to confluency, however they were slower to grow out. MEFs were also grown, post-embryo plating in 5 % O2 conditions, however this did not rescue growth sufficiently to allow for confluent cultures. These results were interesting because they suggested that the defect observed in Fancc''Sodl'' hematopoietic cells also extends to cells of completely different origin. Since it became clear that most cells from Fancc''Sodl''mice could not survive ex vivo for any significant amount of time, we attempted to create primary B M derived-macrophage (BMDM) cell lines by infection of B M cells immediately upon marrow flushing using a J 2 myc/raf virus, (known to infect monocytes specifically). We hypothesized that if we could infect the cells immediately as they were flushed from the marrow, we might be able to minimize the amount of ROS-mediated toxicity from 2 0 % oxygen. While 3 of 5 Fancc''Sodl'' B M cultures were successfully infected with the virus, the monocytes did not proliferate sufficiently to be selected as lines. Qualitative observations indicated that even after viral infection followed by two weeks of monocyte differentiation and macrophage outgrowth, the resultant cells from Fancc''Sodl'' cultures grew significantly slower and were never as confluent as wildtype controls. These results indicate that the 2 0 % O2 culture conditions are likely too toxic for cells from Fancc''Sodl'' mice and studies need to be done either in short term experiments or in vivo, where the concentration of oxygen is less then 5 % O2. 3.4 DISCUSSION It is our hypothesis that superoxide anions are produced in human F A cells at a rate that overwhelms the capacity of the cell to detoxify them and this imbalance leads to ROS-mediated damage and progenitor cell death. This hypothesis is supported by the observations described in 5 2 this chapter using Fancc''Sodl'' mice as a model. The goals of this study were a) to use a functional assay to measure the existence and frequency of early hematopoietic progenitors (CAFC) from the marrow of Fancc''Sodl'' mice, b) to determine if the C A F C were undergoing excess ROS-mediated apoptosis (or whether they were simply not responding to growth factor expansion in vitro), and c) to quantify and attempt to 'rescue' the growth of HPC from liquid cultures in order to delineate the causes of decreased growth. Hematopoiesis is a hierarchial process involving multiple lineages and differentiation states, thus it was important to evaluate the specific population of cells within Fancc''Sodl'' mice that show deregulated growth. We used the C A F C assay system to evaluate the earliest progenitor subset from the marrow of wildtype, Fancc'', Sodl'' and Fancc''Sodl'' mice. Our C A F C data indicate that early progenitor cells do exist in the marrow of Fancc''Sodl'' mice, albeit at lower frequencies then in wildtype cells and that there is an increase in apoptosis in this population of cells, likely due to ROS toxicity. This was demonstrated in two ways, first C A F C frequency can be partially rescued from Fancc''Sodl'' cultures in the presence of 5% 02 This observation highlights the importance of measuring C A F C frequencies at all time points, since we observed a partial rescue of C A F C frequency at days 3-10 in 5% 0 2 from Fancc''Sodl'' cultures that would not have been evident if only day 28 LTC-IC were measured. Furthermore, qualitative observations of the C A F C colonies from Fancc''Sodl''cultures indicate that cellular morphology is severely affected, perhaps reflective of apoptotic membrane changes. Wildtype C A F C colonies are always large, whether these are early, day 7 C A F C or later, day 28 C A F C , and declining C A F C frequency in wildtype cultures is due to a decrease in the number of colonies, and not to the decreased size of the colonies. Therefore, there appear to be two factors at play in decreased Fancc''Sodl'' C A F C frequencies, first, the natural decline in colony number with time that is also observed in wildtype cells and second, the diminished size of the actual C A F C colony with time/well. We hypotheisze that it is the accumulation of superoxide anion over time in vivo that is reducing the C A F C potential to proliferate as well as leading to increased 53 apoptosis. The number of cells required within a colony to be classified as a proper CAPC was arbitrarily selected as 5. This small number was chosen owing to the small CAPC colonies that grew from Fancc'Sodl'' cultures in 20% 02. While these might not technically constitute a CAPC in the classical sense (20 cells are usually considered the minimum number for a CAFC 1M), we wanted to demonstrate that the cells do in fact exist in these cultures and are simply not able to expand normally. While a dramatic repopulating defect has been reported for Fancc'' mice compared to wildtype controls, Fancc'' test cells were able to contribute equally to both the lymphoid and myeloid compartments, indicating that the defect lies within the pluripotent progenitor compartment of Fancc'' mice 9 1 . This in vivo data corroborates our in vitro CAFC results nicely and strongly suggets that pluripotent progenitors are in fact the cells defective in FA. Interestingly, these Fancc'' mice with severe repopulating ability do not naturally develop BM aplasia, and the defect is only observed once the cells have been removed from the donor animal and then transplanted into the recipient. It is possible that the effect observed is due to ROS-mediated toxicity while the cells were processed in in vitro 20% 02 conditions. To address this question, the BM samples should be maintained either in 5% O2, or in the presence of an antioxidant for the duration of their time ex vivo. We chose to 'correct' the ROS overload within Fancc''Sodl'' cells in three ways; by growth in hypoxia, through the addition of a superoxide scavenger (TIRON), or a SOD mimetic (MnTMPyP). These studies required a population of cells that were easier to isolate then CAFC and that could be recovered in sufficient enough numbers. Thus, we used column purified Lin" HPC to examine the effects of the above conditions on cell growth. In keeping with our previous reports of diminished committed progenitor growth and CAFC frequencies from Fancc''Sodl'' mice, we found a severe reduction in HPC growth that was accompanied by an increase in the number of apoptotic nuclei on day 13 of culture. It is clear from these progenitor growth studies that the removal of superoxide, through its specific scavenging (with TIRON or MnTMPyP), 54 diminishes the course of cell death in Fancc'Sodl'' cultures, just as the specific removal of 57 superoxide anions from F A lymphocytes modulated the course of the human disease . These studies provide strong evidence that superoxide anion is a mediator of HPC death in Fancc'' Sodl''HPC cultures. SOD has been given as a potential therapeutic to F A patients with the expectation that it would stimulate marrow growth. 4 F A patients were treated with a 2-week infusion of rh-SOD (25 mg/kg daily) to determine whether rh-SOD had any effect on HPC growth or on the abnormal cellular phenotype. Lymphocyte chromosomal aberrations induced by DEB were decreased during rh-SOD treatment in two patients and B M progenitors were increased in one patient 5 7 . The results were neither convincing enough, nor was B M outgrowth sustained long enough to provoke further study however, disease progression was modulated. There are many potential reasons why the effect of rh-SOD was short-lived in these clinical trials. Native enzymes such as SODs are poor pharmacological tools because there are many limitations to their use as therapeutics. For example, they often have high molecular weights and therefore cannot penetrate cells, limiting the dismutation of superoxide only to the extracellular spaces, and native enzymes are highly susceptible to proteolytic digestion 1 3 3 . The use of a SOD mimetic with high stability and high SOD activity in F A clinical trials might produce a better outcome. In vitro models of oxidative stress have been useful in predicting the use of SOD mimetics (specifically M n metalloporphyrins, such as MnTMPyP) as antioxidants in specific in vivo models of human disease 1 3 3 . Metalloporphyrins have been shown to be protective in in vitro oxidative stress models, and at micromolar levels, protect cultured cells against the toxicity of superoxide generators 1 3 3 . Using MnTMPyP, we were able to partially rescue the growth of HPC from both Fancc'' and Fancc''Sodl'' mice. The correction of HPC growth from Fancc'Sodl'' cultures was not complete, probably due to the fact that superoxide anions are known to readily and sometimes spontaneously dismute to hydrogen peroxide (H 2 0 2 ) which ultimately leads to hydroxyl radical (OH*) 1 3 4 . Another important mechanism by which superoxide anion attenuates 55 disease is through the interaction with nitric oxide (NO) to produce peroxynitrite (ONOO") U 4 . These highly damaging species can lead to lipid peroxidation, protein nitrosylation, induce D N A strand damage which is a trigger for the induction of poly (ADP-ribose) polymerase (PARP), all processes that induce cell death 1 3 5 . Thus, the removal of superoxide anion prevents the formation of cytotoxic OH* and ONOO". The toxicity observed in Fancc''Sodl'' HPC is likely due to the accummulation of damage from all of these species, and the specific scavenging of superoxide alone only rescues damage due to this species and not its breakdown products. In order to completely rescue growth, HPC cultures might need to be treated with a cocktail of antioxidants such as L - N M M A , for NO scavenging, uric acid, for ONOO" scavenging and catalases, for H 2 0 2 scavenging. It is well accepted that low levels of oxidative stress promote cellular proliferation instead of causing cellular death 1 3 4 . This effect occurs through the myriad of signaling roles that exist for ROS in cells. The role for ROS in proliferation was suggested in our HPC cultures grown in the presence of TIRON and MnTMPyP. HPC cultures from wildtype, Fancc'' and Sodr'' mice had a distinct adherent population that was not evident in the Fancc'Sodl'' cultures and growth was fully restored in Fancc'' and Sodl'' HPC cultures. While we did not formally characterize this population (our results are preliminary and strictly qualitative), this differentiated population arose from the initial HPC cells plated. Thus, the resultant marrow hypocellularity and peripheral blood bicytopenia described in Fancc''Sodl'' mice could be due to two related mechanisms, increased apoptosis in the early progenitor compartments as well as decreased proliferative capacity from surviving progenitors. 56 CHAPTER FOUR - FancA1' Sodl'- mice have a less severe phenotype compared to Fancc''Sodl'' Mice 4.1 INTRODUCTION While a role for the F A complex in the nucleus is not disputed, it is becoming increasingly clear that individual F A proteins have important functions that extend beyond the formation of the F A nuclear complex. A significant amount of F A N C A , F A N C G and F A N C C proteins localize to the cytoplasm and the plasma membrane, where they independently interact with various other molecules 1 6 . While information is accumulating regarding these interacting proteins, little is known about the regulatory mechanisms of F A proteins, their interactions in the cytoplasm, or the stimuli required for nuclear translocation of the complex. The F A N C A gene encodes a protein which contains a bipartite nuclear localization signal at its extreme N-terminus, and a partial leucine zipper 2 9 1 3 6 . F A N C A is localized to both the cytoplasm and to the nucleus and a F A N C A mutant with a deleted NLS fails to correct M M C sensitivity, and also fails to bind to F A N C C and F A N C G in the cytoplasm 4 1 . This F A N C A mutant however can still be phosphorylated. These results suggest that the phosphorylation of F A N C A is necessary but not sufficient for its proper cellular function, and that the nuclear localization of F A N C A involves several functional domains 4 1 . The mouse c D N A (Fanca) encodes a protein that shares 65% amino acid sequence identity with human F A N C A , has a ubiquitous pattern of expression in embryonic and adult mouse tissue and its expression in human F A - A lymphoblast cells completely complements the sensitivity to M M C 1 3 7 . Like F A N C C , F A N C A is known to interact with various non-FA proteins, including; the nuclear scaffold protein human oc-spectrin LT 1 3 8 ; the cytoplasmic protein SNX5, required for intracellular receptor trafficking between organelles 1 3 9 ; and BRG1, a component of the human SWI/SNF complex known to be involved in the remodelling of chromatin structure 6 6 . 57 Furthermore, bioinformatic analysis of the coding region of F A N C A has suggested that the protein may have intrinsic peroxidase function 6 1 . A region of the F A N C A protein, encoded by exons 6 to 13 were reported to show limited similarity (4.2%) to the active domain of a superfamily of fungal, plant and bacterial heme (FPBH) peroxidases 6 7 . Within this peroxidase domain, there are six active site amino acid residues that are of major functional importance and of these, three are conserved between the human F A N C A and the F P B H peroxidase motif. Despite the conservation of critical residues, no biochemical function or peroxidase activity has been identified for F A N C A , although this result is complicated by the fact that there is no known substrate for this putative peroxidase, making evaluation of enzymatic activity impossible 6 8 . Given the role of the F A N C C protein in the regulation of both P-450 and GSTP1, known redox regulated proteins, we were interested in examining the possibility that F A N C A may also have a role in regulating the cellular oxidant state. We described in Chapters 2 and 3 that Fancc'' Sodl'' mice have spontaneous B M defects both in vitro and in vivo that exist at the level of the earliest hematopoietic progenitors. To examine whether ROS-mediated F A pathogenesis observed in Fancc'Sodl'' mice is a Fancc-specific effect or a more general F A defect, we generated FancA'''Sodl'' mice and compared the B M phenotype of this strain to the previous Fancc''Sodl'' strain developed in our laboratory. We hypothesized that if the primary defect of the F A disease is to protect the cell from ROS-mediated toxicity, and if F A N C A also has a role in this process, then Fanca-null mice bred with Sodl -deficient mice should result in a similar B M failure phenotype. 58 4.2 MATERIALS AND METHODS Generation of FancA' Sodl'' mice and histological analysis FancA+/~ mice (n=7, backcrossed to C57B1/6) 7 1 were bred with Sodl+/' mice 1 0 8 (n=5, backcrossed to C57B1/6) until mice that were heterozygous at both loci were obtained. Locus-specific PCR was used to genotype mice. Brother-sister matings of FancA+/" Sodl+/' mice were carried out to produce litters having FancA''Sodl'' mice. Knockout mice and littermate controls at 8 weeks of age were used in all experiments. Viral antibody-free mice were housed in the Faculty of Medicine barrier unit according to protocols approved by the Animal Care Committee at the University of Calgary. For light microscopy, tissue samples were fixed in 30% formalin solution and embedded in paraffin and bone sections were first de-calcified before processing. For paraffin-embedded sections; hematoxylin and eosin (H&E) and Masson's trichrome were used. BM Cell Preparation and Clonogenic Assays for committed hematopoietic progenitor cells Mice were sacrificed at 8 weeks of age by C 0 2 asphyxiation. Total B M cells were collected by flushing femurs with cold cdVlEM containing 5% FCS. Cell viability, > 90% in all samples, was determined by trypan blue exclusion. Whole B M cells were plated in 1.1 ml of 1% methylcellulose media supplemented with 10% FCS, 2 m M L-glutamine, 10"4 M 2-mercaptoethanol, 1% bovine serum albumin, 10 pg/ml bovine pancreatic insulin, 200 pg/ml human transferrin, 3 units/ml recombinant human erythropoietin, 10 ng/ml recombinant mouse IL-3, 10 ng/ml recombinant human interleukin-6 and 50 ng/ml recombinant mouse stem cell factor (SCF) (StemCell Technologies, Vancouver, BC). Cells were dispensed using a blunt-ended needle and cultured at a density of 8.5 x 103 cells per 35 mm dish (each sample done in duplicate). Dishes were incubated for 10 days at 37 °C, 5 % C Q 2 in air, > 95 % humidity. 59 Colonies (> 20 cells) were counted on a gridded stage using an inverted light microscope and scored morphologically into C F U - G E M M , C F U - G M / G / M or B F U - E colonies. 4.3 RESULTS Histological analysis of Fane A'''Sodl'' mice. Necropsy and histological analysis of Fane A'''Sodl'' mice revealed modest abnormalities of the liver. On inspection, livers of Fane A'''Sodl'' mice (n=3) did not have the dramatic pale and yellow reticular surface pattern of the Fancc''Sodl'' mice described in Chapter 2. Liver sections were examined by light microscopy, with a typical sample shown in Fig. 21. Liver sections from FaneA+/+Sod1+/+, FancA''', Sodl'', and FancA'Sodl'' mice were stained with Masson's Trichrome. The distinct division between relatively healthy periportal hepatocytes and severely affected centrilobular hepatocytes observed from Fancc'Sodl'' mice was not as obvious in FancA'''Sodl'' mice. Hepatocytes from wildtype, FancA''' and Sodl'' were fairly homogenous with few cytoplasmic vacuoles present. However, liver sections from FancA'''Sodl'' mice did reveal some zone 3 hepatocyte change consisting of multiple cytoplasmic vacuoles (consistent with microvesicular steatosis), that did not displace the nuclei. No inflammatory cell infiltrates were present in the liver, and trichrome stain did not reveal evidence of increased collagen deposition. Oil red O staining (n=2) confirmed the presence of microvesicular steatosis in hepatocytes of FancA'''Sodl'' mice while wildtype controls revealed modest amounts of oil red O-positive droplets distributed in a non-zonal pattern (data not shown due to the low quality of these frozen sections). B M cellularity and body weights are slightly decreased in FancA''Sodl''mice. No developmental defects or gross skeletal abnormalities were detected in FancA'Sodl'' mice. Body weights of FancA''' and Sodl'' mice were not statistically different from 60 FaneA+/+Sod controls (Fig. 22), however both male and female FancA'''Sodl'' mice, had statistically decreased body weights compared to wildtype controls at 8 weeks of age; n=3-4 animals per genotype (p=0.05, 0.008 for male and female respectively). Liver and spleen weights were not increased in any of the mutants as compared to FaneA+/+Sod controls. We also assessed marrow cellularity from FancA+/+Sodl+/+, FancA''', Sodl'' and FancA''Sodl''mice (FigJ 23) and found that the average cell number / femur was slightly decreased (although not significantly) in FancA''' and Sodl'' mice compared to wildtype controls. The average cell number / femur in FancA'''Sodl'' mice was further decreased (3.7 x 107 ± 0.21) compared to wildtype controls (4.9 x 107 ± 0.35), however, this difference was did not reach statistical significance (p=0.06). Colony Forming Assays reveal defective growth from FancA'''Sodl'' committed progenitors. We were primarily interested in the status of committed progenitor growth from FancA''' Sodl''mice and how it compared to the results we obtained from Fancc''Sodl'' mice (see chapter 2). Thus, we plated whole B M into methylcellulose and enumerated the in vitro clonogenic potential of committed myeloid (CFU-GM) and lymphoid (CFU-pre-B) progenitors from FaneA+/+Sod 1+/+, FancA''', Sodl'' and FancA'''Sodl''mice. Fig. 24- represents the average number of progenitors/femur ± S E M from myeloid (a) and pre-B (8) methylcellulose assays for n=3-4 animals per genotype, with each experiment done in duplicate. This data shows that the total number of myeloid and pre-B progenitors/femur from FancA'''Sodl'' mice (p=0.01 and 0.009, respectively) was severely reduced when compared to FaneA+/+Sod 1+/+ controls, where the number of myeloid and pre-B progenitors/femur from Fancc''Sodl'' mice was approximately 15-fold lower than from FaneA+/+Sod 1+/+ controls. The number of colonies obtained from Sodl'' and Fancc'' marrows was also significantly reduced (p=0.009 and p=0.04, respectively) in only the pre-B assays when compared to FancA+/+Sodl+/+ controls. There were no significant differences in Sodl'' and Fancc'' colony formation in the myeloid assays compared to wildtype controls. 61 We also evaluated our methylcellulose results from FancA" Sodl' cultures to determine the frequency of the different cell types arising from a single myeloid progenitor. Thus, the myeloid colonies described in Fig. 24a were scored by cell morphology into C F U - G E M M , C F U -G M / G / M and B F U - E groups and Fig. 25 represents the frequency of progenitors/IO5 B M cells. We found that colonies enumerated from FancA"''Sodl'' samples were mostly erythroid in origin, 57.4 ± 20.44% (p=0.12) with very few C F U - G M / G / M , 8.78 ± 8.0% and C F U - G E M M , 0.46 ± 0.21% colonies being present (p=0.002; p=0.003, respectively). Sodl'', Fancc'1', and Fancc+/+Sodl+/+ progenitors, on the other hand, all gave rise to the different cell types at similar frequencies. 4.4 DISCUSSION The purpose of this study was to determine whether the primary defect in F A is due to a general ROS-deregulation defect or i f this role is specific to F A N C C . Our results indicate that elevated levels of superoxide anion superimposed onto a FancA''' background result in a F A model that is not as severe as the Fancc''Sodl'' mouse described in our earlier studies, however FancA'''Sodl'' mice do demonstrate a significant reduction in clonogenic growth potential of committed progenitors compared to wildtype, FancA''' and Sodl'' controls. The significance of the liver pathology observed from Fancc''Sodl'' mice is unclear since human F A patients have not been reported to have liver pathology (unless anabolic steroids have been administered 2 6 . However, given that C Y T P-450 enzymes are largely expressed, constituitively and inducibly, in the liver 6 2 and that a role for Fancc was identified in the regulation of R E D 5 9 , our observation of a liver defect was of particular interest and indirectly supported the hypothesis that the F A N C C gene product has a role in the regulation of R E D and P-450 mediated-ROS production. While some abnormal liver pathology is evident in FancA'Sodl' ' ' mice, the extent of the defect is not nearly as dramatic as previously observed for the Fancc'' 62 Sodl'' strain. The difference is likely due to the distinct role for F A N C C in R E D regulation. No similar role for F A N C A has been observed 5 3 and the effect seen in FancA''Sodl'' mice may be reflective of purely Sodl'' effects. Sodl'' controls did reveal small amounts of hepatic damage, however, the fact that this phenotype was not greatly exacerbated in the presence of a FancA -null background suggests that FancA may not have as prominent a role in the regulation of the C Y T P-450 system in hepatocytes. It is important to note however, that the Fancc''Sodl'' strain used in Chapters 2 and 3 was of a mixed background and it is thus possible that the phenotype was reflective of a mutation within a modifyer locus or due to some peculiar sensitivity of the Balb/c genetic background. However against this notion, when we backcrossed the Fancc''Sodl'' strain to C57B1/6, (N=5), we found that the liver phenotype persisted as did the B M hypocellularity (n=2-3 mice). The most telling of results obtained from Fancc''Sodl'' mice was the near non-existant clonogenic potential of committed progenitors in methylcellulose. FancA'''Sod1''' mice also showed a significant reduction in committed progenitor frequencies compared to wildtype controls, while both Sodl'' and FancA''' controls revealed a 2-3 fold decrease in progenitor frequency compared to wildtype mice, a result also seen from the Fancc''Sodl'' C F A data in Chapter 2. We can compare the extent of the hematopoietic defect between these strains by assessing the fold decrease in committed progenitors from each double knockout animal to its respective wildtype control. This analysis indicates that the fold decrease of committed progenitor growth from FancA'''Sodl'' mice is 16.6x, and 15.7x for pre-B and myeloid assays respectively, and in Fancc''Sodl'' mice is 66.Ix, and 22.7x, for pre-B and myeloid assays. This comparison suggests that the hematopoietic defect is more severe in Fancc''Sodl'' mice compared to FancA'''Sodl'' mice. This difference in phenotypic severity is reminiscent of the clinical heterogeneity observed in F A patients. It is now well established that F A - C patients tend to have a significantly more severe form of the disease, with the onset of hematological 63 malignancy and B M aplasia occurring at a much younger age compared to other F A groups z o , and this may be reflected in our double knockout strains. The role(s) for each of the F A proteins within the cytoplasm is diverse and it is possible that they do not significantly overlap in function until they are collectively called into the nucleus as a functional complex. Our results suggest that the Fancc protein has a more prominent, or a more direct role, in ROS regulation then the Fanca protein. However, the results are also in keeping with the idea that the F A defect may in fact be a general oxidant stress disorder since specific hematopoietic failure was observed in both knockout strains (over the single mutant controls). While F A N C A may not be directly responsible for the regulation of proteins that generate superoxide anion, it may have a more indirect effect in the regulation of pro-oxidant states within cells. For example, F A N C A has been shown to bind to components of the human SWI/SNF complex 6 6 . It is posssible that the F A N C A protein is partially responsible for opening the chromatin at specific sites for the expression of genes needed to combat pro-oxidant environments. A more detailed analysis of gene expression profiles from HPC cultures of FancA' ''Sodl''' mice may reveal such a mechanism. 64 CHAPTER FIVE - A Novel Role for Nitric Oxide in Mediating Cytokine - Induced Growth Inhibition of Fancc - deficient Bone Marrow Cells. 5.1 INTRODUCTION While the FA genes are ubiquitously expressed in humans and in mice, there is a specific hematopoietic defect within FA individuals that leads to progressive BM failure. In keeping with this observation, a specific role for FANCC in the survival and/or proliferation of hematopoietic progenitor cells (HPC) has been suggested 9 0 . As a potential mechanism to account for the marrow defect in FA, it has been reported that Fancc'' HPC demonstrate increased hypersensitivity to the growth-inhibitory effects of three unrelated pro-inflammatory cytokines: I F N Y , TNFa, and MJPla 7 0' 7 6. Consistent with this result, obtained using murine cells, HPCs from FA-ZVCC-deficient individuals have been shown to upregulate fas and interferon response factor 1 (IRF-1) gene expression at significantly lower doses of I F N Y than required for control cells 9 3 . The apoptotic responses were mediated via the caspase 8-dependent activation of caspase 3 9 4 . Paradoxically however, FANCC cells appear to be hypersensitive to the effects of I F N Y . The activation of STAT1 in response to I F N Y m EBV-transformed FANCC lymphoblast cell lines can be suppressed 9 1 , depending on the nature of the mutation responsible for the loss of FANCC activity 9 5 . We hypothesized that a common mechanism may be responsible for the inhibition of Fancc'' hematopoietic colony formation in response to I F N Y , TNFa and MlPla. Several lines of evidence pointed towards inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO) as a possible candidate for mediating the inhibitory effects of these cytokines. NO, enzymatically generated from L-arginine by one of three NOS isoforms, is a free radical that is also able to react with oxygen to yield other reactive species that range from very stable anions to unstable peroxides 14°. NO is involved in a wide variety of biological processes, for example, NO made by 65 eNOS and nNOS have been implicated in the innate immune response, in tumor killing, control of vascular tone, and in chemotaxis 1 4 1 . NO produced by iNOS in response to factors such as I F N Y , T N F a and Fas-L is seen during infection, inflammation, autoimmunity and apoptosis 1 4 1 . Importantly, NO has also been shown to suppress human hematopoiesis in vitro 1 4 2 . Both fFNy and TNFa , potent inhibitors of hematopoiesis, are known to be capable of inducing iNOS expression and nitric oxide (NO) production in a variety of different cell types 1 4 3 1 4 4 . M l P l a , another suppressor of hematopoiesis 1 4 5 , has also been shown to induce NO generation by human peripheral blood mononucler cells I 4 6 . Furthermore, NO not only amplifies M l P l a responses in lymphocytes, but also increases MIP1 mRNA levels, facilitating the recruitment of polymononuclear leukocytes and macrophages I 4 7 . While much has been published regarding the sensitivity of F A cells to ROS, no information exists in the literature regarding either NO production from FANCC-deficient cells, or the responses of these cells to reactive nitrogen species. We performed experiments to test the hypothesis that an abnormal production of, and/or response to NO might account for the inhibitory effects of proinflammatory factors, I F N Y , M l P l a , and T N F a on Fancc'' B M cells. We observed the following: a) cytokine-dependent inhibition of hematopoietic progenitor growth from murine Fancc'' B M cells was prevented by an iNOS inhibitor; b) Fancc'' hematopoietic progenitors were hypersensitive to NO generating agents; and c) both iNOS gene expression, and NO production were elevated in primary Fancc'' macrophages following exposure to IFNy and I F N Y / L P S 66 5.2 MATERIALS AND METHODS Clonogenic Assays for committed hematopoietic progenitor cells Whole B M cells were plated in 1.1 ml of 1% methylcellulose media supplemented with 10% FCS, 2 m M L-glutamine, 10"4 M 2-mercaptoethanol, 1% bovine serum albumin, 10 pg/ml bovine pancreatic insulin, 200 pg/ml human transferrin, 3 units/ml recombinant human erythropoietin, 10 ng/ml recombinant mouse IL-3, 10 ng/ml recombinant human interleukin-6 and 50 ng/ml recombinant mouse stem cell factor (SCF) (StemCell Technologies, Vancouver, BC). Cells were dispensed using a blunt-ended needle and cultured at a density of 8.5 x 103 cells per 35 mm dish (each sample done in duplicate). Dishes were incubated for 10 days at 37 °C, 5 % CO2 in air, > 95 % humidity. Colonies (> 20 cells) were counted on a gridded stage using an inverted light microscope and scored morphologically into C F U - G E M M , C F U - G M / G / M or B F U -E colonies. Peritoneal Macrophage Isolation 8 week old mice (N=7 on C57/B16 background) were injected with 1 ml of 3 % thioglycollate. On day 5, the mice were sacrificed by CO2 asphyxiation. A small incision was made on the body of the mouse which exposed the peritoneal lining and the skin was gently pulled away such that the peritoneal lining was kept intact. 10 ml of D M E M + 10% FCS was injected into the peritoneum and the media was collected using a 18 gauge needle. The cell suspensions were centrifuged at 1200 rpm for 5 min, resuspended at 1.5 x 106 cells / 2 mis media and grown in a 6 well dish in a humidified chamber in 5% C 0 2 in air. Five hours later the adherent peritoneal macrophages were washed twice with warm PBS to remove the contaminating suspension cells and fresh D M E M + 10% FCS was added. The next day, J F N Y (10 ng/ml) with or without LPS (100 ng/ml) was added to the macrophage cultures. The supernatants were collected for nitrite ELISA tests and lysates were made from the cells. 67 Column Purified Hematopoietic Progenitor (HPC) and BM-Derived Macrophage (BMDM) Isolations Whole B M cells were collected by flushing in a M E M + 5% FCS both femurs from 2 mice per genotype and pooling the samples. Cell viability, > 90% in all samples, was determined by trypan blue exclusion. FJPC isolations were performed as previously described (in Chapter 3). For B M D M cultures, B M samples were centrifuged at 1200 rpm and resuspended at a density of 107 cells/ml in a 10 cm 2 dish in D M E M + 10% FCS + 5% CSF-1 conditioned (cell-free) media. The next day all suspension cells were removed into a sterile 50 ml Falcon tube and the adherent (stromal) cells were discarded. The cells were centrifuged and resuspended in twice the original volume of fresh D M E M + 10% FCS + 5% CSF-1. Cells were plated at a density of 8.5 x 106 cells / well of a 6-well tissue culture dish and were allowed to grow in a humidified chamber in 5% CO2 in air for 8-10 days or until the culture became adherent and confluent. Fresh media was added every third day. Immunoblotting and densitometry Macrophage cells were lysed in Phosphorylation Solubilization Buffer (PSB) (50 mM HEPES, 100 m M NaF, 10 m M Na 4 P 2 0 7 , 2 mM N a 3 V 0 4 , 2 m M E D T A , 2 m M NaMo0 4 , 1% Triton X freshly added, pH 7.35) in the presence of the following additional protease inhibitors Leupeptin (1:1000), Aprotinin (1:1000), PMSF (1:1000) (Roche Diagnostics, Mannheim, Germany). Whole cell lysates were centrifuged for 5 min at 12,000 rpm to remove cellular debris and supematants were collected into a fresh tube and stored at -20°C. Protein concentration was determined by Bradford-method-based assay. Lysate volume corresponding to 40 and 80 pg of total protein (iNOS and Statl blots respectively) was diluted 6:1 with Laemmli sample buffer and the samples were boiled for 5 min prior to electrophoresis. Total cell lysates were separated by SDS-PAGE at 150 V and transferred to PVDF membranes by electroblotting using a semi-dry 68 transfer method at 25 V for 45 min at RT in a solution containing semi-dry transfer buffer (192 mM glycine, 25 mM Tris 10% SDS and 20% methanol). Filters were blocked for 1 hour at RT in TBST (10 mM Tris (pH 8.0), 150 mM NaCl and 0.05% Tween-20) containing 5% BSA. Filters were incubated overnight at 4°C in TBST with 1% BSA with one of the following antibodies; anti-iNOS (1/1000), (Upstate Biotechnology, Lake Placid, NY): anti-Statl (1/1000), anti-P-Statll (1/1000), (Cell Signaling Technology, Beverly, MA) or anti-a-tubulin (1/500) (Sigma, St. Louis MI). After three TBST washes, filters were incubated for 1 h at RT with a horseradish peroxidase-conjugated secondary antibody (Jackson lrnmunoResearch Labs Inc., West Grove, Pennsylvania). Proteins were detected by chemiluminescence (Amersham, Arlington Heights, IL) using Flour-S-Max Imager equipped with densitometry software (Bio-Rad Laboratories Ltd. Mississauga ONT). Nitrite ELISA Supernatants were collected from the previously mentioned stimulated cell cultures and stored at -20°C. 10 pi of 30% (w/v) ZnS0 4 was added to a fresh eppendorf tube containing 250 pi of each supernatant sample, vortexed and incubated at RT for 15 min. The samples were centrifuged at 4000 rpm for 5 min to collect the debris and the clean supernatant was transferred to a fresh eppendorf tube containing 0.5 g cadmium beads. The samples were nutated in the presence of the beads overnight at RT. The next day the samples were transferred to a clean tube, the cadmium beads were removed and the supernatants were centrifuged at 10000 rpm for 5 min. 100 pi of nitrite standards and 100 pi of each sample were loaded in duplicate onto a 96 well ImmunoSorp ELISA plate (NUNC, VWR Inc.). 50 pi of Color Reagent 1 was added to each well and the samples were briefly mixed. 50 pi of Color Reagent 2 was added to each well and the whole plate was incubated at RT for 15 min for the samples to develop. Colour reagents and nitrite standards were provided by Oxford Biomedical Research (Oxford, ML). Absorbance was 69 measured at 540 nm in a Multiskan Ascent Microtiter Plate reader (Dynex Labsystems, Chantilly, Virginia). Data was collected as u M concentrations of nitrite based on a standard curve of nitrite done on each plate and was normalized to total protein. Flow cytometry 1 x 106 cells were resuspended in 500 pL PBS + 2% FCS (FACS buffer) and blocked on ice with 1 pg of anti-FcyRIIb (2.4G2, Pharmingen, Mississauga, ON) for 30 min. The cells were washed once in FACS buffer and then stained with one of the following antibodies for 1 hour at 4°C; 0.5 pg anti-CDllb-FITC, 0.5 pg anti-CD 14-FITC or 0.5 pg anti-CD 119-FITC (Pharmingen, Mississauga, ON). Cells were washed 3x with FACS Buffer and resuspended in 500 pL of buffer before analysis on a FACSort (Becton Dickinson, Mountain View, CA) flow cytometer equipped with CellQuest software (Becton Dickinson). Peritoneal macrophages were immunophenotyped for the following cell surface markers; C D l l b , CD14, and CD119 (JFNyR). There was no difference in the % staining of any of these receptors between Fancc'' and wildtype macrophages (n=3). Chemicals Diethylenetriamine Nitric Oxide Adduct (DETA/NO) was purchased from Sigma-RBI (St. Louis, Missouri). S-nitroso-N-acetyl-D,L-penicillamine (SNAP), and NG-Monomethyl-L-arginine, ( L - N M M A ) , were purchased from Calbiochem (San Diego, CA) . rm IFN-Y, rm TNF-a and rm MEP- la were purchased from R & D Systems Inc. (Minneapolis, MN) . A l l chemicals were diluted in a M E M . 70 5.3 RESULTS Cytokine - inhibited colony growth of Fancc'' B M cells is rescued with L - N M M A . B M cells from wildtype and Fancc'' mice were plated into methylcellulose cultures in the presence of increasing doses of IFNy. Consistent with previous reports 7 0 ' 7 6 , F i g 1 26a demonstrates that Fancc'' B M cells exhibited a dose-dependent inhibition of colony number in response to rPNy (compared to wildtype controls) that was maximal at 1 ng/ml (p = 0.003), (n=4 animals per group). B M cells were plated in the presence of 1 ng/ml I F N Y and increasing concentrations of the i N O S inhibitor, N°-monomethyl-L-arginine , ( L - N M M A ) . A s shown in Eigjj 26B, at 0.1, 0.25 and 0.5 m M L - N M M A there was complete rescue of Fancc'' colony formation in the methylcellulose cultures (n=3). The average Fancc'' colony numbers at these L - N M M A doses are not significantly different from those generated by the wildtype controls (p = 0.75, 0.85 and 0.82 respectively); in contrast, they are significantly different from Fancc'' colony numbers when cells were grown in the presence of 1 ng/ml WNy in the absence of L - N M M A (p = 0.018, 0.025, 0.003). These results suggested that the inhibition of Fancc'' colony formation by IFNy was a consequence o f N O generation. Given this result, and the capacity of two unrelated cytokines to also induce N O production by hematopoietic cells, we examined whether inhibition of colony formation by T N F a and M l P l a was similarly reversible by L - N M M A . B M cells were plated in methylcellulose in the presence of either 0.5 ng/ml T N F a , or 1 ng/ml M l P l a , with or without L - N M M A , n=3 (F|gv«27a and b respectively). F ig . 27a shows that the growth of Fancc'' progenitors when treated with T N F a alone was significantly suppressed as compared to wildtype TNFa-treated cultures (p =0.007). A s seen in the case of the rFNy-treated cultures, TNFa-mediated inhibition of Fancc'' colony growth was reversed in the presence of 0.25 m M L - N M M A (p = 0.36). Growth of T N F a treated Fancc'' cultures was significantly different from Fancc'' cultures grown in the presence 71 of T N F a plus L - N M M A (p = 0.01). Fig. 21b shows that Fancc'' progenitors treated with M l P l a were significantly inhibited as compared to MlPla-treated wildtype cultures (p =0.003; n=4 animals per group). In contrast, growth of Fancc'' B M cells in the presence of M l P l a plus 0.25 mM L - N M M A was no longer significantly different from that of wildtype cells maintained under the same conditions (p = 0.21); also, the average colony number was not significantly different from Fancc'' MlPla-treated cultures (p = 0.29). Increasing L - N M M A to 0.5 m M restored Fancc'' progenitor growth to wildtype levels under the same conditions (p = 0.41); and different from no L - N M M A (p = 0.02). As rescue of M l P l a treated progenitors occurred at a somewhat higher dose of L - N M M A , it suggested that at the concentration tested, M l P l a was generating higher levels of NO than the other two cytokines. Together, these results suggested that NO generation might be a common mechanism through which these three structurally-unrelated polypeptides bring about inhibition of hematopoietic progenitor cell growth. Fancc'' B M cells are hypersensitive to NO donors. Since Fancc'' B M progenitor cells show increased sensitivity (compared to controls) to the above cytokines and growth was reversed by L - N M M A exposure, we hypothesized that Fancc'' B M cells might also be hypersensitive to NO. To test this possibility, colony formation was measured in the presence of two mechanistically-distinct NO donors. B M cells from wildtype and Fancc'' mice were plated in increasing concentrations of S-nitroso-N-acetyl-D,L-penicillamine (SNAP), an NO donor, with colony number displayed as percent of maximal colony number (n=4 animals per group) in Fig. 28a. Although both wildtype and Fancc'' progenitors exhibited a dose-dependent inhibition of colony number in the presence of SNAP, Fancc'' progenitors generated fewer colonies at 0.06 and 0.25 p M SNAP as compared to wildtype controls (p = 0.04 and 0.05, respectively). The reduction in colony number was due to a similar reduction of growth from erythroid as well as G M / G / M progenitors in Fancc'' cultures, 72 while wildtype mice appeared to show a greater inhibitory effect on erythroid colonies as compared to G M / G / M , as previously observed for wildtype mice 1 4 8 . Since SNAP produces large amounts of NO over a wide concentration range, and can also generate/donate additional ROS and sulfhydryls 1 4 0 1 4 9 5 growth inhibition was difficult to attribute solely to NO 1 4 9 . Therefore, wildtype and Fancc'' B M cells were plated in the presence of increasing doses of Diethylene triamine nitric oxide adduct (DETA/NO), a member of the NONOate class of NO donors with a half-life of approximately 20 hr in cell culture and with minimal potential for the generation of additional reactive species 1 4 9 . Fig.J28$ depicts data from four separate experiments in which we observed a strong reduction in Fancc'' B M colony formation at the lowest dose of 5 p M DETA/NO (p = 0.004) that was further reduced at the highest dose tested, 100 p M DETA/NO (p = 0.0009). As seen in Fig. 286, the effect of DETA/NO on wildtype colony formation was minimal at all doses of this agent. These results indicate that committed hematopoietic progenitors of Fancc'' mice are highly sensitive to the growth-inhibitory effects of NO donors. Apoptosis of fENy-treated HPC from Fancc'' mice is inhibited by L - N M M A . To determine whether tENy-mediated NO production inhibited the growth of more primitive hematopoietic progenitor populations (HPC), we cultured column purified HPC in 10 ng/ml fFNy, in the presence and absence of 0.5 mM L - N M M A . After 3 days in culture, cell counts were used to ascertain the effects of these conditions on HPC expansion (Fjg^ 29a), n=4, each count done in duplicate. As expected, I F N Y inhibited the growth of both wildtype, 82% and Fancc'', 58% HPC as compared to the untreated controls (p = 0.04). However, when cultured in the presence of I F N Y P m s the addition of 0.5 mM L - N M M A , cell HPC growth was restored, being.109% for wildtype, and 114% for Fancc'' compared to untreated control populations. To investigate the potential contribution of apoptosis to the inhibitory effect of I F N Y , day 6 HPC were assayed for apoptotic nuclei using a flow cytometry-based T U N E L assay (Fig. 29b), n=4. 73 These experiments revealed that the diminished proliferation of IFNY-treated Fancc'' FfPC was accompanied by a trend towards increased levels of apoptotic cells in the cultures. This effect was blocked by the addition of 0.5 mM L - N M M A to the tFNY-containing cultures of both wildtype and Fancc'' HPC which demonstrated few if any apoptotic nuclei (Fig. 29b). Fancc'' macrophages have elevated expression of iNOS. Given the increased sensitivity of Fancc-deficient cells to NO-generating cytokines, and the ability of L - N M M A to blunt the effects of these, we hypothesized that altered regulation of iNOS might be a feature of these cells. Since progenitor cells that give rise to colonies in the methylcellulose experiments are difficult to purify in sufficient numbers to carry out signal transduction analyses, an alternate BM-derived primary cell source was selected to study the response of the iNOS gene. We first investigated the response of thioglycollate-elicited primary peritoneal macrophages to the combined stimulus of fFNy plus bacterial lipopolysaccharide (LPS) and measured the expression of the iNOS protein. Fig. 30« (top panel) is a representative time-course immunoblot showing iNOS expression in peritoneal macrophages obtained from wildtype and Fancc'' mice following stimulation with 10 ng/ml I F N Y a n d 100 ng/ml LPS. It is evident that induction of iNOS protein occurs more rapidly in the Fancc-deficient cells, and reaches a higher level at the 12 hr time point than that of controls. This is again shown in the densitometry Fig] 30t> which represents the average of five independent experiments, with the data expressed as iNOS expression normalized for loading using a-tubulin. The increase in iNOS is significantly higher in Fancc'' macrophages as compared to wildtype controls at 8, and 12 hours post-stimulation (p = 0.02, 0.04 respectively). This suggests that Fancc-deficient thioglycollate-elicited peritoneal macrophages show an altered regulation of iNOS protein expression following the potent inductive stimulus of I F N Y a n d LPS. The expression of iNOS in peritoneal macrophages from wildtype and Fancc'' mice stimulated only with fFNy alone did not consistently demonstrate increased iNOS expression in 74 the Fancc'' cells, showing this in only three of five independent experiments. The intraperitoneal injection of thioglycollate broth, however, constitutes a pro-inflammatory stimulus that could affect the baseline state of the macrophages, potentially leading to animal-to-animal variation in the responses to a weaker stimulus (for example, I F N Y alone). W e therefore also assessed IFNY-induction of i N O S in BM-derived macrophages ( B M D M ) , cultured from total marrow cells for 7 days, in the absence of any pro-inflammatory stimulus prior to cytokine exposure. A s seen in the immunoblot shown in Fig. 31a (upper panel), Fancc'' B M D M stimulated with 10 ng/ml fFNy expressed higher levels of i N O S than controls. Densitometry analysis in Fig. 31i» shows that i N O S expression was maximal in IFNy-treated Fancc'' B M D M at 8 hours post-stimulation, while the greatest difference over control B M D M was at 5 hours (p = 0.04). This result indicated that Fancc-deficient bone marrow derived monocytic cells were able to generate higher levels of i N O S post-IFNY stimulation than control cells, suggesting a possible explanation for the increased sensitivity of these cells to the growth-inhibitory effects of this cytokine. Fancc'' macrophages have increased nitrite production. T o determine whether the increased levels of i N O S seen in the Fancc-deficient macrophages translated to increased production of N O , we measured levels of nitrite in the supernatants of stimulated macrophages. A s shown in Fig. 32, we found a significant increase in nitrite production from Fancc'' macrophages when these were stimulated with I F N Y p l u s L P S . This difference was statistically distinct from that of wildtype samples at the 8 hrs post-stimulation point (p = 0.04). Fancc'' macrophages stimulated with I F N Y alone revealed an increase in nitrite production compared to wildtype samples at 5 and 8 hours, however, this increase did not reach statistical significance. Thus, there was evidence of a correlation between the levels of i N O S generated and in vitro N O production by the macrophage populations. 75 Phosphorylated Statl is increased in Fancc'' macrophages stimulated with IFNy Several transcription factors, including Statl, are known to regulate expression of the iNOS gene in response to IFNy stimulation 1 4 0 . Given our results which show elevated iNOS expression in Fancc'' cells, we were interested in determining the tyrosine phosphorylation status of Statl in response to IFNy. Thus, peritoneal macrophages from wildtype and Fancc'' mice were stimulated with 10 ng/ml I F N Y a ° d phospho-Statl (P-Statl) levels assessed over time by immunoblotting. Fig. 33a is a representative experiment showing P-Statl levels in wildtype and Fancc'' peritoneal macrophages following stimulation with IFNy (top panel) and normalized for loading using a Statl antibody (lower panel). Densitometry data, shown in Fig. 33b represents four independent experiments with P-Statl expression being normalized for loading using anti-Statl. This data shows that Fancc'' macrophages generate higher levels of P-Statl at 15 min post-stimulation (p = 0.04), as compared to wildtype controls. The possibility of increased cell surface expression of IFNy receptor in Fancc-deficient cells was excluded by flow cytometry using anti-CD119 antibody staining. As Statl can be a positive regulator of iNOS expression 1 5 0 , our results are in keeping with the increased levels of iNOS observed in IFNY" s t i m u i a t ed Fancc-deficient B M cells. Another transcription factor, Hifloc, was recently shown to induce iNOS expression in response to IFNy 1 5 1 . In preliminary experiments, we examined H i f l a protein levels in peritoneal macrophages (n=2) following treatment with 10 ng/ml I F N Y f ° r 14 hrs, in ambient oxygen. We observed an ~2-fold increase in the expression of H i f l a in Fancc'' macrophages over that of wildtype controls (Fig. 34). This transcription factor may act in concert with Statl to mediate the observed effects of I F N Y in Fancc-deficient cells. 76 5.4 DISCUSSION It has been proposed that IFNy ar>d the two other pro-inflammatory cytokines we evaluated, TNF and MlPla, whether released constitutively, or as a result of intercurrent illnesses 3 4 , might play a role in the progressive failure of hematopoiesis seen in human FA 9 3 . Although all murine FA models generated to date lack spontaneous marrow aplasia, increased sensitivity of Fancc'' BM cells to these three cytokines has been demonstrated by both strains 1 0 , 1 6 . Given that NO is suppressive to normal hematopoiesis 1 4 2 , and the fact that all three cytokines are capable of inducing iNOS, it was important to evaluate the effects of the broad-spectrum NOS inhibitor, L-NMMA, on cytokine-inhibited Fancc'' colony formation. Our data support the hypothesis that cytokine inhibited Fancc'' progenitor growth in vitro is mediated primarily through NO generation. The effects of L-NMMA on JENy-mediated inhibition of hematopoietic cells was not confined to committed progenitors assayed in the methylcellulose CFAs, since rescue of more primitive HPC in suspension cultures was also achieved with L-NMMA. The finding that NO donors were inhibitory at lower concentrations to Fancc'' committed progenitor cells in methylcellulose cultures was a novel finding, This suggested that murine Fawcc-deficient cells were more sensitive to the toxic effects of this radical or its derivatives. In keeping with this, we found that the increased number of TUNEL-positive cells in JPNY-treated HPC cultures was returned to normal levels by the presence of L-NMMA. The apparent sensitivity of Fancc-deficient BM progenitors to NO is of considerable interest, and is similar to the well documented sensitivity of FA cells to oxygen 7'8. We were unable to find information about the role of reactive nitrogen species in the pathogenesis of FA, despite evidence that NO is an inhibitor of hematopoiesis 1 4 2 . A considerable amount of experimental evidence indicates that NO has a role in DNA damage, necrosis, and apoptosis I 4 L. NO is a stable free radical that has been detected in many cell types and yields other radicals that range from very stable anions to unstable peroxides 1 4 0 . 77 NO and its derivatives (N 2 0 3 , N0 2 " or NO + ) are known to be genotoxic, causing D N A damage both directly and indirectly. Of particular interest to FA , NO readily reacts with superoxide, which may be elevated in F A cells 5 1 , leading to the formation of the highly reactive entity, peroxynitrite (ONOO") 1 4 1 . ONOO" is capable of causing single and double-strand D N A breaks, oxidative lesions (such as 8-oxoG), induction of PARP, Fe 2 + release, G S H depletion, and cell death , 5 2 " 1 5 4 . NO may also potentially cause damage indirectly by altering the activity of repair molecules, for example, 06-methylguanine-methyltransferase, and Fpg, a bacterial protein responsible for removal of 8-oxoguanine residues from D N A 1 5 5 1 5 6 . Many of the above mentioned lesions have the potential to induce chromosomal aberrations and other mutations that might predispose F A individuals to acute myeloid leukemia. To gain some insight into the cell signal transduction events that might provide a mechanistic explanation for the increased sensitivity of BM-derived cells to I F N Y , a population of primary cells was required that could be obtained in sufficient numbers to permit biochemical analyses. For this reason, peritoneal and BM-derived macrophages were selected for study. iNOS and NO studies are readily performed in macrophages where the stimuli required for iNOS responses have been well characterized 1 4 0 . Furthermore macrophages are appropriate for study in FA, given the possibility that cytokines and NO derived from these cells conceivably play a pathogenic role in F A . Strong stimulation of iNOS expression and subsequent NO production from murine macrophages can be achieved with I F N Y a r j d LPS co-stimulation. Employing this stimulus, we found that Fancc'' peritoneal macrophages generated elevated levels of iNOS expression and NO production as compared to controls. When only I F N Y w a s u s e - d to stimulate Fancc'' peritoneal macrophages, elevated iNOS expression was observed in only three of five independent experiments. This variability may have been due to the fact that the recruitment of macrophages and neutrophils into the peritoneal cavity in response to thioglycollate broth results in the partial activation of these cells, in part owing to chemokine and cytokine production by 78 resident peritoneal macrophages 1 5 7 . For this reason, we were interested in assessing the iNOS response to IFNy in naive cells of the monocyte-macrophage lineage, thus B M D M were obtained for study. In keeping with the results observed when peritoneal macrophages were challenged with the potent I F N Y a ° d LPS combination, B M D M from Fancc'' mice also generated higher levels of iNOS in response to IFNy as compared to controls. These results provide a plausible explanation for the increased sensitivity of Farccc-deficient murine hematopoietic progenitors to I F N Y , namely, that increased levels of iNOS, and NO, in these cells leads to growth inhibition that is greater than that of Fancc-proficient cells. In this regard, it would be very interesting to establish whether Fancc'1inos'' B M cells exhibit resistance to the growth-inhibiting effects of the cytokines tested herein. Cells of the monocyte-macrophage lineage are highly sensitive to a wide variety of stimuli that lead to the induction of iNOS and NO production 1 4 0 . In addition, iNOS expression has been detected in CD34+ progenitor cells following exposure to I F N Y , a ° d NO in this system was shown to exhibit inhibitory effects on proliferation 1 4 2 . Interestingly, the constitutive expression of iNOS was described in B M macrophages of patients with myelodysplastic syndromes (MDS) as well as in idiopathic aplastic anemia 1 5 8 , 1 5 9 . Many contributory factors may be involved in promoting marrow aplasia in humans, while at the same time leaving other tissues uninvolved. These potentially include, a) the uniqueness of the marrow cell populations, many of which possess the ability to generate high levels of myelosuppressing NO, as well as NO-inducing cytokines in response to a wide variety of stimuli, b) the sinusoidal nature of the marrow vascular system, which may favor the accummulaton of NO, chemokines, and cytokines c) and the myelosuppressive potential of NO. The regulation of the iNOS promoter is complex, and regulation of expression occurs at several different levels 1 4 ° . A large number of transcription factor consensus binding-site elements are present within the murine iNOS promoter, including sites for N F - K B , STAT1 and H I F l a 1 4 0 . Since we found that iNOS expression was higher in 79 rFNy-stimulated Fancc-deficient cells, it was important to begin to define signal transduction pathways that might account for this differential effect. Along these lines, we have found that two transcription factors, P-Statl and possibly Ffifla, known positive regulators of iNOS gene expression following IFNy stimulation, were expressed at higher levels in LFNy-treated Fancc'' macrophages than in control cells. The novel finding that H i f l a levels may be altered in Fancc'' cells treated with IFNy suggested a possible contributory mechanism for the increased levels of iNOS seen in Fancc-deficient cells. P-Statl levels were significantly increased in Fancc-deficient cells post-JFNy stimulation for 15 minutes, a finding that contrasts with the reduced levels of JFNy-induced P-Statl that have been seen in some human F A mutant cell lines 9 1 ' 9 5 . There are several potential reasons for this difference. First, we determined the P-Statl response to IFNy in primary murine macrophages rather than EBV-transformed human lymphoblasts; secondly, the murine cells used here were null for the Fancc protein, as compared to human cells which generally harbor point mutations of FANCC91. Thirdly, there exists the possibility that our observations are a characteristic of the predominantly C57B/6 (N=7) genetic background of our Fancc'' mice. Indeed, since all of our findings have been generated through the use of Fancc-deficient primary mouse cells, it would be of considerable interest to determine whether dysregulation of iNOS and NO production and/or the increased sensitivity NO generating compounds are characteristics that are also shared by human F A hematopoietic cells. 80 CHAPTER SIX - DISCUSSION 6.1 Results Summary The primary objective of this thesis was to study the role of increased reactive oxygen and nitrogen species on hematopoiesis in Fancc - deficient mice in vivo. The overall conclusion from this work is that elevated levels of either superoxide anion or nitric oxide lead to aberrant hematopoiesis in Fancc'' cells both in vivo and in vitro that is not evident in the wildtype or single mutant controls. Since the Fancc protein may be responsible for the regulation of redox potential within the cell through its interactions with RED and GSTP1 5 9 ' 6 1 , then a lack of Fancc would be predicted to lead to elevated levels of ROS. However, FA-null mice do not develop 'spontaneous' B M failure unless they have been challenged with M M C in vivo. Due to intrinsic differences between murine and human cells, the levels of ROS that might play a role in marrow aplasia may not be present in Fancc-mx\\ mice unless environmental, or genetic fcators are introduced to create oxidative stress. Thus, we hypothesized that if elevated ROS/RNS were an important pathogenic mechanism in FA, then one would expect that a Fancc'' mouse with an intrinsic pro-oxidant state would resemble human FA. In Chapter 2 we observed that a lack of Sodl, superimposed onto a Fancc - null background results in a hematopoietic phenotype with some similarities to human FA, namely, decreased B M cellularity, reduced peripheral blood R B C and leukocytes, and a complete lack of committed progenitor growth in vitro. Our model is different from the human F A disease in that Fancc'Sodl'' mice develop microvesicular steatosis, and do not develop thrombocytopenia or malignancy with age. It is likely that Fancc'' mice have additional in vivo compensatory mechanisms for dealing with ROS since F A null mice do not spontaneously develop aplasia. It is possible that in humans with FA , a polymorphism or the loss of one allele of a redox or antioxidant gene would result in a pro-oxidant state that might influence disease progression. F A 81 patients and cell lines have not been systematically evaluated for heterozygosity of either other F A genes, or additional candidate genes involved in the regulation of oxidant state. Furthermore, there is the possibility that antioxidant proteins may be damaged, via nitrosylation for example, resulting in diminished activity, and hence a pro-oxidant state in vivo. Factors such as these would help to further explain the heterogeneity differences described for F A in combination with genotype-phenotype correlations that are already known. Since hematopoiesis is hierarchical in nature, we were interested in identifying the specific population of cells that was defective in Fancc''Sodl'' mice and that eventually resulted in diminished peripheral blood counts, B M hypocellularity and reduced committed progenitor numbers. Therefore, in Chapter 3 we investigated whether Fancc is required for normal early progenitor growth in vitro and if the presence of increased intrinsic superoxide anion further affects growth in this compartment. We found that early hematopoietic progenitors do exist in the marrow of Fancc''Sodl'' mice and, the specific scavenging of superoxide anion partially modulated the course of cell death in both C A F C and Lin" HPC cultures, suggesting that superoxide-mediated toxicity was at least partially responsible for both the apoptotic phenotype and the lack of proliferation observed^ We were not able to describe all of the reactive species that were elevated in Fancc''Sodl'' progenitor cells, however, we hypothesize that the dismutation of superoxide anion into H2O2, OH" or ONOO" (through interactions with NO) all contribute to lack of progenitor growth. The major goal in the creation of a FancA'''Sodl'' mouse strain was to determine whether the hematopoietic defect observed in Fancc'Sodl'' mice was specific to the Fancc gene alone, or if the F A disease is due to a more general ROS-mediated toxicity defect. In this preliminary study, we observed that FancA'''Sodl'' mice have a reduction of committed progenitor growth in methylcellulose cultures, although the defect was not as dramatic as that observed for Fancc'' Sodl'' mice. Furthermore, hepatic steatosis did not develop in FancA'''Sodl'' mice, and was 82 presumably due to the function of the Fancc protein in the direct regulation of RED, known to be highly expressed and inducible in the liver. In Chapter 5 we identified a novel mechanism for the observation that F A B M cells are hypersensitive to the effects of inhibitory cytokines in vitro. We were able to show that the growth of B M progenitors can be fully restored to wildtype levels when cytokine-treated cultures were grown in the presence of a NOS inhibitor, L - N M M A . We were also able to show that F A B M progenitors were hypersensitive to NO donors in vitro. These results raise some interesting clues about the source of internal cross-linkers in human F A patients and implicate a new pathway to the pro-oxidant states already described for this disease. 6.2 A Role for FA Proteins in Redox Regulation. Many intriguing questions remain in the field of F A research. Perhaps the most pressing of these has to do with the primary defect in this disease and resolution of the two major hypotheses that exist. The first suggests that the F A proteins are involved in the repair or recognition of specific types of D N A damage, and the lack of an F A protein results in an abnormal repair 'surveillance' system that is manifested primarily by chromosomal aberrations. The observation that the F A proteins co-localize with B R C A 1 at nuclear foci in response to damage induction or that D N A repair proteins co-exist in a large surveillance complex that scans the genome for errors is an intriguing and likely concept. If in fact the F A proteins are part of a large repair machine, they conceivably have a distinct role in the repair of a very specific type of D N A lesion, such as the homologous recombination repair pathway, involved in the correction of cross-links. While several studies have clearly shown a role for the F A protein complex, direct or indirect, in the repair of damaged D N A , the 'damaging' source remains unidentified. D N A cross-link formation does not occur spontaneously in vivo, and if a small subset of cross-links did 83 appear spontaneously, the reason(s) why the B M cells are preferentially affected cannot be explained by this model. It is also clear that the F A proteins have distinct roles outside of the nuclear complex and that these roles are important in the proper function of the cell. The idea that the F A proteins have both nuclear and cytoplasmic functions that are not mutually exclusive is analogous to other chromosomal instability disorders with cancer predisposition such as ataxia telangiectasia where the mutant protein, A T M has a role in both D N A repair and ROS detoxification 1 6 ° . Given the interactions described for F A N C C and F A N C G with cytochrome P-450 enzymes and F A N C C in the regulation of GSTP1, it seems likely that these F A proteins have an indirect role in maintaining cellular oxidant states. Thus a mutation in one of these proteins necessarily leads to elevated ROS levels and an upset in the balance of detoxifying antioxidants. These studies raise the possibility that F A proteins may have a more general role in the regulation of structurally similar proteins that are required for the protection against oxidative stress. In keeping with this hypothesis, and in light of our results in chapter 5 which implicate nitric oxide synthase (NOS) and its product NO in the pathogeneisis of F A , it is important to note that the NOS enzyme has similar functional domains as the cytochrome P-450 reductases 1 6 1 . The N-terminal region of NOS contains a heme oxidase domain and the C-terminus contains a flavin reductase domain, both of which are strictly conserved between the two molecules 1 6 1 . Based on this similarity and our observation that F A B M cells are hypersensitive to NO donor drugs in vitro, it would be worth investigating a possible interaction between F A N C C or F A N C G and NOS. Perhaps the improper regulation of this enzyme within a subset of monocytes in the B M enviornment would lead to slightly elevated levels of NO species. It is known that superoxide anion is a by-product of P-450 reduction 1 1 8 and that P-450 and NOS can exist in similar cells 1 3 4 . Thus, the abnormal regulation of P-450 by either mutant F A N C C or F A N C G proteins could lead to elevated levels of superoxide anion, known to rapidly react with NO to produce the very potent oxidizing by-product peroxynitrite (ONOO). The result is a pro-oxidant state that develops 84 within the cell as a result of ROS and/or RNS build up, that in turn overwhelms the antioxidant systems of the cell leading to cellular damage. 6.3 A Role for NO in FA BM Aplasia It is important to remember that the B M is the most consistently and severely affected tissue in FA. Aplasia is the major cause of morbidity in F A patients and research should be focused on the reasons why this organ is so sensitive to a lack of F A protein function. Explanations for the inhibitory effects of I F N Y o n F A N C C HPC cannot rely primarily on the observation that STAT1 has decreased phosphorylation 9 1 since it is known that IFNy does not always relay its signaling messages through STAT1 3 4 . Furthermore, F A HPC are also hypersensitive to the killing effects of T N F a and M l P l a , which do not signal through STAT1 3 4 . A more general defect must exist within F A HPC or within the B M microenvironment in order to explain the sensitivity of F A B M cells to induced cell death from unrelated cytokines. We have proposed in the second half of this thesis that NO, either from activated macrophages or from natural cellular processes such as C a 2 + signaling, may have a direct role in the selective death of F A HPC. The particular sensitivity of F A cells to D N A interstrand cross-linkers and the fact that this sensitivity exists primarily within the B M microenvironment in vivo, suggests that damage from an endogenous cross-linker that either exists within, or is produced from, cells in the B M of F A patients, leads to eventual B M aplasia. We believe that NO, either alone or in combination with other ROS, may lead to the formation of endogenous cross-linkers initiating the cellular phenotype observed for FA. NO is a good candidate for this role since all mammalian cells can produce low levels of N O and are also influenced by this species 1 4 ° . N O is known to cause mutagenesis leading to cell death, protein and lipid damage and defective cellular signaling. In fact, the majority of the cellular phenotypes described for F A to date can be explained by NO-85 mediated effects (Fig 35). NO can lead to D N A damage either directly or through one of its breakdown products, N2O3 or ONOO". For example, a high level of 8-hydroxy-2-deoxyguanine (8-OHdG) bases have been detected in the D N A of F A cells 5 2 . 8-OHdG is a damaged D N A base considered to be a sensitive marker for oxidative D N A damage and can arise as a result of direct ROS damage as well as from ONOO" induced damage ! 2 0 . Thus ONOO", the product of superoxide anion and NO, and a potent oxidizing agent, can lead to a specific type of D N A damage that is commonly found in F A cells. A study that examined the levels of 8-OHdG in F A cells with and without L - N M M A treatment would help to determine whether endogenous NO levels were increased in F A patients. Perhaps more importantly, NO exposure can also lead to the formation of single-strand breaks, D N A intra- and interstrand cross-links and DNA-protein cross-links 1 2 0 . The direct attack of N2O3 and (to a lesser extent) ONOO" on D N A can lead to D N A deamination with the end result being the formation of abasic sites and the formation of single strand breaks after endonuclease cleavage. While the exact mechanism for cross-link formation from NO treatment is not well understood, it is has been observed in vitro where cross-link formation corresponds to approximatley 6% of the guanine deamination product xanthine 1 2 0 . It is thought that the F A proteins have a role in the repair of double strand breaks (DSB) because increased HR activity has been described in FA-null nuclear extracts 46. A genetic study was done in E.coli to assess the genes that were required for the repair of NO-induced damage: AP-endonuclease-deficient strains and DSB repair-deficient strains were very sensitive to killing by NO, suggesting that recombinational repair must be in place in order to survive NO exposure 1 6 2 . NO is known to regulate the enzyme activity of various proteins (guanylate cyclases, cytochrome P-450 mixed function oxidases, thiols, catalases) by its interaction with Fe-S and heme centers 1 2 0 . Given the observations that F A N C C and F A N C G regulate P-450 enzymes and studies which have identified SOD deficiencies in F A cells without the identification of SOD mutations, it would be interesting to assess peroxynitrite-induced protein damage in F A cells. 86 Using a marker of peroxynitrite-specific protein damage, 3-nitrotyrosine (3-nTyr), antioxidant proteins in F A cells could be studied for increased levels of 3-nTyr. Interestingly, both ONOO" and lipid peroxidation have been indirectly implicated in F A before, with the result that PHGP X , an enzyme responsible for destroying lipid hydroperoxides and malondialdehyde, a product of lipid peroxide degredation, were found to be elevated in SV40-transformed F A fibroblasts 6 3 . Along these lines, the specific nitrosylation of SOD and its resultant inactivation in F A patients might lead to a severe phenotype that was very similar to the Fancc''Sodl'' mouse that was described within this thesis. The existence of modifier genes within complex genetic disorders has been documented and can contribute to the clinical heterogeneity seen in patients with mutations in the same genes 1 6 3 . NO-induced 3-nTyr formation has been shown to effect signaling molecules in cytokine stimulated macrophages. The endogenous production of NO has been shown to inhibit signalling pathways induced by IFNy, by nitrating tyrosine residues in STAT1, thereby preventing phosphorylation 1 6 4 . Similarly, endogenous NO is capable of modifying proteins containing cysteine residues by S-nitrosylation of the thiol group of that particular cysteine. Caspases can be targeted and inactivated by NO via this S-nitrosylation process and upon activation of the Fas apoptotic pathway, the caspase becomes denitrosylated and activated 1 6 5 1 6 6 . NO can regulate CD95 expression on both normal and tumor cells, which can lead to apoptosis through the activation of upstream FLICE caspases l 4 M 6 7 . Furthermore, F A HPC have been shown to have higher levels of fas receptor (CD95) and active caspase 3 upon stimulation with low doses of I F N Y resulting in elevated apoptosis in F A cells 9 4 . Many transcription factors such as N F K B are known to be regulated by the levels of reactive species within the cell 1 6 8 and degregulated levels of ROS in F A cells have been shown to effect N F K B pathways. Constitutive expression of N F K B from untreated F A SV40-transformed fibroblasts occurs through the formation of ROS since the addition of antioxidants and C Y T P-450 inhibitors both reversed this effect 6 3 . This pathway 87 provides an interesting feedback mechanism since NO is also known to stimulate iNOS expression through N F K B 1 6 9 . NO is produced by all mammalian cells at low levels in response to many stimuli and is required for many normal cellular functions 1 4 0 . Recently, NO has been shown to be both necessary and sufficient for the onset of fertilization and is thought to be an important aspect of C a 2 + signaling 1 7 ° . Furthermore, activated macrophages and endothelial cells can induce large amounts of NO through the specific activation of iNOS 1 4 ° . The B M microenvironment consists of many different cytokines required for the proper differentiation and turn-over of particular hematopoietic lineages. These stimuli may initiate signaling cascades that F A cells are particularly sensitive to, such as the I F N Y cascade. Similarly, ROS are known to be involved in growth factor signaling cascades and an upset in this balance may lead to stunted and abnormal differentiation or cellular death 1 3 4 . The wide range of cellular phenotypes observed in F A patients and mice indicates that the F A proteins are involved in a myriad of cellular processes. A l l such processes, while they appear to act independently, likely work in concert to maintain cellular homeostasis, be it D N A damage repair, redox regulation of proteins, or some combination of the two. Although the primary defect in F A remains elusive, deficiencies in pathways such as apoptosis, repair of D N A mutations, cell cycle function or control of ROS/RNS toxicity have been described in F A cells from both human and mouse. F A research has clearly shown, particularly through the study of F A N C C , that the pathogenesis of F A is highly complex with several different proteins having independent roles within different compartments of the cell, that do not necessarily have a common function. Cloning of the genes involved in F A and the subsequent creation of mouse models was an enormous leap forward for F A researchers. Current work should focus on the specific sensitivity and pathology of the B M compartment, on the better understanding of the cytoplasmic roles of the F A proteins, on the specific type of D N A damage that F A cells are 88 required to correct and if this is a direct or indirect role for the F A complex. Only through the understanding of all aspects of this disease will appropriate therapies become available. 89 BIBLIOGRAPHY 1. Fanconi G. Familial constitutional panmyelocytopathy, Fanconi anemia (F.A.). Sem Hematol. 1967;4:233-240 2. Auerbach A, Allen RG. Leukemia and preleukemia in Fanconi anemia patients: a review of the literature and report of the International Fanconi Anemia Registry. Can Genet Cytogehet. 1991;51:1-12 3. Scriver CR. The Metabolic and Molecular Basis of Inherited Disease (ed 7th). New York: McGraw-Hill; 1995 4. Schroeder TM, Anschutz F, Knopp A. [Spontaneous chromosome aberrations in familial panmyelopathy]. Humangenetik. 1964;1:194-196 5. Ishida R, Buchwald M. Susceptibility of Fanconi's anemia lymphoblasts to DNA-cross-linking and alkylating agents. Can Res. 1982;42:4000-4006 6. Auerbach A, Adler B, Chaganti RS. Prenatal and postnatal diagnosis and carrier detection of Fanconi anemia by a cytogenetic method. Pediatrics. 1981 ;67:128 7. Schindler D, Hoehn H. Fanconi anemia mutation causes cellular susceptibility to ambient oxygen. Am J Hum Genet. 1988;43:429-435 8. Joenje H, Arwert F, Eriksson AW, de Koning H, Oostra AB. Oxygen-dependence of chromosomal aberrations in Fanconi's anaemia. Nature. 1981;290:142-143 9. Kubbies M, Schindler D, Hoehn H, Schinzel A, Rabinovitch PS. Endogenous blockage and delay of the chromosome cycle despite normal recruitment and growth phase explain poor proliferation and frequent edomitosis in Fanconi anemia cells. Am J Hum Genet. 1985;37:1022-1030 10. Clarke AA, Philpott NJ, Gordon-Smith EC, Rutherford TR. The sensitivity of Fanconi Anemia group C cells to apoptosis induced by mitomycin C is due to oxygen radical generation, not DNA cross-linking. British J Hematol. 1997;96:240 11. Wright EG. Inherited and Inducible Chromosomal Instability: A Fragile Bridge Between Genome Integrity Mechanisms and Tumorigenesis. J Pathol. 1999;187:19-27 12. Zakrzewski S, Sperling K. Genetic heterogeneity of Fanconi's anemia demonstrated by somatic cell hybrids. Hum Genet. 1980;56:81-84 13. Duckworth-Rysiecki G, Cornish K, Clarke CA, Buchwald M. Identification of two complementation groups in Fanconi anemia. Somat Cell Mol Genet. 1985; 11:35-41 90 14. Strathdee CA, Gavish H, Shannon WR, Buchwald M. Cloning of cDNAs for Fanconi's anaemia by functional complementation. Nature. 1992;356:763-767 15. Strathdee CA, Duncan AM, Buchwald M. Evidence for at least four Fanconi anaemia genes including FACC on chromosome 9. Nat Genet. 1992;1:196-198 16. Joenje H, Patel KJ. The Emerging Genetic and Molecular Basis of Fanconi Anemia. Nat Genet Rev. 2001;2:446-457 17. Lo Ten Foe JR, Rooimans MA, Bosnoyan-Collins L, Alon N, Wijker M, Parker L, Lightfoot J, Carreau M, Callen DF, Savoia A, Cheng NC, van Berkel CG, Strunk MH, Gille JJ, Pals G, Kruyt FA, Pronk JC, Arwert F, Buchwald M, Joenje H. Expression cloning of a cDNA for the major Fanconi anaemia gene, FAA. Nat Genet. 1996;14:320-323 18. Consortium F. Positional cloning of the Fanconi anaemia group A gene. The Fanconi anaemia/breast cancer consortium. Nat Genet. 1996;14:324-328 19. de Winter JP, Waisfisz Q, Rooimans MA, van Berkel CG, Bosnoyan-Collins L, Alon N, Carreau M, Bender O, Demuth I, Schindler D, Pronk JC, Arwert F, Hoehn H, Digweed M, Buchwald M, Joenje H. The Fanconi anaemia group G gene FANCG is identical with XRCC9. Nat Genet. 1998;20:281-283 20. de Winter JP, Rooimans MA, van Der Weel L, van Berkel CG, Alon N, Bosnoyan-Collins L, de Groot J, Zhi Y, Waisfisz Q, Pronk JC, Arwert F, Mathew CG, Scheper RJ, Hoatlin ME, Buchwald M, Joenje H. The Fanconi anaemia gene FANCF encodes a novel protein with homology to ROM. Nat Genet. 2000;24:15-16 21. de Winter JP, Leveille F, van Berkel CG, Rooimans MA, van Der Weel L, Steltenpool J, Demuth I, Morgan NV, Alon N, Bosnoyan-Collins L, Lightfoot J, Leegwater PA, Waisfisz Q, Komatsu K, Arwert F, Pronk JC, Mathew CG, Digweed M, Buchwald M, Joenje H. Isolation of a cDNA representing the Fanconi anemia complementation group E gene. Am J Hum Genet. 2000;67:1306-1308 22. Timmers C, Taniguchi T, Hejna J, Reifsteck C, Lucas L, Bruun D, Thayer M, Cox B, Olson S, D'Andrea AD. Positional cloning of a novel Fanconi anemia gene, FANCD2. Mol Cell. 2001;7:241-248 23. Tipping AJ, Pearson T, Morgan NV, Gibson RA, Kuyt LP, Havenga C, Gluckman E, Joenje H, de Ravel T, Jansen S, Mathew CG. Multiple founder mutations for Fanconi anemia in the Afrikaner population of South Africa. Proc Natl Acad Sci USA. 2001;98:5734-5739 24. Whitney MA, Sato H, Jakobs PM, Gibson RA, Moses RE, Grompe M. A common mutation in the FACC gene causes Fanconi Anaemia in Ashkenazi-Jewish individuals. Nat Genet. 1993;4:202-205 25. Buchwald M. Complementation groups: one or more per gene? Nat Genet. 1995;11:228-230 91 26. Auerbach AD. The International Fanconi Anemia Registry: 20 Year Update. 13th Annual International Fanconi Anemia Scientific Symposium. Portland, Oregon, USA; 2001 27. Krasnoshtein F, Buchwald M. Developmental expression of the Fac gene correlates with congenital defects in Fanconi anemia patients. Hum Mol Genet. 1996;5:85-93 28. Abu-Issa R, Eichele G, Youssoufian H. Expression of the Fanconi anemia group A gene (Fanca) during mouse embryogenesis. Blood. 1999;94:818-824 29. van de Vrugt HJ, Cheng NC, de Vries Y, Rooimans MA, de Groot J, Scheper RJ, Zhi Y, Hoatlin ME, Joenje H, Arwert F. Cloning and characterization of murine fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary. Mamm Genome. 2000;11:326-331 30. van De Vrugt HJ, Koomen M, Beras MA, de Vries Y, Rooimans MA, van Der Weel L, Blom E, de Groot J, Schepers RJ, Stone S, Hoatlin ME, Cheng NC, Joenje H, Arwert F. Characterization, expression and complex formation of the murine Fanconi anaemia gene product Fancg. Genes Cells. 2002;7:333-342 31. Hoatlin ME, Christianson TA, Keeble WW, Hammond AT, Zhi Y, Heinrich MC, Tower PA, Bagby GC, Jr. The Fanconi anemia group C gene product is located in both the nucleus and cytoplasm of human cells. Blood. 1998;91:1418-1425 32. Kupfer GM, Naf D, Suliman A, Pulsipher M, D'Andrea AD. The Fanconi anaemia proteins, FAA and FAC, interact to form a nuclear complex. Nat Genet. 1997;17:487-490 33. Youssoufian H. Cytoplasmic localization of FAC is essential for the correction of a pre-repair defect in Fanconi anemia group C cells. J Clin Invest. 1996;97:2003-2010 34. Fagerlie S, Lensch MW, Pang Q, Bagby GC, Jr. The Fanconi anemia group C gene product: signaling functions in hematopoietic cells. Exp Hematol. 2001;29:1371-1381 35. Waisfisz Q, de Winter JP, Kruyt FA, de Groot J, van der Weel L, Dijkmans LM, Zhi Y, Arwert F, Scheper RJ, Youssoufian H, Hoatlin ME, Joenje H. A physical complex of the Fanconi anemia proteins FANCG/XRCC9 and FANCA. Proc Natl Acad Sci USA. 1999;96:10320-10325 36. Garcia-Higuera I, Kuang Y, Naf D, Wasik J, D'Andrea AD. Fanconi anemia proteins FANCA, FANCC, and FANCG/XRCC9 interact in a functional nuclear complex. Mol Cell Biol. 1999;19:4866-4873 37. Kruyt FA, Abou-Zahr F, Mok H, Youssoufian H. Resistance to mitomycin C requires direct interaction between the Fanconi anemia proteins FANCA and FANCG in the nucleus through an arginine-rich domain. J Biol Chem. 1999;274:34212-34218 92 38. Medhurst A.L., Huber P.A., Waisfisz Q., de Winter J.P., C.G. M. Direct interactions of the five known Fanconi anaemia proteins suggest a common functional pathway. Hum Mol Genet. 2001;10:423-429 39. Grompe M, D'Andrea AD. Fanconi anemia and DNA Repair. Hum Mol Genet. 2001 ;10:2253-2259 40. Qiao F, Moss A, Kupfer GM. Fanconi anemia proteins localize to chromatin and the nuclear matrix in a DNA damage- and cell cycle-regulated manner. J Biol Chem. 2001 ;276:23391-23396 41. Yagasaki H, Adachi D, Oda T, Garcia-Higuera I, Tetteh N, D'Andrea AD, Futaki M, Asano S, Yamashita T. A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA. Blood. 2001;98:3650-3657 42. Otsuki T, Naganshima T, Komatsu K, Kirito K, Furukawa Y, Kobayashi S, Liu JM, Ozawa K. Phosphorylation of Fanconi Anemia Protein, FANCA, is Regulated by Akt Kinase. Biochem Biophy Res Comm. 2002;291:628-634 43. Garcia-Higuera I., Taniguchi T., Ganesan S., Meyn M.S., Timmers C , Hejna J., Grompe M., A.D. DA. Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway. Mol Cell. 2001;7:249-262 44. Buchwald M, Moustacchi E. Is Fanconi anemia caused by a defect in the processing of DNA damage? Mutat Res. 1998;408:75-90 45. Carreau M, Alon N, Bosnoyan-Collins L, Joenje H, Buchwald M. Drug sensitivity spectra in Fanconi anemia lymphoblastoid cell lines of defined complementation groups. Mutat Res. 1999;435:103-109 46. Thyagarajan B, Campbell C. Elevated Homolgous Recombination Activity in Fanconi Anemia Fibroblasts. J Biol Chem. 1997;272:23328-23333 47. Escarceller M, Buchwald M, Singleton BK, Jeggo PA, Jackson SP, Moustacchi E, Papadopoulo D. Fanconi anemia C gene product plays a role in the fidelity of blunt DNA end-joining. J Mol Biol. 1998;279:375-385 48. Papadopoulo D, Guillouf C, Mohrenweiser H, Moustacchi E. Hypomutability in Fanconi anemia cells is associated with increased deletion frequency at the HPRT locus. Proc Natl Acad Sci USA. 1990;87:8383-8387 49. Laquerbe A, Moustacchi E, Fuscoe JC, Papadopoulo D. The molecular mechanism underlying formation of deletions in Fanconi Anemia cells may involve a site-specific recombination. Proc Natl Acad Sci USA. 1995;92:831-835 50. Meyn S. The Fanconi Anemia Protein FANCD2 Associates with Damaged DNA in vivo. 13th Annual International Fanconi Anemia Scientific Symposium. Portland, Oregon, USA; 2001 51. Korkina LG, Samochatova EV, Maschan AA, Suslova TB, Cheremisina ZP, Afanas'ev IB. Release of active oxygen radicals by leukocytes of Fanconi anemia patients. J Leukoc Biol. 1992;52:357-362 93 52. Degan P, Bonassi S, De Caterina M, Korkina LG, Pinto L, Scopacasa F, Zatterale A, Calzone R, Pagano G. In vivo accumulation of 8-hydroxy-2'-deoxyguanosine in DNA correlates with release of reactive oxygen species in Fanconi's anaemia families. Carcinogenesis. 1995;16:735-741 53. Kruyt FA, Youssoufian H. Do Fanconi anemia genes control cell response to cross-linking agents by modulating cytochrome P-450 reductase activity? Drug Resist Updat. 2000;3:211-215 54. Nordenson I. Effect of superoxide dismutase and catalase on spontaneously occurring chromosome breaks in patients with Fanconi's anemia. Hereditas. 1977;86:147-150 55. Nagasawa H, Little JB. Suppression of cytotoxic effect of mitomycin-C by superoxide dismutase in Fanconi's anemia and dyskeratosis congenita fibroblasts. Carcinogenesis. 1983;4:795-799 56. Ruppitsch W, Meisslitzer C, Hirsch-Kauffmann M, Schweiger M. Overexpression of thioredoxin in Fanconi anemia fibroblasts prevents the cytotoxic and DNA damaging effect of mitomycin C and diepoxybutane. FEBS Letters. 1998;422:99-102 57. Liu JM, Auerbach AD, Anderson SM, Green SW, Young NS. A trial of recombinant human superoxide dismutase in patients with Fanconi anaemia. Br J Haematol. 1993;85:406-408 58. Hoshino T., Wang J., Devetten M.P., Iwata N., Kajigaya S., Wise R.J., Liu J.M., H. Y. Molecular chaperone GRP94 binds to the Fanconi anemia group C protein and regulates its intracellular expression. Blood. 1998;91:4379-4386 59. Kruyt FA, Hoshino T, Liu JM, Joseph P, Jaiswal AK, Youssoufian H. Abnormal microsomal detoxification implicated in Fanconi anemia group C by interaction of the FAC protein with NADPH cytochrome P450 reductase. Blood. 1998;92:3050-3056 60. Hoatlin ME, Zhi Y, Ball H, Silvey K, Melnick A, Stone S, Arai S, Hawe N, Owen G, Zelent A, Licht JD. A novel BTB/POZ transcriptional repressor protein interacts with the Fanconi anemia group C protein and PLZF. Blood. 1999;94:3737-3747 61. Cumming RC, Lightfoot J, Beard K, Youssoufian H, O'Brien PJ, Buchwald M. Fanconi anemia group C protein prevents apoptosis in hematopoietic cells through redox regulation of GSTP1. Nat Med. 2001;7:814-820 62. Oinonen T, Lindros KO. Zonation of hepatic cytochrome P-450 expression and regulation. Biochem J. 1998;329:17-35 63. Ruppitsch W, Meisslitzer C, Weirich-Schwaiger H, Klocker H, Scheidereit C, Schweiger M, Hirsch-Kauffmann M. The role of oxygen metabolism for the pathological phenotype of Fanconi anemia. Hum Genet. 1997;99:710-719 94 64. Futaki M, Igarashi T, Watanabe S, Kajigaya S, Tatsuguchi A, Wang J, Liu JM. The FANCG Fanconi anemia protein interacts with CYP2E1: possible role in protection against oxidative DNA damage. Carcinogenesis. 2002;23:67-72 65. Wiseman H., B. H. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J. 1996;313:17-29 66. Otsuki T, Furukawa Y, Ikeda K, Endo H, Yamashita T, Shinohara A, Iwamatsu A, Ozawa K, Liu JM. Fanconi anemia protein, FANCA, associates with BRG1, a component of the human SWI/SNF complex. Hum Mol Genet. 2001;10:2651-2660 67. Mian IS, Moser MJ. The Fanconi anemia complementation group A protein contains a peroxidase domain. Mol Genet Metab. 1998;63:230-234 68. Ren J, Youssoufian H. Functional Analysis of the Putative Peroxidase Domain of FANCA, the Fanconi Anemia Complementation Group A Protein. Mol Genet Metab. 2001 ;72:54-60 69. Chen M, Tomkins DJ, Auerbach W, McKerlie C, Youssoufian H, Liu L, Gan O, Carreau M, Auerbach A, Groves T, Guidos CJ, Freedman MH, Cross J, Percy DH, Dick JE, Joyner AL, Buchwald M. Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia. Nat Genet. 1996;12:448-451'. 70. Whitney MA, Royle G, Low MJ, Kelly MA, Axthelm MK, Reifsteck C, Olson S, Braun RE, Heinrich MC, Rathbun RK, Bagby GC, Grompe M. Germ cell defects and hematopoietic hypersensitivity to gamma-interferon in mice with a targeted disruption of the Fanconi anemia C gene. Blood. 1996;88:49-58 71. Cheng NC, van de Vrugt HJ, van der Valk MA, Oostra AB, Krimpenfort P, de Vries Y, Joenje H, Berns A, Arwert F. Mice with a targeted disruption of the Fanconi anemia homolog Fanca. Hum Mol Genet. 2000;9:1805-1811 72. Yang Y, Kuang Y, De Oca RM, Hays T, Moreau L, Lu N, Seed B, D'Andrea AD. Targeted disruption of the murine Fanconi anemia gene, Fancg/Xrcc9. Blood. 2001;98:3435-3440 73. Koomen M, Cheng NC, van de Vrugt HJ, Godthelp BC, van der Valk MA, Oostra AB, Zdzienicka MZ, Joenje H, Arwert F. Reduced fertility and hypersensitivity to mitomycin C characterize Fancg/Xrcc9 null mice. Hum Mol Genet. 2002;11:273-281 74. Houghtaling S, Timmers C, Reifsteck C, Olson S, Jones S, Grompe M. Fanconi Anemia D2 (Fancd2) Knockout Mice have a Phenotype More Severe Than Fanca and Fancc Mice, but Milder than Brcal Mutants. 13th Annual International Fanconi Anemia Scientific Symposium. Portland, Oregon, USA; 2001 75. Carreau M, Gan Ol, Liu L, Doedens M, McKerlie C, Dick JE, Buchwald M. Bone marrow failure in the Fanconi anemia group C mouse model after DNA damage. Blood. 1998;91:2737-2744 95 76. Haneline LS, Broxmeyer HE, Cooper S, Hangoc G, Carreau M, Buchwald M, Clapp DW. Multiple inhibitory cytokines induce deregulated progenitor growth and apoptosis in hematopoietic cells from Fac-/- mice. Blood. 1998;91:4092-4098 77. Otsuki T, Nagakura S, Wang J, Bloom M, Grompe M, Liu JM. Tumor Necrosis Factor-alpha and CD95 Ligation Suppress Erythropoiesis in Fanconi Anemia C Gene Knockout Mice. J Cell Physiol. 1999;179:79-86 78. van de Vrugt HJ, Cheng NC, Koomen M, Joenje H, Berns A, Arwert F. Mice with Targeted Disruption in Fanca and/or Fancg/Xrcc9. 12th Annual International Fanconi Anemia Scientific Symposium. Amsterdam, The Netherlands; 2000 79. Wang J, Otsuki T, Youssoufian H, Foe JL, Kim S, Devetten M, Yu J, Li Y, Dunn D, Liu JM. Overexpression of the fanconi anemia group C gene (FAC) protects hematopoietic progenitors from death induced by Fas-mediated apoptosis. Can Reas. 1998;58:3538-3541 80. Stark R, Thierry D, Richard P, Gluckman E. Long-term bone marrow culture in Fanconi's anaemia. Br J Haematol. 1993;83:554-559 81. Bagnara GP, Strippoli P, Bonsi L, Brizzi MF, Avanzi GC, Timeus F, Ramenghi U, Piaggio G, Tong J, Podesta M, Paolucci G, Pegoraro L, Gabutti V, Bacigalupo A. Effect of stem cell factor on colony growth from acquired and constitutional (Fanconi) aplastic anemia. Blood. 1992;80:382-387 82. Doneshbod-Skibba G, Martin J, Shahidi N. Myeloid and erythroid colony growth in non-anemic patients with Fanconi's anemia. Brit J Hematol. 1980;44:33 83. Broxmeyer H, Douglas G, Hangoc G, Cooper S, Bard J, English D, Army M, Thomas L, Boyse E. Human unbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells. Proc Natl Acad Sci U S A. 1989;86:3828-3832 84. Butturini A, Gale RP. Long-term bone marrow culture in persons with Fanconi anemia and bone marrow failure. Blood. 1994;83:336-339 85. Rosselli F, Sanceau J, Gluckman E, Wietzerbin J, Moustacchi E. Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia. II. In vitro and in vivo spontaneous overproduction of tumor necrosis factor alpha. Blood. 1994;83:1216-1225 86. Rosselli F, Sanceau J, Wietzerbin J, Moustacchi E. Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia. I. Involvement of interleukin-6. Hum Genet. 1992;89:42-48 87. Schultz JC, Shahidi NT. Tumor necrosis factor-alpha overproduction in Fanconi's anemia. Blood. 1993;42:196-201 9 6 88. Lyman SD, Seaberg M, Hanna R, Zappone J, Brasel K, Abkowitz JL, Prchal JT, Schultz JC, Shahidi NT. Plasma/serum levels of flt3 ligand are low in normal individuals and highly elevated in patients with Fanconi anemia and acquired aplastic anemia. Blood. 1995;86:4091-4096 89. de Cremoux P., Gluckman E., Podgorniak MP., Menier C , Thierry D., Calvo F., G. S. Decreased IL-1 beta and TNF alpha secretion in long-term bone marrow culture supernatant from Fanconi's anaemia patients. Eur J Haematol. 1996;57:202-207 90. Segal G.M., Magenis R.E., Brown M., Keeble W., Smith T.D., Heinrich M.C., Bagby GCJ. Repression of Fanconi anemia gene (FACC) expression inhibits growth of hematopoietic progenitor cells. J Clin Invest. 1994;94:846-852 91. Pang Q, Fagerlie S, Christianson TA, Keeble WW, Faulkner GR, Diaz J, Rathbun RK, Bagby GC. The Fanconi anemia protein FANCC binds to and facilitates the activation of STAT1 by gamma interferon and hematopoietic growth factors. Mol Cell Biol. 2000;20:4724-4735 92. Haneline LS, Gobbett TA, Ramani R, Carreau M, Buchwald M, Yoder MC, Clapp DW. Loss of FancC function results in decreased hematopoietic stem cell repopulating ability. Blood. 1999;94:1-8 93. Rathbun R.K., Faulkner G.R., Ostroski M.H., Christianson T.A., Hughes G., Jones G., Cahn R., Maziarz R., Royle G., Keeble W., Heinrich M.C., Grompe M., Tower P.A., G.C. B. Inactivation of the Fanconi anemia group C gene augments interferon-gamma-induced apoptotic responses in hematopoietic cells. Blood. 1997;90:974-985 94. Rathbun R.K., Christianson T.A., Faulkner G.R., Jones G., Keeble W., O'Dwyer M., G.C. B. Interferon-gamma-induced apoptotic responses of Fanconi anemia group C hematopoietic progenitor cells involve caspase 8-dependent activation of caspase 3 family members. Blood. 2000;96:4204-4211 95. Pang Q, Christianson TA, Keeble WW, Diaz J, Faulkner GR, Reifsteck C, Olson S, Bagby GC. The Fanconi anemia complementation group C gene product: structural evidence of multifunctionality. Blood. 2001;98:1392-1401 96. Pang Q, Keeble WW, Diaz J, Christianson TA, Fagerlie S, Rathbun K, Faulkner GR, O'Dwyer M, Bagby GCJ. Role of double-stranded RNA-dependent protein kinase in mediating hypersensitivity of Fanconi anemia complementation group C cells to interferon gamma, tumor necrosis factor-alpha, and double-stranded RNA. Blood. 2001;97:1644-1652 97. Babior BM. Superoxide: a two-edged sword. Brazilian J Med Biol Res. 1997;30:141-155 98. Chanock SJ, El Benna J, Smith RM, Babior BM. The respiratory burst oxidase. J Biol Chem. 1994;269:24519-245222 99. Suzuki YJ, Forman HJ, Sevanian A. Oxidants as stimulators of signal transduction. Free Rad Biol Med. 1997;22:269-285 97 100. Fridovich I. Superoxide Dismutases. Ann Rev Biochem. 1975;44:147-159 101. Carlioz A, Touati D. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 1986;5:623-630 102. Farr SB, D'Ari R, Touati D. Oxygen-dependent mutagenesis in Escherichia coli lacking superoxide dismutase. Proc Natl Acad Sci, USA. 1986;83:8268-8272 103. Chang EC, Crawford BF, Hong Z, Bilinski T, Kosman DJ. Genetic and biochemical characterization of Cu,Zn superoxide dismutase mutants in Saccharomyces cerevisiae. J Biol Chem. 1991;266:4417-4424 104. Phillips JP, Campbell SD, Michaud D, Charbonneau M, Hilliker AJ. Null mutation of copper-zinc superoxide dismutase in Drosophila confers hypersensitivity to paraquat and reduced longevity. Proc Natl Acad Sci, USA. 1989;86:2761-2765 105. Parkes TL, Elia AJ, Dickinson D, Hilliker AJ, Phillips JP, Boulianne GL. Extension of Drosophila lifespan by overexpression of human SOD1 in motorneurons. Nat Genet. 1998;19:171-174 106. Li Y, Huang TT, Carlson EJ, Melov S, Ursell PC, Olson JL, Noble LJ, Yoshimura MP, Berger C, Chan PH, Wallace DC, Epstein CJ. Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat Genet. 1995;11:376-381 107. Lebovitz RM, Zhang H, Vogel H, Cartwright JJ, Dionne L, Lu N, Huang S, Matzuk MM. Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc Natl Acad Sci, USA. 1996;93:9782-9787 108. Reaume AG, Elliott JL, Hoffman EK, Kowall NW, Ferrante RJ, Siwek DF, Wilcox HM, Flood DG, Beal MF, Brown RHJ, Scott RW, Snider WD. Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat Genet. 1996;13:43-47 109. Ho Y, Gargano M, Cao J, Bronson RT, Heimler I, Hutz RJ. Reduced fertility in female mice lacking coper-zinc superoxide dismutase. J Biol Chem. 1998;273:7765-7769 110. Matzuk MM, Dionne L, Guo Q, Kumar TR, Lebovitz RM. Ovarian function in superoxide dismutase 1 and 2 knockout mice. Endocrinology. 1998;139:4008-4011 111. Shefner JM, Reaume AG, Flood DG, Scott RW, Kowall NW, Ferrante RJ, Siwek DF, Upton-Rice M, Brown RHJ. Mice lacking cytosolic copper/zinc superoxide dismutase display a distinctive motor axonopathy. Neurology. 1999;53:1239-1246 112. Huang TT, Carlson EJ, Leadon SA, Epstein CJ. Relationship of resistance to oxygen free radicals to CuZn-superoxide dismutase activity in transgenic, transfected, and trisomic cells. FASEB J. 1992;6:903-910 98 113. Huang TT, Yasunami M, Carlson EJ, Gillespie AM, Reaume AG, Hoffman EK, Chan PH, Scott RW, Epstein CJ. Superoxide-mediated cytotoxicity in superoxide dismutase-deficient fetal fibroblasts. Arch Biochem Biophy. 1997;344:424-432 114. Rosen DR, Siddique T, Patterson D, Figlewicz DA, Sapp P, Hentati A, Donaldson D, Goto J, O'Regan JP, Deng HX. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature. 1993;362:59-62 115. Ames BM, Shigenaga MK, Hagen TM. Oxidants, antioxidants and the degenerative diseases of aging. Proc Natl Acad Sci, USA. 1993;90:7915-7922 116. Applegate LA, Luscherm P, Tyrrell RM. Induction of heme oxygenase: a general response to oxidant stress in cultured mammalian cells. Can Res. 1991;51:974-978 117. Yeh CC, Wan XS, St Clair DK. Transcriptional regulation of the 5' proximal promoter of the human manganese superoxide dismutase gene. DNA Cell Biol. 1998;17:921-930 118. Casarett LJ, Doull J. Biotransformation of Xenobiotics. Toxicology: The Basic Science of Poisons (ed 5th Edition). New York: McGraw-Hill Company; 1996 119. Jaeschke H. Mechanisms of oxidant stress-induced acute tissue injury. Proc Soc Exp Biol Med. 1995;209:104-111 120. Burney S, Caulfield JL, Niles JC, Wishnok JS, Tannenbaum SR. The Chemistry of DNA damage from nitric oxide and peroxynitrite. Mutat Res. 1999;424:37-49 121. FinkelT. Oxygen radicals and signaling. Curr Opin Cell Biol. 1998;10:248-253 122. Joenje H, Frants RR, Arwert F, de Bruin GJ, Kostense PJ, van de Kamp JJ, de Koning J, Eriksson AW. Erythrocyte superoxide dismutase deficiency in Fanconi's anaemia established by two independent methods of assay. Scand J Clin Lab Invest. 1979;39:759-764. 123. Mavelli I, Ciriolo MR, Rotilio G, De Sole P, Castorino M, Stabile A. Superoxide dismutase, glutathione peroxidase and catalase in oxidative hemolysis. A study of Fanconi's anemia erythrocytes. Biochem Biophys Res Commun. 1982;106:286-290 124. Carreau M, Gan OI, Charbonneau C, Liu L, Rozmahel R, Dick JE, Buchwald M. Progress on Modifier Studies Using the Fancc Knockout Mouse. 12th Annual International Fanconi Anemia Scientific Symposium. Amsterdam, The Netherlands; 2000 125. Hautekeete ML, Degott C, Benhamou JP. Microvesicular steatosis of the liver. Acta Clin Belg. 1990;45:311-326 99 126. Nathan DG, Orkin SH. Hematology of Infancy and Childhood. Vol. 2 (ed 5th). Philadelphia: Saunders; 1998 127. Sattler M, Winkler T, Verma S, Byrne CH, Shrikhande G, Salgia R, Griffin JD. Hematopoietic growth factors signal through the formation of reactive oxygen species. Blood. 1999;93:2928-2935 128. Rhee SG, Bae YS, Lee SR, Kwon J. Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Science. 2000;53:1-6 129. McCord JM, Fridovich I. The biology and pathology of oxygen radicals. Ann Intern Med. 1978;89:122-127 130. Tyler WS, Stohlman FJ, Chovaniec M, Howard D. Effect of a congenital defect in hemopoiesis on myeloid growth and the stem cell (CFU) in an in vivo culture system. Blood. 1976;47:413-421 131. Ploemacher RE, van der Sluijs JP, Voerman JSA, Brons NHC. An in vitro limiting dilution assay of long-term repopulating hematopoietic stem cells in the mouse. Blood. 1989;74:2755-2763 132. Neben S, Anklesaria P, Greenberger J, Mauch P. Quantitation of murine hematopoietic stem cells in vitro by limiting dilution analysis of cobblestone area formation on a clonal stromal cell line. Exp Hematol. 1993;21:438-443 133. Salvemini D, Riley DP, Cuzzocrea S. SOD mimetics are coming of age. Nat Rev Drug Discov. 2002;1:367-374 134. Hancock JT. Superoxide, hydrogen peroxide and nitric oxide as signalling molecules; their production and role in disease. British J Biomed Sci. 1997;54:38-46 135. Toyokuni S. Reactive oxygen species-induced molecular damage and its application in pathology. Pathol Int. 1999;49:91-102 136. Lightfoot J, Alon N, Bosnoyan-Collins L, Buchwald M. Characterization of regions functional in the nuclear localization of the Fanconi anemia group A protein. Hum Mol Genet. 1999;8:1007-1015 137. Wong JC, Alon N, Norga K, Kruyt FA, Youssoufian H, Buchwald M. Cloning and analysis of the mouse Fanconi anemia group A cDNA and an overlapping penta zinc finger cDNA. Genomics. 2000;67:273-283 138. McMahon LW, Walsh CE, Lambert MW. Human alpha spectrin II and the Fanconi anemia proteins FANCA and FANCC interact to form a nuclear complex. J Biol Chem. 1999;274:32904-32908 139. Otsuki T, Kajigaya S, Ozawa K, Liu JM. SNX5, a new member of the sorting nexin family, binds to the Fanconi anemia complementation group A protein. Biochem Biophys Res Commun. 1999;265:630-635 140. MacMicking J., Xie Q.W., C. N. Nitric oxide and macrophage function. Ann Rev Immunol. 1997;15:323-350 100 141. Selleri C , Maciejewski JP. Nitric oxide and cell survival: megakaryocytes say "NO". J Lab Clin Med. 2001;137:225-230 142. Maciejewski J.P., Selleri C , Sato T., Cho H.J., Keefer L.K., Nathan C.F., Young NS. Nitric oxide suppression of human hematopoiesis in vitro. Contribution to inhibitory action of interferon-gamma and tumor necrosis factor-alpha. J Clin Invest. 1995,96:1085-1092 143. Drapier JC, Wietzerbin J, Hibbs JBJ. Interferon-gamma and tumor necrosis factor induce the L-arginine-dependent cytotoxic effector mechanism in murine macrophages. Eur J Immunol. 1988;18:1587-1592 144. Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol. 1988;141:2407-2412 145. Graham GJ, Wright EG, Hewick R, Wolpe SD, Wilkie NM, Donaldson D, Lorimore S, Pragnell IB. Identification and characterization of an inhibitor of haemopoietic stem cell proliferation. Nature. 1990;344:442-444 146. Villalta F, Zhang Y, Bibb KE, Kappes JC, Lima MF. The Cysteine-Cysteine Family of Chemokines RANTES, MlP-la and MlP-lb Induce trypanocidal activity in human macrophages via nitric oxide. Infec Immun. 1998;66:4690-4695 147. Sherry B., Schmidtmayerova H., Zybarth G., Dubrovsky L., Raabe T., M. B. Nitric oxide regulates MlPl-alpha expression in primary macrophages and T lymphocytes: implications for anti-HIV-1 response. Mol (Med. 2000;6:542-549 148. Reykdal S., Abboud C , J. L. Effect of nitric oxide production and oxygen tension on progenitor preservation in ex vivo culture. Exp Hematol. 1999;27:441-450 149. Feelisch M. The use of nitric oxide donors in pharmacological studies. Naunyn-Schmiedeberg's Archives of Pharmacology. 1998;358:113-122 150. Ganster R.W., Taylor B.S., Shao L., Geller DA. Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-kappa B. Proc Natl Acad Sci U S A . 2001;98:8638-8643 151. Melillo G., Musso T., Sica A., Taylor L.S., Cox G.W., L. V. A hypoxia-responsive element mediates a novel pathway of activation of the inducible nitric oxide synthase promoter. J Exp Med. 1995;182:1683-1693 152. Inoue S., Kawanishi S. Oxidative DNA damage induced by simultaneous generation of nitric oxide and superoxide. FEBS Letters. 1995;371:86-88 153. Pieper A.A., Verma A., Zhang J., S.H. S. Poly (ADP-ribose) polymerase, nitric oxide and cell death. Tren Pharmacol Sci. 1999;20:171-181 101 154. Pacelli R., Wink D.A., Cook J.A., Krishna M.C., DeGraff W., Friedman N., Tsokos M., Samuni A., J.B. M. Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia coli. J Exp Med. 1995;182:1469-1479 155. Laval F., Wink DA. Inhibition by nitric oxide of the repair protein, 06-methylguanine-DNA-methyltransferase. Carcinogenesis. 1994;15:443-447 156. Wink D.A., Laval J. The Fpg protein, a DNA repair enzyme, is inhibited by the biomediator nitric oxide in vitro and in vivo. Carcinogenesis. 1994;15:2125-2129 157. Ajuebor M.N., Das A.M., Virag L., Flower R.J., Szabo C , M. P. Role of Resident Peritoneal Macrophages and Mast Cells in Chemokine Production and Neutrophil Migration in Acute Inflammation: Evidence for an Inhibitory Loop Involving Endogenous IL-10. J Immunol. 1999;162:1685-1691 158. Kitagawa M., Takahashi M., Yamaguchi S., Inoue M., Ogawa S., Hirokawa K., R. K. Expression of inducible nitric oxide synthase (iNOS) in bone marrow cells of myelodysplastic syndromes. Leukemia. 1999;13:699-703 159. Young N.S., Maciejewski JP. The Pathophysiology of Acquired Aplastic Anemia. New Eng J Med. 1997;336:1365-1372 160. Rotman G, Shiloh Y. ATM: a mediator of multiple responses to genotoxic stress. Oncogene. 1999;18:6135-6144 161. Zhang J, Martasek P, Paschke R, Shea T, Siler Masters BS, Kim JJ. Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase. J Biol Chem. 2001;276:37506-37513 162. Spek EJ, Wright TL, Stitt MS, Taghizadeh NR, Tannenbaum SR, Marinus MG, Engelward BP. Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide. J Bacteriol. 2001;183:131-138 163. Houlston RS, Tomlinson IPM. Modifier genes in humans: strategies for identification. Eur J Hum Genet. 1998;6:80-88 164. Llovera M, Pearson JD, Moreno C, Riveros-Moreno V. Impaired response to interferon-gamma in activated macrophages due to tyrosine nitration of STAT1 by endogenous nitric oxide. British J Pharmacol. 2001;132:419-426 165. Mohr S, Zech B, Lapetina EG, Brune B. Inhibition of Caspase-3 by S-nitrosylation and oxidation caused by Nitric Oxide. Biochem Biophys Res Commun. 1997;238:387-391 166. Mannick JB, Hausladen A, Liu L, Hess DT, Zeng M, Miao QX, Kane LS, Gow AJ, Stamler JS. Fas-induced caspase denitrosylation. Science. 1999;284:651-654 102 167. Garban HJ, Bonavida B. Nitric oxide inhibits the transcription repressor Yin-Yang 1 binding activity at the silencer region of the Fas promoter: a pivotal role for nitric oxide in the up-regulation of Fas gene expression in human tumor cells. J Immunol. 2001;167:75-81 168. Bunn HF, Poyton RO. Oxygen Sensing and Molecular Adaptation to Hypoxia. Physiol Rev. 1996;76:839-885 169. Umansky V, Hehner SP, Dumont A, Hofmann TG, Schirrmacher V, Droge W, Schmitz ML. Co-stimulatory effect of nitric oxide on endothelial NF-kB implies a physiological self-amplifying mechanism. Eur J Immunol. 1998;28:2276-2282 170. Kuo RC, Baxter GT, Thompson SH, Strieker SA, Patton C, Bonaventura J, Epel D. NO is necessary and sufficient for egg activation at fertilization. Nature. 2000;406:633-636 103 Table I. Clinical and Cellular Phenotypes in Fanconi Anemia Clinical Phenotypes • Growth retardation, short stature • Hypogonadism and reduced fertility • Skeletal malformations (absent or abnormal thumbs and radii) • Microcephaly • Skin hyperpigmentation / cafe au lait spots • Congenital heart disease • Hearing loss • Elevated serum a-fetoprotein Disease Onset • Bone Marrow Failure; Aplastic Anemia • Cancer Predisposition; Acute Myeloid Leukemia and / or Squamous Cell Carcinomas, Solid Tumors Cellular Phenotypes • Spontaneous chromosomal instability (chromatid breaks and interchanges, radial formation) • Spontaneous arrest and delay at the G 2 phase of the cell cycle (4n post-replication) • Hypersensitivity to DNA inter-strand cross-linking agents such as mitomycin C, diepoxybutane, cisplatin, and cyclophosphamide. • Hypersensitivity to elevated oxygen concentrations (35% 02) 104 Table II. Fanconi Anemia Complementation Groups Complementation Group No. of Pathogenic Mutations Chromosomal Location Exons % Identity to Mouse0 Protein Product Evolutionary Conservation Knockout Phenotype FANCA > 100 16q24.3 43 65% 145 kDa Fish" Reduced Fertility FANCB" ? ? ? NA ? ? NA FANCC 12 9q22.3 14 63% 56 kDa Fish" Reduced Fertility F A N C D l 3 ? ? ? NA ? ? NA FANCD2 5 3p25.3 44 NA 145 kDa Worm/Fly Reduced Fertility /Ionizing Radiation FANCE 4 6p21.3 10 60% 53 kDa Fish" NA FANCF 6 l lpl5 1 NA 37 kDa Fishb Reduced Fertility FANCG 18 9pl3 14 83 % 68 kDa XRCC9 Fish" Reduced Fertility Adapted from: Joenje H. and Patel K.J. (2001) Nat Gen Rev. NA, data not available a Remains to be identified b BLAST searches for homologous regions in the FA proteins reveals no significant similarity in non-vertebrate, yeast or bacteria. Significant BLAST hits were identified in fish; Tetraodon nigroviridis (FANCA, FANCE, FANCF), Takifugu rubripes (FANCC) and zebrafish Danio rerio (FANCG). c values represents the % amino acid sequence identity between the mouse cDNA and the human sequence ORF FANCF K1125 ORF 1611 * Mircrodeletion or microinsertion » Nonsense mutation • Missense mutation • Splice site alteration — Deletion o Potymorphism ' | IVS4 »4A -»T1 y=— • « • ORF T A • < ORF |i;63a«f | » + • B V M W I FANCA . 1 1 1 < S 7 «JJJU uj J 11 .14 16 ^HQ{J|}^ ^ »» £ ^ . » IJ » J O l t H I l » S » J J n . « . 41 4> i 41 • ORF •4368 o ooo o • oo • o • • FANCD2 ORF 4356 Figure 1. Mutations Identified in the Fanconi Anemia Genes From: Joenje H. and Patel K.J. (2001) Nat Gen Rev The exon structure of each cloned F A gene is shown as well as the approximate location for each mutation. Mutations surrounded with a box are the most common for that particular gene. The greatest number of pathogenic mutations have been identified in FANCA, and many of these are deletions due to the large number of Alu repeats within the gene. 106 Table III. Current Mouse Models for Fanconi Anemia Model J Genetic Strategy f Similarities to human phenotype Differences from human phenotype Reference Fanca -/- Fanca exons 4-7 replaced with lacZ-neo reduced fertility, cross-linker sensitivity no congenital malformations, hematological abnormalities or tumor formation [Cheng, 2000] Fancc -/- Fancc exons 8 or 9 replaced with neo Reduced fertility, cross-linker and cytokine sensitivity, repopulating ability decreased no congenital malformations, hematological abnormalities or tumor formation [Chen, 1996] [Whitney, 1996] [Haneline, 1998] [Haneline, 1999] Fancg -/- Fancg exons 2-9 replaced with neo reduced fertility, cross-linker and ionizing radiation sensitivity, no Fancd2-L protein isoform no congenital malformations, hematological abnormalities or tumor formation [Yang, 2001] Fancd2 -/- Fancdl exons replaced with neo Reduced fertility, cross-linker and ionizing radiation sensitivity no congenital malformations, hematological abnormalities or tumor formation [Houghtaling, 2001] FANCC transgenic human FANCC ubiquitously overexpressed HPC protected against fas apoptosis N/A [Wang, 1998| FNT Breeding of (hu)TNFcc Tg to Fancc-/- mice BM colony growth is decreased marrow cellularity and peripheral blood counts are normal [Otsuki, 1999] Fanca -/-Fancg -/-double ko a Breeding of the single knockout animals Phenotype same as single knockouts Phenotype same as single knockouts [van de Vragt, 2000] Adapted from Wong et al (2002) Trends in Mol . Med. " results were presented at conference proceedings and have not been published since, information is limited as a result. 108 CN CD L O O o is S g cz cn 7= ro p £ CD | CD i — CN • o • —» z—* CD CN o c^ O CO I CO O CD o o o 00 Q H H I—I H-I CO I 5 o CO CD CD CN ZJ CZ O CD cu cn O ^ ^ o CN CN CN O " IVE L U ES :ACT XYGI PECI DC o CO CO CD CO CC "O o 0_ Q < CN CN "cS ffi 2 'cL co CD QC U CD CO O $ _£0 "SJ "y? c5 - 3 CD O O Q_ CN Q H-l Cu H H H - l CD CZ he "cz 'co CZ cp _ll g CD . > pu an O CD £ 3 Q _ o CD CO H— CO CD CO >^  CD X _ "o CD 0 Oil S9I OL 0 Oil o CO o CD ve CO sp " o —\ CZ O CD CO CD V— odi CO O C L "cz es wn nd o o CO CZ Z3 kd o CO CD co CD CO i_ >, O C Q X 'co o E CD -CZ CD eel ctiv |— CZ co CO CD CO "CO _cz ure mm "5 CO CO CO L U E CD CO CD CO CO CZ o CC o CO Z3 E C C o H H o rt HH 109 Sodl -/- Fancc -/- Fancc+/+ Fancc+/- Fancc-/-Sod1+/+ Sod1+/- Sod1-/-Fig. 4 Body Weights from 8 wk old Fancc'Sodl'' mice. Anaesthetized male (dark bars) and female (gray bars) mice were weighed and the average weight (in grams) ± SEM is represented above. There is a slight decrease in body weight in Fancc"Sodl" mice, however, this difference is not significant (n=5-6). n o Fig. 5 Histological examination reveals zonal hepatic microvesicular steatosis in Fancc-/-Sodl-/- mice. Histology of Fancc-/-Sodl-/- and control livers (magnification 400x). Sections of Fancc-/- (a, e); Sodl-/- (b, f); Fancc+/-Sodl +/- (c, g) and Fancc-/-Sodl-/- (d, h) livers were stained with Masson's trichrome (a-d) and oil red-0 (e-h). While Fancc-/-, Sodl-/- and Fancc+/-Sodl +/-show normal morphology, Fancc-/-Sodl-/- hepatocytes demonstrate a zone 3 abnormality characterized by abundant cytoplasmic vacuolation. Oil red-0 staining demonstrates prominent microvesicular zone 3 lipid accumulation without nuclear displacement in Fancc-/-Sodl-/- mice. Small amounts of lipid droplets are present in Sodl-/- mice. 1 1 1 Fig. 6 Electron Microscopy of hepatocytes from Fancc-/-Sodl-/- mice reveals no increase in organelle damage. Hepatocytes from Fancc+/-Sodl+/- (a and c) and Fancc-/-Sod 1-/-(b and d) mice, a) a normal multi-nucleated centrilobular hepatocyte (5400x) and c) a higher magnification showing normal organelles (1 l,750x). b) microvesicular steatosis in a centrilobular Fancc-/-Sodl-/-hepatocyte (5400x) and d) (1 l,750x). 112 Fancc+/+ Sod1-t/+ Fanoo/- Sod1-A Fancc-/-Sod1-/-Fancc+/-SocH-t/-Fig. 7 Primary hepatocytes from Fancc''Sodl'' mice have increased superoxide levels. The level of superoxide is reflected by the R L U value and was measured using luminol dependent chemiluminescence. Each value represents the mean ± S E M for 5 mice per group, with each sample in triplicate. *, p < 0.05; **, p < 0.001 by the Student's t test. Sodl'" mice have a significant increase (p=0.01) in the R L U value compared to wildtype mice. Fancc'Sodl'mice, had a synergistic increase in R L U values of 1.62 (p=0.0008) compared to wildtype mice. 113 A B C D M n S O D H O - 1 B-tubulin Fig. 8 Liver-specific expression of Mn Sod and HO-1 is increased in Fancc'Sodl" mice. Autoradiographs showing total liver lysates irnmunoblotted with antibodies against M n Sod and HO - 1 , and normalized for loading with anti-tubulin Ab. HO -1 and M n Sod protein expression in A) control, B) Fancc+/+Sodl+/+ and C) Fancc+/'Sodl+/~ livers indicates an increase in protein expression in D) Fancc'Sodl'' samples. 114 Table IV Peripheral Blood Values from 8-10 wk old mice CBC Values § Genotype R B C W B C HGB PLT MCV n (10'7l) (1071) (g/i) (1071) (fL) Fancc+uSodfu 15 10.02 ±0 .27 3.69 ± 1.09 163.4 ±4 .47 605.3 ± 47.2 52.8 ± 0.69 Fancc" Sodf 18 9.66 ± 0.27 6.59 ± 0.86 161.9 ±4 .50 708 ± 49.3 52.3 ± 0.62 Fancc'' 9 9.68 ± 0.20 1.55 ± 0.23 158.6 ±4 .05 435 ± 26.9 53.5 ± 0.42 Sodf 10 8.72 ± 0.42 1.94 ± 0.56 147 ±3 .96 745.5 ±49.1 54.1 ± 1.0 Fancc''Sodf' 9 7.9 ± 0.36 ** 0.96 ± 0.20 * 136 ±4.52*" 719 ±93 .7 58.5 ± 0.68 " Values represent average ± SEM for the indicated number of animals per group , p < 0.05; , p < 0.0005 5 CBCs were quantified using a Sysmex 9500 automated blood analyser. Fancc+/+Sod1+/+ Fancc_/_ Fig. 9 Hypocellulariry and increased fat accumulation in Fancc'Sodl'' bone marrow. Metaphyseal sections of leg bones from Fancc' 'Sodf \ Fancc "'", Sodl "' and Fancc' Sodl" mice (magnification 400x). Marrow fat content (clear areas) is increased in Fancc' Sodl" mice compared to Fancc' ' Sodl' ' controls. Fancc" controls revealed only rare fat cells, while Sodl" mice did show some increase in fat spaces. H6 o d o CD —• o CO JD < < E i l o> tt) T -Q O Q O O C D o C M C M C Q Q L O Q. 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E 2 o o S, co > to < CO 3 CD ° s 17 m re o 0> E 3 0 O 0) 3 O 10 .o < mFancc+/- Sod1+/-• Fancc-/- Sod1-/-Lin+Sca1 + Lin+ckit+ Lin+CD34+ Fig. 10 F A C S analysis of L i n + cells for progenitor markers. Whole B M samples from Fancc Sodl ' and Fancc'Sodl" mice were immunophenotyped by FACS analysis to determine the number of L i n + B M cells also carrying Sea, ckit or CD34 markers on their cell surface. There was approximately a 40% decrease in the absolute number of L i n + cells with any of the above markers from Fancc''"Sodmarrows. Data is from two mice per genotype. 118 Table VI. Pl/Annexin FACS Analysis from total BM samples % Staining Genotype n Annexin+ P l -Annexin+ PI+ Fancc+I- Sod1+/- 5 25.49 ± 4.59 15.52 ±3 .08 Fancc-/- Sod1-/- 5 28.38 ± 3.83 16.44 ±6 .12 FACS Staining was used to quantitate the number of apoptotic cells from total BM sampli of 8-9 wk old mice. Annexin+/PI- values represent cells initiating the apoptotic process and Annexin+/PI+ values represent cells that have already undergone apoptosis and death. 119 Sodl-/- Fancc-/- Fancc+/+ Fancc+/- Fancc-/-Sod1+/+ SodU/- Sodl-/-b) 90 80 70 60 ^ 50 40 -30 -20 -10 -0 • • % G E M M • % G M / G / M • % ERY Sodl-/- Fancc-/- Fancc+/+ Fancc+/-Sod1+/+ Sod1+/-Fancc-A SodU-Fig. 11 Colony Forming Assays reveal abnormal progenitor growth from Fancc'Sodl" mice. a) Colony forming assays reveal decreased numbers of progenitors in Fancc'Sodl'' marrows. Myeloid (dark bars) and Pre-B (hatched bars) CFUs were measured for Sodl'', Fancc''' Fancc+/+Sodl+/+, Fancc+/'Sodl+/' and Fancc''Sodl'' mice. The decrease in the number of progenitors/femur is highly significant when Fancc''Sodl'' mice are compared to Fancc+/+Sodl+/+ controls. Values represent the average number of progenitors/femur of 6 mice per group ± SEM. * = p<0.05, ** = p<0.001. b) Fancc'Sodl'' progenitors fail to generate normal ratios of C F U - G E M M , C F U - G M / G / M and BFU-E. Myeloid colonies from a) were assessed morphologically to determine the cell types contributing to the colonies. C F U - G E M M (dark bars), C F U - G M / G / M (light gray bars) and BFU-E (hatched bars) colonies were scored by eye and the values represent average percent of cell type ± S E M (n = 6-8 mice per group). Ratios of C F U - G M / G / M , and C F U - G E M M were significantly different from Fancc+/+Sodl+/+ controls (p=0.002; and p=0.003, respectively). 120 Table VII. Reagents used to try to rescue colony growth of methylcellulose colonies from Fancc '''Sodl ~'~ mice. R E A G E N T S C E L L U L A R E F F E C T Cell Density • • • 2x 5x lOx Hypoxia • 5 % 0 2 Pan-Caspase Inhibitor • ZVAD-fmk(10, 50 pg) Antioxidants/Drugs • • • • • Ammonium pyrrolidinedithiocarbamate (APDC) (0.1, 1, 10 uM) 4,5 - Dihydroxy - 1,3 - benzene Disulfonic Acid (TIRON) (0.05, 0.5 mM, 5 uM) Superoxide Dismutase (SOD) (100 ng, 1,10,50 pg) ETJK-8, Dihydrate (5, 7, 10, 25 uM) [Mn(III)tetrakis (l-methyl-4-pyridyl)porphyrin pentachloride] (MnTMPyP) (1, 5, 20, 50 uM) • • • • • prevents induction of NO synthetase by inhibiting NOS mRNA translation superoxide anion scavenger scavenger of superoxide anion synthetic complex with SOD and catalase activities SOD mimetic Growth Factors • SCF (50, 100, 200 ng) Each experiment was repeated three times with each sample done in duplicate. A l l tests used littermate Fancc * +Sodl* * or Fancc Sodl*'' mice as controls 121 25 • Sca+/ckit-• Sca+/ckit+ • CD34+/Sca-Fancc+/-Sod1 +/- Fancc-/-Sod1-/-Lin- Subpopulations Fig. 12 FACS of Lin" cells from Fancc Sodl" marrows after column selection. Whole B M samples from Fancc+/~Sodl+/~ and Fancc''Sodl''' mice were depleted of L i n + cells by negative selection and the resultant column purified progenitors were immunophenotyped by FACS analysis. Both the percent staining and the absolute number of Lin" cells with Sea, ckit and/or CD34 markers from Fancc "Sodl ' marrows was similar to Fancc+/'Sodl+/~ controls. Data is from 3 mice per genotype. 60 • Myeloid CFA D PreB CFA Sodl- / - Fancc-/- Fancc+/+ Fancc+/- Fancc-/-Sodl+/+ Sodl+/- Sodl- / -Fig. 13 Colony Forming Assays from aged 6-8 month old mice. Colony forming assays were done from aged cohorts of animals and revealed that the decreased number of progenitors from Fancc'Sodl1' marrows persisted with age. Myeloid (dark bars) and pre-B (hatched bars) CFUs were measured for Sodl'', Fancc'', Fancc+/+Sodl+/+, Fancc+/'Sodl+/' and Fancc''Sodl'' mice. The decrease in the number of progenitors/femur is highly significant when Fancc'" Sodl"'' mice are compared to Fancc+/+Sodl+/+ controls. Values represent the average number of progenitors/femur of 5 mice per group ± SEM. * = p<0.05, ** = p<0.001. w o OQ in < o >. o c 0) 3 CT O o LL < O 00 CM > . (0 o CM > . ns Q Q >. co Q CO >. (0 Q CO = "O CD 0 CD Q . •I—• O c CD o CNJ c o "o O CO o c 00 CNJ o o c oo <M T3 O CO CM 3^ O CO o c 5 <2 c IS « S 2 a, CO > u * cd __, £ r3 CO V3 y '-^  a ca £ a fa ° •2(2 I g i T 3 O 1 ) o a o d v CO O . 2 a a "§ 1 iU 3^ 3^ -3 O Cs § to co Co "'tj "'cj V U CJ cj e K c k k i i ; C C c CO CO u u u g £ i? £ s s « J 43 2 g K u E g g <a ^  ,u a c XI "O ^ a a c CO CCJ jrt * i c u •a H . c C co 2 2. •n cr ca u g U 8 u fS CO CO N bo ,>> >> ca £ c O ca co o cn 6 ca B0 60 00 'cn 'cn S 00 cn * ca ca 1/3 cn a a co co cn t/5 ft Q- rv P P U cn CO O M co "5 6 2 a 6 co ? 124 6 >) o f 03 cn CJ a, o s= u M u > 'in S3 cO <U tl O O = CO c U o rx, < u u i -3 U PQ S 03 _ C <0 *-> Cu -I 8 LO 3 3 • H O e o o U U "2 C CU >» o o CN r> >> 3 •a CO se cu g & eg * r ^ C u >—i fa! "S-•° s a X o 1-O cn u '2 O c u U a, < U •a u lH 3 cn -JJ e U < U > • — •*-> C5 w e CU C/j V i -a CU «8 rr c 43 io o co CD C S O 2 CO o > 4=1 cn SJ 3 U cn TJ C o o § 2 bo -£ & CO 8 M * 1 O -5 -CN CU cj U K c DC • — to o ab 1= | cn U c 125 126 U to < V r> V o 08 yr i 2 I g c« Bo T3 CU c -S to p-to fa U O DH CJ co •£ CO c/5 J= — S3 rt o c^  < cj c S cu 8 M o CO c cu 60 KB >> c> x <m o r-tj cu "3 JS 3 o c o cj o co CJ 5 J5 cj CJ S c o 60 X c o . — 3 60 C cu s a 6 0 ^ CO cj 2 o S .5 C cu © .2 © £ U > U =o CJ a I s CU 6C c 09 -= u 83 o \n 43 a •-c - ^ S3 O O o < CU 'cj T3 CU XJ £ C CO CO co CU >> *-> 5 S3 5 | co r j OH < H u ^ 2 S3 cO •a CJ CJ c CO o •— 08 CJ CO to cu cu-O co CO CJ CU CJ 6 0 .2 CU i . £ 4= © 127 Fig. 16 HPC growth in liquid media reveals abnormal proliferation and increased apoptosis. a) HPC were grown in liquid media supplemented with SCF, FL-3 and IL-6 for 13 days post column isolation and proliferation was determined of triplicate cultures. Data is represented as the average + SEM of triplicate cultures from n=5 mice per genotype, b) FACS analysis of dUTP-positive nuclei from day 13 HPC grown in vitro. FACS data is from 10,000 events. Data represents the average percent staining ± SEM for n=3 per genotype. * p 0.05. Fancc+/+ Sodl-/- Fancc-/- Fancc-/-Sodl+/+ Sodl-/-Fig. 17 Hypoxia partially rescues HPC growth from Fancc Sodl" mice. HPC cultures were grown in liquid media in 20% (dark bars) and 5% (gray bars) oxygen conditions and proliferation was measured on day 9 of culture. Fancc'Sodl'' HPC numbers could be partially rescued to wildtype levels when grown in hypoxic conditions. Data is represented as the average ± S E M of duplicate cultures from n=3-4 mice per genotype. * p 0.05 129 14 0 -I 1 1 1 1 1 0 0.001 0.005 0.5 1 5 Concentration of TIRON (mM) Fig. 18 TIRON partially rescues HPC growth from Fancc''Sodl''' mice. HPC cultures were grown in liquid media in the presence of increasing concentrations of TIRON, a cell permeable superoxide scavenger. Cell number was determined on day 9 of culture. Data is represented as the average ± S E M of duplicate cultures from n=3-4 mice per genotype. * p 0.05 4.5 0 -I 1 1 1 1 1 0 1 2.5 5 10 20 Concentration of MnTMPyP (uM) 2.5 b) !P o C 1.5 i— o n E 3 i 1 o 0.5 — • — -Wt -Wt + MnTMPyP -Dko --0— -Dko + MnTMPyP n=5 Dayl Day3 Day6 Time Day9 Dayl3 Fig. 19 MnTMPyP dose response and partial rescue of HPC proliferation in vitro. a) HPC cultures were grown in liquid media in the presence of increasing concentrations of MnTMPyP, a SOD mimetic. Cell number was determined on day 9 of culture. 5pM was chosen as the concentration which resulted in optimal growth for each genotype. b) HPC were subsequently cultured in the presence of 5 p M MnTMPyP and cell number was determined as above for 13 days post-isolation. Data is represented as the average ± S E M of duplicate cultures from n=3-4 mice per genotype. * p 0.05 1.31 Fancc'Sodl Fancc Sodl o Fig. 20 Primary Mast Cell cultures from Fancc Sodl'' marrows do not grow lit vitro. Whole B M samples from wildtype and Fancc'Sodl'' mice were cultured for 5 weeks in liquid media in the presence of SCF and IL-3 and a representative picture of a typical culture is shown above. Wildtype cultures consisted of a homogenous population of mast cells, while the Fancc'Sodl'' cultures contained a highly heterogenous cell population with many dead cells. Arrows represent an example of a single mast cell. 132 FancA*'* Sod1*/+ FancA'-4i>tk^-?%Vi v-S CAi v Sodf'- FancA'Sodl Fig. 21 Histological examination of FancA'''Sodl'' mice livers. Sections of FancA'/+Sod 1+/+, FancA"'", Sodl'' and FancA'''Sodl'' livers were stained with Masson's trichrome (magnification 400x). FancA''" and FancA+/'Sodl+/' sections reveal normal liver morphology and some Sodl"'' sections have very mild pathology. FancA"'" Sodl''' hepatocytes demonstrate a moderate zone 3 abnormality characterized by cytoplasmic vacuolation. O i l red-0 staining revealed mild microvesicular zone 3 lipid accumulation in FancA'Sodl''' mice and a small amount of lipid droplets are present in Sodl'" mice, however the quality of the photographs was poor. G3 40 • Female FancA+/+ Sod1+/+ FancA- / - S o d l - / - FancA- / - Sod1-/-Fig. 22 Body weights are significantly decreased in FancA SodF' mice. Anaesthetized male (gray bars) and female (dark bars) mice were weighed and the average weight (in grams) ± S E M is represented above. There is a significant decrease in body weight in FancA'''Sod1~'~ mice. n=3-4 mice per genotype. * p 0.05. 134 FancA+/+ Sodl+/+ FancA-/- Sodl -A FancA-/-Sod l -A Fig. 23 Marrow cellularity from FancA Sodl" mice is decreased. Femurs from wildtype, FancA''', Sodl'' and FancA'''Sodl''' mice were flushed and the cellularity per femur measured. FancA'''Sodl''' mice had a statistically significant decrease in femur cellularity compared to wildtype controls. Data is represented as average femur cellularity ± S E M of n=3-4 mice per genotype. * p 0.05. 135 a) 3 E a LL C 0) Ol o ro o 25000 20000 15000 10000 A 5000 1 FancA+/+ Sod1+/+ FancA-/- Sod1-/- FancA-/-Sod1-/-b) 3 E 0) 10 o 'E 0) D) O a "5 o FancA+/+ Sod1+/+ Sod1-/- FancA-/-Sod1-/-Fig. 24 Colony Forming Assays reveal abnormal progenitor growth in FancA'Sodl''' mice. Colony forming assays reveal decreased numbers o f progenitors in FancA'Sodl''' marrows. a) M y e l o i d and b) Pre-B C F U s were measured for Sodl'', FancA~'~' FancA+/+ Sodl+/+, and FaneA''~Sodl''' mice. Both myeloid and pre-B C F U are decreased in FancA'''Sodl'' mice compared to wildtype controls, and the number o f pre-B progenitors/femur is significantly decreased from Sodl'', FancA''"' and FancA'''Sodl'' mice compared to FancA+/+ Sodl+/+ controls. Values represent the average number o f progenitors/femur of 3-4 mice per group ± S E M . * = p<0.05, ** = p<0.001. o o 5 ca c o o o c « 3 a 120 100 A m % GEMM • % GM/G/M • % ERY FancA+/+ Sod1+/+ FancA- / - Sod1- / - FancA- / - Sod1-/-Fig. 25 Frequency of B M progenitors from FancA SodF' mice is abnormal. FancA'1Sod'tA progenitors fail to generate normal ratios of CFU-GEMM, CFU-GM/G/M and BFU-E. Myeloid colonies from Fig. 24a) were assessed morphologically to determine the cell types contributing to the colonies. CFU-GEMM (dark bars), CFU-GM/G/M (light gray bars) and BFU-E (hatched bars) colonies were scored by eye and the values represent the average percent of cell type ± SEM (n = 3-4 mice per group). The ratios of CFU-GM/G/M, and CFU-GEMM were significantly different from FancA+/+Sodl+/+ controls. * p 0.05 137 0-] , . , , . 1 0 0.1 0.25 0.5 1 L-NMMA (mM) + I FNg (1 ng/ml) Fig. 26 Inhibition of Fancc'' colony formation by IFNy is reversed by L-NMMA. a) Colony formation by Fancc'' and littermate control B M progenitor cells plated in methylcellulose in the presence of increasing concentrations of IFNy. There is a dose-dependent inhibition of colony formation from Fancc'' progenitors, b) Colony formation by B M cells from wildtype and Fancc''mice grown in the presence of 1 ng/ml IFNy and with increasing concentrations of L - N M M A . L - N M M A completely rescues colony growth. Data points represent the average number of colonies counted per methylcellulose plate; n = 3 mice. * P < 0.05; ** P < 0.005. a) a 15 H Wildtype Fancc-/• b) 30 25 .n 20 E ° 15 o o » 10 < • MlPla EMIPla + 0.25 mM L-NMMA • MIPla+ 0.5 mM L-NMMA Wildtype Fancc-1-Fig. 27 Inhibited colony formation by T N F a and M l P l a is also reversed by L - N M M A . a) In the presence of 0.5 ng/ml TNFa, there was a reduction in colony formation by Fancc'' B M cells as compared to littermate controls which was reversed by addition of 0.25 mM L-NMMA. b) Reduction in Fancc'' B M cell colony number in the presence of 1 ng/ml MIP1 was partially reversed by the addition of 0.25 mM L-NMMA and completely reversed with 0.5 mM L-NMMA. Data points represent the average number of colonies counted; n = 4 mice. *P<0.05; **Y°<0.005. 139 120 a) 10 100 DETA/NO (uM) Fig. 28 Fancc'' BM progenitors show increased sensitivity to NO-generating drugs. a) Progenitor cell growth in the presence of increasing concentrations of the N O donor, SNAP, b) Growth of progenitor cells in the presence of increasing concentrations of DETA/NO. Percentage maximal colony formation was determined by dividing the number of colonies scored at a given concentration of NO donor by the number of colonies scored in the absence of the N O donor; n = 4 mice per group. * P < 0.05, ** P < 0.005. 140 b) FNg (10 ng/ml) IFNg + 0.5 mM L-NMMA Fig. 29 Inhibition of Fancc'' HPC growth by IFNy was reversed by L-NMMA. a) Fancc'' and wildtype HPC grown in the presence of 10 ng/ml jTNy, with and without the addition of 0.5 mM L-NMMA. Bars indicate percent of control (no cytokine added) for each of the two genotypes, b) Flow cytometry TUNEL analysis showing percentage of HPC cell nuclei that were dUTP-positive staining in untreated, JENy-treated (10 ng/ml), and IFNy plus 0.5 mM L-NMMA. IFNy plus 0.5 mM L-NMMA decreased dUTP-positive numbers to a level similar to that of the untreated group. Flow data was based on 10,000 events, n = 4 mice for each group. * P < 0.05. 141 Wildtype Fancc 0 3 5 8 12 0 3 5 8 12 iNOS 130 kDa a-tubulin 45 Time (hours) Fig. 30 Elevated iNOS expression in stimulated Fancc ' peritoneal macrophages. Peritoneal macrophages were stimulated for 0 - 12 hours with IFNy 10 ng/ml +/- LPS 100 ng/ml, and whole cell lysates assayed for iNOS expression by immunoblotting. a) is a representative filter showing iNOS protein expression in Fancc'' and control peritoneal macrophages following IFNy plus LPS stimulation (top panel), with a-tubulin (bottom panel) as the loading control, b) Densitometric representation of five independent experiments showing a significant difference in iNOS expression between Fancc'' and wildtype peritoneal macrophages at 8 and 12 hours post-stimulation. * P < 0.05. 142 Fig. 31 Elevated iNOS expression in Fancc ' BM-derived macrophages stimulated with IFNy. BM-derived macrophages were stimulated for 0 - 12 hours with IFNy 10 ng/ml and whole cell lysates were assayed for iNOS expression by immunoblotting a) is a representative blot showing iNOS expression in B M D M following IFNy stimulation (top panel), with a-tubulin (bottom panel) as the loading control, b) Densitometric representation of four independent experiments showing a significant increase in iNOS expression within Fancc'' B M D M at 5 hours post-stimulation, with expression of iNOS reaching a maximum at 8 hours. * P < 0.05. 143 0 3 5 8 Time (hrs) Fig. 32 Increased NO production by Fancc'' macrophages. Supernatants from the peritoneal macrophages used in iNOS expression studies (Fig. 30) were harvested and N O levels (as nitrite) were quantitated by ELISA. There was a significant increase in nitrite levels in the supernatants of Fancc'' cells (at 8 hrs) when macrophages were stimulated with IFNy plus LPS as well as when the cells were stimulated with IFNy alone (although the latter did not reach significance). * P < 0.05. 1 4 4 Wildtype Fancc''' 0 5 10 15 0 5 10 15 P-Stat1 120 kDa Total Statl 16 Time upon stimulation with IFNg (min) Fig. 33 Statl phosphorylation is augmented in IFNy-stimulated Fancc" macrophages. Peritoneal macrophages from wildtype and Fancc'' mice were stimulated with IFNy and cell lysates were collected at various time points for Statl immunoblotting analysis, a) is a representative filter showing P-Statl (top panel), and total Statl (bottom panel), b) is densitometry analysis derived from four independent experiments that demonstrates a significant increase in P-Statl signal within Fancc'' macrophages at 15 min post-stimulation with IFNy, * P < 0.05. 145 Fig. 34 H i f l a expression is increased in IFNy stimulated Fancc'' macrophages. Peritoneal macrophages from wildtype and Fancc'' mice were grown in 20% oxygen in the absence or the presence of IFNy and H i f l a protein levels were measured in the nuclear lysates. Data is represented as the average of two experiments and shows an increase in H i f l a protein from Fancc'' macrophage lysates both constitutively and a further increase in the presence of IFNy. 146 y u O CC — O H e« c to < xi o g B CJ J U ° '3 S a -CO >> (H rv CO t/3 i 1 147 

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