ascorbic-acid and Fanconi-Anemia

Hematopoietic stem cell (HSC) attrition is considered the key event underlying progressive BM failure (BMF) in Fanconi anemia (FA), the most frequent inherited BMF disorder in humans. However, despite major advances, how the cellular, biochemical, and molecular alterations reported in FA lead to HSC exhaustion remains poorly understood. Here, we demonstrated in human and mouse cells that loss-of-function of FANCA or FANCC, products of 2 genes affecting more than 80% of FA patients worldwide, is associated with constitutive expression of the transcription factor microphthalmia (MiTF) through the cooperative, unscheduled activation of several stress-signaling pathways, including the SMAD2/3, p38 MAPK, NF-κB, and AKT cascades. We validated the unrestrained Mitf expression downstream of p38 in Fanca-/- mice, which display hallmarks of hematopoietic stress, including loss of HSC quiescence, DNA damage accumulation in HSCs, and reduced HSC repopulation capacity. Importantly, we demonstrated that shRNA-mediated downregulation of Mitf expression or inhibition of p38 signaling rescued HSC quiescence and prevented DNA damage accumulation. Our data support the hypothesis that HSC attrition in FA is the consequence of defects in the DNA-damage response combined with chronic activation of otherwise transiently activated signaling pathways, which jointly prevent the recovery of HSC quiescence.

Topics: Animals; Ascorbic Acid; Bone Marrow Failure Disorders; Cell Line; Cholecalciferol; Dehydroepiandrosterone; DNA Damage; Extracellular Signal-Regulated MAP Kinases; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; Hematopoietic Stem Cells; MAP Kinase Signaling System; Mice; Mice, Knockout; Microphthalmia-Associated Transcription Factor; Nicotinic Acids; Plant Extracts; Smad Proteins

Induction of the Fanconi anemia (FA) DNA repair pathway is a common mechanism by which tumors evolve resistance to DNA crosslinking chemotherapies. Proper execution of the FA pathway requires interaction between the FA complementation group M protein (FANCM) and the RecQ-mediated genome instability protein (RMI) complex, and mutations that disrupt FANCM/RMI interactions sensitize cells to DNA crosslinking agents. Inhibitors that block FANCM/RMI complex formation could be useful therapeutics for resensitizing tumors that have acquired chemotherapeutic resistance. To identify such inhibitors, we have developed and validated high-throughput fluorescence polarization and proximity assays that are sensitive to inhibitors that disrupt interactions between the RMI complex and its binding site on FANCM (a peptide referred to as MM2). A pilot screen of 74,807 small molecules was performed using the fluorescence polarization assay. Hits from the primary screen were further tested using the proximity assay, and an orthogonal proximity assay was used to assess inhibitor selectivity. Direct physical interaction between the RMI complex and the most selective inhibitor identified through the screening process was measured by surface plasmon resonance and isothermal titration calorimetry. Observation of direct binding by this small molecule validates the screening protocol.

Topics: Antineoplastic Agents; DNA Damage; DNA Helicases; DNA Repair; Drug Screening Assays, Antitumor; Fanconi Anemia; High-Throughput Screening Assays; Humans; Multiprotein Complexes; Protein Interaction Maps; RecQ Helicases

Fanconi anemia is a rare genetic disorder with high propensity for development of cancers, such as aplastic anemia, leukemia and head and neck cancers. Collagen digesting matrix metalloproteinase (MMP) enzymes have been implicated in for their role in various malignancies and to promote metastasis. Biological agents that prevent extracellular matrix digestion by the MMPs have been shown to be promising therapeutic approaches to cancer. In this study, we investigated effects of a nutrient mixture (NM) containing, ascorbic acid, lysine, proline and green tea extract, on human FANCA and FANCC lymphoblasts for viability, MMP secretion and invasion.. Human FANCA lymphoblasts GM13022 and HCS536 were challenged with NM at concentration range within 10-1000 µg/ml. Cell toxicity was assessed by Trypan blue dye exclusion test. Invasion was evaluated through Matrigel and gelatinase zymography for MMP activity.. NM was toxic in dose dependent mode to HCS536 cells but not to GM13022 cells. GM13022 cells but not HCS536 cells exhibited MMP-9 secretion, which was inhibited by NM. Matrigel invasion was inhibited in HCS536 cells at 100 and 500 µg/ml by 27% and 93%, respectively. In GM13022 cells, the NM showed completely blocked Matrigel invasion at 500 µg/ml.. NM inhibited MMP secretion and Matrigel invasion in FANCA and inhibited invasion and induced toxicity in FANCC lymphoblasts. These results suggest that the NM may have therapeutic potential in Fanconi anemia associated neoplasia.

Topics: Ascorbic Acid; Blotting, Western; Cell Movement; Cell Proliferation; Fanconi Anemia; Fanconi Anemia Complementation Group A Protein; Fanconi Anemia Complementation Group C Protein; Humans; In Vitro Techniques; Lymphocytes; Lysine; Matrix Metalloproteinases; Plant Extracts; Proline; Tea; Tumor Cells, Cultured

Fanconi Anemia, an autosomal recessive disorder, is characterized by chromosomal abnormality leading to birth defects, progressive bone marrow failure, and a high probability of developing malignancy at an early age. Head and neck squamous cell carcinoma and myeloid leukemia are the major causes of cancer related morbidity and mortality in Fanconi anemia patients.. We investigated the effect of a nutrient mixture on Fanconi Anemia human fibroblast cell lines FA-A:PD20 and FA-A:PD220 on matrix metalloproteinase expression, invasion, cell proliferation, morphology and apoptosis. The cell lines were grown in a modified Dulbecco's Eagle medium and at near confluence were treated with the nutrient mixture at increasing doses: 0; 10; 50; 100; 500; 1000 µg/ml. The cells were also treated with PMA to induce MMP-9 expression.. Zymography demonstrated MMP-2 and PMA-induced MMP-9 activity. The nutrient mixture inhibited expression of both, MMP-2 and MMP-9, in a dose dependent manner with virtually total inhibition observed at 500 µg/ml. Matrigel invasion was inhibited in both cells lines; with 100% inhibition for FA-A:PD20 at 500 µg/ml and 100% inhibition of FA-A:P220 cells at 100 µg/ml. H&E staining did not indicate any change in cell morphology and causes apoptosis at higher doses.. Our data demonstrated that the nutrient mixture inhibited matrix metalloproteinase expression, invasion and induced apoptosis, the important parameters for cancer prevention. The results suggest that the nutrient mixture may have therapeutic potential in Fanconi Anemia associated neoplasia.

Topics: Antioxidants; Apoptosis; Arginine; Ascorbic Acid; Cell Line; Cell Movement; Cell Proliferation; Fanconi Anemia; Humans; Lysine; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Proline; Tea

Some selected oxidative stress parameters were measured in 56 Fanconi anaemia (FA) patients (42 untransplanted and 14 transplanted), 54 FA heterozygotes (parents) and 173 controls. Untransplanted FA patients showed a highly significant increase in leukocyte 8-hydroxy-2'-deoxyguanosine (8-OHdG) (P = 0.00003) and a borderline increase (P = 0.076) in urinary levels of 8-OHdG versus child controls. These increases were more pronounced in female FA patients (P = 0.00005 for leukocyte 8-OHdG and P = 0.021 for urinary 8-OHdG). Female FA patients also displayed a highly significant excess of spontaneous chromosomal breaks versus male patients (P = 0.00026), in the same female:male ratio ( approximately 1.4) as detected for both leukocyte and urine 8-OHdG levels. Plasma methylglyoxal (MGlx) levels were increased in untransplanted FA patients versus child controls (P = 0.032). The increases in leukocyte and urinary 8-OHdG and in MGlx levels were detected in young FA patients (< or =15 years), whereas patients aged 16-29 years failed to display any differences versus controls in the same age group. A significant increase in oxidized:reduced glutathione (GSSG:GSH) ratio was observed (P = 0.046) in the FA patients aged < or =15 years, whereas those aged 16-29 years, both untransplanted and transplanted, displayed a decrease (P = 0.06) in the GSSG:GSH ratio versus the controls of the respective age groups. No significant changes were detected in plasma levels of vitamin C, vitamin E or uric acid. Transplanted FA patients showed lesser alterations in leukocyte 8-OHdG and in GSSG:GSH ratio versus untransplanted patients. The parents of FA patients displayed a significant increase in plasma MGlx levels (P = 0.0014) versus adult controls. The results suggest a gender- and age-related modulation of oxidative stress in FA patients. The observed increase in urinary 8-OHdG in untransplanted FA patients suggests a proficient removal of oxidized DNA bases.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adolescent; Adult; Age Factors; Ascorbic Acid; Case-Control Studies; Child; Child, Preschool; Chromosome Breakage; Chromosomes, Human; Deoxyguanosine; DNA; Fanconi Anemia; Female; Glutathione; Glutathione Disulfide; Heterozygote; Humans; Infant; Leukocytes; Male; Oxidation-Reduction; Oxidative Stress; Pyruvaldehyde; Reactive Oxygen Species; Respiratory Burst; Sex Factors; Transplants; Uric Acid; Vitamin E

Peripheral blood lymphocytes from eight Fanconi anemia (FA) patients, 14 FA heterozygotes, and nine normal subjects have been tested for their susceptibility to chromosomal breakage induction by diepoxybutane (DEB) and by two peroxides. In addition, the effect of five antioxidants was investigated in standard cultures and in cultures stressed either with DEB or with butylhydroperoxide (BHP) or with hydrogen peroxide (H2O2). DEB, BHP, and H2O2 dramatically increased the chromosomal breakage levels in homozygous and heterozygous FA cells. A partial correction of chromosomal instability was obtained by treating the patients' lymphocytes with antioxidants. A "protective" effect was also noted in the DEB or peroxide-stressed lymphocytes of patients and heterozygotes, grown in the presence of antioxidants.

Topics: Anemia, Aplastic; Antioxidants; Ascorbic Acid; Cells, Cultured; Chromosome Aberrations; Cross-Linking Reagents; Cysteine; Epoxy Compounds; Ethers, Cyclic; Fanconi Anemia; Glutathione; Heterozygote; Homozygote; Humans; Hydrogen Peroxide; Lymphocytes; Mercaptoethanol; Peroxides; Tiopronin

Topics: Anemia, Aplastic; Antioxidants; Ascorbic Acid; Chromosome Aberrations; Cysteine; Epoxy Compounds; Ethers, Cyclic; Fanconi Anemia; Humans; Hydrogen Peroxide; Mercaptoethanol; Mutagens; Oxygen Consumption; Peroxides; tert-Butylhydroperoxide