thioguanine-anhydrous and Fanconi-Anemia

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

The thiopurines azathioprine and 6-mercaptopurine have been extensively prescribed as immunosuppressant and anticancer agents for several decades. A third member of the thiopurine family, 6-thioguanine (6-TG), has been used less widely. Although known to be partly dependent on DNA mismatch repair (MMR), the cytotoxicity of 6-TG remains incompletely understood. Here, we describe a novel MMR-independent pathway of 6-TG toxicity. Cell killing depended on two properties of 6-TG: its incorporation into DNA and its ability to act as a source of reactive oxygen species (ROS). ROS targeted DNA 6-TG to generate potentially lethal replication-arresting DNA lesions including interstrand cross-links. These triggered processing by the Fanconi anemia and homologous recombination DNA repair pathways. Allopurinol protected against 6-TG toxicity by acting as a ROS scavenger and preventing DNA damage. Together, our findings provide mechanistic evidence to support the proposed use of thiopurines to treat HR-defective tumors and for the coadministration of 6-TG and allopurinol as an immunomodulation strategy in inflammatory disorders.

Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; Cross-Linking Reagents; DNA; DNA Damage; Fanconi Anemia; Humans; Immunoblotting; Oxidation-Reduction; Reactive Oxygen Species; Thioguanine

Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.

Topics: Animals; Cell Line, Tumor; Cell Survival; Chromosome Aberrations; Cross-Linking Reagents; DNA Repair; Fanconi Anemia; Fibroblasts; Genomic Instability; Humans; Mice; Polydeoxyribonucleotides; Thioguanine; Ultraviolet Rays

The induction of mutants at the hypoxanthine-guanine phosphoribosyltransferase and Na+/K+ ATPase loci by photoaddition of two bifunctional psoralens was compared in normal and in Fanconi's anemia lymphoblasts from the genetic complementation group A. For the two loci, the frequency of mutants was significantly lower in Fanconi's anemia than in normal cells. This is true whether the data are expressed as a function of dose or as a function of survival level. It is suggested that the chromosomal instability characteristic of Fanconi's anemia is responsible for the cancer proneness rather than the mutability at the gene level.

Topics: Anemia, Aplastic; Cell Survival; Fanconi Anemia; Furocoumarins; Humans; Hypoxanthine Phosphoribosyltransferase; Methoxsalen; Mutation; Sodium-Potassium-Exchanging ATPase; Thioguanine; Trioxsalen; Ultraviolet Therapy

Fanconi anemia (FA) is an inherited human disorder associated with a predisposition to cancer and characterized by anomalies in the processing of DNA cross-links and certain monoadducts. We reported previously that the frequency of psoralen-photoinduced mutations at the HPRT locus is lower in FA cells than in normal cells. This hypomutability is shown here to be associated with an increased frequency of deletions in the HPRT gene when either a mixture of cross-links and monoadducts or monoadducts alone are induced. Molecular analysis of mutants in the HPRT gene was carried out. In normal cells the majority of spontaneous and induced mutants are point mutations whereas in FA deletion mutations predominate. In that case a majority of mutants were found to lack individual exons or small clusters of exons whereas in normal cells large (complete or major gene loss) and small deletions are almost equally represented. Thus we propose that the FA defect lies in a mutagenic pathway that, in normal cells, involves bypassing lesions and subsequent gap filling by a recombinational process during replication.

Topics: B-Lymphocytes; Cell Line; Cell Survival; Chromosome Deletion; Dose-Response Relationship, Radiation; Exons; Fanconi Anemia; Humans; Hypoxanthine Phosphoribosyltransferase; Light; Mutagenesis; Reference Values; Thioguanine; Ultraviolet Rays

The incidence of spontaneous 6-thioguanine-resistant (TGr) lymphocytes was studied in the peripheral blood collected from seven Fanconi anemia (FA) patients and five of their heterozygous parents using an autoradiographic or a lymphocyte cloning method. Five of the seven patients showed a significantly elevated incidence of TGr lymphocytes as compared to age- and sex-matched healthy controls. There was, however, no difference between FA heterozygotes and controls. These results suggest some variability among the patients similar to those reported in clinical and cytogenetic investigations. The basis for the increase in TGr cells in the patients is not known, but the inherent genomic instability reflected as increased frequencies of chromosomal aberrations is one possible explanation.

Topics: Adolescent; Adult; Anemia, Aplastic; Autoradiography; Child; Child, Preschool; Chromosome Aberrations; Drug Resistance; Fanconi Anemia; Female; Heterozygote; Humans; Lymphocytes; Male; Mutation; Thioguanine

Fanconi anemia (FA) fibroblasts are known to be exceptionally sensitive to the cytotoxic action of mitomycin C (MMC). The survival of FA cells was enhanced significantly when 0.5 mM caffeine or 0.5 mM adenine was added for 72 h after the cells were exposed to MMC. In other experiments in which MMC was not used, FA fibroblasts were shown to be significantly more sensitive than control cells to 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and 6-azauridine (6-AU). These observations offer a new approach to defining the basic biochemical defect in FA.

Topics: Anemia, Aplastic; Ataxia Telangiectasia; Azauridine; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Fanconi Anemia; Humans; Mercaptopurine; Mitomycins; Skin; Thioguanine