RESUMO
Non-invasive medical imaging techniques such as positron emission tomography (PET) imaging are powerful platforms to track the fate of radiolabeled materials for diagnostic or drug delivery applications. Polymer-based nanocarriers tagged with non-standard PET radionuclides with relatively long half-lives (e.g. 64Cu: t1/2 = 12.7 h, 76Br: t1/2 = 16.2h, 89Zr: t1/2 = 3.3 d, 124I: t1/2 = 4.2 d) may greatly expand applications of nanomedicines in molecular imaging and therapy. However, radiolabeling strategies that ensure stable in vivo association of the radiolabel with the nanocarrier remain a significant challenge. In this study, we covalently attach radioiodine to the core of pre-fabricated nanocarriers. First, we encapsulated polyvinyl phenol within a poly(ethylene glycol) coating using Flash NanoPrecipitation (FNP) to produce stable 75 nm and 120 nm nanocarriers. Following FNP, we radiolabeled the encapsulated polyvinyl phenol with 125I via electrophilic aromatic substitution in high radiochemical yields (> 90%). Biodistribution studies reveal low radioactivity in the thyroid, indicating minimal leaching of the radiolabel in vivo. Further, PEGylated [125I]PVPh nanocarriers exhibited relatively long circulation half-lives (t1/2 α = 2.9 h, t1/2 ß = 34.9 h) and gradual reticuloendothelial clearance, with 31% of injected dose in blood retained at 24 h post-injection.
RESUMO
The rare genetic disorder Fanconi anemia, caused by a deficiency in any of at least thirteen identified genes, is characterized by cellular sensitivity to DNA interstrand crosslinks and genome instability. The excision repair cross complementing protein, ERCC1, first identified as a participant in nucleotide excision repair, appears to also act in crosslink repair, possibly in incision and at a later stage. We have investigated the relationship of ERCC1 to the Fanconi anemia pathway, using depletion of ERCC1 by siRNA in transformed normal human fibroblasts and fibroblasts from Fanconi anemia patients. We find that depletion of ERCC1 does not hinder formation of double strand breaks in crosslink repair as indexed by gammaH2AX. However, the monoubiquitination of FANCD2 protein in response to MMC treatment is decreased and the localization of FANCD2 to nuclear foci is eliminated. Arrest of DNA replication by hydroxyurea, producing double strand breaks without crosslinks, also requires ERRC1 for FANCD2 localization to nuclear foci. Our results support a role for ERCC1 after creation of a double strand break for full activation of the Fanconi anemia pathway.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/metabolismo , Linhagem Celular Transformada , Núcleo Celular/genética , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Mutagênicos/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/genética , Ubiquitinação/efeitos dos fármacosRESUMO
The disease Fanconi anemia is a genome instability syndrome characterized by cellular sensitivity to DNA interstrand cross-linking agents, manifest by decreased cellular survival and chromosomal aberrations after such treatment. There are at least 13 proteins acting in the pathway, with the FANCD2 protein apparently functioning as a late term effecter in the maintenance of genome stability. We find that the chromatin remodeling protein, Tip60, interacts directly with the FANCD2 protein in a yeast two-hybrid system. This interaction has been confirmed by co-immunoprecipitation and co-localization using both endogenous and epitope-tagged FANCD2 and Tip60 from human cells. The observation of decreased cellular survival after exposure to mitomycin C in normal fibroblasts depleted for Tip60 indicates a direct function in interstrand cross-link repair. The coincident function of Tip60 and FANCD2 in one pathway is supported by the finding that depletion of Tip60 in Fanconi anemia cells does not increase sensitivity to DNA cross-links. However, depletion of Tip60 did not reduce monoubiquitination of FANCD2 or its localization to nuclear foci following DNA damage. The observations indicate that Fanconi anemia proteins act in concert with chromatin remodeling functions to maintain genome stability after DNA cross-link damage.
