RESUMO
Human APOBEC3A (A3A) cytidine deaminase shows pro-apoptotic properties resulting from hypermutation of genomic DNA, induction of double-stranded DNA breaks (DSBs) and G1 cell cycle arrest. Given this, we evaluated the antitumor efficacy of A3A by intratumoral electroporation of an A3A expression plasmid. DNA was repeatedly electroporated into B16OVA, B16Luc tumors of C57BL/6J mice as well as the aggressive fibrosarcoma Sarc2 tumor of HLA-A*0201/DRB1*0101 transgenic mice using noninvasive plate electrodes. Intratumoral electroporation of A3A plasmid DNA resulted in regression of ~50% of small B16OVA melanoma tumors that did not rebound in the following 2 months without treatment. Larger or more aggressive tumors escaped regression when so treated. As APOBEC3A was much less efficient in provoking hypermutation and DSBs in B16OVA cells compared with human or quail cells, it is likely that APOBEC3A would be more efficient in a human setting than in a mouse model.
Assuntos
Citidina Desaminase/genética , Eletroporação/métodos , Terapia Genética , Melanoma Experimental/terapia , Plasmídeos/genética , Proteínas/genética , Animais , Feminino , Linfócitos do Interstício Tumoral/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais CultivadasRESUMO
The mouse is widely used as a model for DNA therapy and vaccination even though the efficiency of DNA delivery in higher mammals and humans is much less. The human APOBEC3 (A3) enzymes impact viral genomes by cytidine deamination, which introduces multiple uridine residues into single-stranded DNA, a process known as genetic editing. This initiates rapid DNA catabolism via a uracil DNA glycosylase dependent pathway. In tissue culture, A3A, A3C and A3B can hyperedit transfected plasmid DNA. We explored plasmid catabolism in vivo initiated by A3A, the most efficient of the human enzymes and one that is functionally conserved across most mammals. As rodents do not encode an A3A enzyme, it was possible to explore DNA degradation in the mouse model. Human A3A genetically edits co-electroporated luciferase plasmid DNA in mouse skeletal muscle that initiates DNA degradation resulting in approximately fourfold decrease in bioluminescence. Part of the degradation occurs in the nucleus as indicated by complex hyperedited DNA molecules. As human A3A is strongly upregulated by interferon α and DNA sensing pathways, it is a strong candidate enzyme for restricting plasmid DNA in higher mammals.
Assuntos
Citidina Desaminase/fisiologia , Plasmídeos/genética , Proteínas/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Clivagem do DNA , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Eletroporação , Feminino , Genes Reporter , Luciferases/biossíntese , Luciferases/genética , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Mutação , Taxa de Mutação , TransfecçãoRESUMO
OBJECTIVE: The objective of the study was to characterize the bioactivity of prolactin (PRL) in hyperprolactinaemic patients with prolactinomas, irregular menstrual cycles, regular menstrual cycles and PCOS. METHODS: Serum PRL, biological activity of PRL (after polyethylene glycol (PEG) precipitation) and serum follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), testosterone (T), thyroid-stimulating hormone (TSH) concentrations were measured in all hyperprolactinaemic patients and control group (healthy subjects). Correlations between active PRL (PRL-PEG) and serum FSH, LH, E2, T, TSH concentrations were also evaluated. RESULTS: Prolactinoma is characterized by high serum PRL levels and its high biological activity. Hyperprolactinaemic patients with irregular cycles were characterized by high biological activity of PRL. Patients with hyperprolactinaemia and regular cycles had low biological activity of PRL. CONCLUSIONS: Diagnosis of hyperprolactinaemia should be associated with estimation of PRL biological activity because it is important for type of hyperprolactinaemia management. Low biological activity of PRL does not impair FSH and LH secretion and does not cause hypoestrogenism.