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Mutagenic potential of Bos taurus Papillomavirus type 1E6 recombinant protein: first description 

Araldi RP1, Mazzuchelli-de-Souza J1, Módolo DG1, Souza EB2, Melo TC1, Spadacci-Morena DD3, Magnelli RF1, Carvalho MACR1, Carvalho RF1, Beçak W1, Stocco RC1

1 Laboratório de Genética, 3 Laboratório de Fisiopatologia, Instituto Butantan, Brasil; 2 Genética e Mutagênese, Universidade Estadual Paulista "Júlio de Mesquita Filho" Assis , Brasil

Introduction: Bovine papillomavirus (BPV) is considered a useful model to study Human papillomavirus (HPV) oncogenic process. HPV and BPV interacts with the host chromatin, resulting in DNA damages, which are attributed to E5, E6 and E7 viral oncoproteins activity. Several studies showed different mechanisms of E6 action. However, the mechanisms of BPV E6 oncoprotein per se remain unknown, requiring novel studies. Objectives: This study aimed to evaluate the mutagenic potential of BPV-1 E6 recombinant oncoprotein by the cytokinesis-block micronucleus assay (CBMNA) and comet assay (CA). These noninvasive methods, recommended as part of genotoxicity tests battery for drug validation, when combined, they allow to detect DNA damages as indicative of mutagenesis with high statistical and sensitivity capacity. Methods: BPV-1 E6 recombinant oncoprotein was expressed and purified. A volume of 5 ml of peripheral blood was collected from five asymptomatic calves. The protocols used in this study were approved by the Ethics Committee on Animal Use of Butantan Institute (process 1035/13). Samples were subjected to DNA extraction and molecular diagnosis, which did not reveal presence of BPV sequences. For each sample, three cultures were established: (1) negative control (not treated with any drug), (2) positive control (treated with 50 µg/ml of cyclophosphamide) and (3) experimental group (treated with 1 µg/ml of E6 recombinant oncoprotein). The samples were submitted to the CBMNA and CA. Results and Discussion: CBMNA showed an elevated number of micronucleated lymphocytes, as well as anaphase bridges, in both positive control and in the group treated with E6 recombinant oncoprotein. Results pointed out that the cyclophosphamide and E6 recombinant oncoprotein are able to induce aneugenesis and/or clastogenesis. CA reinforced the CBMNA results, showing DNA degradation. Results show that the BPV E6 oncoprotein induces clastogenesis per se, which is an indicative of genomic instability. These results allowed to better understand the mechanism of cancer promotion associated with the BPV E6 oncoprotein, as well as revealed that this oncoprotein can induce the carcinogenesis per se. E6 recombinant oncoprotein has been suggested as possible vaccine candidate, but modifications are requires to use it as vaccine.

Supported by FAPESP, Butantan Foundation
1035/13



7 - CELLULAR BIOLOGY AND GENETICS 21th Annual Scientific Meeting of Instituto Butantan.