Mutação e superexpressão de p53 em neoplasias mamárias de cadelas

Abstract

The major incidence and the high level of mortality associated to the huge diversity of the cancer phenotypic require numerous strategies for its diagnosis. In this research, the aim was to evaluate the mutation relation in the gene Tp53 with the breast cancer in female dogs, through the immunohistochemistry expression of the protein p53 and of the gene polymorphism in the breast tissues. In the Experiment 1, was studied the immunohistochemistry expression of the protein p53 in relationship to the morphologic aspects of healthy mammary tissues, to classification and grade of tumors and to the ipsilateral inguinal lymph-nodes tissue, metastatic or not. For this purpose 19 breasts from healthy female dogs were used (Group 1- control), 29 samples of breasts with clinical diagnosis of tumor (Group 2), 29 contralateral breasts (Group 3) and 29 lymphnodes (Group 4). The material was analyzed by conventional techniques of microscopy (staining HE) for tissue’s characterization and by immunohistochemistry (estreptavidina-biotina peroxidase method) to evaluate the expression of the protein p53 in the cellular compartment. The contralateral breasts and the lymph-nodes that showed metastasis were also marked, as well as the tissues that still showed normality. There weren´t statistical differences between the staining intensity and the number of cells stained with malignity grade. In the Experiment 2 the aim was to detect mutations in sequences of the exon 8 of the gene Tp53 through the PCR-RFLP technique with specific restriction enzymes to associate with band patterns obtained to the breast tumor type or normal tissues in female dogs. Were used 19 healthy female dogs (control) and 50 female dogs submitted to the tumor exerese from wich samples of tumor tissue and of healthy contralateral mammary glands were obtained. The DNA extraction was done through the fenol-cloroformic method and the PCR-RFLP technique was used for amplifiation and digestion of the exon 8 of the Tp53 with 6 restriction endonucleases, SmaI, DdeI, Rsa, AvaI, BsobI e AluI. The amplified DNA showed only one side of 200 pb in the agarose gel (0,8%). The PCR product showed on average 94% of homology with the known sequence of the gene (GeneBank –AAB42022.1). Among the 6 chosen enzymes, only 4 showed polymorphism (AluI, BsobI, DdeI e SmaI) in the tumor sequence of the exon 8 as to the numbers of pairs of the base (pb) or the quantity of cleavages. The band patterns of each enzyme showed polymorphism among the different groups, but not inside each group (normal female dogs, tumor mammary glands and contralateral breasts). There was´nt any difference among the breast tissues with tumor and the opposite laterals of the same animal, for the enzymes AluI, BsoBI e SmaI, reinforcing the idea that the tissues that didn´t showed visible alterations were already changed at the molecular level and therefore were potentially capable of developing tumors over the time. It is concluded that the gene Tp53 is strongly involved in the breast cancer etiology, and it could be used as a parameter to analyse these tumors´ behavior. The analysis of the protein p53 expression associated to the genetic markers that identify the mutations in the gene sequence could propitiate the early diagnosis of this pathology, allowing the treatment in time of avoiding metastasis and risks of the animal´s survival.