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sghidu 2019 amazon 9 – más de 40. .DESCRIPTION: Nitric oxide (NO) produced by the enzyme nitric oxide synthase (NOS) has been implicated in the pathogenesis of numerous diseases including cancer. NOS expression and activity may be critical to the process of malignant transformation and progression. The mechanism(s) by which NOS expression and activity are altered in neoplastic cells are poorly understood, although recent studies suggest that the constitutive form of NOS (cNOS), which is typically absent from normal tissues, may be aberrantly expressed and activated in neoplastic tissues. The applicant's data demonstrate that the expression of cNOS is regulated by methylation of the cNOS gene promoter, which occurs in an enzyme- specific manner, and that this methylation is enhanced in transformed cells. cNOS is also regulated by protein phosphorylation. The proposed studies will assess the role of both promoter methylation and phosphorylation in regulating cNOS expression in human cancer cells. cNOS expression will be determined by measuring promoter activity and enzyme activity in untransformed and transformed cells. Promoter methylation and protein phosphorylation of the cNOS promoter in transformed cells will be correlated with cNOS expression and enzyme activity. Several specific aims are proposed to test the following hypotheses: Aim 1) NOS expression in transformed cells can be correlated with the extent of cNOS promoter methylation. Aim 2) Transcriptional and post-transcriptional mechanisms regulate cNOS expression in transformed cells. Aim 3) Regulation of cNOS protein expression in transformed cells by changes in its promoter and by protein phosphorylation. In addition, a new technique for nuclear DNA methylation analysis, termed Methylation Sensitive High Resolution Melting (MS-HRM), will be applied to the study of promoter methylation. This technique will be used to test the hypothesis that the cNOS promoter is methylated in transformed cells and that this methylation is increased with tumor progression and degree of malignancy. Because the proposed studies will focus on gene methylation and protein phosphorylation in cancer cells, we anticipate that these studies will help to identify novel targets for chemotherapeutic drugs. The proposed studies will also facilitate our understanding of the role of the cNOS gene in cancer cell biology and in the pathogenesis of cancer.
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