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Nuclear proteome alterations induced by growth factor along cell cycle in mice tumour cells

Grant number: 16/24881-4
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): April 01, 2017
Effective date (End): May 31, 2019
Field of knowledge:Biological Sciences - Biochemistry
Principal researcher:Julia Pinheiro Chagas da Cunha
Grantee:Mariana de Camargo Lopes
Home Institution: Instituto Butantan. Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil
Associated research grant:11/22619-7 - Nuclei and chromatin alterations through cell cycle and senescence in mammalian cells, AP.JP
Associated scholarship(s):17/15835-1 - Cross-talk between signaling pathway and histone modifications in response to growth factor stimulation, BE.EP.DD


Despite being a growth factor, Fibroblast Growth Factor (FGF) 2 has anti-proliferative and anti-tumor activities in some contexts. In adrenocortical mouse (Y1) lineage, FGF-2 delays S-phase progression and permanently blocks cells in the G2/M transition of the cell cycle, leading them to senescence. This project is the continuation of previous project (2015/04867-4) (carried out for 1 year and 8 months) with a budget grant linked to the Young Researcher Project (2011/22619-7) which objective is to study the dynamics of post-translational modifications (MPTs) in histones induced by this growth factor. In summary, we have found that treatment with FGF-2 causes a decrease in acetylation levels of H4K16. As this MPT is associated with DNA damage/repair, we have investigated, by COMETA assays, that FGF-2-stimulated cells show DNA damage possibly due to oxidative stress oxidized bases. Since mitochondria is one of the main sources of Reactive Oxygen Species (ROS) and harbor several sirtuins (enzymes involved in protein deacetylation), we investigated whether FGF-2 could induce mitochondrial alterations that could lead to increased ROS and, consequently, generate DNA damage. However, we did not observe any increase in cytoplasmic or mitochondrial ROS after stimulation with FGF-2. In addition, analyzes of mitochondrial membrane potential (TRME) and calcium homeostasis (Fluo-4AM) did not indicate significant changes in mitochondria. In parallel, we verified by quantitative PCR that the expression levels of the MOF/KAT8 enzyme responsible for the acetylation of H4K16ac, are significantly decreased after treatment with FGF-2. Recently, we analyzed by mass spectrometry MPTs in histones of this lineage and confirmed the decrease in acetylation levels in H4K16 and. In addition, we have identified that H3K4me3, an important transcriptional marker, is also decreased after stimulation. These analyzes suggest that FGF-2 stimulation induces changes in chromatin structure which, directly or indirectly, could affect transcription rates. In the continuation of this project, we intend to extend these proteomic analyzes and to analyze whether the FGF-2 stimulus is inducing global changes in the chromatin structure and at the transcriptional levels. In addition, we intend to generate Y1 cells in which the expression of the MOF/KAT8 enzyme is increased or decreased (by shRNA assays) and to assess whether the presence of high or low levels of H4K16ac may: I. modulate the cytotoxic action of FGF-2; II. Influence DNA repair; III. Change the overall rates of transcription and chromatin structure. In short, we believe that the results generated by this project may, for the first time, describe changes in MPTs in histones induced by FGF-2 and enable a better understanding of phenotypic alterations, which may be dependent on epigenetic alterations. In addition, this project may help elucidate how extracellular signals reach the chromatin. (AU)

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Scientific publications
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
LUND, PEDER J.; LOPES, MARIANA; SIDOLI, SIMONE; CORADIN, MARIEL; DE LUNA VITORINO, FRANCISCA NATHALIA; CHAGAS DA CUNHA, JULIA PINHEIRO; GARCIA, BENJAMIN A.. FGF-2 induces a failure of cell cycle progression in cells harboring amplified K-Ras, revealing new insights into oncogene-induced senescence. MOLECULAR OMICS, v. 17, n. 5, p. 725-739, . (16/24881-4, 15/04867-4, 13/07467-1, 17/15835-1, 18/15553-9, 11/22619-7, 17/18344-9)

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