For decades (Armelin, 1975), our long term goal has been to define the molecular basis of the cell cycle control in mammalian cell lines. In the last 5 years we have been studying the antagonistic interactions between signals initiated in the receptors of FGF2 (Fibroblast Growth Factor 2) and ACTH (Adrenocorticotropic Hormone) or AVP (Arginine Vasopressin) in mouse Y1 adrenocortical cells (Fig 1). FGF2 is a strong mitogen for Y1 cells, whereas ACTH and AVP antagonize FGF2 mitogenic activity. The rationale underlying this approach involved the likely assumption that this experimental study would uncover important reactions comprising the signaling network module that controls the G0->G1->S transition of the cell cycle in Y1 cells. In fact, the results obtained (Lotfi et al, 2000; Lepique et al, 2000; Lotfi & Armelin, 2001; Forti et al, 2002; Schwindt et al, 2003; Rocha et al, 2003) led us to propose a core for the putative control module we have been searching for.

Fig 1. Mouse Y1 adrenocortical cells

            Presently, unpublished results and preliminary observations allow us to amplify the proposed control module to include an extended scheme for the mitogenic pathway triggered by FGF2 to promote the G0?G1?S transition in Y1 cells. This new proposal involves hypotheses whose experimental validation is the aim of this thematic project.

            The experimental plan of this new thematic project involves multiple methodologies: a) recombinant DNA technology applied to mouse cell lines (cloning sequencing of genes and ESTs, construction of expression vectors, transient and permanent transfection of DNA constructs, etc); b) functional knockout of genes/proteins by interference of RNA (RNAi); c) gene expression analysis by microarrays of cDNA; d) analysis of protein complexes by the yeast two-hybrid method or isolation by affinity followed by resolution in SDS-PAGE and mass spectrometry. 
 

References

Armelin, HA (1975) em: Biochemical Actions of Hormones, Ed. G. Litwack.Vol.III, 1-21.

Forti et al (2002) Biochemistry 41, 10133-40.

Lepique et al (2000) Endo Res 26, 825-32.

Lotfi & Armelin (2001) J Endocrinology 168, 381-9.

Lotfi et al (2000) Brazilian J Med Biol Res 33, 1133-40.

Rocha et al (2003) Microscopy Res Tec 15, 61,268-74.

Schwindt et al (2003) Biochemistry 42, 2116-21.