ANALISI DEI MECCANISMI ATTRAVERSO CUI RAS REGOLA LA PROLIFERAZIONE E L’APOPTOSI IN CELLULE DI ADENOCARCINOMA COLORETTALE HT-29

RAS is a family of small proteins with GTPase activity that regulate proliferation, differentiation and apoptosis in all cell types. The three major isoforms of RAS (H-, K- and N-RAS) differ only in the last 25 amino acids which are the site of different post-translational modifications that lead to diverse subcellular localization and efficiency of activation of alternative pathways of signal transduction. This might explain, at least in part, the different biological effects of the RAS isoforms in the cells. RAS mutations are a common event in tumorigenesis and in colorectal carcinomas the mutations in K-RAS are more frequent than mutations in H-RAS. In almost all cases, the genetic alteration is a point mutation in codons 12 or 13, and rarely in codon 61. These mutations lead to a constitutively active protein by inactivating its GTPase activity. However, data indicate in different primary and metastatic tumors that not only mutations of different isoforms of RAS, but also mutations in different codons or different mutations in the same codon of the same isoform of RAS may have diverse biological consequences. To shed more light on the molecular mechanisms responsible for the different effects of RAS mutations, we have used stable clones of HT-29 (a human colorectal adenocarcinoma cell line in which the endogenous RAS genes are wild type) transfected with cDNAs codifying: K-RASG12V (clone K12) and K-RASG13D (clone K13) under the control of a Mifepristone-inducible promoter. We found that the expression of each of these mutated RAS isoforms induces different effects on the growth rate. Cytofluorimetric analysis shows also a differential effect on the cell cycle. In particular, we observe a significant increase in the fraction of cells in the G2/M phase, more relevant in the K13 than in the K12 induced cells. For a better understanding of this phenomenon we investigated the p21CIP1/WAF1 protein intracellular levels by western blot, identifying a similar increase in the amount of p21 in both clones upon induction of the mutated K-RAS isoforms. This increase persisted, and was actually enhanced, even in the presence of inhibitors of the two main signal transduction pathways operating downstream of RAS, the MEK inhibitor PD98059 and the PI3K inhibitor LY294002. B-RAF and RAF-1 expression is instead decreased by both the mutated isoforms of RAS, in growing as well as in starved cells. The two RAS mutations show the ability to differentially influence the pro-apoptotic (MST2-RASSF1A-LATS1) and anti-apoptotic (MST2-RAF-1) pathways. Cellular death was analyzed by propidium iodide and the caspase assay. The data show an increase in total cell death rate in both induced clones, but K-RASG13D preferentially induced a significant increase in necrosis while K-RASG12V in apoptosis, with a less pronounced effect. The K-RASG13D interactome was also analysed and compared to that of unstimulated wild type K-RAS by mass spectrometry in Hke3 cells in starving condition (0% serum).