The Ras oncogene is mutated in about 30% of the human tumors and its mutations are always point mutations concerning codon 12, 13 and 61. These mutations cause in the proteins a reduced GTPase activity, so that they become constitutively active. In human cells there are three main isoforms of Ras (H, K, N-ras) which can trigger alternative pathways of signal trasduction. In order to investigate the effects of expression of different oncogenic Ras isoforms in colorectal carcinoma cells (HT-29), we obtained stable clones of HT-29 cells transfected with cDNAs codifying H-RasG12V and K-Ras G12V called respectively H12 and K12 and K-RasG13D called K13, under the control of an hormone-inducible promoter. We had previously observed changes in shape and growth rate when mutated Ras was expressed. We then analyzed the differential expression of genes such as the cell-cycle inhibitor p21 and the EMT associated gene snail. Q-RT PCR assay showed an increase in p21 level only in H12 cells but not in K13 and K12 compared with the respective controls. Instead, snail mRNA level was increased in all thre clones. We investigated also by Semiquantitative RT-PCR H-RasG12V, p21 and snail levels in H12 cells induced for a long time and we noted a decreased level of H-RASG12V associated with no p21 and snail expression. Our results show that specific mutations of different Ras isoforms have different effects on morphology and gene expression in Ht-29. Other studies are of course necessary to shed more light on the role played by Ras mutations in tumor development.
|Numero di pagine||32|
|Stato di pubblicazione||Published - 2008|