Analyse der Regulationsmechanismen des humanen Tumorsuppressor Gens H-REV107-1

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The H-REV107-1 class II tumor suppressor gene is ubiquitously expressed in normal tissues and down-regulated in human breast, ovarian and lung tumors. H-REV107-1 has the capacity to suppress growth of tumor cells in vitro and in vivo. In A27/80, OVCAR-3, SKOV-3, RCC26, MZ1257 and ROSE199 cell lines H-REV107-1 is up-regulated after treatment with IFN-?. A NIH3T3 cell line harboring an estrogen-inducible IRF-1/hER fusion protein showed a protein synthesis independent up-regulation of H-rev107-1 expression after induction of IRF-1. The parental NIH3T3 cell line showed no induction of H-rev107-1 expression after treatment with estrogen. This demonstrates that H-rev107-1 is a direct target of IRF-1. Furthermore, a time course dependent correlation between H-REV107-1 and IRF-1 expression was confirmed in OVCAR-3 and A27/80 cell lines. Additionally, there was a correlation between H-REV107-1 and IRF-1 expression in the immortalized human ovarian epithelial cell line HOSE and in the carcinoma cell lines SKOV-3 and RCC26. Inhibition of the MEK/ERK pathway in a RAS-transformed derivate of immortalized rat ovarian surface epithelial cells, ROSE199 A2/5, using the MEK1 inhibitor PD 98059, but not the inhibitor of the PI3K pathway LY 294002, leads to a restored expression of H-rev107-1. In the teratocarcinoma cell line PA-1 but not in the A27/80 or OVCAR-3 cell lines, PD 98059 partially restored expression of H-REV107-1. Therefore, H-REV107-1 can be a target of the MEK/ERK-pathway in the ROSE199 A2/5 and the PA-1 cell line. Thus, H-REV107-1 is regulated by at least two different pathways. To understand the regulatory mechanisms of the expression of the H-REV107-1 gene, the putative promoter region was analyzed in silico. The sequence was amplified and cloned into the pGL3-basic Luciferase Reporter plasmid (Promega™). Induction of the promoter constructs with TNF-?, cAMP and IFN-? increased the luciferase activity. Several deletions constructs and constructs with putative transcription factor binding sites mutated were used to narrow down the important regulatory elements of the promoter. While mutations of an IRF-1 binding site and a STAT-binding site as potential targets of the IFN-? signaling pathway did not reduce the luciferase activity, the mutations of a c-Rel binding site and a CREB/ATF-2 binding site decreased the luciferase activity by 91% and 63%, respectively. Co-transfection of the full length promoter construct with a dominant negative IkB expression plasmid, a repressor of NFkB activation, reduced the luciferase activity to 47%. As a result of the investigation H-REV107-1 is directly regulated by IRF-1 and probably indirectly regulated by NF?B and the MEK/ERK signaling pathway. To show a biological function of the CREB/ATF-2 and the c-Rel binding site, an Electro Mobility Shift Assay (EMSA) was performed, using a P32-labeled oligonucleotid, which included the CREB/ATF-2 or the c-Rel binding site. The binding of ATF-2 to the CREB oligonucleotid was demonstrated by the use of a specific antibody. While there was also a specific protein binding to the cRel binding site, none of the used antibodies of the NF?B family resulted in a supershift with the cRel oligonucleotid. The ATF-2 binding site in the posititon – 30 bp - -23 bp of the human, TATA-less H-REV107-1 promoter replaces the TATA-like element, which can be found in the H-rev107-1 promoter of rat and mouse.