Functional Analysis of the Proto-oncogenes Septin9 and Nras

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

Work presented in this thesis concerns the two GTPases SEPTIN9 (SEPT9) and NRAS. Despite their GTPase activity, the two genes are very different. SEPT9 is a part of the cytoskeleton, forming rings and filaments with other septins. It provides stability to the cell and is involved in a number of cellular processes such as cytokinesis and migration. We found reduced substrate attachment in Sept9 depleted immortalized embryonic fibroblasts compared to wild type resulting in cellular instability especially under stressful conditions. Furthermore, Sept9 deletion caused instability of SEPT7 containing filaments. Both SEPT7 and SEPT9 containing filaments were intensified under hypoxia in wild type cells compared to non-hypoxic cells despite their transcriptional downregulation. Due to earlier finding of Sept9 as a frequent integration site in T-cell lymphomas induced by retrovirus, the function of Sept9 in T-cells was investigated in mice with conditional knock out Sept9 alleles. Sept9 deletion in early pre-T-cells caused a reduction in T-cell development in the double negative stage, resulting in decreased numbers of mature T-cells in the periphery. Furthermore, a shift in the distribution of cytotoxic CD8+ T-cell and regulatory CD4+ T-cells was found upon Sept9 deletion probably as a result of reduced proliferation rate in Sept9 depleted CD8+ T-cells compared to wild type. Moreover, Sept9 depleted CD8+ central memory T-cells were upregulated compared to wild type. T-cell lymphoma induction by the murine leukemia virus SL3-3 in heterozygous Sept9 knock out mice did not change the latency time compared to wild type; however, the distribution of T-cell subsets in the thymic tumors was altered. Moreover, Sept9 expression was increased in tumors compared to thymus and spleen non-tumorigenic tissues regardless of genotype indicating an oncogenic role of SEPT9. Nras is a potent proto-oncogene involved in signaling through a number of proliferative pathways. Earlier detected retroviral integration sites resulting in B-cell lymphomas were used to create Nras knock in models harboring the LTR from the murine leukemia virus Akv 1-99 in either sense or antisense direction. In addition a floxed PGK/Tn5 neomycin cassette was inserted. Expression analysis of Nras within knock in was used to study the effect of the LTR. If inserted before the endogenous promoter, the LTR in the antisense direction was found to induce more upregulation of Nras expression compared to the sense orientation. Insertion of the LTR in the sense orientation within intron 1 led to upregulation of Nras in contrast to the antisense orientated LTR, which caused downregulation. Upon excision of the neomycin cassette from the integration within intron 1, upregulation of Nras was found as a result of both the sense and the antisense LTR insertion. Within the intron the sense-orientated LTR caused a higher upregulation, particularly in spleen (25-fold in homozygous mice) compared to the antisense-orientated LTR. The 25-fold upregulation of Nras caused early postnatal lethality of the homozygous mice. A thorough analysis revealed that the homozygous mice suffered from granulocytosis and T-cell expansion within the spleen. The increased population of granulocytes was mainly immature, and subsequently a decrease of monocytes was found, indicating a shift in development as a result of Nras upregulation, possibly forcing the cells in the granulocyte direction. Furthermore, G-csf, Gfi1, RasGRP1, and Sos1 mRNA levels increased and as these factors normally work upstream in the activation process of NRAS, this indicates that upregulation of wild type Nras might cause an auto-regulatory positive feed back loop
UdgivelsesstedAarhus University
ForlagAarhus University, Faculty of Science and Technology
Antal sider234
Rekvirerende organGraduate School of Science and Technology
StatusUdgivet - 17 okt. 2012

Se relationer på Aarhus Universitet Citationsformater

ID: 48401664