The Roles of C-myb protein complex and Cdt1/ Geminin, P53, Cyclin A/Cdk2, P21, P27 for the Mycn amplification in neuroblastoma

Neuroblastoma is the most common tumor in pediatric solid tumors which derive from the peripheral nervous system. MYCN gene amplification give rise to the poor prognosis in neuroblastoma tumors. The mechanism of which the MYCN amplification is formed has been clearly unknown yet. Gene amplifications may form due to tumoral behaviour in human cancers. Increased gene copy numbers occur in also other organisms. Chorion gene copy numbers are regulated by the myb protein complex that bind to ACE3 during the normal developmental stage of Drosophila melanogaster. Myb and E2F1 take part in complex that induce chorion amplification and the myb, E2F2 and l(3)mbt proteins participate in the prevention complex. On the other hand, DUP and geminin play role in general genomic amplification of Drosophila. In this thesis study, myb-like protein complex was hypothesized that may control the MYCN gene amplification. And, the roles of human orthologs that are termed as c-myb, b-myb, a-myb, E2F1, HL3MBTL and hCdt1/gemininin related to defined proteins in Drosophila were investigated in neuroblastoma cell lines. RNA interference was performed by using plasmid vectors that include shRNA sequences specific to c-myb, HL3MBTL, E2F1, hCdt1, p21, geminin, b-myb and control EGFP in Kelly, IMR32, MHH-NB-11 and SIMA cell lines. The mRNA levels of MYCN, c-myb, HL3MBTL, E2F1, hCdt1, p21, geminin, b-myb, reference HPRT1 genes and ratios of the DNA copy numbers of MYCN and reference p53 genes were determined by real time PCR assays during pre- and post-RNA interference. In conclusion, when the c-myb expression was interferenced through shRNA vector system in cell lines, the ratios of MYCN/p53 DNA copy number increased in Kelly (21.09%) and SIMA (104.02%) cell lines and decreased in IMR32 (54.11%) and MHH-NB-11 (78.25%) cell lines. In addition, MYCN gene expression decreased in MHH-NB-11 (66.81%), SIMA (44.44%) and IMR32 (37.43%) cell lines and increased in Kelly (27.14%) cell line. According to present findings, while c-myb acts to decrease the MYCN copy number in Kelly and SIMA, it acts to increase in the IMR32 and MHH-NB-11 cell lines. Consequently, it was seen that c-myb and MYCN amplification are related with each other. C-myb may select as a drug target in neuroblastoma therapy.