Background: Gliomas are the most common and aggressive primary brain tumors for which unfortunately no effective treatment modalities exist despite advances in molecular biology as the knowledge base to unravel the extremely complex molecular mechanisms of tumorigenesis is limited. In this study an attempt has been made to understand the molecular pathological basis of tumorigenesis which led to an identification of an oncogene, CDC2, and an epigenetic strategy has been evaluated to control the tumorigensis by downregulating this oncogene.
Methods: Tissue microarrays were utilized to investigate the expression of genes in a large number of tumor samples and to identify overexpressed genes which could be potentially causing tumorigenesis. Retroviral vectors expressing short hairpin RNAs (shRNAs) targeted against CDC2 were designed and transducted into human glioma cell line ex vivo in order to downregulate the expression of CDC2. Real-Time PCR was used to determine the level of CDC2 mRNA. Western Blotting was used to determine the level of expression of CDC2 protein as measure to quantify down regulation of CDC2 expression along with use of flow cytometry to investigate effect of shRNAs on cell cycles and detection of apoptosis. Following ex vivo study, viral particles containing small interfering RNA for CDC2 were subsequently injected into xenogeneic graft tumor of nude mice and the weight of human glioma xenografts, survival and resulting phenotypic changes of target gene were investigated.
Results: Human glioma tissue microarrays indicated the positive expression rates of CDC2/CyclinB1 with a positive correlation with pathologic grades (r = 0.982, r = 0.959, respectively). Retroviral vectors expressing short hairpin RNAs (shRNAs) against CDC2 caused efficient deletion of CDC2, cellular G2/M arrest concluding in apoptosis and inhibition of proliferation in human glioma cells U251 and SHG-44 cell lines ex vivo. And the viral particles containing small interfering RNA for CDC2 were subsequently injected into subcutaneous and intracranial xenogeneic graft tuomrs of nude mice. For subcutaneous tumors, injection of CDC2-shRNA retroviruses significantly decreased tumor weight and volume compared with control. Immunohistochemistry indicated that CDC2 are negative and TUNEL are positive in tumors treated with recombinant retrovirus. For mice implanted with intracranial gliomas, treatment of CDC2-shRNA retroviruses increased survival times compared with control.
Conclusion: CDC2 gene plays an important role in the proliferation of human gliomas. Downregulation of CDC2 could potentialy inhibit human gliomas cells growth ex vivo and in vivo. From these results, it was suggested that CDC2 might be a potential target on gene therapy of human gliomas.