Development of New EBV-Based Vectors for Stable Expression of Small Interfering RNA to Mimick Human Syndromes: Application to NER Gene Silencing

• Published in: Molecular cancer research 2005-09, Vol.3 (9), p.519-529
• Main Authors: Biard, Denis S F, Despras, Emmanuelle, Sarasin, Alain, Angulo, Jaime F
• Format: Article
• Language: English
• Subjects: Blotting, Southern; Blotting, Western; Cell Cycle - radiation effects; Cell Survival - radiation effects; DNA Repair; DNA-Binding Proteins - genetics; EBV vector; Flow Cytometry; Gene Silencing; Genetic Vectors - physiology; HeLa Cells - physiology; HeLa Cells - radiation effects; Herpesvirus 4, Human - genetics; Humans; KIN17; Nuclear Proteins - genetics; RNA interference; RNA, Small Interfering - genetics; RNA-Binding Proteins; Tumor Stem Cell Assay; Ultraviolet Rays; Xeroderma Pigmentosum Group A Protein; XPA; XPC
• ISSN: 1541-7786; 1557-3125
• DOI: 10.1158/1541-7786.MCR-05-0044

We developed and characterized replicative small interfering RNA (siRNA) vectors for efficient, specific, and long-term gene silencing in human cells. We created stable XPA KD and XPC KD (knockdown) syngeneic cell lines to mimic human cancer-prone syndromes. We also silenced HSA KIN17 . Several clones displaying undetectable protein levels of XPA, XPC, or HSA kin17 were grown for more than 300 days. This stability of gene silencing over several months of culture allows us to assess the specific involvement of these proteins in UVC sensitivity in syngeneic cells. Unlike XPA, HSA KIN17, and XPC gene silencing dramatically impeded HeLa cell growth for several weeks after transfection. As expected, XPA KD and XPC KD HeLa cells were highly UVC sensitive. They presented an impaired unscheduled DNA synthesis after UVC irradiation. Interestingly, XPC KD HeLa clones were more sensitive to UVC than their XPA KD or KIN17 KD counterparts. Hygromycin B withdrawal led to the total disappearance of EBV vectors and the resumption of normal XPA or XPC protein levels. Whereas reverted XPA KD cells recovered a normal UVC sensitivity, XPC KD cells remained highly sensitive, suggestive of irreversible damage following long-term XPC silencing. Our results show that in HeLa cells, HSA kin17 participates indirectly in early events following UVC irradiation, and XPC deficiency strongly affects cell physiology and contributes to UVC sensitivity to a greater extent than does XPA. EBV-based siRNA vectors improve the interest of siRNA by permitting long-term gene silencing without the safety concerns inherent in viral-based siRNA vehicles.