Document Detail


Development of new EBV-based vectors for stable expression of small interfering RNA to mimick human syndromes: application to NER gene silencing.
MedLine Citation:
PMID:  16179499     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Denis S F Biard; Emmanuelle Despras; Alain Sarasin; Jaime F Angulo
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Molecular cancer research : MCR     Volume:  3     ISSN:  1541-7786     ISO Abbreviation:  Mol. Cancer Res.     Publication Date:  2005 Sep 
Date Detail:
Created Date:  2005-09-23     Completed Date:  2005-11-10     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  101150042     Medline TA:  Mol Cancer Res     Country:  United States    
Other Details:
Languages:  eng     Pagination:  519-29     Citation Subset:  IM    
Affiliation:
Laboratoire de Génétique de la Radiosensibilité, Commissariat à l'Energie Atomique, Département de Radiobiologie et de Radiopathologie, Direction des Sciences du Vivant, BP 6, 92265 Fontenay aux Roses, France. denis.biard@cea.fr
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MeSH Terms
Descriptor/Qualifier:
Blotting, Southern
Blotting, Western
Cell Cycle / radiation effects
Cell Survival / radiation effects
DNA Repair*
DNA-Binding Proteins / genetics
Flow Cytometry
Gene Silencing*
Genetic Vectors / physiology*
Hela Cells / physiology,  radiation effects
Herpesvirus 4, Human / genetics*
Humans
Nuclear Proteins / genetics
RNA, Small Interfering / genetics*
RNA-Binding Proteins
Tumor Stem Cell Assay
Ultraviolet Rays*
Xeroderma Pigmentosum Group A Protein
Chemical
Reg. No./Substance:
0/DNA-Binding Proteins; 0/KIN protein, human; 0/Nuclear Proteins; 0/RNA, Small Interfering; 0/RNA-Binding Proteins; 0/XPA protein, human; 0/Xeroderma Pigmentosum Group A Protein; 156533-34-5/XPC protein, human

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