Inhibition of p53 by lentiviral mediated shRNA abrogates G1 arrest and apoptosis in retinal pigmented epithelial cell line.
We silenced p53 gene expression in ARPE-19, a human retinal pigmented epithelial cell line using RNA interference. The effect of silencing the p53 gene in proliferating ARPE-19 cells was studied. Four short hairpin RNAs (shRNAs) targeting different regions of human p53 mRNA were delivered individually into ARPE-19 cells using lentiviral vector to produce stable cell lines. p53 mRNA and protein levels were reduced to varying extents in the four shRNA-transduced ARPE-19 cell lines. The cell line that showed greatest reduction (85-90%) of p53 expression showed decreased p21 promoter activation after DNA damage with camptothecin, etoposide and MMS. Whereas treatment of wild type ARPE-19 cells with camptothecin resulted in apoptosis, silencing p53 expression increased their survival. Cell cycle analyses indicated that irradiation resulted in a G(1) arrest in ARPE-19 cells, and that the arrest was significantly reduced in p53-silenced cells. Thus, p53 plays a central role in the response of ARPE-19 cells to DNA damaging agents that act via different mechanisms. Additionally, ARPE-19 cells with reduced p53 expression behave similar to tumor cell lines with mutated or non-functional p53. The present data demonstrate the utility of lentiviral vectors to create stable isogenic cell lines with reduced expression of a specific gene, thereby permitting the study of the function of a gene, the pathways controlled by it, and the effect of therapeutics on a cell with altered genetic makeup in a pair-wise fashion.
Nair
Ayyappan R
AR
Tranzyme Inc., Research Traingle Park, North Carolina 27709, USA.
Schliekelman
Mark
M
Thomas
Mary Beth
MB
Wakefield
John
J
Jurgensen
Stewart
S
Ramabhadran
Ram
R
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002166:Camptothecin / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D002460:Cell Line; D049109:Cell Proliferation; D004249:DNA Damage; D005047:Etoposide / Q000494:pharmacology; D016193:G1 Phase / Q000502:physiology* / Q000528:radiation effects; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D016158:Genes, p53; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000737:chemistry / Q000166:cytology* / Q000502:physiology; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D018630:Vitreoretinopathy, Proliferative / Q000235:genetics / Q000473:pathology / Q000503:physiopathology
D012333:RNA, Messenger; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; D005047:Etoposide; D002166:Camptothecin