Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways - PubMed (original) (raw)

Synthetic small inhibiting RNAs: efficient tools to inactivate oncogenic mutations and restore p53 pathways

Luis Alfonso Martinez et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Single base pair mutations that alter the function of tumor suppressor genes and oncogenes occur frequently during oncogenesis. The guardian of the genome, p53, is inactivated by point mutation in more than 50% of human cancers. Synthetic small inhibiting RNAs (siRNAs) can suppress gene expression in mammalian cells, although their degree of selectivity might be compromised by an amplification mechanism. Here, we demonstrate that a single base difference in siRNAs discriminates between mutant and WT p53 in cells expressing both forms, resulting in the restoration of WT protein function. Therefore, siRNAs may be used to suppress expression of point-mutated genes and provide the basis for selective and personalized antitumor therapy.

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Figures

Fig 1.

Fig 1.

Selective inhibition of p53 by siRNAs. (a) Sequences of the siRNAs used in this study; mutated nucleotides are indicated in bold. (b) H1299 cells were transfected with expression vectors for WT (WT) or R248W mutant (mt) p53, together with 400 ng of control (c), WT (WT), or mutant siRNA (mt1), as indicated. Cells were harvested 24 h later and analyzed by Western blotting (ib, immunoblot) with the indicated antibodies.

Fig 2.

Fig 2.

Selectivity of mutant p53 inhibition. (a) Schematic model of a putative amplification process that would abolish the specificity of targeting point-mutated sequences: the antisense strand of the siRNA (arrow) is used as a primer by a putative RNA-dependent RNA polymerase for RNA strand elongation, resulting in a double-stranded 5′ portion of the target mRNA; this double-stranded sequence would in turn serve as a substrate for DICER which would result in siRNAs corresponding to the 5′ end of the target, which is common to both WT and mutant siRNAs. The square represents the single base mutation. (b and c) H1299 cells were transfected as in Fig. 1, with WT or R248W mutant (mt) expression vectors, alone or in combination, as indicated, along with the specified siRNAs, and analyzed by Western blotting 24 h later with the indicated antibodies.

Fig 3.

Fig 3.

Inhibition of endogenous mutant p53. (a) Stable transfectants were established from U2OS cells (expressing WT p53) by using a mutant expression vector (R248W) or the corresponding empty vehicle vector (PCDNA). Cells were analyzed by Western blotting with the indicated antibodies. (b_–_d) Fibroblasts from a Li–Fraumeni patient were transfected with siRNAs and analyzed for p53 expression by Western blotting (b), real-time RT-PCR (c), or immunofluorescence (d).

Fig 4.

Fig 4.

Restoration of WT p53 function. (a) H1299 cells were transfected with both WT and mutant p53 vectors, siRNAs as indicated, and an mdm2-luciferase reporter construct along with a β-galactosidase construct as an internal control; cells were harvested 24 h later and extracts were assayed for luciferase activity. Transfections were performed in duplicate. Luciferase assays were performed in triplicate. Results are shown of two independent transfections from a representative experiment. (b) H1299 cells were transfected as in a and analyzed by Western blotting with the indicated antibodies.

Fig 5.

Fig 5.

Immortalized Li–Fraumeni cells (MDAH087) were treated with control or mt1-siRNA as indicated. (a) Western blot analysis. (b) Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) analysis (proportions of TUNEL-positive cells are shown in percentages).

Fig 6.

Fig 6.

Restoration of p53 function in E6-expressing cells. (a) CasKi and SiHa cells that express HPV E6 protein were transfected with indicated siRNAs, treated with doxorubicin, or left untreated for the controls and then analyzed by Western blotting with the indicated antibodies. (b and c) SiHA cells treated as in a were analyzed for cell cycle. The result of a typical experiment is shown.

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