Selective silencing of mammalian gene expression has recently been achieved using short interfering RNA (siRNA). Synthetic siRNA targets homologous mRNA for degradation and the process is highly efficient. Here we demonstrate siRNA silencing of pathogenic viral gene expression. As a well characterized model we chose cervical carcinoma cells positive for human papillomavirus type 16. Over 90% of human cervical cancers are positive for papillomavirus and abnormal cell proliferation is driven by co-operative effects of viral E6 and E7 genes. We sought to silence HPV E6 and E7 gene expression using siRNAs to target the respective viral mRNAs. Our results indicate selective degradation of E6 and E7 mRNAs. Silencing was sustained for at least 4 days following a single dose of siRNA. E6 silencing induced accumulation of cellular p53 protein, transactivation of the cell cycle control p21 gene and reduced cell growth. In contrast, E7 silencing induced apoptotic cell death. HPV-negative cells appeared unaffected by the anti-viral siRNAs. Thus we demonstrate for the first time (i) that siRNA can induce selective silencing of exogenous viral genes in mammalian cells, and (ii) that the process of siRNA interference does not interfere with the recovery of cellular regulatory systems previously inhibited by viral gene expression.

SIRT1 is a conserved NAD-dependent deacetylase that regulates life span in accord with nutritional provision. In mammalian cells, SIRT1 also down-regulates stress-induced p53 and FoxO pathways for apoptosis, thus favoring survival under stress. The functioning of SIRT1 under normal, nonstressed conditions of cell growth is unknown. Here we have asked if SIRT1 has the capacity to influence cell viability in the absence of applied stress. For this purpose we used synthetic small interfering RNA to silence SIRT1 gene expression by RNA interference (RNAi). We show that the process of RNAi, by itself, does not affect cell growth and is not sufficient to activate a cellular stress response (indicated by lack of activation of endogenous p53). We also show that, in the absence of applied stress, SIRT1 silencing induces growth arrest and/or apoptosis in human epithelial cancer cells. In contrast, normal human epithelial cells and normal human diploid fibroblasts seem to be refractory to SIRT1 silencing. Combined gene knockout with RNAi cosilencing experiments indicate that SIRT1 and Bcl-2 may suppress separable apoptotic pathways in the same cell lineage and that the SIRT1-regulated pathway is independent of p53, Bax, and caspase-2. Alternatively, SIRT1 may suppress apoptosis downstream from these apoptotic factors. In either case, we show that FoxO4 (but not FoxO3) is required as proapoptotic mediator. We further identify caspase-3 and caspase-7 as downstream executioners of SIRT1/FoxO4-regulated apoptosis. Our work identifies SIRT1 as a novel target for selective killing of cancer versus noncancer epithelial cells. (Cancer Res 2005; 65(22): 10457-63)

SummaryForkhead box A1 regulates prostate ductal morphogenesis and promotes epithelial cell maturation

Carcinoma-associated fibroblasts (CAFs) play a critical role in malignant progression. Loss of TGF-ϐ receptor II (TGFϐR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity due to loss of TGFϐR2 might contribute to cancer progression, we attenuated TGF-ϐ signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFϐR2 function in 50% of the stromal cell population resulted in malignant transformation of the non-tumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGFϐR2 increased the expression of markers of myofibroblast differentiation [co-expression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-ϐ1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFϐR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-ϐ1, SDF1/CXCL12, and members of the FGF and BMP families. In vivo, tissue recombination of fibroblasts overexpressing TGF-ϐ1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-ϐ signaling to tumor promotion by tumor stromal cells.