Short interfering RNA (siRNA) is widely used for studying gene function and holds great promise as a tool for validating drug targets and treating disease. A critical assumption in these applications is that the effect of siRNA on cells is specific, i.e., limited to the specific knockdown of the target gene. In this article, we characterize the specificity of siRNA by applying gene expression profiling. Several siRNAs were designed against different regions of the same target gene for three different targets. Their effects on cells were compared by using DNA microarrays to generate gene expression signatures. When the siRNA design and transfection conditions were optimized, the signatures for different siRNAs against the same target were shown to correlate very closely, whereas the signatures for different genes revealed no correlation. These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA-mediated gene silencing as a reliable approach for large-scale screening of gene function and drug target validation.DNA microarray ͉ RNA interference ͉ gene knockdown T he use of RNA interference for inhibiting gene expression represents a powerful tool for exploring gene function (1-7), identifying and validating new drug targets, and treating disease (8, 9). The process of RNA interference is mediated by doublestranded RNA, which is cleaved by the enzyme DICER into 21-to 23-nt duplexes containing a 2-nt overhang at the 3Ј end of each strand. These duplexes are incorporated into a protein complex called the RNA-induced silencing complex (RISC). Directed by the antisense strand of the duplex, RISC recognizes and cleaves the target mRNA (for recent reviews, see refs. 1 and 10-13). Although long double-stranded RNAs (Ͼ30 nt) invoke an interferon response, short interfering RNAs (siRNAs) that resemble the products produced by DICER have been reported to specifically inhibit gene expression in many different mammalian cell lines (1-7). It has been shown that even single nucleotide mismatches between the antisense strand of the siRNA and target mRNA can abolish RNA interference (14). In addition, mapping of mRNA cleavage sites has revealed no cleavage sites outside of the region of complementarity (10). However, the specificity of siRNA at the cellular level remains to be comprehensively studied.For siRNAs to be a useful tool in gene knockdown experiments, it is critical that siRNA-mediated transcriptional silencing be specific. It is not enough to simply show that a control siRNA with a scrambled nucleotide sequence fails to knock down the protein of interest or produce the same cellular phenotype. Ideally, the siRNA must not cause any effects other than those related to the knock down of the target gene. There are several types of nonspecific effects that siRNA could potentially display. In addition to the possibility for cross-hybridization of the antisense strand of the siRNA to different mRNAs, siRNAs could bind in a sequencedependent manner to various cellular proteins. I...

ABT-737 is a novel and potent Bcl-2 antagonist with singleagent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X L , Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X L , whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies. [Cancer Res 2007;67(3):1176-83]

A plasmid was constructed with the promoter/regulatory region of the mouse metallothionein-I gene fused to the structural gene of herpesvirus thymidine kinase. When mouse eggs were microinjected with this plasmid and incubated with cadmium (a natural inducer of metallothionein gene transcription) thymidine kinase activity increased approximately 10-fold compared with control eggs not exposed to cadmium. Analysis of a set of deletion mutants revealed that the minimum sequence required for cadmium regulation lies within 90 nucleotides of the transcription start site.