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SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.

Summary Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.

Angiotensinogen (AGT) is the precursor of active vasoconstrictive octapeptide angiotensin II (Ang II) in the renin-angiotensin-aldosterone system. Blocking the AGT-converting enzymes in the pathway and the Ang II receptor through pharmacological agents has been proven to be effective in lowering blood pressure (BP) in hypertensive patients. In this study, we developed chemically modified small interfering RNAs (siRNA) to target hepatic AGT mRNA in rats. Lipid nanoparticle encapsulated siRNAs were efficiently delivered to rat liver and resulted in significant reduction in hepatic Agt mRNA levels and plasma AGT concentration without impairing liver function. Single intravenous injection of Agt siRNA led to significant and sustained BP lowering in spontaneous hypertensive rats and in Sprague-Dawley rats, and the effect was maintained by weekly siRNA dosing. Data presented here provide proof-of-feasibility for the use of siRNA technology for inhibition of peripheral AGT levels via hepatic mRNA silencing with beneficial effects on BP in preclinical rat models. Similar approach could be used for validation of novel hypertension hepatic and extrahepatic targets.

Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).

Reducing circulating LDL-cholesterol (LDL-c) reduces the risk of cardiovascular disease in people with hypercholesterolemia. Current approaches to reduce circulating LDL-c include statins, which inhibit cholesterol synthesis, and ezetimibe, which blocks cholesterol absorption. Both elevate serum PCSK9 protein levels in patients, which could attenuate their efficacy by reducing the amount of cholesterol cleared from circulation. To determine whether PCSK9 inhibition could enhance LDL-c lowering of both statins and ezetimibe, we utilized small interfer­ing RNAs (siRNAs) to knock down Pcsk9, together with ezetimibe, rosuvastatin, and an ezetimibe/rosuvastatin combination in a mouse model with a human-like lipid profile. We found that ezetimibe, rosuvastatin, and ezetimibe/rosuvastatin combined lower serum cholesterol but induce the expression of Pcsk9 as well as the Srebp-2 hepatic cholesterol biosynthesis pathway. Pcsk9 knockdown in combination with either treatment led to greater reductions in serum non-HDL with a near-uniform reduction of all LDL-c subfractions. In addition to reducing serum cholesterol, the combined rosuvastatin/ezetimibe/Pcsk9 siRNA treatment exhibited a significant reduction in serum APOB protein and triglyceride levels. Taken together, these data provide evidence that PCSK9 inhibitors, in combination with current therapies, have the potential to achieve greater reductions in both serum cholesterol and triglycerides.

The use of short interfering RNAs (siRNA) in animals for target validation or as potential therapeutics is hindered by the short physical half-life when delivered as unencapsulated material and in turn the short active half-life of siRNAs in vivo. Here we demonstrate that the character of the two 3'-overhang nucleotides of the guide strand of siRNAs is a determinant of the duration of silencing by siRNAs both in vivo and in tissue culture cells. We demonstrate that deoxyribonucleotides in the guide strand overhang of siRNAs have a negative impact on maintenance of both the in vitro and in vivo activity of siRNAs over time. Overhangs that contain ribonucleotides or 2'-O-methyl modified nucleotides do not demonstrate this same impairment. We also demonstrate that the sequence of an siRNA is a determinant of the duration of silencing of siRNAs directed against the same target even when those siRNAs have equivalent activities in vitro. Our experiments have determined that a measurable duration parameter exists, distinct from both maximum silencing ability and the potency of siRNAs. Our findings provide information on incorporating chemically modified nucleotides into siRNAs for potent, durable therapeutics and also inform on methods used to select siRNAs for therapeutic and research purposes.

The hypoxia-inducible factor (HIF) pathway is essential for cell survival under low oxygen and plays an important role in tumor cell homeostasis. We investigated the function of miR-210, the most prominent microRNA upregulated by hypoxia and a direct transcriptional target of HIFs. miR-210 expression was elevated in multiple cancer types and correlated with metastasis of breast and melanoma tumors. miR-210 overexpression in cancer cell lines bypassed hypoxia-induced cell cycle arrest and partially reversed the hypoxic gene expression signature. We identified MNT, a known MYC antagonist, as a miR-210 target. MNT mRNA contains multiple miR-210 binding sites in the 3' UTR and its knockdown phenocopied miR-210 overexpression. Furthermore, loss of MYC abolished miR-210-mediated override of hypoxia-induced cell cycle arrest. Comparison of miR-210 and MYC overexpression with MNT knockdown signatures also indicated that miR-210 triggered a "MYC-like" transcriptional response. Thus, miR-210 influences the hypoxia response in tumor cells through targeting a key transcriptional repressor of the MYC-MAX network.

High-throughput screening (HTS) of large-scale RNA interference (RNAi) libraries has become an increasingly popular method of functional genomics in recent years. Cell-based assays used for RNAi screening often produce small dynamic ranges and significant variability because of the combination of cellular heterogeneity, transfection efficiency, and the intrinsic nature of the genes being targeted. These properties make reliable hit selection in the RNAi screen a difficult task. The use of robust methods based on median and median absolute deviation (MAD) has been suggested to improve hit selection in such cases, but mean and standard deviation (SD)-based methods are still predominantly used in many RNAi HTS. In an experimental approach to compare these 2 methods, a genome-scale small interfering RNA (siRNA) screen was performed, in which the identification of novel targets increasing the therapeutic index of the chemotherapeutic agent mitomycin C (MMC) was sought. MAD values were resistant to the presence of outliers, and the hits selected by the MAD-based method included all the hits that would be selected by SD-based method as well as a significant number of additional hits. When retested in triplicate, a similar percentage of these siRNAs were shown to genuinely sensitize cells to MMC compared with the hits shared between SD- and MAD-based methods. Confirmed hits were enriched with the genes involved in the DNA damage response and cell cycle regulation, validating the overall hit selection strategy. Finally, computer simulations showed the superiority and generality of the MAD-based method in various RNAi HTS data models. In conclusion, the authors demonstrate that the MAD-based hit selection method rescued physiologically relevant false negatives that would have been missed in the SD-based method, and they believe it to be the desirable 1st-choice hit selection method for RNAi screen results.

RNA interference (RNAi), combined with the availability of genome sequences, provides an unprecedented opportunity for the massive and parallel investigations of gene function. Small interfering RNA (siRNA) represents a popular and quick approach of RNAi for in vitro loss-of-function genetic screens. Efficient transfection of siRNA is critical for unambiguous interpretation of screen results and thus overall success of any siRNA screen. A high-throughput, lipid-based transfection method for siRNA was developed that can process eighty 384-well microplates in triplicate (for a total of 30,720 unique transfections) in 8 h. Transfection throughput was limited only by the speed of robotics, whereas the cost of screening was reduced. As a proof of principle, a genome-scale screen with a library of 22,108 siRNAs was performed to identify the genes sensitizing cells to mitomycin C at concentrations of 0, 20, and 60 nM. Transfection efficiency, performances of control siRNAs, and other quality metrics were monitored and demonstrated that the new, optimized transfection protocol produced high-quality results throughout the screen. (Journal of Biomolecular Screening XXXX:xx-xx)

Clear cell carcinoma of the kidney is a major cause of mortality in patients with von Hippel-Lindau (VHL) disease, which is caused by germ line mutations that inactivate the VHL tumor suppressor gene. Biallelic VHL inactivation, due to mutations or hypermethylation, is also common in sporadic clear cell renal carcinomas. The VHL gene product, pVHL, is part of a ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF) for destruction under well-oxygenated conditions. All VHL mutations linked to classical VHL disease compromise this pVHL function although some missense mutations result in a low risk of kidney cancer (type 2A VHL disease) while others result in a high risk (type 2B VHL disease). We found that type 2A mutants were less defective than type 2B mutants when reintroduced into VHL / renal carcinoma cells with respect to HIF regulation. A stabilized version of HIF2 promoted tumor growth by VHL / cells engineered to produce type 2A mutants, while knock-down of HIF2 in cells producing type 2B mutants had the opposite effect. Therefore, quantitative differences with respect to HIF deregulation are sufficient to account for the differential risks of kidney cancer linked to VHL mutations. von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome caused by germ line mutations of the VHL tumor suppressor gene and is characterized by an increased risk of tumor development in multiple organs, such as hemangioblastoma of the central nervous system, pheochromocytoma of the adrenal glands, and clear cell carcinoma of the kidneys (36). Tumor development occurs when the remaining wild-type allele is inactivated. Kidney cancer develops in 75% of VHL patients by age 60 and is a leading cause of death in this population (70). In keeping with the Knudson two-hit model, somatic VHL mutations leading to VHL inactivation are also common in sporadic clear cell renal cell carcinomas (RCC) and restoration of VHL function in VHL / RCC is sufficient to suppress tumor formation in vivo (21, 30).

Proteolysis of beta-amyloid precursor protein (APP) into amyloid beta peptide (Abeta) by beta- and gamma-secretases is a critical step in the pathogenesis of Alzheimer's Disease (AD), but the pathways regulating secretases are not fully characterized. Ubiquitinylation, which is dysregulated in AD, may affect APP processing. Here, we describe a screen for APP processing modulators using an siRNA library targeting 532 predicted ubiquitin ligases. Seven siRNA pools diminished Abeta production. Of these, siRNAs targeting PPIL2 (hCyp-60) suppressed beta-site cleavage. Knockdown of PPIL2 mRNA decreased BACE1 mRNA, while overexpression of PPIL2 cDNA enhanced BACE1 mRNA levels. Microarray analysis of PPIL2 or BACE1 knockdown indicated that genes affected by BACE1 knockdown are a subset of those dependent upon PPIL2; suggesting that BACE1 expression is downstream of PPIL2. The association of PPIL2 with BACE expression and its requirement for Abeta production suggests new approaches to discover disease modifying agents for AD.

Vpr is a 96-amino-acid protein encoded by human immunodeficiency virus type 1 (HIV-1) that prevents proliferation of infected cells. We have established a system for infection of 100% of a T-cell population with HIV and use this system to show that within the context of HIV-1 infection, Vpr is primarily cytostatic rather than cytotoxic. Vpr acts upstream of dephosphorylation of the mitotic cyclin-dependent kinase, and causes infected cells to accumulate in the G2 stage of the cell cycle. However, some HIV-1 infected cells increase in ploidy and size, accumulating DNA to an 8N level. Furthermore, the mechanism of the Vpr mitotic block is qualitatively different from that of G2 DNA damage checkpoint control.

Analogs of the immunosuppressive cyclic undecapeptide cyclosporin A (CsA) with substitutions in positions 1, 4, 6, and/or 11 were rationally designed to possess substantially diminished or no immunosuppressive activity. When these compounds were assayed for their capacity to interfere with the replication of human immunodeficiency virus, some displayed a potent antiviral activity in newly infected cells. However, only CsA could interfere with virus replication in persistently infected cells. One CsA analog with antiviral activity costimulated the phytohemagglutinin-induced production of interleukin 2 by human lymphocytes. Human immunodeficiency virus particles from drug-exposed cells showed lower infectivity than virions from untreated cells. Thus, these nonimmunosuppressive analogs of CsA constitute a promising class of lead compounds to develop drugs for effective treatment of the acquired immunodeficiency syndrome.

RNA interference is thought to require near-identity between the small interfering RNA (siRNA) and its cognate mRNA. Here, we used gene expression profiling to characterize the specificity of gene silencing by siRNAs in cultured human cells. Transcript profiles revealed siRNA-specific rather than target-specific signatures, including direct silencing of nontargeted genes containing as few as eleven contiguous nucleotides of identity to the siRNA. These results demonstrate that siRNAs may cross-react with targets of limited sequence similarity.

The possible physical association of heat shock proteins (Hsp's) with immunodeficiency viruses (HIV and SIV) has been examined. The virions were purified by a) polyethylene glycol (PEG) precipitation and Sepharose 4B filtration, b) PEG precipitation and centrifugation over a Renografin gradient, or c) PEG precipitation and Matrex Cellufine Sulfate affinity chromatography. Western blotting revealed an Hsp60 related protein associated with HIV and SIV. Other Hsp's (such as Hsp70) were not detected, suggesting a specific interaction between Hsp60 and viral factors.

This study examined 94 injured workers (IWs) treated in a work-hardening program over an 18-month time span. Of the 94 subjects, 78 (82.98 %) were contacted by telephone 1 year after discharge from therapy. Data examined include demographic, therapeutic, and return-to-employment information. Discriminant analyses showed that 74 of the 94 IWs were not working while involved in work hardening. Of these, 70 IWs (94.59 %) were released to gainful employment on discharge. At the time of the follow-up call, 53 of the IWs (67.95 %) were still working. Of the 25 IWs not working, 18 (69.23 %) were involved in litigation. The average subjective pain level for the nonworking group, on a 0-10 scale, was found to be twice that of the working group, 5.35 vs. 2.48 (p

The purpose of this study was to evaluate the use of high resolution LC-MS together with metabolomics and D(4)-cholic acid (D(4)-CA) as a metabolic tracer to measure the metabolism and reconjugation of bile acids (BAs) in vitro and in vivo. Metabolic tracers are very important because they allow for the direct detection (substrate-to-product) of small and significant biological perturbations that may not be apparent when monitoring "static" endogenous levels of particular metabolites. Slc27a5, also known as fatty acid transport protein 5 (FATP5), is the hepatic BA-CoA ligase involved in reconjugating BAs during enterohepatic BA recycling. Using Slc27a5-cKD mice, silencing of ∼90% gene expression was achieved followed by reduction in the reconjugation of D(4)-CA to D(4)-taurocholic acid (D(4)-TCA), as well as other conjugated BA metabolites in plasma (p = 0.0031). The method described allowed a rapid measure of many D(4) and endogenous BA. Analysis of bile resulted in the detection of 39 BA metabolites from a 13 min analytical run. Finally, the utilization of a novel high resolution mass spectrometry method in combination with metabolomics and a stable isotope metabolic tracer allowed for the detection of targeted and untargeted BAs following silencing of the Slc27a5 gene in primary hepatocytes and in mice.

Cancer cells exhibit the ability to proliferate indefinitely, but paradoxically, overexpression of cellular oncogenes in primary cells can result in a rapid and irreversible cell cycle arrest known as oncogene-induced senescence (OIS). However, we have shown that constitutive overexpression of the oncogene c-MYC in primary human foreskin fibroblasts results in a population of cells with unlimited lifespan; these immortalized cells are henceforth referred to as iMYC. Here, in order to further elucidate the mechanisms underlying the immortalization process, a gene expression signature of three independently established iMYC cell lines compared with matched early passage c-MYC overexpressing cells was derived. Network analysis of this “iMYC signature” indicated that a large fraction of the downregulated genes were functionally connected and major nodes centered around the TGFβ, IL-6 and IGF-1 signaling pathways. Here, we focused on the functional validation of the alteration of TGFβ response during c-MYC-mediated immortalization. The results demonstrate loss of sensitivity of iMYC cells to activation of TGFβ signaling upon ligand addition. Furthermore, we show that aberrant regulation of the p27 tumor suppressor protein in iMYC cells is a key event that contributes to loss of response to TGFβ. These findings highlight the potential to reveal key pathways contributing to the self-renewal of cancer cells through functional mining of the unique gene expression signature of cells immortalized by c-MYC.

siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand “seed region,” similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.

microRNAs (miRNAs) are abundant, approximately 21-nucleotide, noncoding regulatory RNAs. Each miRNA may regulate hundreds of mRNA targets, but the identities of these targets and the processes they regulate are poorly understood. Here we have explored the use of microarray profiling and functional screening to identify targets and biological processes triggered by the transfection of human cells with miRNAs. We demonstrate that a family of miRNAs sharing sequence identity with miRNA-16 (miR-16) negatively regulates cellular growth and cell cycle progression. miR-16-down-regulated transcripts were enriched with genes whose silencing by small interfering RNAs causes an accumulation of cells in G(0)/G(1). Simultaneous silencing of these genes was more effective at blocking cell cycle progression than disruption of the individual genes. Thus, miR-16 coordinately regulates targets that may act in concert to control cell cycle progression.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor, consisting of an alpha subunit and a beta subunit, that controls cellular responses to hypoxia. HIFalpha contains two transcriptional activation domains called the N-terminal transactivation domain (NTAD) and the C-terminal transactivation domain (CTAD). HIFalpha is destabilized by prolyl hydroxylation catalyzed by EglN family members. In addition, CTAD function is inhibited by asparagine hydroxylation catalyzed by FIH1. Both hydroxylation reactions are linked to oxygen availability. The von Hippel-Lindau tumor suppressor protein (pVHL) is frequently mutated in kidney cancer and is part of the ubiquitin ligase complex that targets prolyl hydroxylated HIFalpha for destruction. Recent studies suggest that HIF2alpha plays an especially important role in promoting tumor formation by pVHL-defective renal carcinoma cells among the three HIFalpha paralogs. Here we dissected the relative contribution of the two HIF2alpha transactivation domains to hypoxic gene activation and renal carcinogenesis and investigated the regulation of the HIF2alpha CTAD by FIH1. We found that the HIF2alpha NTAD is capable of activating both artificial and naturally occurring HIF-responsive promoters in the absence of the CTAD. Moreover, we found that the HIF2alpha CTAD, in contrast to the HIF1alpha CTAD, is relatively resistant to the inhibitory effects of FIH1 under normoxic conditions and that, perhaps as a result, both the NTAD and CTAD cooperate to promote renal carcinogenesis in vivo.

Rare familial forms of Alzheimer's disease (AD) are thought to be caused by elevated proteolytic production of the Aβ42 peptide from the β-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Aβ42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Aβ42 secretion and identified leucine-rich repeat transmembrane 3 ( LRRTM3 ) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Aβ40, Aβ42, and sAPPβ, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Aβ secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Aβ42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.

Gene silencing by RNA interference is a powerful genetic tool to identify genes involved in specific biological processes in model organisms and human cells (3, 14, 18, 38, 42, 56). It is now possible to perform unbiased genetic screens using cultured human cells. Recently, small-scale siRNA (small interfering RNA) and shRNA (small hairpin RNA) screens in human cells have successfully identified genes that modulate cell growth, apoptosis, chemoresistance, and chemosensitivity (4, 44, 52). While establishing proof-of-concept, most of these early screens tested gene sets too limited in size to provide a comprehensive view of the genes involved in the cellular phenotypes being tested. Several groups have employed nearly genome-wide screens of shRNAs expressed from vectors to identify genes that modify certain phenotypes (6, 40, 49, 59). To date, these shRNA screens have been limited to phenotypes amenable to positive selection, but other approaches like bar code screening have shown promise (6). Systematic well-by-well testing of genomic-scale siRNA or shRNA libraries for modulation of nonselectable phenotypes in human cells has not been widely attempted. Cancer cells undergo numerous genetic changes that drive cellular transformation from normal cell progenitors. Anticancer drug target discovery is now frequently directed toward understanding and exploiting genetic alterations that exist in tumor cells. Knowledge of genetic alterations in tumors may lead to better use of conventional therapeutics or development of new therapeutics that offer better therapeutic windows. Loss-of-function genetic screens can identify genes whose loss of function enhances cytotoxicity of chemotherapeutics in cells with defined genetic lesions. The TP53 transcription factor is a central mediator of the cellular response to DNA damage and a variety of other stresses and is one of the most frequently mutated genes in human cancers (19, 57). TP53 status can also effect the chemosensitivity of tumor cells (15). The identification of genes that, when silenced, selectively enhance the chemosensitivity of TP53 mutant cancer cells but not TP53 wild-type cells would make attractive drug targets. Drugs developed to these genes have the potential to selectively increase the toxicity of the chemotherapeutic in the cancer cell. Genetic screens in model organisms have led to identification of drug enhancers whose loss increases sensitivity to the drug (43, 54, 56, 61). We hypothesized that identification of enhancers for commonly used cancer therapeutics would lead to new strategies for therapeutic application of these drugs and/or identification of new targets for the development of combination chemotherapies. Here we used small- and genome-scale siRNA screens to identify genes that enhance the sensitivity of human tumor cells to the cancer chemotherapeutic cisplatin. The screen hits included known genes that function in DNA repair, cell cycle checkpoints, and survival signaling, as well as genes with no annotated functions. Selected hits from these screens were validated and then tested on matched TP53-positive and TP53-deficient cells to identify enhancers selective for tumor cells that have lost TP53 function. We show that silencing of BRCA1, BRCA2, and certain genes whose products interact with BRCA1/2, selectively enhances cisplatin cytotoxicity in TP53-deficient cells.

Development of effective drugs for treatment of human disease relies on identification of therapeutic molecular targets. The identification of targets to treat human disease has previously relied on genetic screens in model organisms, and less robust or lower throughput approaches in mammalian systems. RNA interference (RNAi) makes possible, for the first time, the use of large-scale functional genomics approaches for target identification in human cells. This remarkable breakthrough has the potential to influence every facet of the drug discovery process, and is poised to revolutionize drug development. Reports of RNAi screens for the identification of novel genes implicated in apoptosis, cell division, and drug resistance support the enormous promise of this technology. Here, we discuss the potential impact of RNAi screens on target identification and validation and consider issues that warrant caution when interpreting RNAi screening results.

HIV-1 infected persons develop a robust CTL response to HIV antigens, yet HIV-1 is able to evade this host response and successfully replicate. The mechanism(s) of evasion is not completely defined but has been suggested to include resistance of infected cells to CTL-mediated apoptosis. The HIV-1 Vpr protein induces G2 arrest by indirectly inhibiting activation of cyclin B/p34cdc2 kinase. Granzyme B, the principle mediator of CTL-induced apoptosis, prematurely activates this same kinase complex. Therefore, we assessed the susceptibility of HIV-1 infected cells to CTL-mediated apoptosis to determine whether the expression of Vpr protected the infected cells from CTL-induced apoptosis. Antigen-specific CD8 + CTL were able to induce apoptosis in HIV-1 infected cells and cells labeled with peptide corresponding to the CTL epitope with equivalent efficiency. This demonstrates that neither HIV-1 Vpr nor any other HIV protein directly inhibits CTL effector functions. Furthermore, we confirm that HIV-1 Nef is able to provide partial protection from CTL recognition of infected cells. Thus, the inability of CTL to control HIV-1 infection is likely not due to direct inhibition of CTL-mediated apoptosis.

Cyclosporin A (CsA) is a potent inhibitor of the NFAT family of transcription factors that enhance T cell activation. The observation that human immunodeficiency virus type 1 (HIV-1)-positive transplant recipients have a reduced HIV-1 viral burden during treatment with CsA suggested that NFAT may play a direct role in enhancing transcription of the HIV-1 viral genome. Two sets of NFAT binding sites were identified in the HIV-1 long terminal repeat (LTR) promoter by in vitro footprinting with full-length recombinant NFAT protein, and gel shift analysis of nuclear protein from polyclonally activated primary CD4 T cells revealed specific binding of NFAT1 to the NFkappaB binding sites of the HIV-1 LTR. Activation of primary CD4 T cells transiently transfected with a HIV-1 LTR luciferase reporter plasmid, lacking the NFAT binding sites in the upstream putative negative regulatory element but maintaining the NFkappaB/NFAT sites, demonstrated increased HIV-1 gene expression when cotransfected with a NFAT1 expression vector. Moreover, CsA, FK506, and a dominant-negative NFAT1 protein independently inhibited HIV-1 LTR promoter activity in CD4 T cells stimulated with phorbol ester and calcium ionophore. In primary human CD4 T cells, CsA also inhibited promoter activity directed by multimers of binding sites for NFAT, while having no effect on NFkappaB multimer-driven promoter activity. Increasing NFAT1 levels in CD4 T cells transiently transfected with a HIV-1 provirus also increased p24 protein expression. Thus, NFAT may be a target for prevention of HIV-1 LTR-directed gene expression in human CD4 T cells.

Apoptosis contributes to the loss of CD4 cells during human immunodeficiency virus type 1 (HIV-1) infection. Although the product of the env gene, gp160/gp120, is known to play a role in cell death mediated by HIV-1, the role of other HIV-1 genes in the process is unclear. We found that HIV-1 lacking the env gene (HIVΔ env ) still induced apoptosis in T-cell lines and primary CD4 T cells. The ability to induce apoptosis was attributable to Tat, a viral regulatory protein. Tat induction of apoptosis was separate from the transactivation function of Tat, required expression of the second exon of Tat, and was associated with the increased expression and activity of caspase-8 (casp-8), a signaling molecule in apoptotic pathways. Moreover, induction of apoptosis could be prevented by treating cells with an inhibitor of casp-8. In addition, we show that HIV-1Δ env infection and Tat expression increased the sensitivity of cells to Fas-mediated apoptosis, an apoptotic pathway that signals via casp-8. The up-regulation of casp-8 by HIV-1 Tat expression may contribute to the increased apoptosis and sensitivity to apoptotic signals observed in the cells of HIV-1-infected persons.

We describe a method for the production of high-titer stocks of human immunodeficiency virus type 1 (HIV-1) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV G). VSV G pseudotypes provide several advantages over other retroviral envelope proteins. The VSV G envelope is mechanically stable, enabling ultracentrifugal concentration of virions to high titers, and VSV G has a broad host range, enabling infection of many mammalian and nonmammalian cell types. VSV G pseudotypes of HIV-1 are useful for the study of HIV infection and replication kinetics and for the study of the function of specific viral proteins. We describe applications for the study of HIV-1 using VSV G pseudotypes. Additionally, we describe a method for pseudotyping retroviral vectors with VSV G. The same advantages of VSV G pseudotypes of HIV-1 apply to retroviral vectors; VSV G pseudotyped retroviral vectors may be used to introduce genes of interest into a wide variety of cell lines.

CCR5 and CXCR4 are the two major coreceptors that have been identified for human immunodeficiency virus (HIV) entry. We have modified several β-galactosidase-based HIV indicator cell lines to express CCR5 and/or CXCR4. Using these new reagents, we have been able to detect all primary isolates tested using one or both of these cell lines. However, there is large variation in the absolute viral infectivity among primary strains. Furthermore, all HIV strains are capable of causing syncytia in the indicator cells when the coreceptor is present regardless of whether they had previously been characterized as “syncytia-inducing” or “non-syncytium-inducing.”

Natural immunity may be involved in controlling viral spread in hosts infected with HIV. A panel of gamma delta T cell receptor-positive lymphocyte clones was isolated from the peripheral blood of healthy HIV- donors and tested for anti-HIV cytotoxic responses. Twelve of 30 (40%) V gamma 9+/V delta 2+ T cell clones, but none of seven V delta 1+ T cell clones, displayed lytic activity against HIV-infected cells. The V gamma 9+/V delta 2+ clones cytotoxic for HIV-infected cells also lysed Daudi cells. However, not all V gamma 9+/V delta 2+ clones which lysed Daudi targets had the capacity to lyse HIV-infected cells. Some of the gamma delta T cell clones were also investigated for potential proliferative responses to HIV-infected cells. One V gamma 9+/V delta 2+ T cell clone (ME8-7) and one V delta 1+ T cell clone (ME18-2) demonstrated proliferative responses towards HIV-infected cells. Another V gamma 9+/V delta 2+ clone (VM39) proliferated in response to cell-free HIV. Taken together, these results provide direct evidence of anti-HIV gamma delta T cell responses in healthy, HIV- persons.

Multiparametric assays generate biological activity profiles that provide valuable insight into complex disease models. The use of multiple assay measurements in RNA interference (RNAi) high-throughput screening (HTS) provides biological signatures produced by knocking down individual genes. This strategy has been applied to a genome-wide high-throughput small interfering RNA (siRNA) screen measuring proteolysis of β-amyloid precursor protein (APP) into amyloid β peptides, a critical step in the pathogenesis of Alzheimer’s disease. The assay measures amounts of secreted Aβ40, Aβ42, sAPPα, and sAPPβ from HEK 293 cells stably expressing an optimized APP construct following siRNA transfection. The effect of each siRNA on the four different APP products was simultaneously measured in order to identify human genes that regulate the amyloidogenic processing of APP. Genes with BACE-like and γ-secretase-like activity profiles were identified for further biological characterization.

We identified gene expression signatures predicting responsiveness to a Kinesin-5 (KIF11) inhibitor (Kinesin-5i) in cultured colon tumor cell lines. Genes predicting resistance to Kinesin-5i were enriched for those from chromosome 20q, a region of frequent amplification in a number of tumor types. siRNAs targeting genes in this chromosomal region identified AURKA, TPX2 and MYBL2 as genes whose disruption enhances response to Kinesin-5i. Taken together, our results show functional interaction between these genes, and suggest that their overexpression is involved in resistance to Kinesin-5i. Furthermore, our results suggest that patients whose tumors overexpress AURKA due to amplification of 20q will more likely resist treatment with Kinesin-5 inhibitor, and that inactivation of AURKA may sensitize these patients to treatment.