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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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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).

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 to matched early passage c-MYC overexpressing cells was derived. Network analysis of this "iMYC signature" indicated that a large fraction of the down-regulated 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.

Small interfering RNAs (siRNAs) are emerging as promising therapeutic tools. However, the widespread clinical application of such molecules as modulators of gene expression is still dependent on several aspects that limit their bioavailability. One of the most promising strategies to overcome the barriers faced by gene silencing molecules involves the use of lipid-based nanoparticles (LNPs) and viral vectors, such as adenoviruses (Ads). The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. In this study, we tested the capability of LNPs and Ad to transduce and treat locally tumors in vivo. Efficient knockdown of a surrogate reporter (luciferase) and therapeutic target genes such as the kinesin spindle protein (KIF11) and polo-like kinase 1 were observed. Most importantly, this activity led to a cell cycle block as a consequence and slowed down tumor progression in tumor-bearing animals. Our data indicate that it is possible to achieve tumor transduction with si/short hairpin RNAs and further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer gene silencing.

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoE⁻/⁻ and low density lipoprotein receptor (LDLr)⁻/⁻ mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETP⁺/⁻ hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETP⁺/⁻ mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.

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.

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.

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.

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.

RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53(+/-) matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity approximately 4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.

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.

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., ((C)2002 Elsevier Science (USA).)

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., (Copyright 2000 Academic Press.)

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 (HIVDeltaenv) 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-1Deltaenv 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 beta-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."

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.

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.

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.

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<.01). It was also discovered that 100 % of the IWs not working at follow up who were previously deemed noncompliant with work hardening were involved in a disputed claim. This study also found that noncompliant individuals were discharged from therapy much earlier than compliant subjects, after 5.5 versus 14.4 visits, and at a much lower cost, $798.39 versus $2,137.01. These results suggest that although "state-of-the-art" programming was administered, behavioral and psychosocial factors (compliance and litigation) confounded the reactivation outcome process. Implications for future research directions are discussed.

Rhesus monkeys (Macaca mulatta) gamma delta T cells were identified using a monoclonal antibody. The relative representation of gamma delta T lymphocytes in the peripheral blood, lymph nodes, and spleen resembles that of Homo sapiens. The analysis of function and specificity revealed further significant similarities between the simian and human gamma delta T-cell systems. Since both human and monkey gamma delta T lymphocytes can effectively lyse cells infected with immunodeficiency viruses, it is possible that the primate gamma delta T-cell systems contribute to antiviral immunosurveillance.