Suppression of Ser/Thr phosphatase 4 (PP4C/PPP4C) mimics a novel post-mitotic action of fostriecin, producing mitotic slippage followed by tetraploid cell death.

UNLABELLED: Fostriecin is a natural product purified from Sterptomyces extracts with antitumor activity sufficient to warrant human clinical trials. Unfortunately, difficulties associated with supply and stable drug formulation stalled further development. At a molecular level, fostriecin is known to act as a catalytic inhibitor of four PPP-family phosphatases, and reports describing the design of molecules in this class suggest derivatives targeting enzymes within the fostriecin-sensitive subfamily can be successful. However, it is not clear if the tumor-selective cytotoxicity of fostriecin results from the inhibition of a specific phosphatase, multiple phosphatases, or a limited subset of fostriecin sensitive phosphatases. How the inhibition of sensitive phosphatases contributes to tumor-selective cytotoxicity is also not clear. Here, high-content time-lapse imaging of live cells revealed novel insight into the cellular actions of fostriecin, showing that fostriecin-induced apoptosis is not simply induced following a sustained mitotic arrest. Rather, apoptosis occurred in an apparent second interphase produced when tetraploid cells undergo mitotic slippage. Comparison of the actions of fostriecin and antisense-oligonucleotides specifically targeting human fostriecin-sensitive phosphatases revealed that the suppression PP4C alone is sufficient to mimic many actions of fostriecin. Importantly, targeted suppression of PP4C induced apoptosis, with death occurring in tetraploid cells following mitotic slippage. This effect was not observed following the suppression of PP1C, PP2AC, or PP5C. These data clarify PP4C as a fostriecin-sensitive phosphatase and demonstrate that the suppression of PP4C triggers mitotic slippage/apoptosis. IMPLICATIONS: Future development of fostriecin class inhibitors should consider PP4C as a potentially important target. Mol Cancer Res; 11(8); 845-55. ©2013 AACR.

Antisense inhibition of connective tissue growth factor (CTGF/CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo.

Augmented expression of connective tissue growth factor (CTGF/CCN2) is observed in healing wounds and in a variety of fibrotic disorders. It appears to enhance many of the effects of transforming growth factor-beta and has been shown to have independent fibrogenic functions. Despite these observations, its importance to dermal wound healing and the transition from wound to scar remains poorly defined. In this study, we use established rabbit models to evaluate the roles of CTGF in dermal wound healing and hypertrophic scarring. We show that CTGF mRNA demonstrates persistent up-regulation in hypertrophic scars. Treatment of wounds with antisense oligonucleotides to CTGF has no measurable effect on early wound closure. However, antisense therapy significantly limits subsequent hypertrophic scarring. Inhibition of CTGF is associated with a marked reduction in the number of myofibroblasts in scars and decreased transcription of TIMP-1 and types I and III collagen. These findings confirm CTGF to be a key mediator of hypertrophic scarring in this model. Its effect on myofibroblasts in this setting suggests a mechanism whereby it plays this role. Its limited participation in early healing implies that it may be a useful and specific target for modulating hypertrophic scarring following injury.

Therapeutic suppression of translation initiation factor eIF4E expression reduces tumor growth without toxicity.

Expression of eukaryotic translation initiation factor 4E (eIF4E) is commonly elevated in human and experimental cancers, promoting angiogenesis and tumor growth. Elevated eIF4E levels selectively increase translation of growth factors important in malignancy (e.g., VEGF, cyclin D1) and is thereby an attractive anticancer therapeutic target. Yet to date, no eIF4E-specific therapy has been developed. Herein we report development of eIF4E-specific antisense oligonucleotides (ASOs) designed to have the necessary tissue stability and nuclease resistance required for systemic anticancer therapy. In mammalian cultured cells, these ASOs specifically targeted the eIF4E mRNA for destruction, repressing expression of eIF4E-regulated proteins (e.g., VEGF, cyclin D1, survivin, c-myc, Bcl-2), inducing apoptosis, and preventing endothelial cells from forming vessel-like structures. Most importantly, intravenous ASO administration selectively and significantly reduced eIF4E expression in human tumor xenografts, significantly suppressing tumor growth. Because these ASOs also target murine eIF4E, we assessed the impact of eIF4E reduction in normal tissues. Despite reducing eIF4E levels by 80% in mouse liver, eIF4E-specific ASO administration did not affect body weight, organ weight, or liver transaminase levels, thereby providing the first in vivo evidence that cancers may be more susceptible to eIF4E inhibition than normal tissues. These data have prompted eIF4E-specific ASO clinical trials for the treatment of human cancers.

Selective down-regulation of glioma-associated oncogene 2 inhibits the proliferation of hepatocellular carcinoma cells.

The sonic hedgehog (Shh) pathway contributes to the initiation and progression of tumors with various origins when aberrantly activated. In this study, we investigated if the Shh pathway is important for the proliferation of hepatocellular carcinoma (HCC) cells and also began to identify which components of the pathway play a pivotal role in the biology of HCC. Expression levels of components in the pathway were measured, and glioma-associated oncogene (Gli) 2 levels were found to be considerably higher in human HCC lines compared with normal liver. Gli2 levels were also higher in tumor tissue from HCC patients compared with normal liver. Antisense oligonucleotides (ASO) were used to specifically down-regulate Gli2, and this led to decreased proliferation of various HCC cell lines. However, inhibition of Gli1 and Gli3 with ASOs did not decrease proliferation in most HCC cell lines and inhibitors targeting the upstream components of the pathway, including smoothened (Smo), displayed antiproliferative effects in only a subset of HCC cell lines. Moreover, in cancer cells harboring Smo mutations or unresponsive to the Smo inhibitor 3-keto-N-aminoethylaminoethylcaproyldihydrocinnamoyl cyclopamine, the Gli2 ASO was still able to inhibit proliferation. The importance of Gli2 in HCC proliferation was further confirmed by the changes in expression levels of genes, such as Bcl-2, c-Myc, and p27, following suppression of Gli2 expression. Taken together, these results suggest that, among the Gli transcription factors, Gli2 plays a predominant role in the proliferation of HCC cells and the suppression of Gli2 expression may provide a useful therapeutic option for the treatment of HCC.

Cantharidin-induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2Aalpha.

Cantharidin, a natural vesicant, inhibits the activity of several PPP family phosphatases, displays antitumor activity, and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, dose-response studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that before apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G(1)-S or S-G(2) phase progression. In contrast, studies with double thymidine-synchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G(2) or early M phase. Immunostaining indicates that cell cycle arrest occurs before the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live cell imaging with time-lapse microscopy shows that cantharidin disrupts the metaphase alignment of chromosomes and produces a prolonged mitotic arrest, with the onset of apoptosis occurring before the onset of anaphase. To explore the contribution of individual phosphatases, antisense oligonucleotides and small interfering RNA were developed to suppress the expression of cantharidin-sensitive phosphatases. The suppression of PP2Aalpha, but not PP2Abeta, is sufficient to induce metaphase arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10- to 15-hour delay, the cells enter anaphase, suggesting that an additional cantharidin-sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis.

Competition for RISC binding predicts in vitro potency of siRNA.

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.

Regulation and targeting of Eg5, a mitotic motor protein in blast crisis CML: overcoming imatinib resistance.

Patients with blast crisis (BC) CML frequently become resistant to Imatinib, a Bcr-Abl tyrosine kinase-targeting agent. Eg5, a microtubule-associated motor protein has been described to be highly expressed in BC CML by microarray analysis (Nowicki et al., Oncogene 2003; 22:3952-63). We investigated the regulation of Eg5 by Bcr-Abl tyrosine kinase and its potential as a therapeutic target in BC CML. Eg5 was highly expressed in all Philadelphia chromosome positive (Ph(+)) cell lines and BC CML patient samples. Inhibition of Bcr-Abl by Imatinib downregulated Eg5 expression in Imatinib-sensitive KBM5 and HL-60p185 cells, but not in Imatinib-resistant KBM5-STI571, harboring a T315I mutation, and Bcr-Abl-negative HL-60 cells. Blocking Eg5 expression with antisense oligonucleotide (Eg5-ASO) or inhibiting its activity with the small-molecule Eg5 inhibitor, S-trityl-L-cysteine induced G(2)/M cell cycle block and subsequent cell death in both Imatinib-sensitive and -resistant cells. Further, Eg5-ASO treatment of SCID mice harboring KBM5 cell xenografts significantly prolonged the median survival of the animals (p = 0.03). Our findings suggest that Eg5 is downstream of and regulated by Bcr-Abl tyrosine kinase in Philadelphia chromosome positive cells. Inhibition of Eg5 expression or its activity blocks cell cycle progression and induces cell death independent of the cellular response to Imatinib. Therefore, Eg5 could be a potential therapeutic target for the treatment of BC CML, in particular Imatinib-resistant BC CML.

Modification of MyD88 mRNA splicing and inhibition of IL-1beta signaling in cell culture and in mice with a 2'-O-methoxyethyl-modified oligonucleotide.

A number of proinflammatory cytokines, including IL-1beta, signal through the adaptor protein MyD88. This signaling leads to phosphorylation of IL-1R-associated kinase-1 (IRAK-1) and, ultimately, activation of the NF-kappaB transcription factor. A splice variant of MyD88 (MyD88(S)), which lacks the ability to couple IRAK-1 to NF-kappaB, has been described. A chemically modified antisense oligonucleotide (ASO) that alters the splicing ratio of MyD88 to MyD88(S) in both cell culture and in animals has been identified. The ASO (ISIS 337846) binds to exon II donor sites in the MyD88 pre-mRNA. By manipulating levels of MyD88 splicing, proinflammatory signaling through the IL-1R has been shown to be diminished, both in cell culture and in mouse liver. To our knowledge, this represents the first example of modulation of RNA splicing of an endogenous gene target in animals after systemic ASO dosing and suggests that this mechanism may be useful as a novel modulator of inflammatory stimuli.

Use of a chemically modified antisense oligonucleotide library to identify and validate Eg5 (kinesin-like 1) as a target for antineoplastic drug development.

A library of 2'-methoxyethyl-modified antisense oligonucleotides (2'MOE ASO) targeting 1,510 different genes has been developed, validated, and used to identify cell cycle regulatory genes. The most effective molecular target identified was Eg5 (kinesin-like-1), which when inhibited gave the largest increase in 4N DNA in various tumor cells. The Eg5 ASO reduced Eg5 levels, inhibited proliferation, increased apoptosis, and altered the expression of other cell cycle proteins, including survivin and Aurora-A. To examine the therapeutic utility of the Eg5 ASO, the compound was also evaluated in xenograft models. Treatment with Eg5 ASO produced a statistically significant reduction of tumor growth, reduction in Eg5 expression in the tumors, and changes in histone phosphorylation, consistent with a loss of Eg5 protein expression. These data show, for the first time, the utility of a 2'MOE ASO library for high-throughput cell culture-based functional assays and suggest that an Eg5 ASO also has potential in a therapeutic strategy.

Defining the transcriptome of accelerated and replicatively senescent keratinocytes reveals links to differentiation, interferon signaling, and Notch related pathways.

Epidermal keratinocytes (KCs) undergo highly orchestrated morphological and molecular changes during transition from proliferative compartment into growth arrested early and late differentiation layers, prior to dying in outermost cornified layers of normal skin. Creation of stratum corneum is vital to barrier function protecting against infection. Transcriptional events in KCs regulating complex processes of differentiation and host defense required to maintain constant epidermal thickness and resistance to infection in either young or aged skin are largely unknown. Furthermore, as terminal differentiation is characterized by irreversible loss of replicative potential culminating in dead layers at the skin surface, this process may be viewed as a form of senescence. However, a complete transcriptional profile of senescent (SN) human KCs has not been previously defined to permit delineation of molecular boundaries involving differentiation and senescence. To fill this void, we utilized global transcriptional analysis of KCs maintained in vitro as either cultures of proliferating (PR) cells, early and late confluent (LC) (accelerated senescence) cultures, or KCs undergoing replicative senescence. Global gene expression profiling revealed early confluent (EC) KCs were somewhat similar to PR KCs, while prominent differences were evident when compared to LC KCs; which were also distinct from replicatively SN KCs. While confluent KCs have in common several genes regulating differentiation with replicatively SN KCs, the latter cells expressed elevated levels of genes involved in interferon signaling and inflammatory pathways. These results provide new insights into cell autonomous transcriptional-based programs operative within KCs contributing to replicative senescence, with partial sharing of genes involved in differentiation. In addition, regulation of KC senescence may involve participation of interferon signaling pathways derived from the important role of KCs in protecting skin from infection. Integrating all of the transcriptional data revealed a key role for Notch receptor mediated signaling in the confluency induced differentiation phenotype using this model system.

Identification of novel PPARgamma target genes in primary human adipocytes.

Adipogenesis is the process by which undifferentiated precursor cells differentiate into fat laden adipocytes. The nuclear proteins peroxisome proliferator-activated receptors (PPARs) play a central role in adipocyte differentiation. The goals of this study were to identify novel PPARgamma responsive genes and to determine their role in regulating human adipocyte differentiation. Affymetrix profiling of gene expression in human adipocytes identified about 1000 genes that were significantly up-regulated subsequent to induction of differentiation. PPARgamma expression was reduced prior to induction of differentiation using a novel, chemically modified antisense oligonucleotide. Affymetrix microarray profiling of these cells identified 278 statistically significantly down-regulated genes. Eight genes were found to contain previously documented PPARgamma recognition element (PPRE) in their upstream nucleotide (promoter) sequence. Four of these genes are novel and have not previously been characterized. Chromatin immuno-precipitation experiments confirmed the binding of PPARgamma to the PPRE of three of these genes. The ortholog of one of these genes, hypothetical protein FLJ 20920, has previously been reported to be involved in the control of body fat composition in Caenorhabditis elegans. Inhibition of expression of this protein was found to also inhibit differentiation of human adipocytes. MAST/MEME algorithm analysis was used to identify novel commonly occurring sequence motifs in the 5' upstream region of transcripts for subset of down-regulated genes, which were grouped according to their sequence similarities. A number of clusters were identified and the largest cluster contained similar motifs from 26 genes with the literature supporting 7 of the 26 genes as being involved in fatty acid metabolism or PPARgamma interaction.

Pharmacologic inhibition of RAF-->MEK-->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1.

Expression of mutationally activated RAS is a feature common to the vast majority of human pancreatic adenocarcinomas. RAS elicits its effects through numerous signaling pathways including the RAF-->mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase [MEK]-->ERK MAP kinase pathway. To assess the role of this pathway in regulating cell proliferation, we tested the effects of pharmacologic inhibition of MEK on human pancreatic cancer cell lines. In eight cell lines tested, MEK inhibition led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest. Concomitant with cell cycle arrest, we observed induced expression of p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. Using both antisense and RNA interference techniques, we assessed the role of p27Kip1 in the observed effects of MEK inhibition on pancreatic cancer cell proliferation. Inhibition of p27Kip1 expression in Mia PaCa-2 cells restored the activity of cyclin/cdk2, phosphorylation of pRb, and E2F activity and partially relieved the effects of U0126 on pancreatic cancer cell cycle arrest. Consistent with the effects of p27Kip1 on cyclin/cdk2 activity, inhibition of CDK2 expression by RNA interference also led to G0-G1 cell cycle arrest. These data suggest that the expression of p27Kip1 is downstream of the RAF-->MEK-->ERK pathway and that the regulated expression of this protein plays an important role in promoting the proliferation of pancreatic cancer cells. Moreover, these data suggest that pharmacologic inhibition of the RAF-->MEK-->ERK signaling pathway alone might tend to have a cytostatic, as opposed to a cytotoxic, effect on pancreatic cancer cells.

Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs.

MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression. The predicted presence of numerous miRNA sequences in higher eukaryotes makes it highly likely that the expression levels of individual miRNA molecules themselves should play an important role in regulating multiple cellular processes. Therefore, determining the pattern of global miRNA expression levels in mammals and other higher eukaryotes is essential to help understand both the mechanism of miRNA transcriptional regulation as well as to help identify miRNA regulated gene expression. Here, we describe a novel method to detect global processed miRNA expression levels in higher eukaryotes, including human, mouse and rats, by using a high-density oligonucleotide array. Array results have been validated by subsequent confirmation of mir expression using northern-blot analysis. Major differences in mir expression have been detected in samples from diverse sources, suggesting highly regulated mir expression, and specific gene regulatory functions for individual miRNA transcripts. For example, five different miRNAs were found to be preferentially expressed in human kidney compared with other human tissues. Comparative analysis of surrounding genomic sequences of the kidney-specific miRNA clusters revealed the occurrence of specific transcription factor binding sites located in conserved phylogenetic foot prints, suggesting that these may be involved in regulating mir expression in kidney.

MicroRNA-143 regulates adipocyte differentiation.

MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.

Constitutive over expression of serine/threonine protein phosphatase 5 (PP5) augments estrogen-dependent tumor growth in mice.

Serine/threonine protein phosphatase 5 (PP5) appears to play an underappreciated role in the regulation of cellular proliferation. In estrogen-responsive cells, PP5 expression is stimulated by 17 beta-estradiol, and in a variety of p53 wild-type tumor cells the suppression of PP5 expression with ISIS 15534 inhibits growth. To further explore the relationship between PP5 and the development of human cancer, here we tested the effect of elevated PP5 expression on tumor growth using a mouse xenograph model and a stable MCF-7 cell line in which the expression of wild-type PP5 was placed under the control of tetracycline-off regulated transactivator and operator plasmids. In the xenograph model a modest two fold increase in PP5 protein levels significantly enhanced the growth rate of estrogen-dependent tumors, suggesting PP5 plays a positive role in tumor development.

Setting sights on the treatment of ocular angiogenesis using antisense oligonucleotides.

The application of antisense technology to study physiological and disease processes continues to mature. Antisense approaches are among the most direct means to use genomic sequence information. When developing therapeutics, applications range from early target validation in discovery to the therapeutic product. In this review, we describe the application of antisense oligonucleotides (ASOs) to identify genes that are important in controlling angiogenesis. High-throughput assays in vitro have been used to evaluate many gene targets. Genes that appear to be important in angiogenesis are then evaluated further in animal models of ocular angiogenesis. The ability of ASOs to reduce target-gene expression in the appropriate cells in the eye raises the possibility that this class of compounds could be used for target validation in vivo, and also be developed as a novel class of therapeutics in their own right.

Use of antisense oligonucleotides in functional genomics and target validation.

With the completion of sequencing of the human genome, a great deal of interest has been shifted toward functional genomics-based research for identification of novel drug targets for treatment of various diseases. The major challenge facing the pharmaceutical industry is to identify disease-causing genes and elucidate additional roles for genes of known functions. Gene functionalization and target validation are probably the most important steps involved in identifying novel potential drug targets. This review focuses on recent advances in antisense technology and its use for rapid identification and validation of new drug targets. The significance and applicability of this technology as a beginning of the drug discovery process are underscored by relevant cell culture-based assays and positive correlation in specific animal disease models. Some of the antisense inhibitors used to validate gene targets are themselves being developed as drugs. The current clinical trials based on such leads that were identified in a very short time further substantiate the importance of antisense technology-based functional genomics as an integral part of target validation and drug target identification.

Down-regulation of connective tissue growth factor and type I collagen mRNA expression by connective tissue growth factor antisense oligonucleotide during experimental liver fibrosis.

Transforming growth factor (TGF)-beta 1 is a major mediator of liver fibrosis. Connective tissue growth factor (CTGF) mediates TGF-beta 1 pro-fibrogenic effects in vitro, but its in vivo role is unknown. Both TGF-beta 1 and CTGF are overexpressed in hepatic stellate cells during liver fibrosis. We have used antisense oligonucleotides to examine the role of CTGF in carbon tetrachloride-induced liver fibrosis in mice. Mice received carbon tetrachloride together with CTGF or TGF-beta 1 antisense oligonucleotides for 2 weeks (preventive model), or carbon tetrachloride for 2 weeks followed by carbon tetrachloride and oligonucleotides for 2 more weeks (curative model). In both models, CTGF and TGF-beta 1 oligonucleotides decreased by more than 50 percent the mRNA expression of their targets. Type I collagen mRNA was also decreased by about 40 percent in the preventive experiment. Tissue inhibitor of matrix metalloproteinase-1 mRNA expression and fibrotic deposition evaluated by Sirius red staining were not modified in any group. In summary, our results suggest that hepatic stellate cells can be targeted in vivo with oligonucleotides, and that reducing CTGF levels can lead to a decrease in fibrogenesis as shown by the reduction in type I collagen expression. The lack of effect on fibrosis may be due to the persistence of high tissue inhibitor of matrix metalloproteinase-1 expression.

Requirement of protein phosphatase 5 in DNA-damage-induced ATM activation.

The checkpoint kinase ATM is centrally involved in the cellular response to DNA double-strand breaks. However, the mechanism of ATM activation during genotoxic stress is only partially understood. Here we report a direct regulatory linkage between the protein serine-threonine phosphatase 5 (PP5) and ATM. PP5 interacts with ATM in a DNA-damage-inducible manner. Reduced expression of PP5 attenuated DNA-damage-induced activation of ATM. Expression of a catalytically inactive PP5 mutant inhibited the phosphorylation of ATM substrates and the autophosphorylation of ATM on Ser 1981, and caused an S-phase checkpoint defect in DNA-damaged cells. Together our findings indicate that PP5 plays an essential role in the activation and checkpoint signaling functions of ATM in cells that have suffered DNA double-strand breaks.

Antisense oligonucleotide-based therapeutics for cancer.

There has been steady progress in antisense technology over the past 14 years. We now have a far better appreciation of the attributes and limitations of the technology. Antisense oligonucleotides have been used to selectively inhibit thousands of genes in mammalian cells, hundreds, if not thousands, of genes in rodents and other species and multiple genes in humans. There are over 20 antisense drugs currently in clinical trials, several of which are showing promising results. Like any other class of drugs in development, there will continue to be successes and failures in the clinic. Despite some disappointments with the technology, it appears to be a valid platform for both drug discovery and as an experimental tool for functionalizing genes. Advances in the medicinal chemistry and formulation of antisense oligonucleotides will further enhance their therapeutic and commercial potential.