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Pharmaceutical manufacturing relies on rigorous methods of quality control of drugs and in particular of the physico-chemical and functional characterizations of monoclonal antibodies. To that end, robust bioassays are very often limited to reporter gene assays and the use of immortalized cell lines that are supposed to mimic immune cells such as natural killer (NK) cells to the detriment of primary materials, which are appreciated for their biological validity but are also difficult to exploit due to the great diversity between individuals. Here, we characterized the phenotype of the peripheral blood circulating cytotoxic cells of 30 healthy donors, in particular the repertoire of cytotoxic markers, using flow cytometry. In parallel, we characterized the antibody-dependent cellular cytotoxicity (ADCC) effector functions of these primary cells by measuring their cytolytic activity against a cancer cell-line expressing HER2 in the presence of trastuzumab and with regards to FCGR3A genotype. We could not establish a correlation or grouping of individuals using the data generated from whole peripheral blood mononuclear cells, however the isolation of the CD56-positive population, which is composed not only of NK cells but also of natural killer T (NKT) and γδ-T cells, as well as subsets of activated cytotoxic T cells, monocytes and dendritic cells, made it possible to standardize the parameters of the ADCC and enhance the overall functional avidity without however eliminating the inter-individual diversity. Finally, the use of primary CD56+ cells in ADCC experiments comparing glycoengineered variants of trastuzumab was conclusive to test the limits of this type of ex vivo system. Although the effector functions of CD56+ cells reflected to some extent the in vitro receptor binding properties and cytolytic activity data using NK92 cells, as previously published, reaching a functional avidity plateau could limit their use in a quality control framework.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Processes that have been linked to aging and cancer include an inflammatory milieu driven by senescent cells. Senescent cells lose the ability to divide, essentially irreversibly, and secrete numerous proteases, cytokines and growth factors, termed the senescence-associated secretory phenotype (SASP). Senescent cells that lack p53 tumor suppressor function show an exaggerated SASP, suggesting the SASP is negatively controlled by p53. Here, we show that increased p53 activity caused by small molecule inhibitors of MDM2, which promotes p53 degradation, reduces inflammatory cytokine production by senescent cells. Upon treatment with the MDM2 inhibitors nutlin-3a or MI-63, human cells acquired a senescence-like growth arrest, but the arrest was reversible. Importantly, the inhibitors reduced expression of the signature SASP factors IL-6 and IL-1α by cells made senescent by genotoxic stimuli, and suppressed the ability of senescent fibroblasts to stimulate breast cancer cell aggressiveness. Our findings suggest that MDM2 inhibitors could reduce cancer progression in part by reducing the pro-inflammatory environment created by senescent cells.

CD133 is widely used as a marker for stem/progenitor cells in many organ systems. Previous studies using in vitro stem cell assays have suggested that the CD133-expressing prostate basal cells may serve as the putative prostate stem cells. However, the precise localization of the CD133-expressing cells and their contributions to adult murine prostate homeostasis in vivo remain undetermined. We show that loss of function of CD133 does not impair murine prostate morphogenesis, homeostasis and regeneration, implying a dispensable role for CD133 in prostate stem cell function. Using a CD133-CreER(T2) model in conjunction with a fluorescent report line, we show that CD133 is not only expressed in a fraction of prostate basal cells, but also in some luminal cells and stromal cells. CD133(+) basal cells possess higher in vitro sphere-forming activities than CD133(-) basal cells. However, the in vivo lineage tracing study reveals that the two cell populations possess the same regenerative capacity and contribute equally to the maintenance of the basal cell lineage. Similarly, CD133(+) and CD133(-) luminal cells are functionally equivalent in maintaining the luminal cell lineage. Collectively, our study demonstrates that CD133 does not enrich for the stem cell activity in vivo in adult murine prostate. This study does not contradict previous reports showing CD133(+) cells as prostate stem cells in vitro. Instead, it highlights a substantial impact of biological contexts on cellular behaviors.

Long non-coding RNAs (lncRNAs) play important roles in all the central aspects of the gene expression cascade with wide ranging effects on cell proliferation and differentiation. As such, lncRNAs are highly promising additions to many cancer biomarkers portfolios and enable more precise diagnostic staging. This, in turn, provides a clearer prognosis and treatment options of different cancers. Direct visualization and quantification of lncRNAs in single cells and in tissue are thus emerging as essential tools for both basic and clinical research. Interestingly, studies have demonstrated that transcript levels are more strongly correlated to clinical traits than the corresponding protein levels. Because RNA biomarkers have the potential to contribute to the development of targeted cancer therapies, there is also an obvious need for a robust and dependable methodology to discover and validate these RNAs. RNA fluorescence in situ hybridization (RNA FISH) provides a powerful means to detect RNA biomarkers in single cells, while still maintaining tissue morphology. Significant advances in RNA FISH technology, such as the hybridization of pools of singly labeled fluorescent 20-mer oligonucleotides to the RNA target, now afford specific and sensitive multiplex detection of RNA to yield information on the RNA's distinct spatial distribution within cells and tissue. Data is presented on the design of probe sets against multiple disease relevant pre-lncRNAs and against the introns of their precursors, as well as on the simultaneous dual-channel imaging of the pre-lncRNAs and mature lncRNAs. Because the absolute majority of post-transcriptional processing, including splicing of mRNAs and lncRNAs, occurs co-transcriptionally, the location of introns can be used as a proxy nuclear location for the encoding gene. We find mature lncRNAs to locate at or close to the site of transcription, but also well away from it. Next, we present data on emerging non-coding RNA biomarkers in breast and prostate cancer tissue and in cell lines utilizing RNA FISH. In summary, the results and tools presented here will contribute to advancing the current capabilities of the detection and treatment of specific cancers, as well as in the continued discovery and development of cancer drug candidates. Citation Format: Arturo V. Orjalo, Jr., Hans E. Johansson. Stellaris® RNA fluorescence in situ hybridization (RNA FISH) for the detection of long non coding RNA biomarkers. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-44.

Diverse cell types have unique transcriptional signatures that are best interrogated at single-cell resolution. Here we describe a novel RNA amplification approach that allows for high fidelity gene profiling of individual cells. This technique significantly diminishes the problem of 3′ bias, enabling detection of all regions of transcripts, including the recognition of mRNA with short or completely absent poly(A) tails, identification of noncoding RNAs, and discovery of the full array of splice isoforms from any given gene product. We assess this technique using statistical and bioinformatics analyses of microarray data to establish the limitations of the method. To demonstrate applicability, we profiled individual cells isolated from the mouse subventricular zone (SVZ)'a well-characterized, discrete yet highly heterogeneous neural structure involved in persistent neurogenesis. Importantly, this method revealed multiple splice variants of key germinal zone gene products within individual cells, as well as an unexpected coexpression of several mRNAs considered markers of distinct and separate SVZ cell types. These findings were independently confirmed using RNA-fluorescence in situ hybridization (RNA-FISH), contributing to the utility of this new technology that offers genomic and transcriptomic analysis of small numbers of dynamic and clinically relevant cells.

The TOR (target of rapamycin) kinase limits longevity by poorly understood mechanisms. Rapamycin suppresses the mammalian TORC1 complex, which regulates translation, and extends lifespan in diverse species, including mice. We show that rapamycin selectively blunts the pro-inflammatory phenotype of senescent cells. Cellular senescence suppresses cancer by preventing cell proliferation. However, as senescent cells accumulate with age, the senescence-associated secretory phenotype (SASP) can disrupt tissues and contribute to age-related pathologies, including cancer. MTOR inhibition suppressed the secretion of inflammatory cytokines by senescent cells. Rapamycin reduced IL6 and other cytokine mRNA levels, but selectively suppressed translation of the membrane-bound cytokine IL1A. Reduced IL1A diminished NF-κB transcriptional activity, which controls much of the SASP; exogenous IL1A restored IL6 secretion to rapamycin-treated cells. Importantly, rapamycin suppressed the ability of senescent fibroblasts to stimulate prostate tumour growth in mice. Thus, rapamycin might ameliorate age-related pathologies, including late-life cancer, by suppressing senescence-associated inflammation.

Nuclear receptors (NRs) are central regulators of pathophysiological processes; however, how their responses intertwine is still not fully understood. The aim of this study was to determine whether and how steroid NRs can influence each other’s activity under co-agonist treatment. We used a unique system consisting of a multicopy integration of an estrogen receptor responsive unit that allows direct visualization and quantification of estrogen receptor alpha (ERα) DNA binding, co-regulator recruitment and transcriptional readout. We find that ERα DNA loading is required for other type I nuclear receptors to be co-recruited after dual agonist treatment. We focused on ERα/glucocorticoid receptor interplay and demonstrated that it requires steroid receptor coactivators (SRC-2, SRC-3) and the mediator component MED14. We then validated this cooperative interplay on endogenous target genes in breast cancer cells. Taken together, this work highlights another layer of mechanistic complexity through which NRs cross-talk with each other on chromatin under multiple hormonal stimuli.

Inflammation underlies most age-related diseases, including cancer, but the etiology is poorly understood. One proposed factor is the presence of senescent cells, which increase with age. The senescence response arrests the proliferation of potentially oncogenic cells, and most senescent cells secrete high levels of proinflammatory cytokines and other proteins. The complex senescence-associated secretory phenotype is likely regulated at multiple levels, most of which are unknown. We show that cell surface-bound IL-1alpha is essential for signaling the senescence-associated secretion of IL-6 and IL-8, 2 proinflammatory cytokines that also reinforce the senescence growth arrest. Senescent human fibroblasts expressed high levels of IL-1alpha mRNA, intracellular protein, and cell surface-associated protein, but secreted very little protein. An IL-1 receptor (IL1R) antagonist, neutralizing IL-1alpha antibodies, and IL-1alpha depletion by RNA interference all markedly reduced senescence-associated IL-6/IL-8 secretion. Depletion of the key IL-1R signaling component IRAK1 also suppressed this secretion, and IL-1alpha neutralizing antibodies prevented IRAK1 degradation, indicating engagement of the IL-1R signaling pathway. Furthermore, IL-1alpha depletion reduced the DNA binding activity of NF-kappaB and C/EBPbeta, which stimulate IL-6/IL-8 transcription. IL-1alpha was a general regulator of senescence-associated IL-6/IL-8 secretion because IL-1alpha blockade reduced IL-6/IL-8 secretion whether cells senesced owing to DNA damage, replicative exhaustion, oncogenic RAS, or chromatin relaxation. Furthermore, conditioned medium from IL-1alpha-depleted senescent cells markedly reduced the IL-6/IL-8-dependent invasiveness of metastatic cancer cells, indicating that IL-1alpha regulates the biological effects of these cytokines. Thus, cell surface IL-1alpha is an essential cell-autonomous regulator of the senescence-associated IL-6/IL-8 cytokine network.

RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the detection and localization of RNA, is becoming an increasingly important complement to other gene expression analysis methods. Especially important for long noncoding RNAs (lncRNAs), RNA FISH adds the ability to distinguish between primary and mature lncRNA transcripts and thus to segregate the site of synthesis from the site of action.We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple primary and mature mRNA and lncRNA gene products and RNA variants in fixed mammalian cells. The technique makes use of fluorescently pre-labeled, short DNA oligonucleotides (circa 20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in fluorescent signals that reveal clusters of RNAs or single RNA molecules as punctate spots without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the counting of single transcripts down to one copy per cell. Additionally, by using probe sets with spectrally distinct fluorophores, multiplex analysis of gene-specific RNAs, or RNA variants, can be achieved. The presented examples illustrate how this method can add temporospatial information between the transcription event and both the location and the endurance of the mature lncRNA. We also briefly discuss post-processing of images and spot counting to demonstrate the capabilities of this method for the statistical analysis of RNA molecules per cell. This information can be utilized to determine both overall gene expression levels and cell-to-cell gene expression variation.

Nuclear pores span the nuclear envelope and act as gated aqueous channels to regulate the transport of macromolecules between the nucleus and cytoplasm, from individual proteins and RNAs to entire viral genomes. By far the largest subunit of the nuclear pore is the Nup107–160 complex, which consists of nine proteins and is critical for nuclear pore assembly. At mitosis, the Nup107–160 complex localizes to kinetochores, suggesting that it may also function in chromosome segregation. To investigate the dual roles of the Nup107–160 complex at the pore and during mitosis, we set out to identify binding partners by immunoprecipitation from both interphase and mitotic Xenopus egg extracts and mass spectrometry. ELYS, a putative transcription factor, was discovered to copurify with the Nup107–160 complex in Xenopus interphase extracts, Xenopus mitotic extracts, and human cell extracts. Indeed, a large fraction of ELYS localizes to the nuclear pore complexes of HeLa cells. Importantly, depletion of ELYS by RNAi leads to severe disruption of nuclear pores in the nuclear envelope, whereas lamin, Ran, and tubulin staining appear normal. At mitosis, ELYS targets to kinetochores, and RNAi depletion from HeLa cells leads to an increase in cytokinesis defects. Thus, we have identified an unexpected member of the nuclear pore and kinetochore that functions in both pore assembly at the nucleus and faithful cell division.

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export. However, these altered pores all still possessed the bulk of pore structure. Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components. The resulting reconstituted nuclei are severely defective for NLS import and DNA replication. Strikingly, they show a profound defect for every tested nucleoporin. Even the integral membrane proteins POM121 and gp210 are absent or unorganized. Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores. We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.

Summary The Escherichia coli cydAB operon, encoding the subunits of the high-affinity cytochrome d oxidase, is maximally transcribed in microaerobiosis as a result of the combined action of the oxygen-responsive regulators Fnr and ArcA. Here, we report that the histone-like protein H-NS is an aerobic repressor of cydAB expression. ArcA is shown to antagonize HNS action to render cydAB expression insensitive to H-NS repression in anaerobiosis. The targets for HNS-mediated aerobic repression are the four oxygenregulated promoters, designated P1, P2, P3 and P4. H-NS control is the result of H-NS binding to an extended region within the cydAB promoter element, including sequences upstream from and overlapping the four regulated promoters. We propose a regulatory model in which oxygen control of cydAB transcription is mediated by three alternative protein‐DNA complexes that are assembled sequentially on the promoter region as the cells are shifted from aerobic to microaerobic and to anaerobic conditions. According to this model, ArcA-P plays a central role in cydAB regulation by antagonizing H-NS repression of cydAB transcription when oxygen becomes limiting. This allows peak gene expression and subsequent repression by Fnr under fully anaerobic conditions.

RNA fluorescence in situ hybridization (FISH) has long been an indispensable tool for the detection and localization of RNA and is increasingly becoming an important complement to other gene expression analysis methods. We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple RNA gene products and RNA variants in fixed mammalian cells. The technique utilizes fluorescently pre-labeled, short DNA oligonucleotides (20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in punctate fluorescent signals representing individual RNA molecules without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the quantification of single RNA transcripts. Additionally, by utilizing probe sets with spectrally distinct fluorophores, multiplex analysis of specific RNAs, or RNA variants, can be achieved. We focus on the detection of a cytoplasmic mRNA and a nuclear long noncoding RNA to illustrate the benefits of this method for cell-specific detection and subcellular localization. Post-processing of images and spot counting is briefly discussed to demonstrate the capabilities of this method for the statistical analysis of RNA molecule number per cell, which is information that can be utilized to determine overall gene expression levels and cell-to-cell gene expression variation.

Although mammalian long non-coding (lnc)RNAs are best known for modulating transcription, their post-transcriptional influence on mRNA splicing, stability and translation is emerging. Here we report a post-translational function for the lncRNA HOTAIR as an inducer of ubiquitin-mediated proteolysis. HOTAIR associates with E3 ubiquitin ligases bearing RNA-binding domains, Dzip3 and Mex3b, as well as with their respective ubiquitination substrates, Ataxin-1 and Snurportin-1. In this manner, HOTAIR facilitates the ubiquitination of Ataxin-1 by Dzip3 and Snurportin-1 by Mex3b in cells and in vitro, and accelerates their degradation. HOTAIR levels are highly upregulated in senescent cells, causing rapid decay of targets Ataxin-1 and Snurportin-1, and preventing premature senescence. These results uncover a role for a lncRNA, HOTAIR, as a platform for protein ubiquitination.

The polyamines, putrescine, spermidine, and spermine, are essential polycations, intimately involved in the regulation of cellular proliferation. Although polyamines exert dynamic effects on the conformation of nucleic acids and macromolecular synthesis in vitro, their specific functions in vivo are poorly understood. We investigated the cellular function of polyamines by overexpression of a key catabolic enzyme, spermidine/spermine N 1 -acetyltransferase 1 (SAT1) in mammalian cells. Transient cotransfection of HeLa cells with GFP and SAT1 vectors suppressed GFP protein expression without lowering its mRNA level, an indication that the block in GFP expression was not at transcription, but at translation. Fluorescence single-cell imaging also revealed specific inhibition of endogenous protein synthesis in the SAT1 overexpressing cells, without any inhibition of synthesis of DNA or RNA. Overexpression of SAT1 using a SAT1 adenovirus led to rapid depletion of cellular spermidine and spermine, total inhibition of protein synthesis, and growth arrest within 24 h. The SAT1 effect is most likely due to depletion of spermidine and spermine, because stable polyamine analogs that are not substrates for SAT1 restored GFP and endogenous protein synthesis. Loss of polysomes with increased 80S monosomes in the polyamine-depleted cells suggests a direct role for polyamines in translation initiation. Our data provide strong evidence for a primary function of polyamines, spermidine and spermine, in translation in mammalian cells.

Senescence is a cellular program that irreversibly arrests the proliferation of damaged cells and induces the secretion of the inflammatory mediators IL- 6 and IL-8 which are part of a larger senescence associated secretory phenotype (SASP). We screened quiescent and senescent human fibroblasts for differentially expressed microRNAS (miRNAs) and found that miRNAs 146a and 146b (miR-146a/b) were significantly elevated during senescence. We suggest that delayed miR-146a/b induction might be a compensatory response to restrain inflammation. Indeed, ectopic expression of miR-146a/b in primary human fibroblasts suppressed IL-6 and IL-8 secretion and downregulated IRAK1, a crucial component of the IL-1 receptor signal transduction pathway. Cells undergoing senescence without induction of a robust SASP did not express miR-146a/b. Further, IL-1alpha neutralizing antibodies abolished both miR-146a/b expression and IL-6 secretion. Our findings expand the biological contexts in which miRNA-146a/b modulates inflammatory responses. They suggest that IL-1 receptor signaling initiates both miR-146a/b upregulation and cytokine secretion, and that miR-146a/b is expressed in response to rising inflammatory cytokine levels as part of a negative feedback loop that restrains excessive SASP activity.

Chronic inflammation is associated with aging and plays a causative role in several age-related diseases such as cancer, atherosclerosis and osteoarthritis. The source of this chronic inflammation is often attributed to the progressive activation of immune cells over time. However, recent studies have shown that the process of cellular senescence, a tumor suppressive stress response that is also associated with aging, entails a striking increase in the secretion of pro-inflammatory proteins and might be an important additional contributor to chronic inflammation. Here, we list the secreted factors that make up the pro-inflammatory phenotype of senescent cells and describe the impact of these factors on tissue homeostasis. We also summarize the cellular pathways/processes that are known to regulate this phenotype – namely, the DNA damage response, microRNAs, key transcription factors and kinases and chromatin remodeling.

Stellaris ™ fluorescence in situ hybridization (FISH) probes: a powerful tool for mRNA detection

RNA biomarkers are discovered and validated through advanced transcriptome analysis at a fast pace. These markers that allow the interrogation of the active genes hold promise for more precise staging, prognosis, and treatment of cancer. Long RNA markers comprise pre-mRNAs found in the cell nucleus, long non-coding RNAs (lncRNAs) that may be found in both the nucleus and the cytoplasm, as well as mature and mostly cytoplasmic mRNAs. Importantly, comparative studies in mouse have demonstrated that transcript levels are more strongly correlated with clinical traits than the corresponding protein levels. Because lncRNA and mRNA biomarkers have the potential to contribute to the development of targeted cancer therapies, there is an obvious need for a robust and dependable methodology to reliably detect and validate these RNA targets. RNA fluorescence in situ hybridization (RNA FISH) provides a powerful means to detect specific RNAs in single cells, while still maintaining tissue morphology. Significant advances in RNA FISH technology, such as the hybridization of multiple, fluorescently labeled 20-mer oligonucleotides to the RNA target, allow for the detection of single RNAs and yields information on the RNA's distinct spatial distribution within tissues and even within cells. In this study, we investigate emerging RNA biomarkers in prostate cancer cell lines and tissue utilizing RNA FISH. We also examine established breast cancer biomarkers such as human epidermal growth factor receptor 2 (HER2 / ERBB2), estrogen receptor α (ER α / ESR1), and progesterone receptor (PR / PGR) in breast cancer tumors and cell lines. The results and tools presented here will contribute to advancing the current capabilities of the detection and treatment of specific cancers, as well as in the continued discovery and development of cancer drug candidates. Citation Format: Hans E. Johansson, Arturo V. Orjalo, Sally R. Coassin, Raymund Yin. Detection and validation of novel RNA cancer biomarkers by single molecule RNA fluorescence in situ hybridization (smRNA FISH). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3399. doi:10.1158/1538-7445.AM2014-3399

The genome is extensively transcribed, yet more than 50 % of RNA transcripts lack protein coding potential. In recent years, long noncoding RNAs (lncRNAs) have been shown to play extensive roles in all the central aspects of gene function including imprinting, epigenetic regulation, transcription, splicing and nuclear/cytoplasmic trafficking, translation and RNA stability, with wide ranging effects on cell cycle and differentiation. Due to the increasingly central role of lncRNAs in gene transcription and disease states, it is important to develop tools to allow direct visualization and quantification of lncRNAs that could be useful both in the basic science arena and in the clinical setting with possible uses both in diagnostics and prognostics. We recently streamlined both the design and synthesis of fluorescently labeled probe sets containing multiple tiled 20-mer GC-balanced oligonucleotides for use in RNA fluorescence in situ hybridization (RNA FISH). In contrast to traditional cell-disruptive RNA analyses (e.g. northern blotting and qPCR) that report relative population averages of RNA content, single molecule RNA FISH affords specific and sensitive detection of RNAs and quantitatively establishes the intracellular localization of the target lncRNAs. We have validated an imaging based platform to perform RNA FISH in both manual (coverslip) and automated (384 well) formats that allows tremendous multiplexing and throughput using high throughput microscopy. Moreover, we developed high content analysis routines that permit quantification of RNA particles at the single cell level and determine their localization and pattern (texture). As specific lncRNAs (e.g. MALAT1) are known to target specific intranuclear domains (e.g. splicing speckles) we developed classification methods based upon sets of intensity and texture features to compare intracellular patterns of novel/less characterized ncRNAs. Moreover, this framework will also permit to address changes in lncRNA expression and patterns during the cell cycle since large number of cells can be analyzed simultaneously. We are now expanding our analysis to primary tumor tissues where lncRNAs patterns will be correlated with known FISH-compatible biomarkers, thus providing a picture simultaneously for intra- and inter-cell variation. In summary, the results and tools presented here will enable the study of disease related lncRNAs in a high content, high throughput mode. These efforts will enable gaining better understanding of how expression and localization of lncRNAs may contribute to the diseased phenotype, and add a large selection of novel biomarkers that can be interrogated for improved diagnosis, prognosis, and therapeutic choice. Citation Format: Hans E. Johansson, Arturo V. Orjalo, Sally R. Coassin, Jerry. L. Ruth, Fabio Stossi, Michael A. Mancini. High-throughput imaging and high content analysis of disease relevant lncRNAs examined by RNA fluorescence in situ hybridization (RNA FISH). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1940. doi:10.1158/1538-7445.AM2013-1940

The fate of any given mRNA has remained difficult to experimentally follow and has left mechanisms of unexpected gene expression phenomena in the dark. Examples are differential stability of mRNA expression from different promoters, and inhibition of expression in trans from co-transfected plasmids encoding N1-spermidine/spermine, acetyltransferase and a second unrelated gene. Recent advances in single molecule fluorescence in situ hybridization (smRNA FISH) now bring such central aspects of cellular mRNA metabolism into view. Streamlining both the design and synthesis of probe sets of multiple tiled singly labeled 20-mer oligonucleotides with balanced GC content have made most mRNAs targetable. In contrast to traditional cell-disruptive RNA analyses - northern blotting, QPCR, and microarrays - that yield average and relative information on the content of the probed mRNA, smRNA FISH affords discrete and cell-specific copy numbers. That added spatial dimension can be applied to provide cell-to-cell variability and the actual range of mRNA numbers in a given cell within the field of focus, or to examine defined subsets of cells. The hypusine pathway genes (EIF5A1, EIF5A2, DHPS, DOHH, and SAT1) are essential with deep evolutionary conservation. The gene products are tightly linked to the growth of the cell with aberrant expression in cells that have lost proliferation control, and in several cancers. SAT1, encoding N1-spermidine/spermine, acetyl-transferase, in particular plays a pivotal function in regulating polyamine levels and in turn determining the cells’ proliferative state. Recently, vectors episomally expressing SAT1 were found to inhibit the expression from co-transfected plasmids. Inhibition only occurred with enzymatically active SAT1. To gain a better understanding of how the expression of the hypusine pathway genes may contribute to the transformed phenotype, the fate of these mRNAs and those of unrelated reporter genes was examined by smRNA FISH. Single cell expression data for the hypusine pathway genes is presented and compared with previous results from northern analyses. Data is also presented on the further use of the probe sets to examine changes in the numerical and spatial expression in cells after manipulation of the hypusine status. Lastly, data on the expression - mRNA and protein - in cells co-transfected with vectors for SAT1 and unrelated reporter genes is presented, distinguishing between post-transcriptional events that may cause the inhibition. In summary, we show smRNA FISH as a suitable tool to disseminate post-transcriptional events for genes relevant to polyamine homeostasis and regulated protein synthesis in normal and aberrant cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4209. doi:1538-7445.AM2012-4209

An attractive therapeutic approach against cancers with wild-type (wt) p53 is the emergent class of small molecule MDM2 inhibitors, including the imidazoline nutlin-3 and more potent spiro-oxindole MI-63 (Ascenta Therapeutics). Both can rapidly increase intracellular p53wt levels and induce apoptosis or a senescence growth arrest, depending on the neoplastic status of the target cell. A critical question in the clinical development of such MDM2 inhibitors is their potential for normal tissue toxicity. In the absence of MDM2, p53 activation can cause fatal normal tissue pathology; and MDM2 inhibition can induce p53-dependent cell senescence in mouse fibroblasts. To assess the effects of MI-63 effects in human cells, we compared treatment (2.5-10 uM, 1-7 d) responses in p53wt malignant human mammary epithelial cells (MCF7) with those of p53wt normal human foreskin fibroblasts (HCA2). As expected, MI-63 had little effect on p53mut human cancer cells (MDA-231). However, against p53wt cancer cells and fibroblasts, MI-63 produced a dose-dependent increase in both nuclear p53 levels and reporter gene (p21) expression within 24 h. MCF7 cells responded with a dose- and time-dependent increase in apoptosis after 2 d exposure (vital dye uptake, cleaved PARP). By contrast, HCA2 cells responded with partially reversible growth arrest and a senescent-like phenotype (increased ROS, SOD2, and SA-βgal) within 3 days. A common early (24 h) MI-63 response by both the malignant and normal cells was nuclear loss of high-mobility group box-1 protein (HMGB1) associated with its active extracellular secretion. Acetylation of this damage-reporting cytokine, thought to be required for its secretion, was detected by western and mass spectrometry analyses. Although HMGB1 is generally pro-inflammatory and may be a harbinger of the senescence-associated secretory phenotype (SASP), MI-63 treated fibroblasts failed to exhibit induction of DNA damage foci or other features characteristic of SASP including IL-6 secretion. Surprisingly, MI-63 treatment of HCA2 beginning 24 or 48 h after a senescence-inducing dose of ionizing radiation (10 Gy) prevented enlarged cell morphology and produced a >80% reduction in SASP associated IL-6 secretion. These findings indicate that upregulation of p53wt in proliferating fibroblasts by MI-63, in the absence of DNA damage or other cell stresses, induces an incomplete senescence like growth arrest without a SASP response, consistent with our finding that p53 restrains SASP. In contrast, MI-63 upregulation of p53wt in malignant epithelial cells caused apoptotic cell death. The early release of HMGB1 from these dying cancer cells may modulate host innate immune response and/or recruit tumor infiltrating inflammatory cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4507. doi:10.1158/1538-7445.AM2011-4507