Androgen Receptor Activation Induces Senescence in Thyroid Cancer Cells.

Thyroid cancer (TC) is the most common endocrine malignancy, with an approximately three-fold higher incidence in women. TCGA data indicate that androgen receptor (AR) RNA is significantly downregulated in PTC. In this study, AR-expressing 8505C (anaplastic TC) (84E7) and K1 (papillary TC) cells experienced an 80% decrease in proliferation over 6 days of exposure to physiological levels of 5α-dihydrotestosterone (DHT). In 84E7, continuous AR activation resulted in G1 growth arrest, accompanied by a flattened, vacuolized cell morphology, with enlargement of the cell and the nuclear area, which is indicative of senescence; this was substantiated by an increase in senescence-associated ß-galactosidase activity, total RNA and protein content, and reactive oxygen species. Additionally, the expression of tumor suppressor proteins p16, p21, and p27 was significantly increased. A non-inflammatory senescence-associated secretory profile was induced, significantly decreasing inflammatory cytokines and chemokines such as IL-6, IL-8, TNF, RANTES, and MCP-1; this is consistent with the lower incidence of thyroid inflammation and cancer in men. Migration increased six-fold, which is consistent with the clinical observation of increased lymph node metastasis in men. Proteolytic invasion potential was not significantly altered, which is consistent with unchanged MMP/TIMP expression. Our studies provide evidence that the induction of senescence is a novel function of AR activation in thyroid cancer cells, and may underlie the protective role of AR activation in the decreased incidence of TC in men.

MicroRNA-133a-Dependent Inhibition of Proximal Tubule Angiotensinogen by Renal TNF (Tumor Necrosis Factor).

We showed that intrarenal suppression of TNF (tumor necrosis factor) production under low salt (LS) conditions increases renal cortical AGT (angiotensinogen) mRNA and protein expression. Intrarenal injection of murine recombinant TNF attenuated increases of AGT in mice ingesting LS. Moreover, AGT mRNA and protein expression increased ≈6-fold and 2-fold, respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6-TNF-ex4). Silencing of TNF under normal salt and high salt (HS) conditions also resulted in increased AGT expression. Since renal TNF production decreases in response to LS and increases in response to HS, the data suggest that alterations in TNF production under these conditions modulate the degree of AGT expression. We also tested the hypothesis that TNF inhibits intrarenal AGT expression by a mechanism involving miR-133a. Expression of miR-133a decreased in mice given LS and increased in response to HS for 7 days. Intrarenal silencing of TNF reversed the effects of HS on miR-133a-dependent AGT expression. In contrast, intrarenal TNF administration increased miR-133a expression in the kidney. Collectively, the data suggest that miR-133a is a salt-sensitive microRNA that inhibits AGT in the kidney and is increased by TNF. The HS-induced increase in blood pressure observed following silencing of TNF was markedly reduced upon intrarenal administration of miR-133a suggesting that intrinsic effects of TNF in the kidney to limit the blood pressure response to HS include an increase in miR-133a, which suppresses AGT expression.

Berberine affects mitochondrial activity and cell growth of leukemic cells from chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (CLL) results from accumulation of leukemic cells that are subject to iterative re-activation cycles and clonal expansion in lymphoid tissues. The effects of the well-tolerated alkaloid Berberine (BRB), used for treating metabolic disorders, were studied on ex-vivo leukemic cells activated in vitro by microenvironment stimuli. BRB decreased expression of survival/proliferation-associated molecules (e.g. Mcl-1/Bcl-xL) and inhibited stimulation-induced cell cycle entry, irrespective of TP53 alterations or chromosomal abnormalities. CLL cells rely on oxidative phosphorylation for their bioenergetics, particularly during the activation process. In this context, BRB triggered mitochondrial dysfunction and aberrant cellular energetic metabolism. Decreased ATP production and NADH recycling, associated with mitochondrial uncoupling, were not compensated by increased lactic fermentation. Antioxidant defenses were affected and could not correct the altered intracellular redox homeostasis. The data thus indicated that the cytotoxic/cytostatic action of BRB at 10-30 µM might be mediated, at least in part, by BRB-induced impairment of oxidative phosphorylation and the associated increment of oxidative damage, with consequent inhibition of cell activation and eventual cell death. Bioenergetics and cell survival were instead unaffected in normal B lymphocytes at the same BRB concentrations. Interestingly, BRB lowered the apoptotic threshold of ABT-199/Venetoclax, a promising BH3-mimetic whose cytotoxic activity is counteracted by high Mcl-1/Bcl-xL expression and increased mitochondrial oxidative phosphorylation. Our results indicate that, while CLL cells are in the process of building their survival and cycling armamentarium, the presence of BRB affects this process.

Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies.

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5' or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5' ends.

40 Years of My Venture with CYTOMETRY.

Briefly depicted are the publications in CYTOMETRY that received the highest frequency of citations. Among them are seminal papers describing application of metachromatic fluorochrome acridine orange to differentially stain DNA versus RNA or to analyze susceptibility of DNA in situ to denaturation; both features being markers of different sections of the cell cycle including identification of noncycling quiescent cells. The papers reviewing detection of cyclins D1, E, A or B1, each in relation to cell cycle phase, were also among the highly cited ones. The highest citation rates received publications describing development of the TUNEL methodology to detect apoptotic DNA fragmentation, and more recently expression of ϒH2AX to reveal DNA damage. © 2020 International Society for Advancement of Cytometry.

Declining BRCA-Mediated DNA Repair in Sperm Aging and its Prevention by Sphingosine-1-Phosphate.

Recent data suggest that paternal age can have major impact on reproductive outcomes, and with increased age, there is increased likelihood of chromosomal abnormalities in the sperm. Here, we studied DNA damage and repair as a function of male aging and assessed whether sphingosine-1-phosphate (S1P), a ceramide-induced death inhibitor, can prevent sperm aging by enhancing DNA double-strand breaks (DSB) repair. We observed a significant increase in DNA damage with age and this increase was associated with a decline in the expression of key DNA DSB repair genes in mouse sperm. The haploinsufficiency of BRCA1 male mice sperm showed significantly increased DNA damage and apoptosis, along with decreased chromatin integrity when compared to similar age wild type (WT) mice. Furthermore, haploinsufficiency of BRCA1 male mice had lower sperm count and smaller litter size when crossed with WT females. The resulting embryos had a higher probability of growth arrest and reduced implantation. S1P treatment decreased genotoxic-stress-induced DNA damage in sperm and enhanced the expressions of key DNA repair genes such as BRCA1. Co-treatment with an ATM inhibitor reversed the effects of S1P, implying that the impact of S1P on DNA repair is via the ATM-mediated pathway. Our findings indicate a key role for DNA damage repair mechanism in the maintenance of sperm integrity and suggest that S1P can improve DNA repair in sperm. Further translational studies are warranted to determine the clinical significance of these findings and whether S1P can delay male reproductive aging. There is mounting evidence that sperm quality declines with age, similar to that of the oocyte. However, the reasons behind this decline are poorly understood and there is no medical intervention to improve sperm quality. Our study suggests a strong role for DNA damage repair in maintenance of sperm quality, and for the first time, a potential pharmaceutical approach to prevent sperm aging.

Kinetic analysis of IFIT1 and IFIT5 interactions with different native and engineered RNAs and its consequences for designing mRNA-based therapeutics.

In response to foreign RNA, cellular antiviral mechanisms stimulate high expression of interferon-induced proteins with tetratricopeptide repeats (IFITs). Two members of the IFIT protein family, IFIT1 and IFIT5, are capable of binding the very terminal 5' end of mRNA. In eukaryotes, these mRNA termini contain a cap structure (m7GpppN, cap 0) that is often subjected to further modifications. Here, we performed a thorough examination of IFIT1 and IFIT5 binding to a wide spectrum of differently capped as well as fully uncapped mRNAs. The kinetic analysis of IFIT1 and IFIT5 interactions with mRNA ligands indicates that the cap structure modifications considerably influence the stability of IFIT1/RNA complexes. The most stable complexes were formed between IFIT1 and GpppG/A- and m7GpppG/A-RNAs. Unexpectedly, we found that NAD+- and NADH-capped RNAs associate with IFIT5 with kinetic parameters comparable to pppG-RNA. Finally, we measured interactions of IFIT1 with mRNAs bearing modified synthetic cap analogs that start to become the important tools in biotechnological and medicinal research. We found that incorporation of modified cap analogs to the RNA protects the latter, to a certain degree, from the translational inhibition caused by IFIT1 protein.

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure.

The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain single- versus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single- versus double-stranded DNA with acridine orange.

Two forms of human DNA polymerase δ: Who does what and why?

DNA polymerase δ (Pol δ) plays a central role in lagging strand DNA synthesis in eukaryotic cells, as well as an important role in DNA repair processes. Human Pol δ4 is a heterotetramer of four subunits, the smallest of which is p12. Pol δ3 is a trimeric form that is generated in vivo by the degradation of the p12 subunit in response to DNA damage, and during entry into S-phase. The biochemical properties of the two forms of Pol δ, as well as the changes in their distribution during the cell cycle, are reviewed from the perspective of understanding their respective cellular functions. Biochemical and cellular studies support a role for Pol δ3 in gap filling during DNA repair, and in Okazaki fragment synthesis during DNA replication. Recent studies of cells in which p12 expression is ablated, and are therefore null for Pol δ4, show that Pol δ4 is not required for cell viability. These cells have a defect in homologous recombination, revealing a specific role for Pol δ4 that cannot be performed by Pol δ3. Pol δ4 activity is required for D-loop displacement synthesis in HR. The reasons why Pol δ4 but not Pol δ3 can perform this function are discussed, as well as the question of whether helicase action is needed for efficient D-loop displacement synthesis. Pol δ4 is largely present in the G1 and G2/M phases of the cell cycle and is low in S phase. This is discussed in relation to the availability of Pol δ4 as an additional layer of regulation for HR activity during cell cycle progression.

Detection of Histone H2AX Phosphorylation on Ser-139 as an Indicator of DNA Damage.

This unit describes immunocytochemical detection of histone H2AX phosphorylated on Ser-139 (γH2AX) to reveal DNA damage, particularly when the damage involves the presence of DNA double-strand breaks (DSBs). These breaks often result from DNA damage induced by ionizing radiation or by treatment with anticancer drugs such as DNA topoisomerase inhibitors. Furthermore, DSBs are generated in the course of DNA fragmentation during apoptosis. The unit presents strategies to distinguish radiation- or drug-induced DNA breaks from those intrinsically formed in untreated cells or associated with apoptosis. The protocol describes immunocytochemical detection of γH2AX combined with measurement of DNA content to identify cells that have DNA damage and concurrently to assess their cell-cycle phase. The detection is based on indirect immunofluorescence using FITC- or Alexa Fluor 488-labeled antibody, with DNA counterstained with propidium iodide and cellular RNA removed with RNase A. © 2019 by John Wiley & Sons, Inc.

Direct Inhibition of MmpL3 by Novel Antitubercular Compounds.

MmpL3, an essential transporter involved in the export of mycolic acids, is the proposed target of a number of antimycobacterial inhibitors under development. Whether MmpL3 serves as the direct target of these compounds, however, has been called into question after the discovery that some of them dissipated the proton motive force from which MmpL transporters derive their energy. Using a combination of in vitro and whole-cell-based approaches, we here provide evidence that five structurally distinct MmpL3 inhibitor series, three of which impact proton motive force in Mycobacterium tuberculosis, directly interact with MmpL3. Medium- to high-throughput assays based on these approaches were developed to facilitate the future screening and optimization of MmpL3 inhibitors. The promiscuity of MmpL3 as a drug target and the mechanisms through which missense mutations located in a transmembrane region of this transporter may confer cross-resistance to a variety of chemical scaffolds are discussed in light of the exquisite vulnerability of MmpL3, its apparent mechanisms of interaction with inhibitors, and evidence of conformational changes induced both by the inhibitors and one of the most commonly identified resistance mutations in MmpL3.

Identification of Binding Sites for Efflux Pump Inhibitors of the AcrAB-TolC Component AcrA.

The overexpression of multidrug efflux pumps is an important mechanism of clinical resistance in Gram-negative bacteria. Recently, four small molecules were discovered that inhibit efflux in Escherichia coli and interact with the AcrAB-TolC efflux pump component AcrA. However, the binding site(s) for these molecules was not determined. Here, we combine ensemble docking and molecular dynamics simulations with tryptophan fluorescence spectroscopy, site-directed mutagenesis, and antibiotic susceptibility assays to probe binding sites and effects of binding of these molecules. We conclude that clorobiocin and SLU-258 likely bind at a site located between the lipoyl and ß-barrel domains of AcrA.

Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs.

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F>640 ) within these organelles. Activation of TG2 was detected in the same cell subpopulation by the evidence of crosslinking of cytoplasmic proteins revealed by the increased intensity of the side light scatter (SSC) as well as following cell lysis by detergent, by its red fluorescence after staining by sulforhodamine 101. Because at low AO concentration nuclear DNA of the lysed cells was stoichiometrically stained green (F530 ) its quantity provided information on effects of the drug treatments on cell cycle in relation to activation of TG2. The data reveal that activation of lysosomal proton pump was evident in subpopulations of cells treated with 2-deoxyglucose plus berberine, topotecan, etoposide and mitoxantrone but not with ranpirnase. The collapse of lysosomal proton pump possibly reporting rupture of these organelles was observed in definite cell subpopulations after treatment with each of the studied drugs. Because regardless of the inducer of apoptosis TG2 activation invariably was correlated with lysosomes rupture it is likely that it was triggered by calcium ions or protons released from the ruptured lysosomes. This new methodological approach offers the means to investigate mechanisms and factors affecting autophagic lysosomes proton pump activity vis-à-vis TG2 activation that are common in several pathological states. © 2019 International Society for Advancement of Cytometry.

Discovery of a novel DNA polymerase inhibitor and characterization of its antiproliferative properties.

Chromosomal duplication is targeted by various chemotherapeutic agents for the treatment of cancer. However, there is no specific inhibitor of DNA polymerases that is viable for cancer management. Through structure-based in silico screening of the ZINC database, we identified a specific inhibitor of DNA polymerase δ. The discovered inhibitor, Zelpolib, is projected to bind to the active site of Pol δ when it is actively engaged in DNA replication through interactions with DNA template and primer. Zelpolib shows robust inhibition of Pol δ activity in reconstituted DNA replication assays. Under cellular conditions, Zelpolib is taken up readily by cancer cells and inhibits DNA replication in assays to assess global DNA synthesis or single-molecule bases by DNA fiber fluorography. In addition, we show that Zelpolib displays superior antiproliferative properties to methotrexate, 5-flourouracil, and cisplatin in triple-negative breast cancer cell line, pancreatic cancer cell line and platinum-resistant pancreatic cancer cell line. Pol δ is not only involved in DNA replication, it is also a key component in many DNA repair pathways. Pol δ is the key enzyme responsible for D-loop extension during homologous recombination. Indeed, Zelpolib shows robust inhibition of homologous recombination repair of DNA double-strand breaks and induces "BRCAness" in HR-proficient cancer cells and enhances their sensitivity to PARP inhibitors.

Upregulation of PD­L1 expression by resveratrol and piceatannol in breast and colorectal cancer cells occurs via HDAC3/p300­mediated NF­κB signaling.

Programmed cell death ligand 1 (PD­L1) expressed in cancer cells interacting with its receptor programmed cell death 1 (PD­1) expressed in immune cells represents a regulatory axis linked to the suppression and evasion of host immune functions. The blockade of PD­1/PD­L1 interaction using monoclonal antibodies has emerged as an effective therapy for several solid tumors; however, durable response has been observed in a subset of patients with PD­L1-positive tumors. Thus, the understanding of the mechanisms responsible for the expression of PD­L1 in tumor cells may help to improve the response to PD­L1 blockade therapies. In this study, we investigated whether resveratrol, a grape-derived stilbenoid with immunoregulatory activity, modulates the expression of PD­L1 in breast and colorectal cancer cells. The surface expression of PD­L1 was determined by flow cytometry in cancer cells treated with resveratrol and/or piceatannol. Each stilbenoid alone induced PD­L1 and when used in combination, elicited a synergistic upregulation of PD­L1 in some cell lines. The induction of PD­L1 by the combined use of stilbenoids was most pronounced in the Cal51 triple-negative breast cancer (TNBC) and SW620 colon cancer cells. The observed induction of PD­L1 was transcriptionally mediated by nuclear factor (NF)-κB, as shown by NF­κB reporter assays, the nuclear accumulation of the p65 subunit of NF­κB, inhibition by the IKK inhibitor, BMS­345541, and histone the modification inhibitors, resminostat, entinostat or anacardic acid. Combined treatment with resveratrol and piceatannol also decreased tumor cell survival as indicated by the upregulation of the DNA damaging marker, γH2AX, the cleavage of caspase 3, the downregulation of the survival markers, p38-MAPK/c­Myc, and G1-to-S cell cycle arrest.

Nuclear cytometry and chromatin organization.

The nuclear-targeting chemical probe, for the detection and quantification of DNA within cells, has been a mainstay of cytometry-from the colorimetric Feulgen stain to smart fluorescent agents with tuned functionality. The level of nuclear structure and function at which the probe aims to readout, or indeed at which a DNA-targeted drug acts, is shadowed by a wide range of detection modalities and analytical methods. These methods are invariably limited in terms of the resolution attainable versus the volume occupied by targeted chromatin structures. The scalar challenge arises from the need to understand the extent and different levels of compaction of genomic DNA and how such structures can be re-modeled, reported, or even perturbed by both probes and drugs. Nuclear cytometry can report on the complex levels of chromatin order, disorder, disassembly, and even active disruption by probes and drugs. Nuclear probes can report defining features of clinical and therapeutic interest as in NETosis and other cell death processes. New cytometric approaches continue to bridge the scalar challenges of analyzing chromatin organization. Advances in super-resolution microscopy address the resolution and depth of analysis issues in cellular systems. Typical of recent insights into chromatin organization enabled by exploiting a DNA interacting probe is ChromEM tomography (ChromEMT). ChromEMT uses the unique properties of the anthraquinone-based cytometric dye DRAQ5™ to reveal that local and global 3D chromatin structures effect differences in compaction. The focus of this review is nuclear and chromatin cytometry, with linked reference to DNA targeting probes and drugs as exemplified by the anthracenediones.

Analysis of Cellular DNA Content by Flow Cytometry.

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.

Analysis of Cellular DNA Content by Flow Cytometry.

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc.

Nuclear Gene 33/Mig6 regulates the DNA damage response through an ATM serine/threonine kinase-dependent mechanism.

Gene 33 (Mig6, ERRFI1) is an adaptor protein with multiple cellular functions. We recently linked Gene 33 to the DNA damage response (DDR) induced by hexavalent chromium (Cr(VI)), but the molecular mechanism remains unknown. Here we show that ectopic expression of Gene 33 triggers DDR in an ATM serine/threonine kinase (ATM)-dependent fashion and through pathways dependent or not dependent on ABL proto-oncogene 1 non-receptor tyrosine kinase (c-Abl). We observed the clear presence of Gene 33 in the nucleus and chromatin fractions of the cell. We also found that the nuclear localization of Gene 33 is regulated by its 14-3-3-binding domain and that the chromatin localization of Gene 33 is partially dependent on its ErbB-binding domain. Our data further indicated that Gene 33 may regulate the targeting of c-Abl to chromatin. Moreover, we observed a clear association of Gene 33 with histone H2AX and that ectopic expression of Gene 33 promotes the interaction between ATM and histone H2AX without triggering DNA damage. In summary, our results reveal nuclear functions of Gene 33 that regulate DDR. The nuclear localization of Gene 33 also provides a spatial explanation of the previously reported regulation of apoptosis by Gene 33 via the c-Abl/p73 pathway. On the basis of these findings and our previous studies, we propose that Gene 33 is a proximal regulator of DDR that promotes DNA repair.