DNA REPAIR PATHWAYS INVOLVED IN THE FORMATION OF ANAPHASE BRIDGES

Chromosomal alterations can arise from numerous events, including errors during cell division or repair of damaged DNA. Of these errors, segregational defects such as anaphase bridges and multipolar spindles play a major role in chromosomal instability, leading to tumorigenesis. Bridges can theoretically be produced by several mechanisms including telomere-telomere fusion, persistence of chromatid cohesion into anaphase or repair of broken DNA ends. DNA damage can induce anaphase bridges following exposure to agents such as hydrogen peroxide or ionizing radiation (IR). Our hypothesis is that while the majority of double strand breaks (DSBs) are repaired, to restore the original chromosome structure, incorrect fusion events also occur leading to bridging and that bridge formation allows cells to bypass the apoptotic pathways that are activated in response to DNA damage. To test this, we set out to determine what pathways the cells use to heal the damage and form bridges. Our data suggest that neither of the two major pathways used by the cell for repair of double strand breaks, homologous recombination (HR) and non-homologous end joining (NHEJ), is required for bridge formation. In fact, the NHEJ pathway seems to play a role in the prevention of bridges. When NHEJ is compromised, the cell appears to use HR to repair the break, resulting in increased anaphase bridge formation. Moreover, intrinsic NHEJ activity of different cell lines appears to be correlated with induction of bridges from DNA damage. Our preliminary data also suggest that cell lines with high levels of bridging are capable of apoptosis, yet further experiments are required to see if blocking bridging can enhance cell death.Multipolar spindles are aberrant mitotic figures when a cell divides into two or more poles, which can lead to uneven segregation of the chromosomes. In our studies, we found that IR treatment can lead to an increase in multipolarity shortly after treatment and changes the distribution of spindle pole components. Initial observations on the splitting of centrosomal proteins following IR treatment are presented

Dual LSD1 and HDAC6 Inhibition Induces Doxorubicin Sensitivity in Acute Myeloid Leukemia Cells

Defects in epigenetic pathways are key drivers of oncogenic cell proliferation. We developed a LSD1/HDAC6 multitargeting inhibitor (iDual), a hydroxamic acid analogue of the clinical candidate LSD1 inhibitor GSK2879552. iDual inhibits both targets with IC50 values of 540, 110, and 290 nM, respectively, against LSD1, HDAC6, and HDAC8. We compared its activity to structurally similar control probes that act by HDAC or LSD1 inhibition alone, as well as an inactive null compound. iDual inhibited the growth of leukemia cell lines at a higher level than GSK2879552 with micromolar IC50 values. Dual engagement with LSD1 and HDAC6 was supported by dose dependent increases in substrate levels, biomarkers, and cellular thermal shift assay. Both histone methylation and acetylation of tubulin were increased, while acetylated histone levels were only mildly affected, indicating selectivity for HDAC6. Downstream gene expression (CD11b, CD86, p21) was also elevated in response to iDual treatment. Remarkably, iDual synergized with doxorubicin, triggering significant levels of apoptosis with a sublethal concentration of the drug. While mechanistic studies did not reveal changes in DNA repair or drug efflux pathways, the expression of AGPAT9, ALOX5, BTG1, HIPK2, IFI44L, and LRP1, previously implicated in doxorubicin sensitivity, was significantly elevated

Solution chemical properties and anticancer potential of 8-hydroxyquinoline hydrazones and their oxidovanadium(IV) complexes

Funding Information: This work was supported by Centro de Química Estrutural, which is financed by national funds from Fundação para a Ciência e Tecnologia (FCT), projects UIDB/00100/2020, UIDP/00100/2020 and LA/P/0056/2020, and Programa Operacional Regional de Lisboa 2020. We also thank project PTDC/QUI-QIN/0586/2020 and N. Ribeiro acknowledges FCT for SFRH/BD/135797/2018 grant. The Portuguese NMR and Mass spectrometry IST-UL are acknowledged for the access to the equipment. This work was supported by the Portuguese-Hungarian Scientific & Technological CooperationTÉT-PT-2018-00002, ÚNKP-21-3-SZTE-455 (to V. Pósa) New National Excellence Program Ministry of Human Capacities. The support of the ‘Lendület’ Programme (ELKH, LP2019-6/2019) and the COST ActionCA18202, NECTAR-Network for Equilibria and Chemical Thermodynamics Advanced Research is also acknowledged. This work was also supported by Koç University School of Medicine (KUSOM) and the authors gratefully acknowledge use of the services and facilities of the Koç University Research Center for Translational Medicine (KUTTAM), funded by the Presidency of Turkey, Presidency of Strategy and Budget. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Presidency of Strategy and Budget. G. Sciortino, V. Ugone, E. Garribba thank Fondazione di Sardegna (grant FdSGarribba2017) and Regione Autonoma della Sardegna (grant RASSR79857); G. Sciortino also thanks MICINN’ Juan de la Cierva program, FJC2019-039135-I for the financial support. L. Maia thanks the Associate Laboratory for Green Chemistry - LAQV, which is financed by national funds from Fundação para a Ciência e a Tecnologia, MCTES (FCT/MCTES; UIDB/50006/2020 and UIDP/50006/2020). Funding Information: This work was supported by Centro de Química Estrutural , which is financed by national funds from Fundação para a Ciência e Tecnologia (FCT), projects UIDB/00100/2020 , UIDP/00100/2020 and LA/P/0056/2020 , and Programa Operacional Regional de Lisboa 2020 . We also thank project PTDC/QUI-QIN/0586/2020 and N. Ribeiro acknowledges FCT for SFRH/BD/135797/2018 grant. The Portuguese NMR and Mass spectrometry IST-UL are acknowledged for the access to the equipment. This work was supported by the Portuguese-Hungarian Scientific & Technological Cooperation TÉT-PT-2018-00002 , ÚNKP-21-3-SZTE-455 (to V. Pósa) New National Excellence Program Ministry of Human Capacities . The support of the ‘Lendület’ Programme (ELKH, LP2019-6/2019 ) and the COST Action CA18202 , NECTAR-Network for Equilibria and Chemical Thermodynamics Advanced Research is also acknowledged. This work was also supported by Koç University School of Medicine (KUSOM) and the authors gratefully acknowledge use of the services and facilities of the Koç University Research Center for Translational Medicine (KUTTAM), funded by the Presidency of Turkey, Presidency of Strategy and Budget. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Presidency of Strategy and Budget. G. Sciortino, V. Ugone, E. Garribba thank Fondazione di Sardegna (grant FdSGarribba2017 ) and Regione Autonoma della Sardegna (grant RASSR79857 ); G. Sciortino also thanks MICINN’ Juan de la Cierva program, FJC2019-039135-I for the financial support. L. Maia thanks the Associate Laboratory for Green Chemistry - LAQV , which is financed by national funds from Fundação para a Ciência e a Tecnologia, MCTES (FCT/MCTES; UIDB/50006/2020 and UIDP/50006/2020 ). Publisher Copyright: © 2022 Elsevier Inc.We report the synthesis and characterization of a family of benzohydrazones (Ln, n = 1–6) derived from 2-carbaldehyde-8-hydroxyquinoline and benzylhydrazides containing different substituents in the para position. Their oxidovanadium(IV) complexes were prepared and compounds with 1:1 and 1:2 metal-to-ligand stoichiometry were obtained. All compounds were characterized by elemental analyses and mass spectrometry as well as FTIR, UV–visible absorption, NMR (ligand precursors) and EPR (complexes) spectroscopies, and by DFT computational methods. Proton dissociation constants, lipophilicity and solubility in aqueous media were determined for all ligand precursors. Complex formation with V(IV)O was evaluated by spectrophotometry for L4 (Me-substituted) and L6 (OH-substituted) and formation constants for mono [VO(HL)]+, [VO(L)] and bis [VO(HL)2], [VO(HL)(L)]−, [VO(L)2]2− complexes were determined. EPR spectroscopy indicates the formation of [VO(HL)]+ and [VO(HL)2], with this latter being the major species at the physiological pH. Noteworthy, the EPR data suggest a different behaviour for L4 and L6, which confirm the results obtained in the solid state. The antiproliferative activity of all compounds was evaluated in malignant melanoma (A-375) and lung (A-549) cancer cells. All complexes show much higher activity on A-375 (IC50 20 μM). Complex 3 (F-substituted) shows the lowest IC50 on both cell lines and lower than cisplatin (in A-375). Studies identified this compound as the one showing the highest increase in Annexin-V staining, caspase activity and induction of double stranded breaks, corroborating the cytotoxicity results. The mechanism of action of the complexes involves reactive oxygen species (ROS) induced DNA damage, and cell death by apoptosis.publishersversionpublishe

Promising anticancer agents based on 8-hydroxyquinoline hydrazone copper(II) complexes

We report the synthesis and characterization of a group of benzoylhydrazones (L n ) derived from 2-carbaldehyde-8-hydroxyquinoline and benzylhydrazides containing distinct para substituents (R = H, Cl, F, CH 3 , OCH 3 , OH and NH 2 , for L 1-7 , respectively; in L 8 isonicotinohydrazide was used instead of benzylhydrazide). Cu(II) complexes were prepared by reaction of each benzoylhydrazone with Cu(II) acetate. All compounds were characterized by elemental analysis and mass spectrometry as well as by FTIR, UV-visible absorption, NMR or electron paramagnetic resonance spectroscopies. Complexes isolated in the solid state ( 1–8 ) are either formulated as [Cu(HL)acetate] (with L 1 and L 4 ) or as [Cu(L n )] 3 ( n = 2, 3, 5, 6, 7 and 8). Single crystal X-ray diffraction studies were done for L 5 and [Cu(L 5 )] 3 , confirming the trinuclear formulation of several complexes. Proton dissociation constants, lipophilicity and solubility were determined for all free ligands by UV-Vis spectrophotometry in 30% (v/v) DMSO/H 2 O. Formation constants were determined for [Cu(LH)], [Cu(L)] and [Cu(LH −1 )] for L = L 1 , L 5 and L 6 , and also [Cu(LH −2 )] for L = L 6 , and binding modes are proposed, [Cu(L)] predominating at physiological pH. The redox properties of complexes formed with L 1 , L 5 and L 6 are investigated by cyclic voltammetry; the formal redox potentials fall in the range of +377 to +395 mV vs. NHE. The binding of the Cu(II)-complexes to bovine serum albumin was evaluated by fluorescence spectroscopy, showing moderate-to-strong interaction and suggesting formation of a ground state complex. The interaction of L 1 , L 3 , L 5 and L 7 , and of the corresponding complexes with calf thymus DNA was evaluated by thermal denaturation. The antiproliferative activity of all compounds was evaluated in malignant melanoma (A-375) and lung (A-549) cancer cells. The complexes show higher activity than the corresponding free ligand, and most complexes are more active than cisplatin. Compounds 1, 3, 5 , and 8 were selected for additional studies: while these complexes induce reactive oxygen species and double-strand breaks in both cancer cells, their ability to induce cell-death by apoptosis varies. Within the set of compounds tested, 8 emerges as the most promising one, presenting low IC 50 values, and high induction of oxidative stress and DNA damage, which eventually lead to high rates of apoptosis

Epigenetic-focused CRISPR/Cas9 screen identifies (absent, small, or homeotic)2-like protein (ASH2L) as a regulator of glioblastoma cell survival

Background: Glioblastoma is the most common and aggressive primary brain tumor with extremely poor prognosis, highlighting an urgent need for developing novel treatment options. Identifying epigenetic vulnerabilities of cancer cells can provide excellent therapeutic intervention points for various types of cancers. Method: In this study, we investigated epigenetic regulators of glioblastoma cell survival through CRISPR/Cas9 based genetic ablation screens using a customized sgRNA library EpiDoKOL, which targets critical functional domains of chromatin modifiers. Results: Screens conducted in multiple cell lines revealed ASH2L, a histone lysine methyltransferase complex subunit, as a major regulator of glioblastoma cell viability. ASH2L depletion led to cell cycle arrest and apoptosis. RNA sequencing and greenCUT&RUN together identified a set of cell cycle regulatory genes, such as TRA2B, BARD1, KIF20B, ARID4A and SMARCC1 that were downregulated upon ASH2L depletion. Mass spectrometry analysis revealed the interaction partners of ASH2L in glioblastoma cell lines as SET1/MLL family members including SETD1A, SETD1B, MLL1 and MLL2. We further showed that glioblastoma cells had a differential dependency on expression of SET1/MLL family members for survival. The growth of ASH2L-depleted glioblastoma cells was markedly slower than controls in orthotopic in vivo models. TCGA analysis showed high ASH2L expression in glioblastoma compared to low grade gliomas and immunohistochemical analysis revealed significant ASH2L expression in glioblastoma tissues, attesting to its clinical relevance. Therefore, high throughput, robust and affordable screens with focused libraries, such as EpiDoKOL, holds great promise to enable rapid discovery of novel epigenetic regulators of cancer cell survival, such as ASH2L. Conclusion: Together, we suggest that targeting ASH2L could serve as a new therapeutic opportunity for glioblastoma

A Glance at the methods for detection of apoptosis qualitatively and quantitatively

While programmed cell death, apoptosis, occurs as a necessary and natural event for multicellular organisms, necrosis is a form of unplanned cell death as a result of pathological or chemical trauma. There are numerous molecular and morphological differences between these two forms of cell death, whose decision is based on the type and dose of the stress. As apoptosis is critical for homeostasis of an organism, i.e. for development to adult stage, disease progression, or response to different stimuli, it is being studied more extensively in the area of basic research and clinics, and the need for quick detection of apoptosis and well established criteria for the discrimination of apoptosis have also gained more popularity. Here, we review our knowledge on the most commonly used methods for both qualitatively and quantitatively measuring apoptosis, including morphological imaging (i.e. through light, fluorescence, phase contrast or electron microscopy), immunohistochemical (i.e. Annexin V-FITC, TUNEL, M30 antigen or caspase 3 detection), biochemical (i.e. DNA-or protein-based electrophoresis or flow cytometry-based methods), immunological (i.e. ELISA), and molecular biology techniques (i.e. array-based techniques) while focusing on the differences for distinction between the two forms of cell death. Indeed, one has to confirm that cell death occurs through apoptosis based on more than one of these protocols depending on the specific purpose of the user

A Glance at the methods for detection of apoptosis qualitatively and quantitatively

While programmed cell death, apoptosis, occurs as a necessary and natural event for multicellular organisms, necrosis is a form of unplanned cell death as a result of pathological or chemical trauma. There are numerous molecular and morphological differences between these two forms of cell death, whose decision is based on the type and dose of the stress. As apoptosis is critical for homeostasis of an organism, i.e. for development to adult stage, disease progression, or response to different stimuli, it is being studied more extensively in the area of basic research and clinics, and the need for quick detection of apoptosis and well established criteria for the discrimination of apoptosis have also gained more popularity. Here, we review our knowledge on the most commonly used methods for both qualitatively and quantitatively measuring apoptosis, including morphological imaging (i.e. through light, fluorescence, phase contrast or electron microscopy), immunohistochemical (i.e. Annexin V-FITC, TUNEL, M30 antigen or caspase 3 detection), biochemical (i.e. DNA-or protein-based electrophoresis or flow cytometry-based methods), immunological (i.e. ELISA), and molecular biology techniques (i.e. array-based techniques) while focusing on the differences for distinction between the two forms of cell death. Indeed, one has to confirm that cell death occurs through apoptosis based on more than one of these protocols depending on the specific purpose of the user

Prolonged overexpression of PLK4 leads to formation of centriole rosette clusters that are connected via canonical centrosome linker proteins

Abstract Centrosome amplification is a hallmark of cancer and PLK4 is one of the responsible factors for cancer associated centrosome amplification. Increased PLK4 levels was also shown to contribute to generation of cells with centriole amplification in mammalian tissues as olfactory neuron progenitor cells. PLK4 overexpression generates centriole rosette (CR) structures which harbor more than two centrioles each. Long term PLK4 overexpression results with centrosome amplification, but the maturation of amplified centrioles in CRs and linking of PLK4 induced amplified centrosomes has not yet been investigated in detail. Here, we show evidence for generation of large clustered centrosomes which have more than 2 centriole rosettes and define these structures as centriole rosette clusters (CRCs) in cells that have high PLK4 levels for 2 consecutive cell cycles. In addition, we show that PLK4 induced CRs follow normal centrosomal maturation processes and generate CRC structures that are inter-connected with canonical centrosomal linker proteins as C-Nap1, Rootletin and Cep68 in the second cell cycle after PLK4 induction. Increased PLK4 levels in cells with C-Nap1 and Rootletin knock-out resulted with distanced CRs and CRCs in interphase, while Nek2 knock-out inhibited separation of CRCs in prometaphase, providing functional evidence for the binding of CRC structures with centrosomal linker proteins. Taken together, these results suggest a cell cycle dependent model for PLK4 induced centrosome amplification which occurs in 2 consecutive cell cycles: (i) CR state in the first cell cycle, and (ii) CRC state in the second cell cycle

Smooth Muscle Cells Isolated from Thoracic Aortic Aneurysms Exhibit Increased Genomic Damage, but Similar Tendency for Apoptosis

Aortic aneurysms (AA) are characterized by structural deterioration leading to progressive dilation. During the development of AA, two key structural changes are pronounced, one being degradation of extracellular matrix and the other loss of smooth muscle cells (SMCs) through apoptosis. Reactive oxygen species (ROS) are produced above physiological levels in dilated (aneurismal) part of the aorta compared to the nondilated part and they are known to be associated with both the extracellular matrix degradation and the loss of SMCs. In this study, we hypothesized that aneurismal SMCs are more prone to apoptosis and that at least some cells undergo apoptosis due to elevated ROS in the aortic wall. To test this hypothesis, we first isolated SMCs from thoracic aneurismal tissue and compared their apoptotic tendency with normal SMCs in response to H2O2, oxidized sterol, or UV treatment. Exposed cells exhibited morphological changes characteristic of apoptosis, such as cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. Terminal deoxynucleotidyl transferased UTP nick end labeling (TUNEL) further confirmed the fragmentation of nuclear DNA in these cells. Vascular SMCs were analyzed for their micronuclei (MN) and binucleate (BN) frequency as indicators of genomic abnormality. These data were then compared to patient parameters, including age, gender, hypertension, or aortic diameter for existing correlations. While the tendency for apoptosis was not significantly different compared to normal cells, both the %MN and %BN were higher in aneurismal SMCs. The data suggest that there is increased DNA damage in TAA samples, which might play a pivotal role in disease development