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Tyrosyl DNA phosphodiesterase 1 as a putative target for camptothecin-based antitumor treatment
No full textLe camptotecine sono agenti citotossici con un ampio spettro di attività antitumorale e hanno
un meccanismo d’azione peculiare poiché inibiscono la DNA topoisomerasi IB (Topo I), un
enzima coinvolto nella regolazione di funzioni cellulari fondamentali quali la replicazione del
DNA, la trascrizione, la ricombinazione e la riparazione del danno. La Topo I catalizza
cambiamenti della topologia del DNA attraverso il taglio transiente e la ricucitura dell’acido
nucleico. Tale reazione porta alla formazione di un complesso covalente enzima-DNA, il
“cleavable complex” attraverso il gruppo idrossile di una tirosina dell’enzima, legato
covalentemente all’estremità 3’ del DNA. Le camptotecine avvelenano la Topo I stabilizzando
il complesso DNA-enzima, evento che – in cellule tumorali proliferanti - causa un arresto
della replicazione e la formazione di rotture irreversibili nel DNA. Per la natura del complesso
DNA-Topo I, la tirosil DNA fosfodiesterasi 1 (TDP1), che catalizza l’idrolisi del legame tra il
residuo tirosilico e il gruppo 3’ fosfato è coinvolta nella riparazione dei complessi covalenti
Topo I-DNA e rappresenta un potenziale bersaglio di modulazione dell’attività delle
camptotecine.
Sulla base di queste premesse, lo scopo di questo studio era la definizione del potenziale
interesse di TDP1 quale bersaglio nella terapia dei tumori, con particolare riferimento al suo
ruolo nella resistenza cellulare a gimatecan, una camptotecina attualmente in sperimentazione
clinica. Poiché in linee cellulari di carcinoma ovarico selezionate nel nostro laboratorio per
resistenza a gimatecan (IGROV-1CPT/H and IGROV-1CPT/L), la resistenza era associata ad
aumentati livelli di TDP1, il significato di questo fattore è stato inizialmente esplorato in tali
modelli cellulari. Le varianti resistenti avevano un grado di resistenza a gimatecan di circa 6 e
presentavano cross-resistenza ad altre camptotecine (topotecan, SN38) e a inibitori della DNA
topoisomerasi II. Inoltre, le cellule resistenti erano caratterizzate da una maggior capacità di
riparare le rotture a singolo filamento indotte nel DNA da gimatecan e presentavano una
ridotta frequenza di rotture a doppio filamento. Il silenziamento di TDP1 produceva un
aumento delle rotture doppie in cellule resistenti trattate con gimatecan, come dimostrato
mediante immunofluorescenza con anticorpo anti istone fosfo-H2AX.
La linea cellulare di osteosarcoma U2-OS, particolarmente adatta a studi funzionali è stata
successivamente impiegata per studiare se TDP1 potesse costituire un bersaglio di
modulazione dell’efficacia delle camptotecine. L’inattivazione di TDP1 attraverso trasfezione
di “small-interfering RNAs” sintetici o mediante l’espressione stabile di microRNA specifici
non produceva un cambiamento della sensibilità cellulare a gimatecan. Tuttavia,
l’inattivazione simultanea di altri “pathways” coinvolti con TDP1 nella risposta cellulare agli
inibitori di Topo I risultava in una marcata sensibilizzazione a gimatecan e suggeriva una
cooperazione tra XRCC1 - una proteina “scaffold” coinvolta nella riparazione delle rotture al
DNA - e TDP1 nella risposta a bassi livelli di danno indotti dal farmaco.
Inoltre, cellule U2-OS sovra-esprimenti TDP1 presentavano una modesta resistenza a
gimatecan, osservata impiegando il test di inibizione della crescita delle colonie. Questo
comportamento si associava a una diminuzione dei livelli di rotture a singolo filamento dopo
esposizione al farmaco.
Nel complesso, i nostri risultati suggeriscono che TDP1 abbia un ruolo nell’acquisizione della
resistenza cellulare alle camptotecine, come dimostrato dalla sua aumentata espressione in
cellule resistenti e come documentato dai ridotti livelli di danno in cellule che sovraesprimono
TDP1. Tuttavia, TDP1 di per se sembra non essere in grado di conferire elevati
livelli di resistenza, come dimostrato mediante approcci funzionali. Il ruolo della proteina
nella riparazione del danno appare pertanto non essenziale. La cooperazione tra TDP1 ed altri
“pathways” nella regolazione della sensibilità alle camptotecine è l’evento più probabile, ma
rimangono da definire i fattori più critici nella risposta cellulare alle camptotecine ed in
particolare a gimatecan.
In conclusione, TDP1 per se non sembra essere un bersaglio promettente nell’ambito delle
terapie basate sull’impiego di camptotecine. Tuttavia, è prevedibile che l’inibizione di TDP1
in cellule tumorali con difetti in altri “pathways” rilevanti per la riparazione del danno
mediato da Topo I possa favorire la morte cellulare dopo esposizione alle camptotecine.
Poiché i tumori possono avere difetti nei “checkpoint” cellulari, l’inattivazione simultanea di
TDP1 e altri fattori critici potrebbe contribuire a chiarire gli aspetti più rilevanti della
riparazione del danno mediato da Topo I.Camptothecins are cytotoxic agents with a wide spectrum of antitumor activity and are
endowed with a unique mechanism of action because they inhibit DNA topoisomerase IB
(Topo I), an enzyme implicated in crucial cellular processes such as DNA replication,
transcription, recombination and repair. Topo I catalyzes changes in the DNA topology by
transiently breaking and resealing the nucleic acid backbone. The enzyme forms a covalent
bond at the 3’ terminus of the nicked DNA. Such a reaction leads to the formation of a Topo I
covalent complex (i.e cleavable complex) through a tyrosine hydroxyl group of the enzyme.
Camptothecins poison Topo I by stabilizing the DNA-Topo I complex, thereby leading - in
proliferating tumor cells – to replication fork arrest and generation of irreversible DNA strand
breaks. Because of the features of the Topo I-DNA lesions, Tyrosyl DNA phosphodiesterase 1
(TDP1), which catalyses the hydrolysis of 3’ phosphotyrosyl bonds is implicated in repair of
Topo I-DNA covalent complexes and represents a putative target for modulating the activity
of camptothecins.
Thus, the aim of the present study was to define the potential interest of TDP1 as a target in
antitumor therapy with particular reference to its role in cellular resistance to gimatecan, a
camptothecin currently undergoing clinical evaluation. Since in ovarian carcinoma cell lines
selected for resistance to gimatecan (IGROV-1CPT/H and IGROV-1CPT/L), resistance was
associated with increased levels of TDP1, the significance of this factor has been initially
explored in these ovarian carcinoma cell lines. The selected sublines exhibited around 6-fold
resistance to gimatecan, cross-resistance to other camptothecins (topotecan, SN38) and to
DNA topoisomerase II inhibitors. The resistant cells displayed an increased capability to
repair gimatecan-induced single-strand breaks and a reduced amount of double-strand breaks.
TDP1 silencing resulted in an increased amount of gimatecan-induced double-strand breaks,
as assessed by staining of phosphorylated H2AX histone in resistant cells.
The U2-OS cell line that is very suitable for loss and gain of function studies was then
employed to investigate whether TDP1 may be a target for modulating the efficacy of
camptothecins. Knocking down of TDP1 either by transfection of synthetic small interfering
RNAs or by stable expression of exogenous TDP1 tailored miRNAs did not produce a change
in cell sensitivity to gimatecan. However, co-targeting of other pathways cooperating with
TDP1 in cell response to Topo I poisons indicated that XRCC1 – a scaffold protein implicated
in DNA strand break repair - may cooperate with TDP1 at least in the presence of low levels
of DNA damage. Mild resistance to gimatecan was observed in cells transfected with TDP1
cDNA using clonogenic assays, and this feature was associated with a decreased level of
single-strand breaks after drug exposure.
Overall, our findings support that TDP1 participates in cellular resistance to camptothecins as
suggested by increased expression in resistant cells, and as supported by reduced levels of
damage in cells transfected with TDP1 full-lenght cDNA. However, TDP1 alone cannot
account for relevant levels of resistance as documented by loss and gain of function
approaches. Its role in repair of Topo I-mediated DNA lesions appears dispensable. The
cooperation between TDP1 and other pathways in regulating sensitivity to camptothecins is
the most likely event, but critical inter-players of TDP1 in cell response to gimatecan still
need to be identified.
In conclusion, TDP1 per se does not appear to be an attractive target for camptothecin-based
antitumor treatment. However, it is conceivable that inhibition of TDP1 in tumor cells
exhibiting defects in other pathways playing a role in repair of Topo I-mediated damage could
favour cell death after exposure to camptothecins. Since tumor cells may exhibit checkpoint
defects, the interest of co-targeting of TDP1 and other putative co-targets should be pursued
and is expected to further dissect out the most critical aspects of repair of Topo I-mediated
damage
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
The mTOR/4E-BP1/eIF4E signalling pathway as source of cancer drug targets
No full text: The mechanistic/mammalian target of rapamycin (mTOR) is the crucial hub of signalling pathways that regulate essential steps in the cell life cycle. Once incorporated in the mTORC1 complex, mTOR phosphorylates the eukaryotic initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1), which then releases eIF4E. When not bound to 4E-BPs, eIF4E recognizes the mRNA 5'-cap structure and, together with eIF4A and eIF4G, it forms the eIF4F complex that recruits the ribosome on the mRNA. Under normal conditions, the cellular concentration of eIF4E is very low, making eIF4E the limiting factor in the initiation of protein synthesis. The vast majority of cancer types are characterized by the simultaneous deregulation of the mTOR/4E-BP1 signaling pathway and upregulation of eIF4E, which lead to an increased expression of cancer-promoting genes and deregulated cellular growth. Over the last decades, a growing number of selective inhibitors of the mTOR/4E-BP1/eIF4E pathway have been discovered or designed. Several inhibitors with encouraging preclinical results have been tested in clinical trials. This review summarizes the most recent research on drug development against mTOR, 4E-BP1 and eIF4E, describing the design rationale and the available structural and functional data on the most promising compounds
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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