Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a major dose-limiting side effect of several anticancer drugs. The prevalence of CIPN ranges from one-third to two-thirds of cancer patients. CIPN can persist for months to years after completion of chemotherapy. Despite the efficacious use of paclitaxel in the treatment of tumors, it can induce many sensory symptoms, such as paresthesia, numbness, tingling and burning pain, and mechanical and cold allodynia, which typically are present in the hands and feet. Similar to other types of chronic pain, paclitaxel-induced CIPN is comorbid with depression and anxiety in cancer survivors, and paclitaxel induces changes in affect-like behavior in cancer-free animal models, suggesting that paclitaxel can cause long-lasting changes in mood, reducing the quality of life. While adjuvant therapies, such as duloxetine, tricyclic antidepressants, and gabapentin are prescribed to treat CIPN symptoms, none of these compounds can consistently reverse or prevent the development of CIPN. With no FDA-approved medication to treat CIPN, the purpose of the dissertation was to: i) characterize and develop a mouse model of paclitaxel-induced CIPN, ii) identify putative targets for CIPN treatment, and iii) test novel compounds for their ability to prevent and reverse CIPN in C57BL/6J mice. In the first Aim, we demonstrate that paclitaxel induces time- and dose-dependent hypersensitivity (mechanical and cold), which is potentiated by combination therapy with the chemotherapeutic carboplatin. In addition, paclitaxel-treated mice show changes in affect-like behaviors (anxiety-like, depression-like). In the second Aim, we used the prototypic nicotinic receptor (nAChR) agonist nicotine to reverse or prevent paclitaxel-induced mechanical hypersensitivity and degeneration of Intra-Epidermal Nerve Fibers (IENFs). Further, we discovered that nicotine’s antinociceptive effects in this mouse model of CIPN are mediated by the nicotinic receptor subtype α7. The third Aim used genetic and pharmacological approaches to dissect the role of α7 on the development and maintenance of paclitaxel-induced CIPN. Null mutant α7 mice (KO) hastens the onset, increases the magnitude, and delays the recovery of paclitaxel-induced mechanical hypersensitivity, as compared to littermate wildtype controls, whereas the selective α7 silent agonist R-47 to reverses and prevents paclitaxel-induced CIPN in C57BL/6J mice. We also examined the impact of R-47 on the paclitaxel-induced reduction of intraepidermal nerve fiber (IENF), as well as microglial morphology in the dorsal horn of the spinal cord. The data show that R-47 prevents paclitaxel-induced changes in microglial morphology and mechanical hypersensitivity behavior, without producing tolerance upon repeated administration. Finally, R-47 induces preference using the conditioned place test in paclitaxel-treated mice but vehicle-treated animals, suggesting that R-47 is a viable candidate for ongoing, spontaneous pain, with limited risk of abuse potential. Overall, these results support that the α7 nAChR subtype is an important target for the treatment and prevention of CIPN.

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 305-320).

published_or_final_version / abstract / Pharmacology / Master / Master of Philosophy

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on January 6, 2009). Research advisor: George C Wood, Ph.D. Document formatted into pages (xii, 126 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 112-126).

Thesis (Ph. D.)--Lehigh University, 1998. / Includes vita. Bibliography: leaves 102-110.

Thesis (Ph. D.)--Ohio State University, 2006. / Available online via OhioLINK's ETD Center; full text release delayed at author's request until 2009 Apr 14

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Οι μικροσωληνίσκοι της μιτωτικής ατράκτου είναι υπεύθυνοι για τον αποχωρισμό των αδελφών χρωματιδίων κατά την Ανάφαση. Τροποποίηση ή καταστολή της δυναμικής του δικτύου των μικροσωληνίσκων συνεπάγεται αναστολή της κυτταρικής διαίρεσης μέσω ενεργοποίησης του σημείου ελέγχου της Μίτωσης. Έτσι, ενώσεις που προσδένονται στο μόριο της β-τουμπουλίνης και σταθεροποιούν τους μικροσωληνίσκους, όπως το paclitaxel και η griseofulvin, ή τους αποσταθεροποιούν, όπως η nocodazole, αποτελούν αποτελεσματικά φάρμακα έναντι του καρκίνου, αλλά και άλλων ασθενειών. Η πρόσδεση των φαρμάκων στους μικροσωληνίσκους έχει ως αποτέλεσμα, την αναστολή της αλληλεπίδρασης μικροσωληνίσκων-κινητοχώρου, βλάβες στη λειτουργία της μιτωτικής ατράκτου και ανώμαλο χρωμοσωματικό αποχωρισμό, με συνέπεια την εμφάνιση ανευπλοειδικών κυττάρων. Στόχος της παρούσας εργασίας είναι η συγκριτική διερεύνηση της ανευπλοειδογόνου δράσης των φαρμακευτικών ενώσεων nocodazole, paclitaxel και griseofulvin σε τρία κυτταρικά συστήματα in vitro: στους μυοβλάστες ποντικού C2C12, στους ινοβλάστες ανθρώπου HFFF2 και στα καρκινικά επιθηλιακά κύτταρα ανθρώπου MCF-7. Η συγκριτική ανάλυση πραγματοποιήθηκε με τη μελέτη επαγωγής μικροπυρήνων και του μηχανισμού προέλευσης τους, μέσω της διπλής ανοσοσήμανσης πρωτεϊνών του κινητοχώρου (CREST) και του δικτύου του κυτταροσκελετού (α- τουμπουλίνη). Ακολούθησε μελέτη της ακεραιότητας της μιτωτικής συσκευής, μέσω συνδυασμένης ανοσοσήμανσης πρωτεϊνών του κεντροσώματος (γ- τουμπουλίνη & Aurora A) και του δικτύου των μικροσωληνίσκων (β- τουμπουλίνη). Τέλος, πραγματοποιήθηκε μελέτη της έκφρασης πρωτεϊνών που συμμετέχουν στο χρωμοσωματικό αποχωρισμό, όπως οι Aurora A, β- και γ-τουμπουλίνη, μέσω της μεθόδου της ανοσοαποτύπωσης (Western Blot Analysis). Με την ολοκλήρωση της μελέτης παρατηρείται ότι και οι τρεις χημικές ενώσεις: επάγουν το φαινόμενο της χρωμοσωματικής καθυστέρησης, κατάσταση που υποδηλώνει την ύπαρξη ολόκληρου χρωμοσώματος, με αύξηση της συχνότητας των μικροπυρήνων με κινητοχώρο στο εσωτερικό τους και στις τρεις κυτταρικές σειρές. Επίσης, προκαλούν αύξηση του ποσοστού πολυπύρηνων μεσοφασικών κυττάρων και στα τρία κυτταρικά συστήματα. Προκαλούν αύξηση του μιτωτικού δείκτη και συσσώρευση των κυττάρων στο στάδιο της Μετάφασης στα κύτταρα C2C12, HFFF2 και MCF-7. Η συσσώρευση αυτή συνοδεύεται με ταυτόχρονη μείωση των Ανατελοφάσεων. Αποδιοργανώνουν, ακόμα, το δίκτυο των μικροσωληνίσκων, τόσο στα μεσοφασικά όσο και στα μιτωτικά κύτταρα. Ειδικότερα, η nocodazole επάγει την αύξηση του ποσοστού μεσοφασικών κυττάρων με πολύ συμπυκνωμένο δίκτυο και κατεστραμμένο (στικτό) γενετικό υλικό στα κύτταρα ποντικού C2C12. Επίσης, και οι τρεις ενώσεις επηρεάζουν τον κεντροσωματικό πολλαπλασιασμό, με την εμφάνιση ανώμαλων Μεταφάσεων και στις τρεις κυτταρικές σειρές. Η nocodazole επάγει το σχηματισμό μονοπολικών Μεταφάσεων, αντίθετα το paclitaxel σχηματίζει πολυπολικές Μεταφάσεις. Η griseofulvin παρουσιάζει κυτταρική εξειδίκευση με την πρόκληση μονοπολικών Μεταφάσεων στα κύτταρα C2C12, πολυπολικών Μεταφάσεων στα καρκινικά κύτταρα MCF-7 και κατά πλειονότητα μη-ομαδοποιημένων Μεταφάσεων στα κύτταρα HFFF2. Οι εν λόγω μελετηθείσες χημικές ενώσεις τροποποιούν επίσης την έκφραση των πρωτεϊνών Aurora A, β- & γ-τουμπουλίνης και στα τρία κυτταρικά συστήματα. Κύριο χαρακτηριστικό της δράσης της nocodazole είναι η μείωση της έκφρασης των πρωτεϊνών Aurora A και β-τουμπουλίνης. Αντίθετα το paclitaxel και η griseofulvin οδηγούν σε υπερέκφραση των Aurora A, β- και γ-τουμπουλίνη. Και οι τρεις φαρμακευτικές ενώσεις παρουσιάζουν κυτταρική εξειδίκευση ως προς τον επηρεασμό της έκφρασης των πρωτεϊνών που μελετήθηκαν. Τα ευρήματα αυτά υποδηλώνουν τη συμμετοχή των τριών πρωτεϊνών στην εκδήλωση της ανευπλοειδογόνου δράσης των τριών χημικών ενώσεων. Τα παραπάνω συμπεράσματα επιβεβαιώνουν και ερμηνεύουν την ανευπλοειδογόνο δράση των φαρμακευτικών ενώσεων, nocodazole, paclitaxel και griseofulvin. / The microtubules of the mitotic apparatus are responsible for the chromosome segregation during Anaphase. Modification or inhibition of the dynamics of the microtubules results to the activation of the mitotic checkpoint. Chemicals that bind at the molecule of β-tubulin and stabilize microtubules, like paclitaxel and griseofulvin or destabilize them, like nocodazole, act as drugs against cancer or other diseases. This binding results to the inhibition of microtubule-kinetochore interactions, damage of the organization of the mitotic spindle, abnormal chromosome segregation and thus the generation of aneuploid cells. In the present study we comparatively investigated the aneugenic potential of three chemical compounds, nocodazole, paclitaxel and griseofulvin in three cell lines in vitro: C2C12 mouse fibroblasts, HFFF2 human myoblasts and MCF-7 human epithelial cancer cells. The comparative analysis was established by three different experimental procedures. The study of micronucleus induction and their generation mechanism was accomplished by CREST analysis in combination with immunostaining of α- tubulin. The ability of the chemicals to affect the organization of mitotic apparatus was investigated by double immunofluorescence of microtubule network (β-tubulin) and centrosomes (γ-tubulin & Aurora A) in all cell lines. Finally, to understand further the mechanisms by which nocodazole, paclitaxel and griseofulvin exert their aneugenic potential, we examined the ability of these compounds to modulate the expression of proteins that participate in chromosome segregation, such as Aurora A, β- and γ-tubulin, by Western blot analysis in C2C12, HFFF2 and MCF-7 cells. Our results revealed that the three chemical compounds:  Induce chromosome delay, showing a high frequency of micronuclei with kinetochore, indicating the presence of intact chromosome in every studied cell line. In addition, all the drugs evoke induction of multinucleated cells in all cell lines.  Increase the mitotic index with simultaneous Metaphase arrest. The cell accumulation at the Metaphase is accompanied with reduction of Anatelophases in all cell lines.  Promote disorganization of the microtubule network. Especially, nocodazole causes induction of abnormal interphase cells with very condensed network (bundled cells) and punctuated genetic material in C2C12 mouse cell line.  Affect the centrosome proliferation, increasing the frequency of abnormal Metaphases in the three cell lines. Nocodazole induces high frequency of monopolar Metaphases, whereas paclitaxel generates polypolar Metaphases. Griseofulvin produces monopolar Metaphases in C2C12 cells, polypolar Metaphases in cancer MCF-7 cells and mainly non-congressed Metaphases in HFFF2 cells, exhibiting cell specificity.  Modify the expression of the proteins Aurora A, β- and γ-tubulin in all cell lines. Nocodazole reduces Aurora’s A expression in C2C12, HFFF2 and MCF-7 cell lines. The same is observed for β- tubulin’s expression, as for C2C12 and MCF-7 cells. Paclitaxel exerts its aneugenic potential by enhancing the expression of Aurora A and γ-tubulin in all cell lines, whereas enhancing β- tubulin’s expression is only noticed in C2C12 and MCF-7 cells. Griseofulvin increases the expression of Aurora A and β-tubulin in human cancer cells MCF-7. As for γ-tubulin’s expression, is elevated only in the human cell lines HFFF2 and MCF-7. Thus, we suggest that the activity of the pharmaceutical compounds on the expression of the above proteins, exhibit cell specificity. These findings implicate that the aneugenic potential of the studied drugs is mediated through changes in the expression of these proteins. The conclusions above confirm and explain the aneugenic potential of the pharmaceutical compounds, nocodazole, paclitaxel and griseofulvin.

Plants produce a wide variety of natural products that can be exploited for medicinal purposes. Paclitaxel is a key anti-cancer drug originally isolated from the bark of Taxus spp. that is currently approved for use in the treatment of breast, lung and non-small cell cancers, AIDS-related Kaposi's sarcoma and coronary artery disease. Worldwide demand for paclitaxel is high and plant cell culture (PCC) is an attractive production route. Cultured cambial meristematic cells (CMCs) provide a good platform from which to increase drug production, as they possess superior growth properties on an industrial scale compared to typical dedifferentiated cell culture. Elicitors, such as methyl-jasmonate (MeJA), can up-regulate paclitaxel production in PCC, however the effect is only transient. Identification and characterisation of the key transcriptional regulators that control MeJA induced metabolic reprogramming can provide potential tools to manipulate Taxus CMC culture to produce more paclitaxel. Roche454 sequencing was employed to establish the basic transcriptomic profile of Taxus cuspidata CMCs, which was then utilized as a reference to observe the transcriptional profile of CMCs at three time points after MeJA elicitation (0.5, 2 and 12 h). Analysis of the transcriptional regulatory network identified 19 transcription factors (TFs) that were significantly up-regulated at an early time point (0.5 h) after elicitation. These TFs came from five families – AP2, MYB, NAC, bHLH and WRKY – that are well known to regulate secondary plant metabolism. An Arabidopsis thaliana transient expression assay (TEA) was employed to investigate the regulatory activity of these 19TFs against 10 paclitaxel biosynthetic promoters. The TEA screen identified 79 significant interactions with every promoter interacting with at least three TFs, which could activate or repress activity. A MYB TF was identified that could up-regulate eight out of the ten promoters tested, indicating it maybe a potential overall regulator of paclitaxel biosynthesis. In vitro electromobility shift assays established the possible binding site for this TF as an AC element, with the consensus sequence of A(A/C)C. Repressors of promoter activity were also identified, for example an AP2 TF which contains the well-established ERF associated amphiphilic repression (EAR) motif. The activity of the EAR domain was explored in vivo using a TEA assay and site directed mutagenesis mutants. Activity was lost when the mutation occurred within the domain suggesting the TF was working as an active repressor. TFs can work individually or in combination to achieve metabolic reprogramming after MeJA elicitation. One of the best characterised examples of plant combinatorial control is between particular sub classes of MYB and bHLH TFs. However investigation into possible interactions between the T. cuspidata MYB and bHLH TFs in vivo using yeast two hybrid and TEAs found few combinations that led to a significant change in regulatory activity. The regulatory activity of WRKY TFs was shown to be post-translationally controlled when the TEAs were treated with MeJA, however the mechanism by which this occurs remains to be elucidated. The interactions identified between the 19 TFs and the paclitaxel biosynthetic promoters can be exploited in the future to produce superior Taxus CMC lines with increased paclitaxel yields.

L'objectif de cette thèse est de proposer des modèles mathématiques permettant de décrire l'instabilité dynamique d'une population de microtubules (MTs) et l'effet de médicaments sur cette instabilité. L'instabilité dynamique des MTs joue un rôle extrêmement important dans les processus de la mitose et de la migration cellulaire et donc dans la progression tumorale. L'instabilité dynamique est un processus complexe qui implique différents états de la tubuline (polymérisée ou non-polymérisée, tubuline-GTP ou tubuline-GDP qui correspondent à deux états énergétiques différents des dimères) et qui résulte de processus chimiques (polymérisation, dépolymérisation, hydrolyse, recyclage, nucléation) liant ces différents états de la tubuline. Décrire cette complexité par le biais de modèles mathématiques permet alors de tester des hypothèses biologiques quant à l'impact de chacun de ces processus et l'action de molécules anti-MTs. De récents travaux suggèrent que le "vieillissement" des MTs impacte leur dynamique. Nous avons testé dans ce travail l'hypothèse que ce "vieillissement" accélère l'hydrolyse du GTP au sein de la tubuline. Nous avons construit de nouveaux modèles couplant deux équations de transport multi-D avec deux équations différentielles ordinaires impliquant des termes intégraux. Nous avons calibrer notre nouveau modèle à partir des données expérimentales; tester l'hypothèse biologique sur le mécanisme du processus de vieillissement; analyser la sensibilité du modèle par rapport aux paramètres décrivant les processus; tester différentes hypothèses quant l'effet des médicaments anti-MTs. / The aim of this thesis is to design new mathematical models that are able to appropriately describe dynamic instability of a population of microtubules (MTs) and effect of drugs on MT dynamics. MT dynamic instability play an important role in the processes of mitosis and cell migration and, thus, in cancer progression. Dynamic instability is a complex process that involves different states of tubulin (polymerized or non-polymerized, GTP-tubulin or GDPtubulin that correspond to two different energetic states of tubulin dimers) that resulted from chemical processes (polymerization, depolymerization, hydrolysis, recycling, nucleation) linking these different states of tubulin. Description of this complexity by mathematical models enables one to test biological hypotheses concerning the impact of each process and action of drugs on microtubule dynamics. Recent observations show that MT dynamics depends on aging of MT. One of the aims of the work is to test the hypothesis that MT aging results from the acceleration of the GTP hydrolysis. We construct for that new models that couple two multidimensional transport equations with two ordinary differential equations involving integral terms. We have calibrated our models on the basis of experimental data; tested biological hypothesis on mechanism of aging process; performed a sensitivity analysis of the model with respect to parameters describing chemical processes; and tested hypotheses concerning actions of drugs.