No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Vincristine is metabolized by the cytochrome P450 3A subfamily of enzymes possibly including CYP3A5, a genetically polymorphic enzyme. The contribution of CYP3A5 to the metabolism of vincristine was quantified by various in vitro models: cDNA-expressed enzymes, human liver microsomes, and human hepatocytes. With these models, the major CYP metabolite of vincristine, M1, was identified and extensively characterized. The rates of M1 formation in the cDNA-expressed enzyme models were at least 7-fold higher with CYP3A5 than CYP3A4; approximately 90% of the hepatic metabolism was predicted to be CYP3A5-mediated. For human liver microsomes with high CYP3A5 expression, the CYP3A5 contribution was substantial, approximately 80%. Human hepatocytes with at least one CYP3A5*1 allele also metabolized vincristine, albeit at a slower rate (10-fold) than human liver microsomes. The CYP3A5 low-expressing hepatocytes did not metabolize vincristine. We conclude that for high CYP3A5 expressers, the majority of the CYP metabolism is mediated by CYP3A5. By in vitro/in vivo scaling with microsomes, the hepatic clearances of high CYP3A5 expressers are predicted to have a 5-fold higher hepatic clearance than low expressers. However, the role of metabolism in the systemic clearance of vincristine is unknown. To study the disposition of vincristine in vivo, a sensitive and selective LC/MS/MS assay was validated for the quantification of vincristine and M1 quantification in human plasma. Vincristine and M1 were identified and quantified in select pediatric plasma and urine samples. For future large-scale clinical studies, the vincristine and M1 concentrations in plasma will be quantified to understand the role of CYP3A5 genotype in vincristine pharmacokinetics. For patients that are CYP3A5 high expressers, the systemic clearance of vincristine may be higher than that of low CYP3A5 expressers. Thus, CYP3A5 genotype may be an important determinant of inter-individual variability in clinical outcomes.

Thesis (M.S.)--State University of New York at Binghamton, Chemistry Dept., 2006. / Includes bibliographical references.

Thesis (Ph. D.)--Indiana University, 2007. / Title from screen (viewed on November 16, 2007). Department of Pharmacology & Toxicology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Stephen D. Hall, Lisa M. Kamendulis, Sherry F. Queener, Leonard C. Erickson, Steven A. Wrighton. Includes vitae. Includes bibliographical references (leaves 195-203).

La vincristine est un vinca-alcaloïde très largement utilisé et reste incontournable dans la prise en chargethérapeutique de nombreux cancers, notamment en pédiatrie. Ce médicament présente une neurotoxicitédose-limitant, très variable d’un individu à l’autre, difficilement prévisible, ayant ainsi des conséquencescliniques directes sur l’efficacité thérapeutique. Cette variabilité pourraient avoir une originepharmacocinétique et pharmacogénétique, en partie liée au métabolisme hépatique intense de la vincristinepar les enzymes CYP3A4 et CYP3A5 et à son transport par la P-glycoprotéine (codée par le gène ABCB1).En effet, de nombreux polymorphismes génétiques des gènes CYP3A4, CYP3A5 et ABCB1 ont été décritsdont certains sont associés à d’importantes différences d’expression et/ou de fonctionnalité de la protéine.Nous avons donc développé une méthode de dosage sensible et hautement spécifique de la vincristine parLC-MSMS, adaptée à la réalisation d’une étude de pharmacocinétique-pharmacogénétique dans unepopulation pédiatrique atteinte de tumeurs solides.Nous avons montré que les différents facteurs démographiques, thérapeutiques et génétiques étudiés(allèles CYP3A5*3, CYP3A4*1B ; mutations ABCB1 C1236T, G2677T(A) et C3435T) ne sont pasprédictifs de la variabilité de la pharmacocinétique de la vincristine observée dans notre populationpédiatrique. Ces résultats sont renforcés par l’observation d’une large variabilité pharmacocinétique entreles cures pour un même patient, qui ne peut être expliquée par des facteurs génétiques. Nous avons pu chezcertains patients, évaluer l’accumulation intracellulaire de la vincristine. Une importante variabilité estégalement retrouvée au niveau cellulaire et nos résultats montrent deux profils d’accumulationintracellulaire de vincristine très distincts qui pourraient avoir une origine génétique. En effet, les patientsporteurs du génotype hétérozygote d’ABCB1 présentaient une tendance à une plus forte accumulation surles temps précoces.Néanmoins, l’incidence de survenue d’une neurotoxicité n’a pas non plus été associée à des différencesd’exposition plasmatique ou d’accumulation intracellulaire de vincristine ou encore aux polymorphismesgénétiques étudiés du CYP3A4, CYP3A5 et d’ABCB1 dans notre population. La pertinence clinique d’unedifférence d’accumulation intracellulaire de la vincristine reste à être évaluée sur un effectif beaucoup plusimportant. La variabilité de la neurotoxicité de la vincristine ne semble donc pas être prédite par desfacteurs génétiques affectant la pharmacocinétique et pourrait donc avoir notamment une originepharmacodynamique, liée à une modification de sa sensibilité chez certains patients. / Vincristine is a natural vinca-alkaloid widely used in many chemotherapy regimens for paediatric tumourdiseases. The most frequent and clinically relevant side-effect of vincristine is a dose-limitingneurotoxicity which is unpredictable and characterized by a great variability between patients. Thisvariability could have a pharmacokinetic and pharmacogenetic origin, partly due to the intense hepaticmetabolism of vincristine through both CYP3A4 and CYP3A5 and a transport by P-glycoprotein (encodedby the ABCB1 gene). Indeed, several genetic polymorphisms have been described for CYP3A4, CYP3A5and ABCB1 which could affect the expression and/or the functionality of the protein. We have firstdeveloped a sensitive and highly specific LC-MS/MS method for vincristine quantification, suitable for apharmacokinetics-pharmacogenetics study in paediatric patients treated for solid tumours diseases.Our results showed that demographic, therapeutic and genetic factors assessed (CYP3A5*3, CYP3A4*1Balleles and ABCB1 C1236T, G2677T (A) and C3435T mutations) are not predictive of vincristinepharmacokinetics variability observed in our paediatric population. These results are enhanced by theobservation of a wide inter-course variability which cannot be explained by genetic factors. In somepatients, we were able to evaluate the vincristine intracellular concentration and we observed similarly alarge inter-patient variability. Two populations emerge according with the intracellular vincristineaccumulation and this dichotomic distribution could have a genetic origin. Indeed, a tendency to a greateraccumulation of intracellular vincristine was observed in patients with ABCB1 CGC-TTT diplotype in theearly post-dose period.Nevertheless, the incidence of neurotoxicity in our population has not been associated to differences inplasma exposure and intracellular accumulation of vincristine and to the assessed genetic polymorphismsof CYP3A4, CYP3A5 and ABCB1. The clinical relevance of differences in the intracellular accumulation ofvincristine remains to be evaluated on a larger cohort of patients. The variability of vincristineneurotoxicity does not seem to be explained by genetic factors affecting its pharmacokinetics and thereforecould have a pharmacodynamic origin, probably linked to a modification of vincristine sensitivity inpatients.

A aneuploidia é uma característica dos tumores sólidos. Ela pode ser resultado de diferentes erros durante a mitose, como a amplificação centrossômica, mitoses multipolares, e anormalidades durante a citocinese. Hoje se sabe que a aneuploidia pode estar relacionada à supressão ou progressão tumoral dependendo do grau da aneuploidia e do contexto genético das células, e assim esforços vem sendo feitos a fim de elucidar quais erros durante a mitose estão relacionados à formação de células aneuploides viáveis e inviáveis. Estudos prévios do nosso grupo mostraram que tratamentos de células em cultura com fibras de crisotila e com vincristina levam a formação de células aneuploides. Agora direcionados nossos esforços para elucidar os mecanismos envolvidos na formação dessas células, investigando alterações nos centrossomos, número de cromossomos, e origens e destinos de mitoses multipolares após o tratamento com crisotila e com vincristina. As fibras de crisotila, em linhagens de células tumorais e normais, levaram a padrões de localização alterados de proteínas relacionadas à abscisão durante a citocinese, e ocorreu a regressão deste processo e consequente formação de apenas uma célula-filha com o dobro do conteúdo de cromossomos e de centrossomos. Nas duas linhagens estudadas essas células tetraploides progrediram no ciclo celular, gerando mitoses multipolares e consequente formação de células aneuploides. O tratamento com vincristina levou a respostas similares e também distintas em células normais e tumorais. Durante a retenção em metáfase ocorreu a fragmentação da matriz pericentriolar, e as células foram encaminhadas à morte celular ou à saída da mitose sem a ocorrência de divisão celular. Entretanto, células de origem normal tetraploides não progrediram no ciclo celular e não formaram mitoses multipolares, enquanto na linhagem tumoral as células apresentaram aumento da expressão de Aurora A e células com conteúdo cromossômico aumentado e aneuploide em mitoses multipolares. As mitoses multipolares formaram uma, duas ou três células e apresentaram diversas anormalidades no processo de divisão. As alterações observadas no número e composição dos centrossomos após o tratamento com as duas linhagens indicaram que processos de amplificação centrossômica ocorreram após o tratamento. Os dados foram compatíveis com a reduplicação dos centrossomos e com a formação de centríolos a partir do aumento da matriz pericentriolar. Os dados reunidos mostram que apenas células tumorais foram capazes de proliferar mesmo após diferentes erros mitóticos, enquanto células normais puderam apenas superar os erros ocasionados pelas fibras de crisotila / Aneuploidy is a feature of solid tumors. Aneuploid cells result from errors during mitosis, such as centrosome amplification, multipolar mitosis and cytokinesis abnormalities. The capability of aneuploidy to promote and to suppress tumorigenesis has driven the efforts to characterize mitotic errors that form viable and not viable aneuploid cells. We have previously shown that chrysotile, an asbestos fiber, and vincristine, a chemotherapeutic agent, are able to induce aneuploidy. Now we directed our focus to discover possible mechanisms involved in aneuploid cell formation. Herein we evaluated centrosome morphology, chromosome number, and origins and fates of multipolar mitosis after chrysotile and vincristine treatment. Chrysotile fibers, in normal and cancer cells, led to mislocalization of proteins involved in abscission, which resulted in cytokinesis regression and tetraploid cells. These cells were able to enter cell cycle, giving rise to multipolar mitosis and aneuploid cells. Vincristine treatment led to specific and common responses in normal and cancer cells. During metaphase arrest, pericentrosomal matrix was fragmented, and the cells could be conducted to mitotic slippage in both lineages. However, normal tetraploid cells could not progress through cell cycle and neither to form multipolar mitosis, while cancer tetraploid cells showed Aurora A overexpression, structural and numerical centrosome abnormalities, multipolar mitosis and high levels of aneuploidy. The results showed that cancer cells could proliferate even after several mitotic errors, while normal cells could only overcome errors induced by chrysotile treatment

A aneuploidia é uma característica dos tumores sólidos. Ela pode ser resultado de diferentes erros durante a mitose, como a amplificação centrossômica, mitoses multipolares, e anormalidades durante a citocinese. Hoje se sabe que a aneuploidia pode estar relacionada à supressão ou progressão tumoral dependendo do grau da aneuploidia e do contexto genético das células, e assim esforços vem sendo feitos a fim de elucidar quais erros durante a mitose estão relacionados à formação de células aneuploides viáveis e inviáveis. Estudos prévios do nosso grupo mostraram que tratamentos de células em cultura com fibras de crisotila e com vincristina levam a formação de células aneuploides. Agora direcionados nossos esforços para elucidar os mecanismos envolvidos na formação dessas células, investigando alterações nos centrossomos, número de cromossomos, e origens e destinos de mitoses multipolares após o tratamento com crisotila e com vincristina. As fibras de crisotila, em linhagens de células tumorais e normais, levaram a padrões de localização alterados de proteínas relacionadas à abscisão durante a citocinese, e ocorreu a regressão deste processo e consequente formação de apenas uma célula-filha com o dobro do conteúdo de cromossomos e de centrossomos. Nas duas linhagens estudadas essas células tetraploides progrediram no ciclo celular, gerando mitoses multipolares e consequente formação de células aneuploides. O tratamento com vincristina levou a respostas similares e também distintas em células normais e tumorais. Durante a retenção em metáfase ocorreu a fragmentação da matriz pericentriolar, e as células foram encaminhadas à morte celular ou à saída da mitose sem a ocorrência de divisão celular. Entretanto, células de origem normal tetraploides não progrediram no ciclo celular e não formaram mitoses multipolares, enquanto na linhagem tumoral as células apresentaram aumento da expressão de Aurora A e células com conteúdo cromossômico aumentado e aneuploide em mitoses multipolares. As mitoses multipolares formaram uma, duas ou três células e apresentaram diversas anormalidades no processo de divisão. As alterações observadas no número e composição dos centrossomos após o tratamento com as duas linhagens indicaram que processos de amplificação centrossômica ocorreram após o tratamento. Os dados foram compatíveis com a reduplicação dos centrossomos e com a formação de centríolos a partir do aumento da matriz pericentriolar. Os dados reunidos mostram que apenas células tumorais foram capazes de proliferar mesmo após diferentes erros mitóticos, enquanto células normais puderam apenas superar os erros ocasionados pelas fibras de crisotila / Aneuploidy is a feature of solid tumors. Aneuploid cells result from errors during mitosis, such as centrosome amplification, multipolar mitosis and cytokinesis abnormalities. The capability of aneuploidy to promote and to suppress tumorigenesis has driven the efforts to characterize mitotic errors that form viable and not viable aneuploid cells. We have previously shown that chrysotile, an asbestos fiber, and vincristine, a chemotherapeutic agent, are able to induce aneuploidy. Now we directed our focus to discover possible mechanisms involved in aneuploid cell formation. Herein we evaluated centrosome morphology, chromosome number, and origins and fates of multipolar mitosis after chrysotile and vincristine treatment. Chrysotile fibers, in normal and cancer cells, led to mislocalization of proteins involved in abscission, which resulted in cytokinesis regression and tetraploid cells. These cells were able to enter cell cycle, giving rise to multipolar mitosis and aneuploid cells. Vincristine treatment led to specific and common responses in normal and cancer cells. During metaphase arrest, pericentrosomal matrix was fragmented, and the cells could be conducted to mitotic slippage in both lineages. However, normal tetraploid cells could not progress through cell cycle and neither to form multipolar mitosis, while cancer tetraploid cells showed Aurora A overexpression, structural and numerical centrosome abnormalities, multipolar mitosis and high levels of aneuploidy. The results showed that cancer cells could proliferate even after several mitotic errors, while normal cells could only overcome errors induced by chrysotile treatment

No description available.

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

No description available.