This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Neuroblastoma (NB) is the most common extracranial solid tumor diagnosed in the first year of life. The disease is characterized by lack of somatic mutations and high genomic instability including the amplification of MYCN which correlates with poor clinical outcome. These features of NB suggest a pivotal role of transcriptome dynamics in this disease. Analyses of 1089 NB patients show that an overall increase in m6A-regulatory factors correlates with the worst prognosis. Notably, the m6A reader YTHDF1 is upregulated in stage 4 NB corresponding to the highest degree of MYCN amplification. In line with these notions, we observed a positive correlation between MYCN and YTHDF1 by analyzing 33 NB cell lines with different MYCN amplification status. Consistent with this observation, we demonstrated that MYCN knockdown is accompanied by a decrease in all the YTHDF proteins. Furthermore, we demonstrate a loss of proliferating activity in a MYCN-amplified cell line upon knocking out all the YTHDF paralogs and show that the loss in proliferation is mediated by downregulation of DNA replication and chromatin remodeling upon YTHDF depletion. Retinoic acid (RA) is a key agent employed in NB therapy; it induces cell differentiation limiting the proliferation of malignant cells. However, therapeutic protocols adopting RA are limited to maintenance therapy. Thereby, identifying new therapeutic strategies that synergistically enhance RA response is essential to expand the therapeutic window for this drug. Since m6A is a well-known regulator of cellular differentiation, we hypothesized that the protein involved in m6A regulation could act in concert with RA in driving this process. We demonstrate that YTHDF depletion potentiates the response to RA enriching the key processes required for differentiation and that this correlates with an enhanced pro-neural of the YTHDF depleted cells. Together, our findings suggest that targeting the YTHDF family in MYCN-amplified NB represents a promising therapeutic opportunity to control the progression of this disease.

A isquemia cerebral causada pela parada cardíaca leva ao desapareciemnto neuronal. studamos os mecanismos de morte celular envolvidos na isquemia in vitro em linhagem de neuroblastoma.O insulto isquêmicao foi reproduzido cultivando as células sem fatores de crescimento, sem glicose e em embiente hipóxico produzido por um sistema de anaerobiose. Os resultados sugerem que a privação de oxigênio, glicose e fatores de cresciemtno do meio de cultura reproduzem o fenômeno semelhante a isquemia. INvestigamos ainda a participação de processo apoptótico e sua modulação térmica. Observams que a hipotermia produz neuroproteção, enquanto a hipertermia agrava o processo de morte celular por apoptose. / Cardiac arrest causes cerebral ischemia and neuronal disappearance. We investigate celular death mechanisms elucidated by a model of ischemia in neuroblastoma cell line. The ischemic insult was reproduced by deprivation of growth factors and glucose in a hypoxic environment produced by an anaerobiosis system. Our results validate the experimental model and revel the participation of an apoptotic process in the celular loss induced by ischemia. We also demonstrated that hypothermia can be used as a neuroprotector agent whereas hyperthermia aggavates celular damage.

Despite the identification of MYCN amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed. Here, by combining a whole genome shRNA library screen and Master Regulator Inference Algorithm (MARINa) analysis, we identified Transcription Factor Activating Protein 4 (TFAP4) as a novel synthetic lethal interactor with MYCN amplification in neuroblastoma. Silencing TFAP4 selectively inhibits MYCN amplified neuroblastoma growth both in vitro and in xenograft mice models. TFAP4 expression is inversely correlated with patient survival in MYCN-high neuroblastoma. Mechanistically, silencing TFAP4 induces neuroblastoma differentiation, as seen by increased neurite outgrowth, and up-regulation of neuronal markers. TFAP4 regulates a downstream signature similar to the signature of the oncogene anaplastic lymphoma kinase (ALK). Taken together, our results validate TFAP4 as an important master regulator in MYCN amplified neuroblastoma and a novel synthetic interactor with MYCN amplification. Thus, TFAP4 may be a novel drug target for neuroblastoma treatment.

Neuroblastoma is one of the most common solid tumors in patients below the age of 15. In this PhD project, the possible anti-tumor effects of the soy isoflavones on neuroblastoma cells have been investigated. A number of aspects of neuroblastoma cancer cell biology, including cell proliferation, cell cycle regulation, cell differentiation and apoptosis, intracellular signaling mechanisms, tumor invasiveness and metastatic properties, have been examined. Furthermore, novel antitumor properties of the soy isoflavones on neuroblastoma have also been quested for, hoping that through this PhD project, a better understanding of the potential anti-tumor effects of soy isoflavones on neuroblastoma cells can be obtained. In Chapter Three, we have demonstrated that the major soy isoflavones daidzein and genistein exerted potent anti-proliferative effect on murine neuroblastoma Neuro-2a (BU-1) cells. These two compounds were shown to modulate cell cycle distribution in BU-1 cells in different ways, possible through their differential effects on the expression of cell cycle regulatory proteins such as cyclins and cyclin-dependent kinase inhibitors. The anti-tumor effect of daidzein was found to be fairly specific to tumor cells, as daidzein only exhibited little, if any, direct cytotoxicity to normal murine cells such as bone marrow cells, macrophages, and thymocytes. Furthermore, the anti-proliferative effect of daidzein was unlikely to be attributed to its direct cytotoxicity to the BU-1 cells, but might be coupled with its ability to trigger the apoptosis and differentiation in the neuroblastoma cells. Our results show that daidzein could induce DNA fragmentation, ultrastructural changes, and the enrichment of cytoplasmic mono- and oligo-nucleosomes in the treated-BU-1 cells. Moreover, daidzein was shown to induce neuronal differentiation in the BU-1 cells, as indicated by morphological changes, and increased expression of the neuronal differentiation marker microtubule-associated protein-2, and acetylcholine esterase activity. In addition, we have also demonstrated that the signals of daidzein might be mediated by the estrogen receptor and nuclear factor-kappa B pathways. In Chapter Four, daidzein was shown to exert a differential an proliferative effect on three human neuroblastoma cell lines, including SK-N-DZ and SH-SY5Y. The most sensitive cell line, LA-N-1, was chosen for further mechanistic studies. It was found that daidzein-induced growth-inhibitory effect was coupled with the induction of neurite outgrowth and altered mRNA expression of the N-myc-related transcription factors. Moreover, daidzein was observed to modulate the invasiveness and metastatic properties of LA-N-1 cells, as indicated by the reduction of colony-forming ability, cell migratory ability, in vivo tumorigenicity, tumor vascularity, and angiogenic factors expression. Our results clearly suggest that the major soy isoflavone daidzein can exert its pleiotropic anti-tumor effects on both marine and human neuroblastoma cells. In Chapter Five, we isolated two stable actively proliferating subclones from the human neuroblastoma SH-SY5Y cells. We compared the sensitivities of the parental SH-SY5Y cells and the active subclones to the growth inhibition exerted by a number of conventional cancer chemotherapeutic agents and the soy isoflavone derivatives. We found that the major soy isoflavones daidzein and genistein were rather selective to the active subclones and this phenomenon was not observed with other chemotherapeutic agents. The anti-tumor action mechanisms of genistein on the most active subclone, designated as SH-SY5Y cl.6 cells, were examined in detail. It was found that genistein could induce apoptosis in SH-SY5Y cl.6 cells, as indicated by the induction of ultrastructural changes, phosphatidylserine externalization, and cytoplasmic enrichment of mono- and oligo-nucleosomes. The genistein-induced apoptosis in SH-SY5Y cl.6 cells was found to be both mitochondria and caspases-dependent, as mitochondrial membrane depolarization, cytosolic release of apoptotic mitochondrial factors and activation of caspase-3 were observed. To sum up, our results show that the major soy isoflavones, particularly daidzein and genistein, exhibit pleiotropic anti-tumor effects on both murine and human neuroblastoma cells, and they are also selective to the actively proliferating human neuroblastoma cells. (Abstract shortened by UMI.) / Lo, Fai-Hang. / "September 2007." / Adviser: Leung Kwok-Nam. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0949. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 255-287). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Neuroblastoma (NB) é a forma mais indiferenciada de tumores neuroblásticos e a principal causa de morte por câncer pediátrico. Alterações epigenéticas interagem em todas as etapas do desenvolvimento do câncer, promovendo a progressão tumoral. A remodelação da cromatina é influenciada pela acetilação de histonas e a metilação de DNA. Acetiltransferases de histona (HATs), desacetilases de histonas (HDAC) e metiltransferase de DNA (DNMTs) são alvos de estratégias terapêuticas em tumores. Os retinoides agem nas vias de diferenciação celular, anti-proliferação e pró-apoptose. Nesse trabalho, é proposto que a combinação desses moduladores epigenéticos e de diferenciação em linhagens de células de NB humano é mais efetiva que os agentes isolados. Os tratamentos induziram mudanças na expressão de marcadores de diferenciação e indiferenciação, como c-Myc, β-3tubulina, NeuN e Bmi1, e alterações morfológicas nas duas linhagens celulares utilizadas, SK-N-BE(2) e SH-SY5Y. Os dados encontrados podem contribuir para uma melhor compreensão dos mecanismos moleculares dos moduladores retinoides e epigenéticos em NB capazes de acrescentar melhorias nas atuais estratégias terapêuticas. / Neuroblastoma (NB) is the most undifferentiated form of neuroblastic tumors and the leading cause of death from pediatric cancer. Epigenetic changes interact at all stages of cancer development, promoting tumor progression. Chromatin remodeling is influenced by histone acetylation and DNA methylation. Histone acetyltransferases (HATs), histone deacetylases (HDAC), and DNA methyltransferase (DNMTs) are targets for therapeutic strategies in cancer. Retinoids act on cell differentiation pathways and display anti-proliferation and pro-apoptotic actions. In the present research we examined the effects of combining epigenetic modulators and a retinoid receptor agonist in human NB cells. The retinoid all trans-retinoic acid (ATRA) combined with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase was more effective than any drug given alone in impairing the proliferation of SH-SY5Y and SK-N-BE(2) NB cells. In addition, the treatments induced differential changes in the expression of differentiation markers including c-Myc, β-3tubulin, NeuN and Bmi1, and morphological changes in SK-N-BE(2) e SH-SY5Y cell lines. The data contribute to a better understanding of the molecular mechanisms of retinoid modulators and epigenetic in NB able to add improvements in current therapeutic strategies.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Neuroblastoma (NB) é a forma mais indiferenciada de tumores neuroblásticos e a principal causa de morte por câncer pediátrico. Alterações epigenéticas interagem em todas as etapas do desenvolvimento do câncer, promovendo a progressão tumoral. A remodelação da cromatina é influenciada pela acetilação de histonas e a metilação de DNA. Acetiltransferases de histona (HATs), desacetilases de histonas (HDAC) e metiltransferase de DNA (DNMTs) são alvos de estratégias terapêuticas em tumores. Os retinoides agem nas vias de diferenciação celular, anti-proliferação e pró-apoptose. Nesse trabalho, é proposto que a combinação desses moduladores epigenéticos e de diferenciação em linhagens de células de NB humano é mais efetiva que os agentes isolados. Os tratamentos induziram mudanças na expressão de marcadores de diferenciação e indiferenciação, como c-Myc, β-3tubulina, NeuN e Bmi1, e alterações morfológicas nas duas linhagens celulares utilizadas, SK-N-BE(2) e SH-SY5Y. Os dados encontrados podem contribuir para uma melhor compreensão dos mecanismos moleculares dos moduladores retinoides e epigenéticos em NB capazes de acrescentar melhorias nas atuais estratégias terapêuticas. / Neuroblastoma (NB) is the most undifferentiated form of neuroblastic tumors and the leading cause of death from pediatric cancer. Epigenetic changes interact at all stages of cancer development, promoting tumor progression. Chromatin remodeling is influenced by histone acetylation and DNA methylation. Histone acetyltransferases (HATs), histone deacetylases (HDAC), and DNA methyltransferase (DNMTs) are targets for therapeutic strategies in cancer. Retinoids act on cell differentiation pathways and display anti-proliferation and pro-apoptotic actions. In the present research we examined the effects of combining epigenetic modulators and a retinoid receptor agonist in human NB cells. The retinoid all trans-retinoic acid (ATRA) combined with inhibitors of either histone deacetylases (HDACs) or DNA methyltransferase was more effective than any drug given alone in impairing the proliferation of SH-SY5Y and SK-N-BE(2) NB cells. In addition, the treatments induced differential changes in the expression of differentiation markers including c-Myc, β-3tubulin, NeuN and Bmi1, and morphological changes in SK-N-BE(2) e SH-SY5Y cell lines. The data contribute to a better understanding of the molecular mechanisms of retinoid modulators and epigenetic in NB able to add improvements in current therapeutic strategies.