Das BCL-2 Gen wurde als Onkogen der t (14;18)(q32;q21)-Translokation bei follikulären Non- Hodgkin-Lymphomen identifiziert. Die biologische Wirkung des BCL-2 Proteins liegt in der Hemmung der Apoptose. Bei der AML wird eine vermehrte BCL-2 Expression und eine dem- entsprechend verminderte Apoptose bei unreifen malignen myeloischen Vorläuferzellen gefun- den. Diese Krankheit ist teilweise auch chemoresistent. Goldstandard der Induktionstherapie bei AML ist eine Kombination aus Ara-C und Idarubicin, welche Doppel- und Einzelstrang- brüche der DNA induzieren. Apoptose der Leukämiezellen wird durch Schädigung der DNA ausgelöst. BCL-2 kann die Zellen durch Hemmung der Apoptose schützen, indem es die Cy- tochrom-C-Freisetzung blockiert. Darüber hinaus befinden sich die BCL-2- überexprimierenden Zellen in der G0-Phase und sprechen dabei schlecht auf die Chemothera- pie an. Deshalb stellt BCL-2 den Leukämiezellen "doppelten" Schutz zur Verfügung. BCL-2 spielt somit eine wichtige Rolle bei der Chemoresistenz. Ob ein Therapieprotokoll in der Be- handlung der AML effektiv ist, schlägt sich in der Kinetik der zunehmenden oder abnehmen- den BCL-2-Transkripte nieder. Zur Kontrolle des BCL-2-Transkriptes ist die quantitative PCR der qualitativen PCR überlegen. Die Quantifizierung dieses Transkriptes wurde mittels Echtzeit-Fluoreszenz-PCR realisiert. Bei der Echtzeit-Fluoreszenz-PCR wird die Reaktion im geschlossenen Reaktionsgefäß durchge- führt, sodass die Gefahr von Kontamination minimiert werden kann. Da keine Post-PCR Schrit- te nötig sind, wird die Überprüfung zahlreicher Proben durch ein 96-well-Format innerhalb eines Laufes ermöglicht. Die Echtzeit-Fluoreszenz-PCR garantiert ihre Spezifität durch eine spezifi- sche Sonde-Zielsequenz-Bindung und erlaubt eine exakte Quantifizierung der BCL-2- Transkriptzahl. In der vorliegenden Arbeit wurde die BCL-2-Expression in 53 AML-Fällen mittels Echtzeit- Fluoreszenz-PCR untersucht. Das ?-Actin Gen wurde als Referenzgen benutzt. Für die BCL-2- Expression wurde eine Ratio aus der Transkriptzahl des BCL-2 Gens und des ?-Actin Gens ge- bildet. Bei 53 AML-Fällen, die den sieben AML-Subtypen zugeordnet werden konnten(FAB M0-M7), konnte eine BCL-2-Expression nachgewiesen werden. Trotz der unterschiedlich hohen BCL-2-Expression bei diesen Patienten, ergab sich keine signifikante Korrelation zwischen der BCL-2-Expression und den FAB-Subtypen. Außerdem wurde die BCL-2-Expression in T- Zellen, B-Zellen und Granulozyten aus 5 AML-Patienten nachgewiesen. Die BCL-2-Expression wurde nicht von den Subpoplationen der mononukleären Zellen wie z.B. T-Zellen, B-Zellen, Granulozyten beeinflusst. Bei sieben Patienten wurden Proben im Verlauf untersucht. Dabei korrelierte eine hohe oder ansteigende BCL-2-Expression mit einem Rückfall der AML. Die Anzahl der untersuchten Proben im Verlauf ist jedoch zu klein, um definitive Schlußfolgerungen zu ziehen. Eine prospektive Untersuchung von größeren Patientenzahlen erscheint sinnvoll. / The bcl-2 oncogene was discovered by virtue of its association with the translocation, t(14;18) (q32;q21), observed in most follicular lymphomas. The bcl-2 protein is a 26 kDa integral membrane protein which functions by enhancing cell viability through the inhibition of apoptotic death. Acute myeloid leukemia is a lethal malignant disease characterized by an abnormal proliferation and differentiation of myeloid progenitor cells. The bcl-2 oncogene contributes to leukemogenesis by prolonging the life span of defected progenitor cells. Although the expression of bcl-2 in blast cells of acute myeloid leukemia is heterogeneous, a significant proportion of blast cells are shown to have high bcl-2 levels. The highest bcl-2 levels are found in cells that grow autonomously in vitro and also in blast cells expressing the CD34 surface antigen. These groups of AML patients are tranditionally the ones in which the prognosis is poor, because most of the chemotherapeutic agents like cytosine-arabinoside (Ara-C) exert their effect by triggering apoptosis. The high level of the bcl-2 gene that inhibits apoptosis is implicated in the resistance of AML blast cells to chemotherapy and leads to unfavorable prognosis. In this study, a real time fluorescence PCR assay was used to monitor the expression of the bcl-2 transcript in the therapeutic course of AML patients. By applying this rapid new developed quantitative method, the changes of the bcl-2 transcript with chemotherapy can help to evaluate the efficacy of therapeutic interventions in AML. The real time fluorescence PCR has many advantages over traditional measures. First, the assay is extremely rapid because post-PCR processing steps are unnecessary. All relevant data are collected real time during the course of a 2h PCR cycle program; data analysis can be completed in less than 10 min. Second, the assay from reaction set-up to data collection and analysis is a closed-tube system, which reduces the risk of false positive resulting from PCR product carry- over contamination and eliminates variation from additional pipetting steps. Finally, the real time fluorescence PCR is highly specific for the gene target of interest. Here the expression levels of the bcl-2 gene were measured in 53 patients with acute myeloid leukemia and normalized by ?-actin, a house-keeping gene expression as endogenous reference. The bcl-2/?-actin ratio from the 53 patients with AML was various, but not related to FAB subtypes. And also, this transcript ratio was not affected by mononucleated cell types. The samples from seven patients were measured to evaluate the association between the bcl-2 expression and the responsiveness of AML patients to the chemotherapy. The high or gradual elevation of the bcl-2 expression demonstrated the loss of effect in update-therapy protocol and the relapse in AML patients. Although the amount of samples are not large enough to reach the final conclusion, it is of significance that a number of patients will be analyzed in the future.

As estatísticas relacionadas aos cânceres hematológicos indicam que a incidência e mortalidade dessas doenças têm aumentado ao longo dos anos. Embora a maioria dos casos de linfomas e leucemias não possua etiologia definida, sugere-se que fatores genéticos possam estar envolvidos. Nesse contexto, destaca-se a família de proteínas Bcl-2, divididas em anti e pró-apoptóticas. Os genes Bcl-2 e Bcl-XL, membros de uma nova classe de oncogenes, que atuam no mecanismo de morte celular das células cancerígenas, sobretudo apoptose, a qual é controlada por numerosos sinais intra e extracelulares. Uma nova estratégia para o tratamento desta doença inclui a terapia gênica mediada por RNA de interferência, que silencia importantes genes, a exemplo dos genes da família Bcl-2. Visto que o silenciamento isolado de um único gene pode não ter resultados expressivos, o presente trabalho teve por objetivo desenhar um RNA de interferência (RNAi) homólogo a dois tipos distintos de RNA mensageiro (RNAm) e inibir simultaneamente os genes Bcl-2 e Bcl-XL,assim como testar a inibição isolada dos mesmos. Amostras de linhagem tumoral Jurkat e Granta-519 foram avaliadas após transfecção com os seguintes RNA:i Bcl-2,Bcl-XL, Bcl-2/Bcl-XL,Bcl-2+Bcl-XL e scramble. Os nossos achados evidenciam que, na linhagem Granta-519, a sequência do RNAi Bcl-2 inibe, isoladamente ou conjugado ao Bcl-XL, o gene Bcl-2. Deste modo, o RNAi Bcl-2 apresenta-se mais eficiente no mecanismo de silenciamento gênico, uma vez que propicia a morte celular frente a toxicidade do quimioterápico etoposide. / The hematological cancer statistics indicates that its incidence and mortality have increased over the years. Although most cases of lymphomas and leukemias has no definite etiology however is suggested that genetic factors may be involved. In this context there is the Bcl-2 proteins family divided into anti-apoptotic and pro-apoptotic which Bcl-2 and Bcl-XL genes are members of a new class of oncogenes that act in cancer cells death mechanisms, especially apoptosis, that is controlled by numerous intra-and extracellular signals. Among new strategies to treat hematological cancer includes gene therapy mediated by RNA interference, which can decrease expression of genes like Bcl-2 family components. Studies of single gene silencing have not shown significant results so this study aimed to design an RNA interference (iRNA) homologous to two distinct types of messenger RNA (mRNA) and inhibit both genes Bcl-2 and Bcl-XL -XL as well as test the inhibition. Commercial cells Jurkat and Granta-519 were evaluated after transfection with iRNA as follows: Bcl-2, Bcl-XL, Bcl-2/Bcl-XL, Bcl-2+Bcl-XL and scramble. Our findings show that in Granta-519 cell line Bcl-2 RNAi sequence inhibits, alone or conjugated to Bcl-XL, Bcl-2 gene. Thus RNAi Bcl-2 appears more effective in gene silencing mechanism as it promotes cell death due chemotherapeutic agent etoposide toxicity.

Neste trabalho foram analisados 30 casos de linfomas de células B da região oral, fixados em solução de formaldeído e incluídos em parafina, através da técnica de imuno-histoquímica para as proteínas c-Myc, Bcl-2, Bcl-6 e ciclina D1. Dos casos analisados 40% foram positivos para a marcação para c-Myc, 33,3% para a marcação para ciclina D1, 83,3% para a marcação para Bcl-2 e 53,3% para a marcação para Bcl-6. Todos os casos foram diagnosticados como linfomas difusos de grandes células B, o subtipo de linfoma com a maior casuística. A análise destas proteínas é de fundamental importância para o diagnóstico e direcionamento do tratamento de doenças hematopoiéticas, pois estão envolvidas em vários processos de controle da transcrição gênica, do ciclo celular e dos processos apoptóticos e o aumento do conhecimento sobre sua ação em diferentes subtipos de linfomas pode corroborar outros estudos. / In this study, 30 cases of formalin-fixed and paraffin-embedded B-cell lymphomas of the oral region were submitted to immunohistochemistry for the detection of proteins c-Myc, Bcl-2, Bcl-6 and cyclin D1. Fourty percent (40%) of the studied cases were positive for c-Myc, 10% for cyclin D1, 83.3% for Bcl-2 and 53.3% for Bcl-6. The analysis of these proteins has fundamental importance for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription and cell cycle. All cases were diffuse large B-cell lymphomas, the subtype with the highest incidence. The analysis of these proteins is very important for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription, cell cycle and apoptotic processes and an increase in the knowledge of their action in different subtypes of lymphomas can corroborate to other studies.

Neste trabalho foram analisados 30 casos de linfomas de células B da região oral, fixados em solução de formaldeído e incluídos em parafina, através da técnica de imuno-histoquímica para as proteínas c-Myc, Bcl-2, Bcl-6 e ciclina D1. Dos casos analisados 40% foram positivos para a marcação para c-Myc, 33,3% para a marcação para ciclina D1, 83,3% para a marcação para Bcl-2 e 53,3% para a marcação para Bcl-6. Todos os casos foram diagnosticados como linfomas difusos de grandes células B, o subtipo de linfoma com a maior casuística. A análise destas proteínas é de fundamental importância para o diagnóstico e direcionamento do tratamento de doenças hematopoiéticas, pois estão envolvidas em vários processos de controle da transcrição gênica, do ciclo celular e dos processos apoptóticos e o aumento do conhecimento sobre sua ação em diferentes subtipos de linfomas pode corroborar outros estudos. / In this study, 30 cases of formalin-fixed and paraffin-embedded B-cell lymphomas of the oral region were submitted to immunohistochemistry for the detection of proteins c-Myc, Bcl-2, Bcl-6 and cyclin D1. Fourty percent (40%) of the studied cases were positive for c-Myc, 10% for cyclin D1, 83.3% for Bcl-2 and 53.3% for Bcl-6. The analysis of these proteins has fundamental importance for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription and cell cycle. All cases were diffuse large B-cell lymphomas, the subtype with the highest incidence. The analysis of these proteins is very important for the diagnosis and treatment course of hematopoietic diseases, because they are involved in various processes controlling gene transcription, cell cycle and apoptotic processes and an increase in the knowledge of their action in different subtypes of lymphomas can corroborate to other studies.

Para que ocorra a transição do epitélio normal de laringe para carcinoma escamoso é necessário um processo de múltiplas etapas tal como exposição prolongada ao fumo e álcool e uma possível associação à infecção pelo HPV. Vários tipos de marcadores moleculares vêm sendo estudados na carcinogênese da laringe, entre eles proteínas associadas a apoptose (bcl-2 e PARP-1) assim como proteínas envolvidas em múltiplos processos biológicos como a Galectina-3. Neste estudo foram realizadas análise imunoistoquímica quantitativa e qualitativa para bcl-2, PARP-1 e galectina-3 em 65 pacientes diagnosticados com câncer de laringe subdivididos em: carcinoma de laringe in situ (CLIS), carcinoma de laringe com metástase (CLM), sem metástase (CLS) e linfonodos cervicais (LC). A detecção e tipificação do HPV foram realizadas pela reação em cadeia da polimerase (PCR) e os tipos de HPV avaliados foram HPV 6, 11, 16, 18, 31 e 33. Na avaliação quantitativa de galectina-3 observou-se um significativo aumento de expressão no carcinoma invasivo de laringe (CLS e CLM) quando comparado com carcinoma in situ (CLIS), podendo concluir que essa proteína seria um bom marcador pra progressão de câncer de laringe. Para as proteínas PARP-1 e bcl-2 não houve diferença nos níveis de expressão nos grupos analisados. Na análise qualitativa PARP-1 apresentou uma homogeneidade de marcação tanto alta como baixa entre os grupos. Em relação à Galectina-3 observou-se um predomínio de casos com alta expressão, diferentemente da proteína bcl-2 onde o predomínio foi de baixa expressão em todos os casos de carcinoma de laringe e seus respectivos linfonodos metastáticos. Dos 65 pacientes, 55 (84,6%), foram positivos para beta-globina e 7 (12.7%) dos 55 pacientes foram positivos para HPV. Não foi possível verificar quaisquer correlações entre as proteínas Galectina-3, bcl-2 e PARP-1 e o HPV devido ao baixo índice de casos positivos. / To occur the transition from normal epithelium to squamous cell carcinoma is a necessary a for multiple stages process, such as smoking and alcohol abuse and a possible association with HPV infection. Several types of molecular markers have been studied in cancer of larynx, including proteins associated with apoptosis (bcl-2 and PARP-1) and proteins involved in many biological process such as galectin-3. In this study, analyses of qualitative and quantitative immunohistochemistry was performed for bcl-2, PARP-1 and galectin-3 in 65 patients diagnosed with laryngeal squamous cell carcinoma divided into in situ laryngeal carcinomas(LSCCS), laryngeal squamols cells carcinomas without metastases (LSCCWT) and with metastasis (LSCCW) and cervical lymph nodes (CL). HPV detection and typing was performed by PCR and the HPV types evaluated were HPV 6, 11, 16, 18, 31 and 33. In quantitative of galectin-3 there was observed a significant increase of expression in invasive laryngeal squamous cell carcinoma (LSCCWT and LSCCW) compared with in situ laryngeal carcinomas (LSCCS), may indicating that this protein could be a good marker for progression of laryngeal carcinoma. For PARP-1 and bcl-2 protein there was no difference in the levels of expression in all groups studied. In qualitative analysis PARP-1 showed a homogenous immunolabeling in both high and low among the groups. In relation to Galectin-3, it was observed a predominance of cases with high expression, unlike the protein bcl-2 where the expression prevalence was low in all cases of laryngeal carcinoma and their metastatic lymph nodes. Of the 65 patients, 55 (84.6%) were positive for beta-globin and 7 (12.7%) of 55 patients were positive for HPV. Because of a low incidence of HPV in the cases studied, it was not possible correlate the proteins bcl-2, PARP-1 and Galectin-3 with the presence of HPV.

Thesis (M.Sc.)--Memorial University of Newfoundland, Faculty of Medicine, 1999. / Bibliography: leaves 98-114.

No description available.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 5 uppsatser.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 112-125). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;