Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

No description available.

L'instabilité chromosomique, marque des cellules tumorales, peut trouver sa source dans un défaut d'initiation de la réplication. Ceci a été illustré chez la levure Saccharomyces cerevisiae et concorde avec l'observation de mutations de régulateurs de la transition G1/S dans un grand nombre de tumeurs. Toutefois, les mécanismes par lesquels cette instabilité survient n'ont pas encore été clairement définis. Pour résoudre cette question, nous avons utilisé le mutant de levure cdc6-1 où la formation des complexes pré-réplicatifs est graduellement affectée avec l'augmentation de la température. Nous avons mis en évidence que l'allongement de la durée de la réplication qui en suit induit des cassures de l'ADN (DSB) seulement à l'entrée en mitose. Par combinaisons de mutants, nous avons vu que la condensation des chromosomes est en partie responsable de ces DSB. Ces DSB sont signalées à la cellule via la protéine Rad9, protéine adaptatrice du checkpoint de dommages à l'ADN. De façon concordante, nous avons observé une activation de la protéine effectrice de ce checkpoint Rad53 à l'entrée en mitose. La viabilité des cellules cdc6-1 repose sur les protéines de checkpoint Chk1 et Rad53 ainsi que sur la présence de cohésines et des topoisomérases Top2 et Top3. Selon nous, la réplication prolongée par diminution du nombre d'origines n'est pas détectée par les cellules comme un stress réplicatif. Lors de l'entrée en mitose, la condensation des chromosomes transformerait les fourches de réplication en structures reconnues et clivées par les nucléases Mus81-Mms4 et Yen1, qui sont activées en mitose, dirigeant ces régions sous-répliquées vers la réparation par recombinaison. Ce sont les coupures induites en mitose, non la progression des fourches, qui activent le checkpoint. Nous proposons que la sous-réplication de segments d'ADN consécutive à un défaut de licensing des origines favorise la recombinaison non homologue et génère l'instabilité chromosomique, à l'image des sites fragiles communs qui sont le siège de remaniements récurrents lors de la cancérogenèse. / Chromosome instability (CIN), a hallmark of cancer cells, can take its roots in the G1 phase of the cell cycle, when replication origins are licensed. This has been illustrated in the yeast Saccharomyces cerevisiae and is consistent with the fact that a vast number of tumors presents mutations in G1/S transition regulators. However the mechanisms by which this instability occurs are still not well established. Using the yeast cdc6-1 mutant in which preRC formation can be decreased gradually with temperature, we show that cells replicating from fewer origins undergo massive DNA double-strand break (DSB) formation in mitosis. Blocking mitotic entry by Swe1 overexpression or Clb1-4 depletion, and inactivation of Cdc5 (Polo) both suppress DSB formation in cdc6-1 cells, demonstrating that DSBs do not stem from collapsed forks but are actively induced during mitosis. DSB formation is dependent on chromosome condensation and the Mus81-Yen1 structure-specific endonucleases. These DSBs then trigger the Rad9 DNA damage checkpoint. Accordingly, Rad53 phosphorylation is detected only after entry into mitosis. We propose that cells replicating their DNA from fewer origins enter mitosis undetected, then condense their chromosomes and cleave unreplicated regions by Mus81-Yen1 for repair by recombination. The viability of cdc6-1 cells at semi-permissive temperature relies on Chk1 and Rad53, as well as on cohesins and topoisomerases Top2 and Top3. Cleavage of under replicated DNA segments in mitosis may favor non-homologous repair pathways leading to chromosome rearrangements, as seen for common fragile sites that co-localize with recurrent breakpoints in cancer.

Childhood sexual abuse (CSA) is a stressful life experience with lasting/far-reaching health and psychopathological consequences. Our laboratory recently identified a significantly increased frequency of acquired chromosomal anomalies (assessed using the cytokinesis-blocked micronucleus assay) in adult female twins exposed to CSA when compared to their unexposed co-twin. The primary aim of this study was to evaluate potential mechanism(s) underlying the observed increases in levels of micronuclei in an expanded group of 90 female identical twins (61 CSA+ females and 29 CSA- females [including a total of 27 MZ co-twin pairs]) using fluorescence in situ hybridization (FISH) methodologies, with PNA probes specific for the centromeric and telomeric regions of all chromosomes coupled with the standard CBMN assay, we were able to characterize the chromosomal contents of MN and, thus, gain insight into the mechanisms underlying MN formation. By scoring 100 MN per study participant for the number of centromeric signal(s) and/or telomeric signal(s) present, we categorized the MN as harboring either: (1) terminal fragments (only a telomeric signal); (2) acentric interstitial fragments (no telomeric or centromeric signal); (3) centric interstitial fragments (only a centromeric signal); or (4) an intact chromosome(s) or chromatid(s). We identified elevated frequencies of intact chromosome-derived MN in CSA+ women as compared to CSA- women (P=0.014), implicating chromosome loss as a mechanism potentially underlying the increased frequencies of MN identified in adult females with a history of CSA. MN containing fragmented chromosomes were also observed in all of the study participants evaluated; however MN containing terminal fragments and MN containing acentric interstitial fragments were seen less frequently in CSA+ women compared to CSA- women. This study represents the first time that the chromosomal contents of MN have been evaluated in individuals in the context of a psychosocial factor. As chromosomal loss and breakage contributes to the development of age-related health problems, these observations provide important insight into the biological mechanisms that may underlie the latent morbidity and psychopathology associated with childhood adversity. Future studies aimed at understanding the biological impact of early-life trauma could determine if the observed increase in acquired chromosomal abnormalities results in detectable somatic clonal mosaicism. This knowledge could ultimately be used to develop screening tools to identify individuals “at risk” for negative health outcomes in adulthood.

Introdução: A síndrome de Bloom (SB) é uma síndrome de instabilidade cromossômica rara, transmitida por herança autossômica recessiva. Caracteriza-se por deficiência de crescimento pré e pós-natal, microcefalia, hipoplasia malar, eritema telangiectásico em face e comprometimento do sistema imunológico. Os pacientes com SB apresentam predisposição aumentada para o desenvolvimento de neoplasias em idade precoce, sendo esta, a principal causa de óbito. No estudo citogenético observa-se aumento de quebras cromossômicas espontâneas e trocas entre cromátides irmãs (TCI), que é utilizada como marcador diagnóstico para a SB. Essas alterações são causadas por um defeito no mecanismo de reparo do DNA, decorrente de uma mutação no gene BLM. Objetivos: Realizar o estudo citogenético de trocas entre cromátides irmãs para o diagnóstico de pacientes com suspeita clínica de SB; caracterizar os aspectos clínicos e avaliar a evolução de pacientes com SB. Métodos: Foram estudados nove pacientes (4 M e 5 F) pertencentes a oito famílias com suspeita clínica de SB utilizando preparações cromossômicas tratadas com 5- bromo-2-desoxiuridina (BrdU) e coloração Hoechst - Giemsa para visualização diferencial das cromátides irmãs e análise de freqüência de TCI. Resultados e Discussão: Todos os pacientes foram positivos para a pesquisa de TCI cuja freqüência variou de 45,2 a 61,3 TCI/metáfase. A idade dos pacientes ao diagnóstico variou de 1a1m até 11a (média de 4a6m). O principal motivo do encaminhamento foi o déficit de crescimento e apenas um paciente foi encaminhado por apresentar lesões cutâneas. Todos apresentaram deficiência de crescimento pré e pós-natal, microcefalia e hipoplasia malar. O eritema esteve presente em 8/9 pacientes. Manchas café-au-lait e/ou manchas hipocrômicas foram observadas em sete pacientes. Um paciente apresentou agenesia unilateral da fíbula, encurtamento da tíbia e agenesia do 5° artelho, associado à hipoplasia renal. As infecções de repetição foram relatadas em 8/9 pacientes, sendo principalmente pneumonia e diarreia. Deficiência de imunoglobulinas foi observada em 6/9 pacientes, principalmente: deficiência de IgG (3/6), de IgA (2/6) e de IgM (1/6). A consanguinidade entre os pais foi encontrada em 4/8 famílias, apenas uma família apresentou dois filhos afetados. Duas pacientes (2/9) evoluíram com tumor de Wilms (TW), uma aos 3a6m e a outra aos 3a11m. Houve recidiva em uma paciente que faleceu aos cinco anos. A outra paciente evoluiu bem e atualmente está com 20 anos. Conclusão: O diagnóstico da SB deve ser feito precocemente baseado na avaliação clínica. A pesquisa citogenética de TCI, que é de baixo custo e fácil aplicação, é fundamental para a confirmação diagnóstica. A freqüência aumentada para o desenvolvimento de neoplasias em idade precoce, alerta para um rastreamento das neoplasias mais comuns como linfoma, leucemia e tumor de Wilms. / Introduction: Bloom syndrome (BS) is a rare chromosomal instability syndrome, transmitted by autosomal recessive inheritance. It´s characterized by pre and postnatal growth deficiency, microcephaly, malar hypoplasia, telangiectatic erythema on the face and impaired immune system. BS patients present an increased predisposition to develop cancer at early age, which is the main cause of death. In the cytogenetic exam is observed an increase of spontaneous breaks and sister chromatid exchange (SCE) that is used as a diagnostic biomarker for the BS. These changes are caused by a defect in DNA repair mechanism, due to mutations in the BLM gene. Objectives: Perform the cytogenetic study of sister chromatid exchange for the clinical diagnosis of BS patients; to characterize the clinical aspects and assess the follow-up of patients. Methods: Nine patients (4 M, 5 F) from eight families with clinical diagnoses of BS were studied using standard chromosome preparations treated with 5-bromo-2-deoxyuridine (BrdU) and Hoechst-Giemsa differential staining for visualization and analysis of frequency of SCE. Results and Discussion: All patients were positive for the presence of SCE with the frequency ranged from 45.2 to 61.3 SCE/metaphase. The age at diagnosis ranged from 1y1mo to 11y (mean 4y6mo). The main reason for referral was growth deficit except one due to skin lesions. All patients presented pre and post-natal growth deficiency, microcephaly and malar hypoplasia. The erythema was present in 8/9 patients. Cafe-au-lait spots and/or hypochromic spots were observed in seven patients. One patient had unilateral agenesis of the fibula, shortening tibia and agenesis of the 5th toe associated with renal hypoplasia. The recurrent infections were reported in 8/9 patients, mainly pneumonia and diarrhea. Immunoglobulin deficiency was observed in 6/9 patients such as IgG (3/6), IgA (2/6) and IgM (1/6). The parental consanguinity was found in 4/8 families, one family had two affected. Two patients (2/9) developed Wilms tumor (WT), one at 3y6mo and another at 3y11mo. There was recurrence in one patient who died at five years. The other patient is well at 20 years old. Conclusion: The diagnosis of BS should be done early based in clinical findings. The cytogenetic for SCE exam is essential for diagnostic confirmation, which is low cost and easy application. The screening for the most common malignancies such as lymphoma, leukemia and WT must be done due to increased predisposition for cancer development at an early age

Introdução: A síndrome de Bloom (SB) é uma síndrome de instabilidade cromossômica rara, transmitida por herança autossômica recessiva. Caracteriza-se por deficiência de crescimento pré e pós-natal, microcefalia, hipoplasia malar, eritema telangiectásico em face e comprometimento do sistema imunológico. Os pacientes com SB apresentam predisposição aumentada para o desenvolvimento de neoplasias em idade precoce, sendo esta, a principal causa de óbito. No estudo citogenético observa-se aumento de quebras cromossômicas espontâneas e trocas entre cromátides irmãs (TCI), que é utilizada como marcador diagnóstico para a SB. Essas alterações são causadas por um defeito no mecanismo de reparo do DNA, decorrente de uma mutação no gene BLM. Objetivos: Realizar o estudo citogenético de trocas entre cromátides irmãs para o diagnóstico de pacientes com suspeita clínica de SB; caracterizar os aspectos clínicos e avaliar a evolução de pacientes com SB. Métodos: Foram estudados nove pacientes (4 M e 5 F) pertencentes a oito famílias com suspeita clínica de SB utilizando preparações cromossômicas tratadas com 5- bromo-2-desoxiuridina (BrdU) e coloração Hoechst - Giemsa para visualização diferencial das cromátides irmãs e análise de freqüência de TCI. Resultados e Discussão: Todos os pacientes foram positivos para a pesquisa de TCI cuja freqüência variou de 45,2 a 61,3 TCI/metáfase. A idade dos pacientes ao diagnóstico variou de 1a1m até 11a (média de 4a6m). O principal motivo do encaminhamento foi o déficit de crescimento e apenas um paciente foi encaminhado por apresentar lesões cutâneas. Todos apresentaram deficiência de crescimento pré e pós-natal, microcefalia e hipoplasia malar. O eritema esteve presente em 8/9 pacientes. Manchas café-au-lait e/ou manchas hipocrômicas foram observadas em sete pacientes. Um paciente apresentou agenesia unilateral da fíbula, encurtamento da tíbia e agenesia do 5° artelho, associado à hipoplasia renal. As infecções de repetição foram relatadas em 8/9 pacientes, sendo principalmente pneumonia e diarreia. Deficiência de imunoglobulinas foi observada em 6/9 pacientes, principalmente: deficiência de IgG (3/6), de IgA (2/6) e de IgM (1/6). A consanguinidade entre os pais foi encontrada em 4/8 famílias, apenas uma família apresentou dois filhos afetados. Duas pacientes (2/9) evoluíram com tumor de Wilms (TW), uma aos 3a6m e a outra aos 3a11m. Houve recidiva em uma paciente que faleceu aos cinco anos. A outra paciente evoluiu bem e atualmente está com 20 anos. Conclusão: O diagnóstico da SB deve ser feito precocemente baseado na avaliação clínica. A pesquisa citogenética de TCI, que é de baixo custo e fácil aplicação, é fundamental para a confirmação diagnóstica. A freqüência aumentada para o desenvolvimento de neoplasias em idade precoce, alerta para um rastreamento das neoplasias mais comuns como linfoma, leucemia e tumor de Wilms. / Introduction: Bloom syndrome (BS) is a rare chromosomal instability syndrome, transmitted by autosomal recessive inheritance. It´s characterized by pre and postnatal growth deficiency, microcephaly, malar hypoplasia, telangiectatic erythema on the face and impaired immune system. BS patients present an increased predisposition to develop cancer at early age, which is the main cause of death. In the cytogenetic exam is observed an increase of spontaneous breaks and sister chromatid exchange (SCE) that is used as a diagnostic biomarker for the BS. These changes are caused by a defect in DNA repair mechanism, due to mutations in the BLM gene. Objectives: Perform the cytogenetic study of sister chromatid exchange for the clinical diagnosis of BS patients; to characterize the clinical aspects and assess the follow-up of patients. Methods: Nine patients (4 M, 5 F) from eight families with clinical diagnoses of BS were studied using standard chromosome preparations treated with 5-bromo-2-deoxyuridine (BrdU) and Hoechst-Giemsa differential staining for visualization and analysis of frequency of SCE. Results and Discussion: All patients were positive for the presence of SCE with the frequency ranged from 45.2 to 61.3 SCE/metaphase. The age at diagnosis ranged from 1y1mo to 11y (mean 4y6mo). The main reason for referral was growth deficit except one due to skin lesions. All patients presented pre and post-natal growth deficiency, microcephaly and malar hypoplasia. The erythema was present in 8/9 patients. Cafe-au-lait spots and/or hypochromic spots were observed in seven patients. One patient had unilateral agenesis of the fibula, shortening tibia and agenesis of the 5th toe associated with renal hypoplasia. The recurrent infections were reported in 8/9 patients, mainly pneumonia and diarrhea. Immunoglobulin deficiency was observed in 6/9 patients such as IgG (3/6), IgA (2/6) and IgM (1/6). The parental consanguinity was found in 4/8 families, one family had two affected. Two patients (2/9) developed Wilms tumor (WT), one at 3y6mo and another at 3y11mo. There was recurrence in one patient who died at five years. The other patient is well at 20 years old. Conclusion: The diagnosis of BS should be done early based in clinical findings. The cytogenetic for SCE exam is essential for diagnostic confirmation, which is low cost and easy application. The screening for the most common malignancies such as lymphoma, leukemia and WT must be done due to increased predisposition for cancer development at an early age

Durante a divisão celular as Aurora-quinases (AURKA e AURKB) participam da formação e controle das fibras do fuso mitótico enquanto as isoformas proteicas (DIDO1, DIDO2 e DIDO3), originadas do splicing alternativo do gene DIDO, auxiliam na junção dos microtúbulos aos cinetócoros. Portanto, ambas são relevantes na regulação do ciclo celular. Interessantemente, a superexpressão (ou o ganho de função) das AURKs ou a baixa expressão (ou perda de função) das isoformas de DIDO estão ambos associados com amplificação dos centrossomos e à instabilidade cromossômica (CIN), com consequente aneuploidia. Dentre as doenças hematológicas com registros de CIN, a leucemia linfoide crônica (LLC) pode apresentar amplificação dos centrossomos e alteração nos níveis de expressão das AURKs acarretando aneuplodias. Apesar disso, não existem estudos avaliando a potencial associação destes genes com CIN na LLC. Avaliando seus níveis de expressão gênica em amostras de LLC de pacientes com ou sem aberrações cromossômicas, mostramos que o aumento dos níveis de AURKA e AURKB e, inversamente, a redução dos níveis das variantes de DIDO, são significativamente associados com ganhos cromossômicos e com aumento da contagem de glóbulos brancos (WBC). Claramente, amostras de LLC sem qualquer anormalidade citogenética apresentam níveis de expressão semelhantes às amostras que contêm aberrações não-numéricas. O achado de que níveis de expressão de AURKs e variantes de DIDO são completamente opostos, mostrando um padrão discreto de inter-relação, levou-nos a investigar o potencial mecanismo regulatório por trás disso. Tendo em vista que outros, anteriormente, mostraram que o cluster oncogênico miR-17~92 é significativamente hiper-regulado em células de pacientes com LLC purificadas expressando genes IGHV não mutados (em comparação com células mutadas de pacientes) e, que o miR-17 é expresso em níveis significativamente mais elevados em células IGHV não mutadas ou ZAP-70 positivas (mau prognóstico geralmente associada à CIN), resolvemos investigar o potencial de regulação negativa dos microRNAs deste cluster sobre as variantes de DIDO. Além disso, com base no mecanismo regulatório já descrito pelo qual a superexpressão de AURKA induz a transcrição do cluster miR-17~92, mediada por E2F1 (com uma correlação entre as expressões de ambas as proteínas em diferentes tipos de câncer), decidimos investigar este eixo regulatório em LLC. Notavelmente, todas as variantes de DIDO apresentam-se preditas como fortes alvos de vários microRNAs deste cluster oncogênico. Mostramos, então, que amostras de LLC com baixa expressão de DIDO, além dos já mencionados níveis elevados de AURK, exibiram níveis significativamente mais elevados do fator de transcrição E2F1 e de seu alvo transcricional, o transcrito primário do miR-17~92 (MIR17HG). Além disso, por meio do uso da linhagem de celular NTERA-2, como modelo experimental, mostramos que o siRNA nocaute para AURKA (nos níveis transcricional e proteico, como confirmado por qPCR e western blot) é acompanhada por uma significativa redução de E2F1 e também de MIR17HG. Ainda, a transfecção de células NTERA-2 com sintéticos microRNAs miméticos do cluster miR-17~92 (ou seja, 19a-miR, miR-20a e miR-92a) resultou em uma clara e significativa redução dos níveis de transcrição de todas as variantes de DIDO. Por fim, a inibição do siRNA especifico para a variante DIDO3 (mas não às outras variantes) levou a uma redução significativa dos níveis de transcrição de todas as variantes de DIDO, indicando um mecanismo adicional contribuindo para a downregulação dos transcritos de DIDO. Ao todo, nossos resultados demonstram a existência de um potencial mecanismo regulatório interconectado entre AURK e DIDO, associado à CIN e maior contagem de WBC na LLC. Mais importante, os níveis de expressão elevada de AURKs e os baixos níveis associados das variantes de DIDO são especificamente relacionados com anormalidades citogenéticas apresentando ganhos cromossomais, com destaque para o mecanismo celular específico, subjacente à CIN, observado neste grupo distinto LLC. Dado o papel central da CIN na gênese e progressão do câncer, esses achados provavelmente terão um impacto importante no prognóstico ou tratamento da LLC. / During cell cycle division Aurora kinases (AURKA and AURKB) participate in the formation and control of mitotic spindle fibers, while, protein isoforms (DIDO1, DIDO2 and DIDO3), derived by alternative splicing of the DIDO gene, assist at the junction of microtubules to kinetochores. Thus, both are relevant to cell cycle maintenance. Interestingly, overexpression (or gain of function) of AURKs or low expression (or loss of function of DIDO) are both associated with centrosomal amplification and chromosomal instability (CIN), leading to aneuploidy. Among hematological diseases with CIN records, chronic lymphocytic leukemia (CLL) can display centrosome amplification and changes in AURKs expression levels leading to aneuploidy. Despite this, there are no studies evaluating the potential association of these genes with CIN in CLL. By evaluating their gene expression levels in CLL samples from patients with or without chromosomal aberrations, we show that increased levels of AURKA and AURKB and, conversely, reduced levels of DIDO variants, are both significantly associated with chromosomal gains and with increased white blood cell (WBC) counts. Clearly, CLL samples without any cytogenetic abnormality had expression levels similar to samples mostly harboring non-numerical aberrations. The finding that the expression levels of AURKs and DIDO variants are completely opposed, showing a discrete inter-related pattern, led us to investigate the potential regulatory mechanism behind this. Given that other have previously shown that the oncogenic miR-17~92 cluster is significantly upregulated in purified CLL patient cells expressing unmutated IGHV genes (as compared to mutated patient cells), and that miR-17 is expressed at significantly higher levels in unmutated or ZAP-70 high cases (bad prognostic cases generally associated with chromosomal instability), we investigated the potential negative regulation of DIDO variants by microRNAs from this cluster. In addition, based on the already described regulatory mechanism by which AURKA overexpression induces the E2F1-mediated transcription upregulation of the miR-17~92 cluster (with an observed expression correlation of both proteins in cancer specimens); we decided to investigate this regulatory axis in CLL. Notably, we found that all DIDO variants are predicted to be heavily targeted by several miRs of this oncogenic cluster. We show that CLL samples with low DIDO expression, in addition to the already mentioned AURK high levels, displayed significant higher levels of the transcription factor E2F1 and of its transcriptional target, the miR-17~92 primary transcript (MIR17HG). Moreover, by using the NTERA-2 cell line as a model, we show that siRNA knockdown of AURKA (at the transcript and protein level, as confirmed by qPCR and western blot) is accompanied by a striking significant reduction of E2F1 and also of MIR17HG. Furthermore, transfection of NTERA-2 cells with synthetic mimics of the miR-17~92 cluster (namely, miR-19a, miR-20a and miR-92a) results in a clear and significant reduction in the transcript levels of all DIDO variants. Finally, specific siRNA inhibition of the DIDO3 variant (but not the others) led to a significant reduction in the transcript levels of all DIDO variants, indicating an additional mechanism contributing to the downregulation of DIDO transcripts. Altogether, our results demonstrate the existence of a potential interconnected regulatory mechanism between AURK and DIDO, associated with CIN and higher WBC counts in CLL. More importantly, the high expression levels of AURKs and the associated low levels of DIDO variants are specifically associated with cytogenetic abnormalities presenting chromosomal gains, highlighting the specific cellular mechanism underlying the CIN observed in this distinct CLL group. Given the central role of CIN in cancer genesis and progression, these findings will likely have an important impact on prognosis or treatment of CLL.

Durante a divisão celular as Aurora-quinases (AURKA e AURKB) participam da formação e controle das fibras do fuso mitótico enquanto as isoformas proteicas (DIDO1, DIDO2 e DIDO3), originadas do splicing alternativo do gene DIDO, auxiliam na junção dos microtúbulos aos cinetócoros. Portanto, ambas são relevantes na regulação do ciclo celular. Interessantemente, a superexpressão (ou o ganho de função) das AURKs ou a baixa expressão (ou perda de função) das isoformas de DIDO estão ambos associados com amplificação dos centrossomos e à instabilidade cromossômica (CIN), com consequente aneuploidia. Dentre as doenças hematológicas com registros de CIN, a leucemia linfoide crônica (LLC) pode apresentar amplificação dos centrossomos e alteração nos níveis de expressão das AURKs acarretando aneuplodias. Apesar disso, não existem estudos avaliando a potencial associação destes genes com CIN na LLC. Avaliando seus níveis de expressão gênica em amostras de LLC de pacientes com ou sem aberrações cromossômicas, mostramos que o aumento dos níveis de AURKA e AURKB e, inversamente, a redução dos níveis das variantes de DIDO, são significativamente associados com ganhos cromossômicos e com aumento da contagem de glóbulos brancos (WBC). Claramente, amostras de LLC sem qualquer anormalidade citogenética apresentam níveis de expressão semelhantes às amostras que contêm aberrações não-numéricas. O achado de que níveis de expressão de AURKs e variantes de DIDO são completamente opostos, mostrando um padrão discreto de inter-relação, levou-nos a investigar o potencial mecanismo regulatório por trás disso. Tendo em vista que outros, anteriormente, mostraram que o cluster oncogênico miR-17~92 é significativamente hiper-regulado em células de pacientes com LLC purificadas expressando genes IGHV não mutados (em comparação com células mutadas de pacientes) e, que o miR-17 é expresso em níveis significativamente mais elevados em células IGHV não mutadas ou ZAP-70 positivas (mau prognóstico geralmente associada à CIN), resolvemos investigar o potencial de regulação negativa dos microRNAs deste cluster sobre as variantes de DIDO. Além disso, com base no mecanismo regulatório já descrito pelo qual a superexpressão de AURKA induz a transcrição do cluster miR-17~92, mediada por E2F1 (com uma correlação entre as expressões de ambas as proteínas em diferentes tipos de câncer), decidimos investigar este eixo regulatório em LLC. Notavelmente, todas as variantes de DIDO apresentam-se preditas como fortes alvos de vários microRNAs deste cluster oncogênico. Mostramos, então, que amostras de LLC com baixa expressão de DIDO, além dos já mencionados níveis elevados de AURK, exibiram níveis significativamente mais elevados do fator de transcrição E2F1 e de seu alvo transcricional, o transcrito primário do miR-17~92 (MIR17HG). Além disso, por meio do uso da linhagem de celular NTERA-2, como modelo experimental, mostramos que o siRNA nocaute para AURKA (nos níveis transcricional e proteico, como confirmado por qPCR e western blot) é acompanhada por uma significativa redução de E2F1 e também de MIR17HG. Ainda, a transfecção de células NTERA-2 com sintéticos microRNAs miméticos do cluster miR-17~92 (ou seja, 19a-miR, miR-20a e miR-92a) resultou em uma clara e significativa redução dos níveis de transcrição de todas as variantes de DIDO. Por fim, a inibição do siRNA especifico para a variante DIDO3 (mas não às outras variantes) levou a uma redução significativa dos níveis de transcrição de todas as variantes de DIDO, indicando um mecanismo adicional contribuindo para a downregulação dos transcritos de DIDO. Ao todo, nossos resultados demonstram a existência de um potencial mecanismo regulatório interconectado entre AURK e DIDO, associado à CIN e maior contagem de WBC na LLC. Mais importante, os níveis de expressão elevada de AURKs e os baixos níveis associados das variantes de DIDO são especificamente relacionados com anormalidades citogenéticas apresentando ganhos cromossomais, com destaque para o mecanismo celular específico, subjacente à CIN, observado neste grupo distinto LLC. Dado o papel central da CIN na gênese e progressão do câncer, esses achados provavelmente terão um impacto importante no prognóstico ou tratamento da LLC. / During cell cycle division Aurora kinases (AURKA and AURKB) participate in the formation and control of mitotic spindle fibers, while, protein isoforms (DIDO1, DIDO2 and DIDO3), derived by alternative splicing of the DIDO gene, assist at the junction of microtubules to kinetochores. Thus, both are relevant to cell cycle maintenance. Interestingly, overexpression (or gain of function) of AURKs or low expression (or loss of function of DIDO) are both associated with centrosomal amplification and chromosomal instability (CIN), leading to aneuploidy. Among hematological diseases with CIN records, chronic lymphocytic leukemia (CLL) can display centrosome amplification and changes in AURKs expression levels leading to aneuploidy. Despite this, there are no studies evaluating the potential association of these genes with CIN in CLL. By evaluating their gene expression levels in CLL samples from patients with or without chromosomal aberrations, we show that increased levels of AURKA and AURKB and, conversely, reduced levels of DIDO variants, are both significantly associated with chromosomal gains and with increased white blood cell (WBC) counts. Clearly, CLL samples without any cytogenetic abnormality had expression levels similar to samples mostly harboring non-numerical aberrations. The finding that the expression levels of AURKs and DIDO variants are completely opposed, showing a discrete inter-related pattern, led us to investigate the potential regulatory mechanism behind this. Given that other have previously shown that the oncogenic miR-17~92 cluster is significantly upregulated in purified CLL patient cells expressing unmutated IGHV genes (as compared to mutated patient cells), and that miR-17 is expressed at significantly higher levels in unmutated or ZAP-70 high cases (bad prognostic cases generally associated with chromosomal instability), we investigated the potential negative regulation of DIDO variants by microRNAs from this cluster. In addition, based on the already described regulatory mechanism by which AURKA overexpression induces the E2F1-mediated transcription upregulation of the miR-17~92 cluster (with an observed expression correlation of both proteins in cancer specimens); we decided to investigate this regulatory axis in CLL. Notably, we found that all DIDO variants are predicted to be heavily targeted by several miRs of this oncogenic cluster. We show that CLL samples with low DIDO expression, in addition to the already mentioned AURK high levels, displayed significant higher levels of the transcription factor E2F1 and of its transcriptional target, the miR-17~92 primary transcript (MIR17HG). Moreover, by using the NTERA-2 cell line as a model, we show that siRNA knockdown of AURKA (at the transcript and protein level, as confirmed by qPCR and western blot) is accompanied by a striking significant reduction of E2F1 and also of MIR17HG. Furthermore, transfection of NTERA-2 cells with synthetic mimics of the miR-17~92 cluster (namely, miR-19a, miR-20a and miR-92a) results in a clear and significant reduction in the transcript levels of all DIDO variants. Finally, specific siRNA inhibition of the DIDO3 variant (but not the others) led to a significant reduction in the transcript levels of all DIDO variants, indicating an additional mechanism contributing to the downregulation of DIDO transcripts. Altogether, our results demonstrate the existence of a potential interconnected regulatory mechanism between AURK and DIDO, associated with CIN and higher WBC counts in CLL. More importantly, the high expression levels of AURKs and the associated low levels of DIDO variants are specifically associated with cytogenetic abnormalities presenting chromosomal gains, highlighting the specific cellular mechanism underlying the CIN observed in this distinct CLL group. Given the central role of CIN in cancer genesis and progression, these findings will likely have an important impact on prognosis or treatment of CLL.