Objective: Two chromosomal instability (CIN) pathways are described in glioblastoma multiforme (GBM), type 1 and type 2, which can be observed in up to 70% of the cases. Microsatellite instability (MSI) plays a pathogenic role in sporadic cancers such as colon, gastric and endometrial carcinomas with deficient mismatch repair (MMR). We aimed to perform a comprehensive analysis of the relationship between CIN and MSI mechanisms in sporadic glioblastomas. Methods: 129 GBMs were examined (109 newly diagnosed and 20 relapses) investigating MSI, immunohistochemical expression of MMR proteins as well as sequencing and promoter methylation of hMLH1. We characterized the molecular changes frequently correlated with CIN in MSI+ GBMs and compared them with 26 microsatellite-stable tumors. Results: Low-level MSI was observed in 11 of 129 (8.5%) cases and was higher in relapses than in primary GBMs (25 vs. 5.5%, p = 0.027). High-level MSI was not found in any case. A deficient expression of MLH1 and PMS2 without hMLH1 inactivation was observed only in one giant cell GBM. 55% of the MSI+ GBMs showed a profile which did not correspond to one of the known CIN pathways. An inverse association was observed between MSI and mutations of both p53 and PTEN. Conclusions: Our data suggest that CIN and MSI contribute to the genomic instability in GBMs via independent pathways. Since MSI was significantly more frequent in relapses, it might play a role in the malignant progression of GBM. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Recent studies from our laboratory on the aromatic amine carcinogen, 4-aminobiphenyl (ABP) have shown a significantly lower prevalence of ABP-induced liver tumors in male mice lacking the N-acetyltransferases, and a dramatically lower prevalence in females than in males, but no association of tumor prevalence with strain or sex differences in levels of acute ABP-induced DNA damage. This thesis aimed to investigate the possible involvement of acute cytotoxic effects of ABP in the development of a tumor-promoting inflammatory environment. We found that wild-type male mice showed higher acute hepatotoxicity to ABP, as well as, a possible trend towards higher serum levels of the pro-inflammatory cytokine interleukin 6. This correspondence between acute ABP cytotoxicity and inflammatory response with ultimate tumor growth is consistent with a model whereby ABP not only initiates cells by damaging DNA but also promotes tumor growth in a gender-selective fashion that may be governed by gonadal hormone influences.

Recent studies from our laboratory on the aromatic amine carcinogen, 4-aminobiphenyl (ABP) have shown a significantly lower prevalence of ABP-induced liver tumors in male mice lacking the N-acetyltransferases, and a dramatically lower prevalence in females than in males, but no association of tumor prevalence with strain or sex differences in levels of acute ABP-induced DNA damage. This thesis aimed to investigate the possible involvement of acute cytotoxic effects of ABP in the development of a tumor-promoting inflammatory environment. We found that wild-type male mice showed higher acute hepatotoxicity to ABP, as well as, a possible trend towards higher serum levels of the pro-inflammatory cytokine interleukin 6. This correspondence between acute ABP cytotoxicity and inflammatory response with ultimate tumor growth is consistent with a model whereby ABP not only initiates cells by damaging DNA but also promotes tumor growth in a gender-selective fashion that may be governed by gonadal hormone influences.

Le virus de la maladie de Marek (MDV) est un alphaherpesvirus à l’origine du développement de lymphomes chez les volailles. A ce jour, l’origine du développement des tumeurs induites par le MDV est encore peu connue malgré l’identification de plusieurs oncoprotéines virales. Au vu de l’implication des dommages à l’ADN dans la pathogenèse de plusieurs virus notamment les herpesvirus, mon projet de thèse avait pour objectif de déterminer l’impact de l’apparition de lésions d’ADN sur le cycle viral du MDV. Nous avons montré que l’infection cytolytique de MDV s’accompagne d’une accumulation de lésions dans l’ADN cellulaire de lymphocytes et cellules fibroblastiques de poulet. La phase de latente de l’infection MDV n’affecte pas l’intégrité de l’ADN des lymphocytes alors que la réactivation du virus conduit à l’apparition de lésions d’ADN. De plus, en utilisant une approche originale in vivo, nous avons confirmé le rôle essentiel de la protéine virale VP22 dans l’induction de ces dommages. Nous avons pu établir que l’induction des dommages à l’ADN au cours de l’infection et/ou la réponse cellulaire engendrée sont non seulement favorables à la réplication du virus mais également que l’apparition de ces lésions est étroitement liée au pouvoir oncogène du MDV. / Marek’s disease virus (MDV) is an alphaherpesvirus responsible of T lymphoma in chickens. Mechanisms leading to cellular transformation mediated by MDV are still incompletely understood. DNA damage and the associated cellular response participate actively in the life cycle of viruses, especially herpesviruses. Here, we aimed at deciphering the role of DNA damages in MDV pathogenesis. We show that MDV lytic infection leads to DNA lesions in lymphocytes and fibroblasts of chickens. Moreover, we demonstrated that MDV latently-infected lymphocytes exhibits undamaged DNA whereas MDV reactivation leads to an onset of DNA lesions. Also, using an original in vivo approach, we objectified the role of VP22 on DNA damages induction. Finally, we established that DNA damage and/or the associated DNA damage response are not only benefic to MDV replication but also that the DNA lesions onset might participate to MDV oncogenicity.

A galectina-1 (LGALS1) participa em diferentes etapas da neoplasia, mas sua função na leucemia mielóide crônica (LMC) é desconhecida. Neste trabalho, a expressão etópica de BCR-ABL selvagem, mas não de BCR-ABL quinase deficiente, em linhagens celulares hematopoéticas, resultou em aumento de LGALS1. O efeito foi revertido com a inibição da tirosina quinase pelo mesilato de imatinibe. Este resultado indicou que a galectina-1 é modulada pela atividade tirosina-quinase de BCR-ABL. Em pacientes com LMC, a maior expressão de LGALS1 foi correlacionada a altos níveis de BCR-ABL, progressão da doença e a um tempo de sobrevida menor. Adicionalmente, as células K562 com LGALS1 inibida por RNA de interferência exibiram crescimento mais lento do que as células K562 com LGALS1 intacta, em camundongos nude. Portanto, o pior prognóstico de pacientes com altos níveis de galectina-1 sugere um efeito cooperativo de galectina-1 na tumorigênese de BCR-ABL reforçando o conceito de que a galectina-1 é um forte candidato para intervenção terapêutica na LMC. / Galectin-1 (LGALS1) participates in different steps of neoplasia but its role in chronic myeloid leukemia (CML) is unknown. In this work, ectopic expression of wild-type but not kinase-deficient BCR-ABL in different hematopoietic cells resulted in LGALS1 upregulation. Tyrosine-kinase inhibition by imatinib mesylate reversed this effect. This result indicate that galectin-1 is modulated by tyrosine kinase activity. In CML patients, the elevated expression of LGALS1 was correlated with high BCR-ABL levels, disease progression and shorter survival time. Additionally, in nude mice, LGALS1-deficient K562 cells obtained by RNA interference were less efficient in tumor formation than control K562 cells. Therefore, the worst prognosis in patients bearing high LGALS1 levels suggests a cooperative role for galectin-1 in BCR-ABL-positive leukemia and support the concept that galectin-1 is a strong candidate for CML therapeutic intervention.

Mdm2 and Mdm4 are critical negative regulators of the p53 tumor suppressor. Mdm4-null mutants are severely anemic and exhibit impaired proliferation of the fetal liver erythroid lineage cells. This phenotype may indicate a cell-intrinsic function of Mdm4 in erythropoiesis. In contrast, red blood cell count was nearly normal in mice engineered to express low levels of Mdm2, suggesting that Mdm2 might be dispensable for red cell production. In the first part of the thesis, we further explore the tissue-specific functions of Mdm2 and Mdm4 in the erythroid lineage by crossing the conditional Mdm4 and Mdm2 alleles to an erythroid-specific-cre (EpoRGFP-Cre ) knock-in allele. Our data show that Mdm2 is required for rescuing erythroid progenitors from p53-mediated apoptosis during primitive erythropoiesis. In contrast, Mdm4 is only required for the high erythropoietic rate during embryonic definitive erythropoiesis. Thus, in this particular cellular context, interestingly, Mdm4 only contributes to p53 regulation at a specific phase of the differientation program.<p><p>Moreover, a large body of evidence indicates that aberrant expression of either MDM2 or MDM4 impairs p53 tumor suppression function and consequently favors tumor formation. Overexpression of MDM2 was observed in 10% of 8000 human cancers from various sites, including lung or stomach, and MDM4 was found amplified and/or overexpressed in 10-20% of over 800 diverse tumors including lung, colon, stomach and breast cancers. Remarkably, selective MDM4 amplification occurs in about 65% of human retinoblastomas. In contrast, MDM2 amplifications are relatively rare (about 5%) in retinoblastomas, indicating that depending on the tumor context (cell type, initiating oncogene, …), MDM4, rather than MDM2, overexpression might be selected for as a more efficient mean of suppression of p53 function. As part of a large effort to better understand why different cell types require distinct combinations of mutations to form tumours, we will examine the molecular basis for selective up-regulation of Mdm4 in retinoblastomas. In this context, we have successfully generated 2 conditional transgenic mouse lines expressing either mycMdm2 or mycMdm4 driven by the PCAGGs promoters in the ROSA26 locus. Since a cassette containing a floxed transcriptional stop element is inserted upstream of the transgenes, we can achieve tissue-specific expression and spatio-temporal regulation of the transgenes by using different Cre and CreER. By the use of N-terminal myc-tag fused with the transgenes, we are able to compare the expression levels of the transgenes. Finally, due to C-terminal IRES-GFP element, we can easily identify transgene expressing cells. One of our aims is to use this Mdm4 conditional transgenic mouse line as the first, non-chimeric, mouse model of retinoblastoma that can be used as an appropriate preclinical model to improve treatment of this disease.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

The Ste20-like kinase SLK plays a pivotal role in cell migration and focal adhesion turnover. SLK activity is regulated by the LIM domain-binding proteins Ldb1/2. In addition to playing role in tumor initiation and progression, these proteins have been demonstrated to interact with LMO4. Therefore, this project assessed the ability of LMO4 to interact and regulate SLK activity. Results show that LMO4 can directly bind to SLK and activate its kinase activity. LMO4 can be co-precipitated with SLK following the induction of cell migration by scratch wounding. Cre deletion of LMO4 inhibits cell migration and SLK activation, and impairs Ldb1 and SLK recruitment to the leading edge of migrating cells. Src/Yes/Fyn-deficient cells (SYF) express very low levels of LMO4 and do not recruit SLK to the leading edge. Src-family kinase inhibition impairs SLK recruitment to the leading edge, suggesting that both expression of LMO4 and the recruitment of SLK to the leading edge require c-Src activity. In conclusion, cell migration and activation of SLK requires its recruitment to the leading edge by LMO4 in a Src-dependent manner. This study also investigated whether LMO4 deletion through MMTV-Cre-driven excision would impair mammary tumorigenesis in a PyMT mouse model of breast cancer. No difference in Overall Survival was observed between animals with and without LMO4 expression. Western blot analysis and IHC showed that tumors expressed LMO4 protein in animals genotyped as Cre-positive. This result suggests that expression of LMO4 is required for tumor initiation in the PyMT model of murine mammary carcinoma. This project has established a novel cytosolic role for the transcriptional co-activator LMO4 and validated it’s involvement in the regulation of SLK and cell migration. This pathway may provide a novel therapeutic strategy as LMO4 appears to be critical to the initiation and progression of breast cancer.

The content of the thesis is a description of intracellular processes responsible for cell cycle regulation and reactions of cells to external and internal stimuli. Thoroughly described are important signaling pathways with appropriate methods, which can be used to simulate them in silico. From these cellular processes, a cell cycle model is created and implemented in a tool programmed in C ++ with OpenGL used for visualization. The model is then tested for various cell processes including HeLa cells growth. Finally, the results are compared with the behavior of living cells.

Objective: Two chromosomal instability (CIN) pathways are described in glioblastoma multiforme (GBM), type 1 and type 2, which can be observed in up to 70% of the cases. Microsatellite instability (MSI) plays a pathogenic role in sporadic cancers such as colon, gastric and endometrial carcinomas with deficient mismatch repair (MMR). We aimed to perform a comprehensive analysis of the relationship between CIN and MSI mechanisms in sporadic glioblastomas. Methods: 129 GBMs were examined (109 newly diagnosed and 20 relapses) investigating MSI, immunohistochemical expression of MMR proteins as well as sequencing and promoter methylation of hMLH1. We characterized the molecular changes frequently correlated with CIN in MSI+ GBMs and compared them with 26 microsatellite-stable tumors. Results: Low-level MSI was observed in 11 of 129 (8.5%) cases and was higher in relapses than in primary GBMs (25 vs. 5.5%, p = 0.027). High-level MSI was not found in any case. A deficient expression of MLH1 and PMS2 without hMLH1 inactivation was observed only in one giant cell GBM. 55% of the MSI+ GBMs showed a profile which did not correspond to one of the known CIN pathways. An inverse association was observed between MSI and mutations of both p53 and PTEN. Conclusions: Our data suggest that CIN and MSI contribute to the genomic instability in GBMs via independent pathways. Since MSI was significantly more frequent in relapses, it might play a role in the malignant progression of GBM. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Von Hippel-Lindau (VHL) syndrome is an autosomal dominant predisposition to cancer in neurological tissues, the kidneys, adrenal glands, pancreas, and liver, including neurological hemangioblastoma (HB), pheochromocytoma (PCC), pancreatic neuroendocrine tumors (PNET), pancreatic and renal cysts, and clear cell renal cell carcinoma (ccRCC). The disease process follows Knudson's two-hit model, requiring spontaneous loss or mutation of a normal VHL tumor suppressor allele to induce expression of the disease. VHL syndrome principally involves dysregulation of oxygen sensing pathways including the Hypoxia Inducible Factor (HIF)-Vascular Endothelial Growth Factor-A (VEGF-A) and HIF-Erythropoietin (EPO) pathways. RNA sequencing (RNA-Seq) data from our previously published experiments revealed a potentially novel VEGF-A splice variant with excision of the VEGF Receptor-1 (VEGFR-1)/Flt-1 binding domain, rendering this isoform resistant to native down-regulation. Additionally, phenotypic changes were observed in adult VHL mutant mice, specifically very red appearing extremities with prominently visible vasculature. In order to determine the etiology of this phenotype, we observed red blood cell count, Epo gene expression levels, and arterialization of the blood vessels in these experimental mice as compared to littermate controls. Current research into the VEGF-A isoform is ongoing in the lab, and preliminary evidence for the etiology of the apparent chronic erythema phenotype is inconclusive due to lack of experimental replicates due to COVID-19 quarantine orders. / Master of Science / Von Hippel-Lindau (VHL) syndrome is characterized by cancer development primarily in the brain and spinal cord, kidneys, adrenal glands, pancreas, and liver. VHL syndrome involves mutations which render the VHL gene dysfunctional. Since the VHL gene's normal role is one of preventing cancer development, sensing oxygen levels, and impacting blood vessel development, it follows that the loss of this gene results in tumor development with a rich blood vessel network. One of the downstream effectors of this process is a signaling molecule called Vascular Endothelial Growth Factor-A (VEGF-A). Our lab found a unique variant of VEGF-A, which may be overactive in the body in the setting of VHL disease. Additionally, we noted that our VHL mutant mice turned very red, and we sought to identify the biological cause of this phenomenon. In order to determine the cause of this redness, we studied red blood cell counts and their regulatory gene (Erythropoietin, EPO), as well as potential blood vessel abnormalities using high-power microscopy.