Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michal Rataj Supervisor: Doc. PharmDr. Martin Beránek Ph.D. Title of diploma thesis: Genetic polymorphism in the NBS1 gene for diagnosis and treatment of patients with cervical carcinoma The aim of this diploma thesis is to find optimal methods for screening of mutation 657del5 and estimate frequency of heterozygotes and homozygotes for the mutation 657del5 in population of the Czech republic. In the first section of the theoretical part is comprehensively pointed out the effect of factors affecting the integrity of genetic information and the formation of mutations in DNA. On the contrary, the second section devotes to the ability of cells to respond to this damage. In detail, the thesis devotes to the NBS1 gene and its product nibrin. In the complex MRE11/Rad50/NBN nibrin is an important member of the mechanisms of repair of double strand breaks NHEJ (non-homologous end joining) and HR (homologous recombination). The thesis is focused on nibrin and its functions, but also to mutations that prevent these functions and causes genetic disease Nijmegen breakage syndrome. Nibrin is translated from the sequence of the NBS1 gene. Gene NBS1 appears in population with several various...

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Telomerase is a ribonucleoprotein complex that catalyses telomeric repeat addition at the ends of chromosomes. The catalytic subunit, hTERT, acts as a key determinant for telomerase activity control; the induction of hTERT expression is required for telomerase activity. hTERT participates in cellular immortalization and is elevated in certain malignant tissues. Several tumours exhibit telomerase activity, which contributes to the infinite proliferation capacity that promotes tumour progression. Renal cell carcinoma (RCC) represents 2% of all adult malignancies and has a high mortality rate. The WHO classifies RCC into several sub-types based on cytogenetic aberrations and morphological features; the most prevalent sub-types are clear cell (ccRCC), papillary (pRCC), and chromophobe RCC (chRCC). The aims of this thesis were to study the expression patterns of various signalling molecules, to elucidate the functional links among them, and to define the roles of these signalling molecules in the regulation of hTERT gene expression and telomerase activity in RCC. The first paper included in this thesis revealed mRNA overexpression of DJ-1 (a PTEN inhibitor), cMyc, and hTERT in clinical ccRCC samples compared to tumour-free kidney cortex tissues. Significant, positive correlations were detected for DJ-1, cMyc, and hTERT mRNA levels in ccRCC, but not in pRCC. In vitro knockdown of DJ-1 by siRNA in ccRCC cells induced downregulation of p-Akt, cMyc, hTERT, and telomerase activity. Forced overexpression of DJ-1 in an ovarian carcinoma cell line was followed by increased hTERT promoter activity, which appeared to be dependent on cMYC binding to the promoter. Collectively, the in vitro studies verified a functional link among DJ-1, cMyc, and hTERT as implied in the clinical ccRCC samples. The second paper included in this thesis demonstrated overexpression of NBS1 mRNA levels in ccRCC compared to the kidney cortex. NBS1 mRNA levels exhibited significant, positive correlations with DJ-1, cMyc, and S phase, but not with hTERT. In vitro experiments suggested that DJ-1 could regulate NBS1 gene expression. The role of the hTERT transcriptional repressor WT1 in RCC was evaluated in the third paper included in this thesis. ccRCC samples displayed low WT1 mRNA levels compared to kidney cortex samples. Interestingly, WT1 expression was negatively associated with hTERT and cMyc both of which were elevated in ccRCC. Forced overexpression of WT1 isoforms in a ccRCC cell line increased the expression of several negative transcriptional regulators of hTERT and diminished the expression of hTERT positive regulators. In consequence, hTERT mRNA levels and telomerase activity were reduced. Chromatin immunoprecipitation verified direct binding of WT1 to the cMyc, Smad3, and hTERT promoters. Taken together, these data suggested that in ccRCC, WT1 affects hTERT at the transcriptional level via a combined effect on both positive and negative regulators. In conclusion, DJ-1 can regulate hTERT and telomerase activity through the PI3K pathway encompassing PTEN, NBS1, p-Akt, and cMyc in ccRCC, but not in pRCC. WT1 negatively regulates hTERT and telomerase activity directly and indirectly through multiple pathways in ccRCC.

Histone deacetylases (HDACs) are a family of enzymes whose functions have been overwhelmingly associated with gene expression and chromatin dynamics by modifying the histone tails. In recent years, intensive studies have demonstrated that many non-histone proteins also could serve as substrates for HDACs. And their functions and activities have been found to be regulated by posttranslational acetylation on the ε-amino group of lysines. Here, we report that two DNA repair factors including NBS1 (Nijmegen breakage syndrome 1) and ATDC (Ataxia-Telangiectasia Group D Complementing) are acetylated proteins. SIRT1 could maintain NBS1 in a hypoacetylated state, which is required for ionizing radiation-induced NBS1 Ser343 phosphorylation. And by modulating the acetylation of ATDC, HDAC9 could prevent ATDC-p53 complex formation, promoting IR-induced cell death. These data suggest HDACs play much wider roles in cells in addition to their transcriptional repression function.

Abstract Mutations in BRCA1 and BRCA2 explain only about 20% of familial aggregation of breast cancer, suggesting involvement of additional susceptibility genes. In this study five DNA damage response genes, ATM, ATR, MRE11, NBS1 and RAD50, were considered as putative candidates to explain some of the remaining familial breast cancer risk, and were screened for germline mutations in families displaying genetic predisposition. Analysis of ATM indicated that clearly pathogenic mutations seem to be restricted to those reported in ataxia-telangiectasia (A-T). However, a cancer risk modifying effect was suggested for a combination of two ATM polymorphisms, 5557G>A and IVS38-8T>C, as this allele seemed to associate with bilateral breast cancer (OR 10.2, 95% CI 3.1–33.8, p = 0.001). The relevance of ATM mutations, originally identified in Finnish A-T patients, in breast cancer susceptibility was evaluated by a large case-control study. Two such alleles, 6903insA and 7570G>C, in addition to 8734A>G previously associated with breast cancer susceptibility, were observed. The overall mutation frequency in unselected cases (7/1124) was higher than in controls (1/1107), but a significantly elevated frequency was observed only in familial cases (6/541, p = 0.006, OR 12.4, 95% CI 1.5–103.3). These three mutations showed founder effects in their geographical occurrence, and had different functional consequences at protein level. In ATR no disease-related mutations were observed, suggesting that it is not a breast cancer susceptibility gene. The mutation screening of the Mre11 complex genes, MRE11, NBS1 and RAD50, revealed two novel potentially breast cancer associated alleles: NBS1 Leu150Phe and RAD50 687delT were observed in 2.0% (3/151) of the studied families. The subsequent study of newly diagnosed, unselected breast cancer cases indicated that RAD50 687delT is a relatively common low-penetrance susceptibility allele in Northern Finland (cases 8/317 vs. controls 6/1000, OR 4.3, 95% CI 1.5–12.5, p = 0.008). NBS1 Leu150Phe (2/317) together with a novel RAD50 IVS3-1G>A mutation (1/317) was also observed, both being absent from controls. Loss of the wild-type allele was not observed in the tumors of the studied mutation carriers, but they all showed an increase in chromosomal instability of peripheral T-lymphocytes. This suggests an effect for RAD50 and NBS1 haploinsufficiency on genomic integrity and susceptibility to cancer.

Protection of the genome from carcinogenic consequences of DNA double-strand breaks (DSBs) is accomplished through the pathways of non-homologous end-joining (NHEJ) or homologous recombinational repair. Five human proteins with homology to Rad51 known as the Rad51 paralogs, Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, whose loss of function in cell lines leads to high chromosomal instability. Previous studies have shown Rad51C participate in two paralog protein complexes, one containing Rad51B, Rad51C, Rad51D and XRCC2 (BCDX2) and the other containing only Rad51C and XRCC3 (CX3). However, the only structural data available is the crystal structure of RecA, the bacterial homolog, the determination of the N-terminus of human Rad51 by NMR, and the crystal structure of Pyroccocus furious Rad51. Currently the Alvinlla pompejana Rad51C has been cloned, expressed and is currently being crystallized in the Tainer laboratory (UC Berkeley) since the human Rad51C protein has proven too difficult to be utilized. To test functional association of Hs Rad51B and Hs XRCC3 to Ap Rad51C. The human proteins were heterologously expressed in Pichia pastoris and the other expressed in E. coli. The proteins were extracted and interaction was tested through co-immunoprecipitation. Initial results depict weak binding or an unstable interaction between Hs Rad51B and Ap Rad51C. Hs XRCC3 and Ap Rad51C interaction remains unclear and requires further testing. Additionally, we have utilized a cellular model of HNSCC to identify whether the down-regulation of Rad51 after application of VD 3 is concomitant with the down-regulation of NBS1. NBS1 is a DNA repair protein involved in both pathways of DNA double-strand break repair, non-homologous end-joining and homologous recombinational repair. It has recently been demonstrated that NBS1 binds to Rad51 aiding in its localization to sites of DNA damage. VD 3 is a potential chemopreventive agent in the treatment of head and neck cancer. For the in vitro model Rad51 and NBS1 protein were both extracted from SCC25 and MCF-7 cancer cell lines were treated with 100 nM of VD 3 . For the in vivo model hamsters cheek pouch tissue sections with VD 3 treated and DMBA over the course of 14 weeks were used. Rad51 and NBS1 staining is restricted to the nuclei of the basal cell layer of the epithelium in VD 3 treated animals as compared to untreated controls where staining is evident throughout the dysplastic epithelium and is not restricted to nuclei. Unlike the western blot data of Rad51 that shows similar downregulation as the immunocytochemistry, the western blot analysis of NBS1 is unclear. However, the immunocytochemistry suggests that NBS1 is also downregulated by VD 3 in vivo, and therefore, it may be implied that both the HRR and NHEJ pathways are involved in the cellular effects of VD 3 in HNSCC.

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Das Nijmegen-Breakage-Syndrom (NBS) ist ein autosomal-rezessiv vererbtes Chromosomenbruchsyndrom, dem in > 90% der Patienten eine 5bp-Deletion im Nbs1-Gen zugrundeliegt. Klinisches Hauptmerkmal ist ein stark erhöhtes Krebsrisiko, insbesondere für B-Zell-Lymphome. Bereits bekannt ist die Funktion des entsprechenden Genprodukts, Nibrin, bei den für die Krebsprävention wichtigen Mechanismen der DNA-Reparatur und der Zellzykluskontrolle. Daneben spielt die Apoptose eine essentielle Rolle bei der Krebsentstehung. Zu untersuchen ob Nibrin auch hier Funktionen übernimmt war Gegenstand dieser Arbeit. Eine Störung der Apoptose könnte dabei mitverantwortlich für das hohe Krebsrisiko der NBS-Patienten sein. Kern der Arbeit war die Untersuchung von NBS-B-Lymphozyten hinsichtlich ihrer Fähigkeit, nach einer DNA-Schädigung die Apoptose zu induzieren. Hierzu wurde in den entsprechenden Zellen mittels Bleomycin der Apoptoseprozess ausgelöst und die prozentualen Apoptoseraten durchflusszytometrisch bestimmt. Die Mehrheit der NBS-Zelllinien zeigte eine Störung in der Apoptoseinduktion im Sinne signifikant verminderter Apoptoseraten. Dies weist auf eine Funktion des Nibrins bei der Induktion der Apoptose hin. Andere NBS-Zelllinien zeigten normale Apoptoseindices. Dies könnte auf dem individuellen genetischen Hintergrund der Zellen beruhen, der auch für die erhebliche klinische Variabilität des Krankheitsbildes verantwortlich ist. Eine Korrelation der Apoptoseraten mit der Krebsinzidenz zeigte, dass alle Patienten mit reduzierten Apoptoseraten bereits Lymphome entwickelt hatten, während Patienten mit normalen Apoptoseindices bisher keine Lymphome aufwiesen. Möglicherweise gibt es also generell zwei Gruppen von NBS-Patienten - Patienten mit höherem und mit niedrigerem Entartungsrisiko, wobei eine verminderte Apoptoseinduktion als Risikofaktor für Krebs angesehen werden könnte. / The human genetic disorder, Nijmegen-Breakage-Syndrome (NBS), is characterised by an in increased risk for cancer, particularly B-cell-lymphoma. The Nbs1-gene codes for a protein, Nibrin, involved in the processing/repair of DNA double strand breaks and in cell cycle checkpoints - mechanisms relevant for cancer-prevention. As a third mechanism, apoptosis is important in preventing cancer. To evaluate whether Nibrin plays a role in this process was the aim of this study. Failure of apoptosis-induction could be another factor responsible for the high cancer risk in NBS. For this purpose we examined a set of NBS-B-cell-lines for their capacitiy to enter into apoptosis after a DNA-damaging treatment with Bleomycin. The majority of NBS-cell-lines showed a deficiency in apoptosis-induction. This may indicate a function of Nibrin in mechanisms of apoptosis-regulation. Some NBS-cell-lines showed a proficient apoptotic response, though. The reason may be found in the variable genetic background of the cell lines, also responsible for the high clinical variability of the disease. Correlation of apoptosis rates with cancer incidence showed that all patients deficient in apoptosis had already developed B-cell-lymphoma, whereas patients with normal rates had not developed lymphoma so far. Possibly there are two groups of NBS-patients- patients with higher and with lower risk of malignancy, with reduced apoptotic rates being a risk-factor for the development of cancer in NBS.

Apie 5–10 proc. visų krūties navikų atvejų sudaro paveldimas vėžys. BRCA1 ir BRCA2 genai yra patys svarbiausi polinkį susirgti krūties vėžiu sąlygojantys genai. Kiti reikšmingai su padidėjusia krūties navikų išsivystymo rizika susiję – CHEK2 ir NBS1 genai. Šiame darbe ištirtos paveldimos dažniausiai Europos regione nustatomos šių genų mutacijos. Nustatytas BRCA1 ir CHEK2 genų mutacijų dažnis tarp jaunų krūties vėžiu susirgusių moterų, ištyrinėtos jų sąsajos su pacientės amžiumi, naviko klinikinėmis ir morfologinėmis savybėmis. Išanalizuota šeiminės anamnezės prognozinė vertė nustatant paveldimus BRCA1 ir CHEK2 genų pokyčius. Pirmą kartą Lietuvoje įsisavintas CHEK2 bei NBS1 genų tyrimas, nustatyta, kokios CHEK2 geno mutacijos dažniausios. Nors NBS1 geno mutacijų nerasta, bet įsisavinta metodika, kuri bus panaudota ateities tyrimams. Sukurtas kompleksinis BRCA1 bei CHEK2 genų mutacijų radimo prognozavimo modelis. Šiandien klinikinėje praktikoje panašūs modeliai naudojami įvertinti BRCA1/2 genų mutacijų tikimybę. Jų pritaikomumas ir specifiškumas skirtingose etninėse grupėse gali skirtis. Naudojant tirtų pacienčių charakteristikas, įtraukiant ne tik šeiminę anamnezę, pacientės ypatybes, bet ir klinikinius bei molekulinius navikų požymius, sukurti mūsų regionui pritaikyti modeliai bei nustatyti kriterijai, kurie padės atrinkti pacientes genetiniam konsultavimui dėl BRCA1 bei CHEK2 genų mutacijų. Šis naujas požiūris turi didžiulę praktinę naudą. / Approximately 5–10% of all breast cancer cases are considered to be hereditary. BRCA1 and BRCA2 genes are the most important breast cancer predisposing genes. Other genes significantly linked with an increased risk of breast tumors are CHEK2 and NBS1 gene. In this scientific work were studied the most prevalent in European region mutations of these genes. The rate of BRCA1 and CHEK2 gene mutations in young women with breast cancer was evaluated and the relationships between these mutations and patient's age, clinical and morphological tumor features are examined. The prognostic value of family history was analyzed when forecasting hereditary BRCA1 and CHEK2 gene mutations. For the first time in Lithuania the CHEK2, NBS1 genes tests were applied and the evaluation of which CHEK2 gene mutations are most prevalent was obtained. Although NBS1 gene mutations were not found, but applied test technique will be used in future research. There was created a prognostic model for determination of BRCA1 and CHEK2 gene mutations. In today's clinical practice similar models are used to assess the likelihood of the BRCA1/2 mutation. Their applicability and specificity in different ethnic groups may vary. Applying the studied data there was created a model adapted to our region. Testing patients, there were considered not only family medical history and personal characteristics, but also the clinical and molecular features of tumors. The criteria have been found which will help in selecting... [to full text]

Le parvovirus humain Adeno-Associé (AAV) est un Dependoparvovirus qui ne peut accomplir son cycle réplicatif qu’en présence d’un virus auxiliaire tel que l’Adénovirus (AdV) ou le virus de l’Herpès Simplex de type 1 (HSV-1). En absence de virus auxiliaire, l’AAV va persister sous forme épisomale ou intégrée. Cette intégration survient de façon préférentielle dans un locus spécifique, au site AAVS1, présent sur le chromosome 19 du génome humain.Des travaux précédents ont porté sur l’étude du contrôle de la réplication de l’AAV par les facteurs cellulaires de réparation des cassures d’ADN. En particulier, le complexe MRN (Mre11/Rad50/Nbs1), un senseur majeur des cassures de l’ADN double brin (CDB), a été montré comme pouvant inhiber les réplications virales de l’AAV et de l’AdV lors d’une co-Infection. L’AdV est capable de contrer cet effet en induisant la délocalisation et la dégradation de MRN. A l’opposé, MRN participe de façon positive à la réplication de l’HSV-1 et se retrouve localisé dans les centres de réplication viraux (CR) de l’AAV induits par HSV-1. Ceci nous a conduits à explorer plus en détail le rôle de ce complexe sur la réplication de l’AAV en présence d’HSV-1. Les résultats obtenus indiquent, qu’en absence de MRN, la réplication du génome de l’AAV est réduite de façon significative dans des cellules co-Infectées avec le virus HSV-1, sauvage ou muté pour son activité polymérase. Cette diminution est spécifique à l’AAV sauvage car aucune perturbation n’est observée sur la réplication des vecteurs AAV recombinants lorsque MRN est absent. La régulation positive de la réplication de l’AAV par MRN est dépendante de l’activité de pontage de l’ADN exercée par Rad50. De façon intéressante, l’absence de MRN inhibe également de façon significative l’intégration préférentielle de l’AAV au site AAVS1, que ce soit en absence ou en présence d’HSV-1.Ce travail de thèse suggère que le complexe MRN régulerait de façon différentielle la réplication de l’AAV en fonction du virus auxiliaire qui l’accompagne et identifie, pour la première fois, MRN comme un facteur clé pour l’intégration du génome de l’AAV au site AAVS1. / Adeno-Associated Virus (AAV) is a helper dependent Dependoparvovirus that requires co-Infection with adenovirus (AdV) or herpes simplex virus type 1 (HSV-1) to productively replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Integration occcurs preferentially at a specific locus called AAVS1 and based on human chromosome 19.Previous studies have analyzed the DNA damage response induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DNA damage response induced by double-Stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during co-Infection. AdV counteracts this effect by inducing the delocalization and degradation of MRN. In contrast, MRN favors HSV-1 replication and our previous studies showed that it was recruited to AAV replication compartments that were induced in the presence of HSV-1. In this study we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after co-Infection with polymerase deleted or wild type HSV-1. This reduction was specific of wild type AAV since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering and nuclease activities. Importantly, knockdown of MRN could also negatively regulate AAV site-Specific integration within the human AAVS1 site, an event which occurred at a significant level during AAV replication induced by co-Infection with HSV-1. Altogether, this work demonstrates that MRN can differentially regulate AAV replication depending on the helper virus which is present and identifies a new function of this DNA repair complex during site-Specific integration of the AAV genome.