|
|
Molecular mechanisms of sensitivity and resistance towards chemotherapeutics in most frequent solid cancers
Čumová, Andrea ; Vodička, Pavel (advisor) ; Černá, Marie (referee) ; Hlaváč, Viktor (referee)
Despite the great effort, the main obstacle to cancer therapy represents low response towards common chemotherapeutics and/or resistance. Chemoresistance causes cancer relapse and formation of metastases, dramatically challenging the prognosis of patients. It is estimated, that about 90% of cancer mortality can be directly or indirectly attributed to chemoresistance. There are several intrinsic or acquired cellular mechanisms of tumor chemoresistance, with DNA repair being one of the key culprits affecting the response towards chemotherapeutics in cancer cells. This is based on the fundamental principle of their action, as the majority of chemotherapeutics are designed to increase DNA damage and to suppress DNA repair or DNA damage response, ultimately triggering the death of malignant cells. Consequently, understanding the complex mechanisms of DNA repair and its regulation is essential for more targeted and effective treatment of cancer patients. In this dissertation Thesis, we attempted to elucidate some of the regulatory mechanisms of DNA repair and their effects on response to common chemotherapeutics. We confirmed that single nucleotide polymorphisms in microRNA binding sites of DNA repair genes may influence the patient's survival and response to cancer therapy. We investigated the role of...
|
|
|
Virus interaction with the cellular DNA damage response
Lemberková, Eva ; Šroller, Vojtěch (advisor) ; Kadlečková, Dominika (referee)
Maintenance of genomic integrity is an essential mechanism for every cell. Genomic integrity is disturbed by diverse exogenous or endogenous effects influencing the cell and causing damage of its DNA. Cellular mechanisms capable of fixing these disturbances in structure or sequence are indispensable because damaged genetic information can later cause expression of damaged proteins or inaccurate segregation of chromosomes to daughter cells. Therefore, many effective mechanisms for fixing wide range of types of DNA damage have evolved. This thesis focuses mainly on the eucaryotic MRN complex, which plays an important role in detection and repair of double strand breaks. Many viral families try to block these cellular repair mechanisms because they are activated soon after viral infection. One of the reasons for their activation is the resemblance of some viral genomes to the cellular DNA with double strand breaks. Thus, in many cases, the cell ends up inhibiting the life cycle of the virus by attempting to repair viral genomes. However, there are viruses that use cellular repair mechanisms for the replication of their genome, making these mechanisms essential for their own growth. Key words: DNA damage response, homologous recombination, non-homologous end-joining, MRN complex, Adenoviridae,...
|
|
|
Molecular mechanisms of Wip1 phosphatase function in DNA damage response
Štorchová, Radka ; Macůrek, Libor (advisor) ; Fajkus, Jiří (referee) ; Líčeníková Hořejší, Zuzana (referee)
Human cells are constantly exposed to diverse factors causing DNA lesions, which activate the DNA damage response (DDR). Depending on the severity of DNA damage, DDR can promote temporary cell cycle arrest (checkpoint), permanent growth arrest (senescence) or programmed cell death (apoptosis). DDR signalling is regulated by a cascade of post- translational modifications, where key mediators are represented by protein kinases ATM, ATR and DNA-PK. Wip1 phosphatase (encoded by PPM1D gene) plays an important role in DDR termination by dephosphorylation of many targets of these kinases. In this thesis, we investigated checkpoint-independent functions of PPM1D in cells and described several new substrates. We discovered, that PPM1D interacts with the shelterin complex and localizes at telomeres. PPM1D dephosphorylates the shelterin component TRF2 at S410. TRF2 S410 phosphorylation enhanced TRF2 interaction with TIN2, indirectly also increasing recruitment of TPP1 to telomeres. Importantly, cells over expressing PPM1D showed increased number of telomeric fusions. These findings might be very relevant for some cancer types, in particular those expressing high levels of PPM1D or carrying C-terminally truncated mutations in PPM1D. To validate the published substrates and to detect possible new targets of...
|
|
|
The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes.
Mašín, Martin ; Jirkovská, Anna (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Mašín Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes. DNA Topoisomerases comprise a family of enzymes that are able to alter DNA topology by transient single- or double-strand breaks (DSB) during fundamental processes such as replication and transcription. Inhibition of topoisomerase II (TOP II) is the main mechanism of action of some antitumour drugs, such as anthracyclines (ANT; e.g., daunorubicin). They stabilize the DNA-TOP II complex, leading to the formation of DSBs and later to apoptosis. Other inhibitors, that interact with the enzyme without the DSB formation, can modulate the effect of ANT. In this thesis, we studied the DNA damage caused by daunorubicin (DAU) and its main metabolite daunorubicinol (DAUnol) and the effect of two naturally-derived compounds and TOP II catalytic inhibitors resveratrol (RES) and gambogic acid (GA) in neonatal rat cardiomyocytes. The DNA damage was determined as the extent of histone H2AX phosphorylation (γ-H2AX) and by Comet Assay. It can be concluded that both DAU and DAUnol (1,2 μM) exhibit DNA damage that is...
|
|
|
The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes.
Mašín, Martin ; Jirkovská, Anna (advisor) ; Pávek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Martin Mašín Supervisor: PharmDr. Anna Jirkovská, Ph.D. Title of diploma thesis: The effect of resveratrol and gambogic acid on the DNA damage caused by daunorubicin in neonatal rat cardiomyocytes. DNA Topoisomerases comprise a family of enzymes that are able to alter DNA topology by transient single- or double-strand breaks (DSB) during fundamental processes such as replication and transcription. Inhibition of topoisomerase II (TOP II) is the main mechanism of action of some antitumour drugs, such as anthracyclines (ANT; e.g., daunorubicin). They stabilize the DNA-TOP II complex, leading to the formation of DSBs and later to apoptosis. Other inhibitors, that interact with the enzyme without the DSB formation, can modulate the effect of ANT. In this thesis, we studied the DNA damage caused by daunorubicin (DAU) and its main metabolite daunorubicinol (DAUnol) and the effect of two naturally-derived compounds and TOP II catalytic inhibitors resveratrol (RES) and gambogic acid (GA) in neonatal rat cardiomyocytes. The DNA damage was determined as the extent of histone H2AX phosphorylation (γ-H2AX) and by Comet Assay. It can be concluded that both DAU and DAUnol (1,2 μM) exhibit DNA damage that is...
|
|
|
Cell response to genotoxic stress-based anti-cancer therapies
Imrichová, Terezie ; Hodný, Zdeněk (advisor) ; Rossmeislová, Lenka (referee) ; Rotrekl, Vladimír (referee)
The dissertation deals with a cell response to genotoxic stress, specifically to anti-cancer treatments with a genotoxic mechanism of action. In principle, cells can respond to these perturbing stimuli in several ways: in case of severe DNA damage, they usually undergo apoptosis or enter senescence. In case of minor DNA damage, or upon defective checkpoint mechanisms, they may continue the cell cycle, either with successfully repaired DNA or with mutations of various kind. Thanks to selection pressure, the mutations that provide cells with a certain growth advantage under conditions of continuing genotoxic stress, gradually accumulate and render the tumor treatment-resistant. In my thesis, I focus on several aspects of this whole process. First, I participated in a characterization of a radioresistant and anoikis-resistant population of prostate cancer cells. This population was generated by irradiating cells 35 times by 2 Gy, a regime used in clinics. After this treatment, a population of low-adherent cells emerged that demonstrated increased expression of EMT- and stem cell markers. The low-adherent state of these cells was maintained by Snail signaling and their anoikis resistance by ERK1/2 signaling. Interestingly, after a protracted period of time, these cells were able to re-adhere and...
|
|
|
Molecular mechanisms of checkpoint signalling and termination
Benada, Jan ; Macůrek, Libor (advisor) ; Brábek, Jan (referee) ; Truksa, Jaroslav (referee)
Cells employ an extensive signalling network to protect their genome integrity, termed DNA damage response (DDR). The DDR can trigger cell cycle checkpoints which prevent cell cycle progression and allow repair of DNA damage. The failures in these safeguarding mechanism are represented by serious human malignancies, most predominantly by cancer development. This work aims to contribute to the understanding of how do the cells negatively regulate DDR and cell cycle checkpoint signalling. We focused mainly on Wip1 (PPM1D) phosphatase, which is a major negative regulator of DDR and is indispensable for checkpoint recovery. Firstly, we have shown that Wip1 is degraded during mitosis in APC-Cdc20 dependent manner. Moreover, Wip1 is phosphorylated at multiple residues during mitosis, resulting in inhibition of its enzymatic activity. We suggest that the abrogation of Wip1 activity enables cells to react adequately even to low levels of DNA damage encountered during unperturbed mitosis. In the following publication, we have investigated why the mitotic cells trigger only early events of DDR and do not proceed to the recruitment of DNA repair factors such as 53BP1. We showed that 53BP1 is phosphorylated within its ubiquitination-dependent recruitment domain by CDK1 and Plk1. These phosphorylations prevents...
|
|
|
Posttranlational protein modifications in response to DNA damage
Kroupa, Michal ; Hodný, Zdeněk (advisor) ; Novotný, Marian (referee)
- 5 - Abstract Thousands of DNA lessions occur in each cell every day of which the most toxic are double-strand breaks (DSBs). Signaling of their presence and subsequent repair are mediated by so-called DNA-damage response mechanism (DDR), which involves accumulation of many effector proteins into DSBs sites. These molecular accumulation at DSBs are termed DNA damage foci. Depending on presence of sister chromatid, DSBs are repaired by two major mechanisms: by homologous recombination and by non-homologous end joining. Both pathways lead to activation of checkpoint kinases (Chk1 or Chk2) which iniciate checkpoints in cell cycle and allow repair of damaged DNA. Signaling of DNA damage and activation of these pathways are regulated by posttranslational protein modifications. These enzymatic reactions involve mainly phosphorylation, ubiquitination and sumoylation. Recently it was shown that ubiquitination of damaged chromatin is a prerequisite for sumoylation of tumor supressors BRCA1 and 53BP1. Failure in DNA damage recognizing mechanisms caused by disorders such as modifications or mutations of 53BP1 and BRCA1 genes can lead to subsequent disruption of genomic integrity and then a high risk for selection of cell clones with tumorigenic potencial. Current research is focused on regulation of posttranslational...
|
|
|
Implication of eukaryotic DNA repair machinery in viral replication
Hron, Tomáš ; Španielová, Hana (advisor) ; Harant, Karel (referee)
Eukaryotic DNA damage response is an important mechanism which ensures genome stability. Its components are also mobilized during viral infection as a reaction against extraneous nucleic acid. Additionally, DNA repair machinery seems to be activated by some viruses purposely to provide their replication. This activation is mediated mainly by viral proteins which are able to interact with cellular factors. In many cases, key components of DNA damage mechanisms are associated with viral replication centre and likely participate in this process. Furthermore, cellular DNA damage signaling is exploited to provide competent environment for viral reproduction. However, particular mechanisms how these cellular factors participate in viral infection are still largely unclear. In this thesis, the principles of relationship between viral infection and eukaryotic DNA damage response are summarized and main viral families which are known to activate and utilize these mechanisms for its genom replication are described.
|
|
|
Mechanisms of phenotypic plasticity induced by genotoxic stress
Přibyl, Miroslav ; Hodný, Zdeněk (advisor) ; Remešová, Hana (referee) ; Vomastek, Tomáš (referee)
Therapy resistance of malignant cells represents the main reason responsible for the failure of cancer therapy. The growth of malignant cells at primary tumour sites but most importantly the dissemination of tumour cells and their growth at secondary sites, are the main reasons why patients eventually succumb to the disease. Even novel immune-based therapies find their limitation in most tumour types. The therapy resistance is mediated by the tumour cells but also by other cellular components of the tumour microenvironment. Understanding the tumour cells mechanisms and the tumour microenvironment features responsible for therapy resistance enables the development of novel therapeutic strategies. Here, we show that ionizing irradiation, 5-azacytidine, and IFNγ treatments induced expression of suprabasin (SBSN) and therapy-resistant low-adherent phenotype in cancer cells. Knockdown of SBSN resulted in suppression of the phenotype. Next, we identified aberrantly elevated SBSN in the bone marrow of a subgroup of myelodysplastic syndromes (MDS) patients. SBSN was expressed by myeloid-derived suppressor cells (MDSCs) and showed significant anti-correlation with T cell abundance and CCL2 levels, hence promises a prognostic value in clinical use. We compiled the most of the relevant knowledge of SBSN...
|
guest ::
