CRISPR-Cas9 as a Tool in Cancer Therapy.

BACKGROUND: Genome editing using CRISPR-Cas9 has become one of the basic methods of biological research over a short period of time. This recently discovered system of adaptive immunity of bacteria has been adapted to the needs of science and has become a valuable tool for DNA manipulation. Its simplicity and reliability have contributed to widespread use of the method. Genome editing refers to targeted modifications of genomic DNA with single base pair accuracy. CRISPR-Cas9 differs significantly from previous technologies in the simplicity of directing the enzyme to the target sequence. In the field of cancer research, CRISPR-Cas9 has enabled the development of a number of models for the study of carcinogenesis and drug testing. From a therapeutic point of view, CRISPR-Cas9 has been applied in the field of immunotherapy, especially in ex vivo genetic modifications of the T-cells of patients. AIM: Currently, several clinical trials are trying to verify the therapeutic potential of CRISPR-Cas9. Based on these studies, we have summarised the strategies used in the preparation of therapeutic tools useful in cancer therapy. CONCLUSION: CRISPR-Cas9 appears to be crucial in basic research, particularly in the study of the function of individual genes involved in carcinogenesis. However, it will still be necessary to optimise the efficacy, safety and specificity of CRISPR-Cas9 before it is used in clinical practice.

Synthetic Lethality - Its Current Application and Potential in Oncological Treatment.

BACKGROUND: Synthetic lethality is a gene interaction where a defect in one of the interacting genes is compatible with cell viability, whereas the disruption of both genes leads to cell death. The discovery of the lethal effect of poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2 mutant cells has opened an important direction in the development of targeted therapy in oncology. The PARP inhibitor olaparib has become the first registered drug for recurrent high-grade serous ovarian cancer treatment based on synthetic lethality that has reached the clinic. Current research focuses on the combination of PARP inhibitors and inhibitors of kinases, which control the cell cycle, to prevent or overcome resistance to PARP inhibitors. There are also ongoing clinical trials which examine PARP inhibitor treatment in other types of cancers including tumours presenting the so-called BRCAness phenotype. Screenings for new synthetic lethalities which could serve as potential targets for new drug development have improved with the CRISPR/Cas9 technology, but another key problem persists in the screening efforts, namely the incomplete penetrance of synthetic lethality throughout a tumour cell population. PURPOSE: This paper summarises the current application of synthetic lethality principles in oncology and discusses the challenges in research focused on potential new drugs based on synthetic lethality.

Glycosylation as an Important Regulator of Antibody Function.

BACKGROUND: The glycosylation of constant regions of antibodies significantly affects their interaction capabilities with immune cells. It is a modification that, in addition to the bio-logical activity of antibodies, has an impact on their conformation, stability, solubility, secretion, pharmaco-kinetics, and immunogenicity. The location of glycosylations on the molecule is essential for the proper function of the antibody, as is the structure of the individual glycans. Changes in the glycosylation profiles of antibodies have been described in some physiological processes like pregnancy or ageing, but also in many pathological conditions such as rheumatoid arthritis or gastric, lung and prostate tumours. There are still several unexplained mechanisms that control the glycosylation of antibodies or immune responses, which in turn are regulated by these modifications. Multiple sources describe the importance of some specific glycosylations as potential bio-markers. PURPOSE: The aim of this review is to summarise and present the knowledge of the glycosylation of antibodies and to highlight their influence on immune responses and their role dur-ing dis-ease. Their importance is also underlined by the fact that the most of these therapeutic antibodies used and developed are modified by glycosylation. The targeted introduction of appropriate glycosylations, which can promote activities such as antibody--dependent cellular cytotoxicity, antibody--dependent cellular phagocytosis or complement--dependent cytotoxicity, have improved the ability of these antibodies to kill pathogens or tumour cells. Therefore, more attention is be-ing paid to this area. In the future, more effective tools for dia-gnos-ing and treat-ing certain dis-eases can be created with better knowledge.

Protein Ubiquitination Research in Oncology.

BACKGROUND: Ubiquitination is a vital posttranslational protein modification involved in the regulation of many eukaryotic signalling pathways. Aberrant ubiquitin signalling is known to be a molecular causality of certain cancer, neurodegenerative, immune system or cardiovascular diseases. The recent development of mass spectrometry methods enables qualitative and quantitative ubiquitination analysis in biological material from cancer patients. Research of ubiquitination may clarify the molecular cause of aberrant changes in the protein level of tumour suppressors or oncogenes. PURPOSE: We aim to explain the meaning and importance of ubiquitination in certain molecular processes taking place in the human body. We hereby emphasise the connection between ubiquitination and malignant processes. A literature search is followed by introducing our mass spectrometry platform intended for ubiquitin identification via diglycyl remnants in the CHIP protein sequence. The aim is to introduce tandem mass spectrometry identification of ubiquitin modification, ubiquitination tandem mass spectra validation and the time-dependent manner of CHIP ubiquitination to the reader. CONCLUSION: A literature search familiarises the reader with known mechanisms of aberrant ubiquitination in malignant diseases. A successfully optimised mass spectrometry platform could serve as a potent tool for determining ubiquitin position in proteins that are a part of real tumour samples.

Oncogenic Viral Protein Interactions with p53 Family Proteins.

BACKGROUND: Cellular transformation induced by oncogenic viruses is a complex process including viral molecules, host cells and environmental factors. Viruses alone are unable to reproduce and thus they need a host to use their signalling, proteosynthetic and metabolic pathways. One target host molecule is the p53 tumour suppressor. Viral proteins functionally inactivate p53 and deregulate the expression of proteins active during apoptosis, cell proliferation and DNA damage response. Hepatitis virus B HbX protein and hepatitis virus C proteins NS2 and NS5A interact with p53 and prevent its localisation to the nucleus and thus reduce its transcriptional activity. Another mechanism lies in elevated p53 degradation caused by the BZLF1 protein of the Epstein-Barr virus, the LANA protein of the Kaposi sarcoma virus and human papilloma virus E6. The Merkel cell polyomavirus large T antigen does not interact directly with p53, however it acts through downregulation of p53 mediated transcription. The tax protein of human T cell lymphotropic virus type 1 modifies p53 posttranslationally and thus blocks its interaction with other factors of transcription machinery. Due to its tumour suppressor function and role in the maintenance of the genome integrity, the p53 protein is one of the best studied proteins. Following this, evolutionary homologues with important developmental functions p63 and p73 are intensively studied as well. Their roles in oncogenesis have not been clarified yet. PURPOSE: This review describes some of their known interactions with oncogenic viral proteins.

Cooperation of Genomic, Transcriptomics and Proteomic Methods in the Detection of Mutated Proteins.

BACKGROUND: Current anti-tumour therapy is characterised by high non-specificity due to the diverse nature of tumours, which can significantly reduce its efficiency. The massive development of genomic, transcriptomic, and proteomic methods has enabled the detailed characterisation of individual tumours at the genome, transcriptome and proteome levels. Whole-genome sequencing, whole-transcriptome sequencing and exome sequencing can be listed as examples of genomics and transcriptomics methods. Those methods are suitable for detecting single-nucleotide polymorphisms. In the case of proteomic methods, where a peptide library is available, it is possible to detect mutated proteins in a bio-logical sample. Also important is software that interprets and visualises the results or facilitates conversion between data formats that are specific to the method. The combination of methods can in principle increase the likelihood of detecting new neoantigens and design-specific anti-tumour therapy. AIM: The article primarily describes the bio-informatics analysis of samples using the methods of genomics, transcriptomics and proteomics, and the possible problems which must be considered during the analysis. The article includes a description of TransPEM software designed to convert the results from the analysis of single nucleotide polymorphisms into a peptide library of sequences useful for the detection of neopeptides using proteomic methods. The publication is accompanied by a brief description of the proteomics methods using this peptide library and the summary of its limitations.

Intrinsic proteotoxic stress levels vary and act as a predictive marker for sensitivity of cancer cells to Hsp90 inhibition.

Response of tumours to Hsp90 inhibitors is highly variable and their clinical effects are unpredictable, emphasising the need for a predictive marker. We postulated that sensitivity to Hsp90 inhibitors is connected to basal proteotoxic stress that makes cells dependent on Hsp90. Therefore, we assessed HSF1 as a general sensor of proteotoxic stress and correlated its activity with sensitivity to three separate small molecule Hsp90 inhibitors in seven breast cancer cell lines representing each of the different cancer subtypes. Flow cytometry was used to analyse the viability of breast cancer cell lines after Hsp90 inhibition. HSF1 activity was characterised by Ser326 phosphorylation and the transactivation capacity of HSF1 was determined by qPCR analysis of the ratios of HSF1-dependent (HOP, Hsp70) and HSF1-independent (CHIP) chaperones and cochaperone mRNAs. We show that the sensitivity of breast cancer cell lines to Hsp90 inhibition is highly variable. The basal levels of phosphorylated HSF1 also vary between cell lines and the magnitude of change in HSF1 phosphorylation after Hsp90 inhibition showed a negative correlation with sensitivity to Hsp90 inhibitors. Similarly, the basal transactivation capacity of HSF1, determined by the ratio of Hsp70 or HOP mRNA to CHIP mRNA level, is directly proportional to sensitivity to Hsp90 inhibitors. Increasing basal HSF1 activity by prior heat shock sensitised cells to Hsp90 inhibition. These results demonstrate that endogenous HSF1 activity varies between individual cancer cell lines and inversely reflects their sensitivity to Hsp90 inhibitors, suggesting that basal proteotoxic stress is an important and generalised predictor of response. Mechanistically, the data indicate that high endogenous proteotoxic stress levels sensitise to Hsp90 inhibition due to the inability to respond adequately to further proteotoxic stress. HSF1 activity therefore represents a potential biomarker for therapy with Hsp90 inhibitors, which may be useful for the rational design of future clinical studies.

[Application of PLA Method for Detection of p53/p63/p73 Complexes in Situ in Tumour Cells and Tumour Tissue]. / Vyuzití metody PLA pro detekci komplexu p53/p63/p73 in situ v nádorových bunkách a nádorové tkáni.

BACKGROUND: PLA (proximity ligation assay) can be used for detection of protein-protein interactions in situ directly in cells and tissues. Due to its high sensitivity and specificity it is useful for detection, localization and quantification of protein complexes with single molecule resolution. One of the mechanisms of mutated p53 gain of function is formation of proten-protein complexes with other members of p53 family - p63 and p73. These interactions influences chemosensitivity and invasivity of cancer cells and this is why these complexes are potential targets of anti-cancer therapy. The aim of this work is to detect p53/p63/p73 interactions in situ in tumour cells and tumour tissue using PLA method. MATERIAL AND METHODS: Unique in-house antibodies for specific detection of p63 and p73 isoforms were developed and characterized. Potein complexes were detected using PLA in established cell lines SVK14, HCC1806 and FaDu and in paraffin sections of colorectal carcinoma tissue. Cell lines were also processed to paraffin blocks. RESULTS: p53/T-antigen and ΔNp63/T-antigen protein complexes were detected in SVK14 cells using PLA. Interactions of ΔNp63 and TAp73 isoforms were found in HCC1806 cell line with endogenous expression of these proteins. In FaDu cell line mut-p53/TAp73 complex was localized but not mut-p53/ΔNp63 complex. p53 tetramer was detected directly in colorectal cancer tissue. CONCLUSION: During development of PLA method for detection of protein complexes between p53 family members we detected interactions of p53 and p63 with T-antigen and mut-p53 and ΔNp63 with TAp73 tumour suppressor in tumour cell lines and p53 tetramers in paraffin sections of colorectal cancer tissue. PLA will be further used for detection of p53/p63, p53/p73 and p63/p73 interactions in tumour tissues and it could be also used for screening of compounds that can block formation of p53/p63/p73 protein complexes.Key words: p53 protein family - protein interaction mapping - immunofluorescence This work was supported by MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

[Ascites May Provide Useful Information for Diagnosis of Ovarian Cancer]. / Ascitická tekutina u nádoru vajecníku muze poskytnout informace vhodné pro diagnostiku.

BACKGROUND: Ovarian cancer is the most lethal gynecological cancer, almost 80% of all patients succumb the disease within 5 years of diagnosis. High mortality is caused especially by nonspecific symptoms, diagnosis in late stages and the absence of a specific biomarker. Currently, the most common diagnostic biomarkers are the membrane glycoprotein Cancer Antigen 125 (CA 125), the Human Epididymal Protein 4 (HE4) and the Carcinoembryonic Antigen (CAE). None of these biomarkers is specific only for ovarian cancer and increased levels may be caused by other diseases. Therefore, current research is focused on finding new biomarkers for diagnosis and prognosis of ovarian cancer. Interesting clinical material is ascites, the fluid accumulated in abdominal cavity, which is typical for ovarian cancer and it is present in almost 90% of all cases of stage III and IV. MATERIAL AND METHODS: For this study, samples of ascites from patients with benign and malignant ovarian tumors were used. For full glycomic and proteomic analysis, only 5 µL of ascites were used. Glycans were released from proteins by the enzyme PNGase F and proteins were digested to peptides by trypsin. Samples were purified and measured using a mass spectrometer. RESULTS: Glycan and protein profiles of patients with benign and malignant ovarian cancer were compared. In patient with a benign tumor, more simple glycans with lowm/z were increased while in the patient with a malignant tumor, higher, more complex glycans were increased. In the malignat tumor in comparison to benign tumor, 127 unique proteins were identified, especially proteins of the annexin, mucin and peroxiredoxin families. CONCLUSION: This investigation is a pilot study focused on comparison of protein and glycan composition of ascites in patients with benign and malignant ovarian cancer. Significant differences were found on both glycan and protein levels. Results will be verified on a larger set of patients and compared with a set of control samples.Key words: glycomics - proteomics - ascitic fluid - ovarian cancer This study was supported by projects of the Ministry of Education Youth and Sports - National Sustainability Program I - LO1413; Ministry of Health, Czech Republic - conceptual development of research organization (MMCI, 00209805); Czech Science Foundation 16-04496S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

[Diagnostic and Therapeutic Potential of Membrane HSP90]. / Diagnostický a terapeutický potenciál membránového HSP90.

BACKGROUND: Heat shock protein (HSP90) is a molecular chaperone involved in maintaining protein homeostasis by modulating stability of de novo synthesized proteins. Neoplastic cells with high metabolic rate have higher expression of HSP90 and develop so called "chaperone addiction". Specific inhibition of HSP90 has been therefore discussed as a viable therapeutic strategy and several inhibitors of HSP90 have already entered clinical trials. Recently, a novel role for HSP90 was found on plasma membrane of cancer cells. Since then, extracellular HSP90 has been implicated in increased tumor invasiveness and metastasis, but better understanding of its regulation is needed to fully explore its potential in early detection of malignity and import of specific HSP90 inhibitors. We have therefore analyzed correlation of extracellular HSP90 level with import of fluorescently-labeled inhibitor of HSP90 and total expression of HSP90. METHODS: Flow cytometry was used to analyze cell uptake of FITC-Geldanamycin as well as level of extracellular HSP90, while total expression of HSP90 was analyzed by SDS-PAGE and subsequently Western blotting. Data was then subjected to statistical analysis to analyze possible correlation. RESULTS: We have analyzed import of fluorescently labeled HSP90 inhibitor together with total and membrane level of HSP90 on a panel of selected breast carcinoma cell lines (BT-474, BT-549, BT-20, MCF-7, MDA-MB-468, SK-BR-3 a T-47D). Acquired data were subjected to statistical analysis that revealed a correlation between total and membrane level of HSP90 as well as correlation of ectopic HSP90 with uptake of HSP90 inhibitor. CONCLUSIONS: Our analysis has shown that import of HSP90 inhibitors is likely dependent on membrane level of HSP90 as well as its total expression, and therefore can potentially reflect HSP90 addiction of cancer cells.Key words: breast neoplasms - HSP90 - heat shock proteins - geldanamycin This work was supported by MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.

p53 binds the mdmx mRNA and controls its translation.

MDMX and MDM2 are two nonredundant essential regulators of p53 tumor suppressor activity. MDM2 controls p53 expression levels, whereas MDMX is predominantly a negative regulator of p53 trans-activity. The feedback loops between MDM2 and p53 are well studied and involve both negative and positive regulation on transcriptional, translational and post-translational levels but little is known on the regulatory pathways between p53 and MDMX. Here we show that overexpression of p53 suppresses mdmx mRNA translation in vitro and in cell-based assays. The core domain of p53 binds the 5' untranslated region (UTR) of the mdmx mRNA in a zinc-dependent manner that together with a trans-suppression domain located in p53 N-terminus controls MDMX synthesis. This interaction can be visualized in the nuclear and cytoplasmic compartment. Fusion of the mdmx 5'UTR to the ovalbumin open reading frame leads to suppression of ovalbumin synthesis. Interestingly, the transcription inactive p53 mutant R273H has a different RNA-binding profile compared with the wild-type p53 and differentiates the synthesis of MDMX isoforms. This study describes p53 as a trans-suppressor of the mdmx mRNA and adds a further level to the intricate feedback system that exist between p53 and its key regulatory factors and emphasizes the important role of mRNA translation control in regulating protein expression in the p53 pathway.

[Endoplasmic Reticulum Chaperones at the Tumor Cell Surface and in the Extracellular Space]. / Chaperony endoplazmatického retikula na povrchu nádorové bunky a v extracelulárním prostredí.

BACKGROUND: Endoplasmic reticulum chaperones are stress induced proteins capable of translocation into cytosol, cell membrane or extracellular space. The chaperones are transported from the endoplasmic reticulum particularly under endoplasmic reticulum stress conditions, while their constitutive extracellular expression was found in many cancers. Cell surface or extracellular endoplasmic reticulum chaperones take up distinct functions compared to their endoplasmic reticulum resident variants because they act like multifunctional receptors and thus affect cell signaling and proliferation. AIM: The presented review focuses primarily on endoplasmic reticulum chaperones expression on the cell surface of cancer cells and into extracellular space. The work describes possible mechanisms of chaperones translocation to the cancer cell surface, including KDEL transport mechanism and retrotranslocation and the influence of chaperone posttranslation modifications on their localization within the cell. Well described cancer cell surface endoplasmic reticulum chaperones include GRP78, GRP94, calreticulin and calnexin that are involved in cancer cell signaling in different ways. The attention is also paid to immunogenic properties of membrane-localized chaperones for their ability to participate in immune reactions. They can take part in innate and adaptive immune response through their interaction with toll-like receptors or during the antigen presentation as well as in tumor-specific immunity. The expression of endoplasmic reticulum chaperones on the cancer cells surface is potentially exploitable in specific antitumor therapy as well as vaccine therapy, thus the final part of this review is dedicated to this topic.Key words: endoplasmic reticulum - glucose-regulated proteins - molecular chaperones - KDEL sequence - immunobiologyThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 17. 5. 2016Accepted: 20. 7. 2016.

[Impact of HSP90 Inhibition on Viability and Cell Cycle in Relation to p53 Status]. / Vplyv inhibície HSP90 na viabilitu a bunkový cyklus v závislosti od stavu p53.

BACKGROUND: Chaperone system inhibition is a recent promising strategy for cancer treatment that exploits increased metabolic needs required for rapid proliferation as well as higher level of proteotoxic stress in neoplastic cells. Chaperone HSP90 plays a key role in proper folding of many de novo synthesized proteins, so-called clients, including tumor suppressor p53 which is commonly mutated in majority of cancers. Aim of this work was therefore to understand the impact of HSP90 inhibition by NVP-AUY922 on breast cancer cell lines with wild-type and mutated p53. METHODS: Flow cytometry was used to analyze cell viability by fluorescein diacetate assay and changes in cell cycle. Western blotting was used to analyze expression of p53 and p21 proteins. RESULTS: Analysis of cell viability after HSP90 inhibition revealed higher sensitivity of cell line with wild-type p53. Cell cycle analysis then showed that both cell lines undergo increase in G2/M block of the cell cycle, but wild-type cell line had also substantial decrease in proliferative capacity of treated cells. We also observed increased expression of negative cell cycle regulator p21 in cell line with wild-type p53. CONCLUSIONS: Since p21 is directly regulated by p53, our results suggest that mutation status of p53 can be important factor in treatment of breast cancer cells by HSP90 chaperone inhibition and that wild-type p53 can increase sensitivity to HSP90 inhibition.Key words: breast cancer - cell cycle - chaperone - HSP90 - TP53 - p21 - p53 - NVP-AUY922This work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 17. 5. 2016.

[Mechanisms of Protein Homeostasis Regulation in Cancer Development]. / Mechanizmy regulace proteinové homeostázy ve vývoji nádorových onemocnení.

BACKGROUND: The proteome of eukaryotic cells represents a complex system. Its components are exposed to various intrinsic and extrinsic stresses. Therefore, the function of the cellular proteome is dependent on the existence of compensatory mechanisms balancing the inner protein homeostasis - proteostasis. These mechanisms involve the network of molecular chaperones and transcriptional program regulating their expression. The process of cancerogenesis is accompanied by significant changes in the intracellular milieu of cancer cells - temperature, pH, availability of nutrients. On the one hand, these changes represent a consequence of the deregulated growth of the tumor tissue; on the other hand, they can be a source of selection pressure, which allows the emergence of resistant and aggressive tumor cell populations. Description of the proteostatic apparatus components and the mechanism of their involvement in the tumor tissue development is provided in this review article. AIM: This review focuses on the description of two causally linked groups of proteostatic events; their mutual coordination is crucial to the process of tumor cell and by extension the entire tumor tissue response to environmental and internal stress factors. The first group of these processes is represented by the "executory" role of molecular chaperones from HSP70, HSP90 and so-called small molecular chaperone protein families. These proteins are involved in maintaining stability of cellular proteins essential for proliferation, apoptosis, senescence, migration and phenotypic plasticity of tumor cells. The second group of the described processes comprises the posttranslational control of the "systemic" role of the transcription factor HSF1 in regulating the gene expression of molecular chaperones and other genes specifically regulated by this transcription factor in the tumor and stromal cells.Key words: molecular chaperones - heat-shock factor 1 - cancer - protein homeostasisThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 15. 5. 2016Accepted: 25. 5. 2016.

[Molecular Mechanisms of Carcinogenesis of Epithelial Ovarian Cancers]. / Molekulární podstata kancerogeneze epiteliálních ovariálních karcinomu.

BACKGROUND: Epithelial ovarian carcinomas are one of the most common causes of death among gynecologic malignancies in the Czech population. This group of tumors is characterized by considerable heterogeneity in terms of its pathogenesis and response to therapy. It is questionable whether advances in the elucidation of molecular pathogenesis of various types of epithelial ovarian carcinomas can contribute to application of personalized targeted therapy. AIMS: This work aims to summarize current knowledge on carcinogenesis and molecular basis of epithelial ovarian cancers and point out their potential applications in clinical practice. The characterization of the epithelial ovarian carcinomas is based on a dualistic model, which divides these tumors into two groups based on their different origins and mechanisms of carcinogenesis. Type I includes low-grade serous carcinomas, endometrioid carcinomas, mucinous carcinomas and Brenner tumor. Type II then comprises high-grade serous carcinomas. CONCLUSION: The new findings acquired by next generation sequencing revealed major differences in the genetic alterations in both groups of tumors. Differences in genetic instability between the two groups of tumors determine the mechanisms of their carcinogenesis and show new ways for application of targeted therapy. Deficient homologous recombination and high genetic instability in type II tumors is a prerequisite for efficient application of platinum cytostatics and PARP (poly-ADP ribose polymerase) inhibitors. On the other hand, carcinogenesis of the less aggressive, but often resistant type I tumous is dependent on the activation of signaling pathways PI3K/AKT and RAS/BRAF/MEK/ERK pathway. Targeted inhibition of these pathways could efficiently improve therapy of type I tumors and decrease serious adverse side effects.Key words: ovarian cancer - high-grade serous ovarian carcinoma - low-grade ovarian carcinoma - endometrioid carcinoma - mucinous carcinoma - malignant transformation - genetic instabilityWe would like to thank M.Sc. Eva Michalova for critical reading of the manuscript.This work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 7. 8. 2016Accepted: 29. 8. 2016.

[The Role of PD-1/PD-L1 Signaling Pathway in Antitumor Immune Response]. / Úloha PD-1/PD-L1 signalizace v protinádorové imunitní odpovedi.

BACKGROUND: Correct function of the immune system depends on close cooperation between stimulation and inhibition signals, which protect an organism from outside microorganisms and other agents, but also protects healthy tissues against possible self-destructing attacks of the immune system. However, the inhibitory mechanisms can be abused by cancer cells that evade immune responses and, in fact, they help develop cancer. Therefore, one of the characteristics of cancer cells is the ability to evade immune recognition. Immunotherapy is a treatment method that stimulates the immune system to fight cancer. The checkpoints of the immune system can be considered as effective and specific therapeutic targets. Programmed cell death signaling pathway (PD-1/PD-L1) is one of the most discussed inhibition pathways in recent years. Blockage of PD-1/PD-L1 interaction restores mechanisms of immune response and increases antitumor immune activity. Monoclonal antibodies blocking PD-1 receptor or its ligand PD-L1 have already shown clinical efficacy. However, it is important to carry out research to explore the mechanisms of PD-1/PD-L1 pathway to find new factors, which influence its activity and, of course, to illuminate the variability of this pathway which naturally originates in the diversity of the tumor milieu. Obtained results could be utilized to achieve maximal anticancer effect after inhibition of PD-1/PD-L1 signaling pathway useful in clinical practice. AIM: The aim of the article is to summarize current knowledge about PD-1/PD-L1 signaling pathway and to discuss its role in antitumor immune response.Key words: programmed cell death pathway - tumor escape - PD-1 - PD-L1 - CD274This work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 6. 2016Accepted: 4. 8. 2016.

[Utilization of Hydrogen/Deuterium Exchange in Biopharmaceutical Industry]. / Vyuzití metody vodík/deuteriové výmeny v biofarmaceutickém prumyslu.

BACKGROUND: The development of biopharmaceutics is the fastest growing segment of the present pharmaceutical industry. The analysis of proteins therapeutics is a challenging task due to their large size and complexity of spatial structure. Any changes in the primary, secondary, tertiary or quaternary protein structure can have huge impact on their function, efficiency and toxicity. Mass spectrometry proved itself to be a powerful tool for analysis of primary protein structure (amino acid sequence) and thanks to the development of new techniques in last years it is able to analyse higher order protein structures. One of these new techniques is hydrogen/deuterium exchange (HDX). HDX is based on exchange of amid protons with deuterium from solution on the protein backbone chain. Protons on the surface of protein are exchanging with deuterium much faster than protons buried inside of protein. HDX results could provide information about spatial protein structure and also about protein-protein interactions and protein-ligand interactions. Furthermore, by analysing of deuterium exchange in different time points this method could give information about dynamic changes of protein structure and dynamics of proteins interactions. Because of possibilities of this method, HDX become attractive method for characterization of protein biopharmaceuticals. AIMS: This review article is focused on the utilization of mass spectrometry in biopharmaceutical industry and mainly on HDX method and its applications.Key words: mass spectrometry - proteomics - protein conformation - drug discovery - drug industry - hydrogen/deuterium exchangeThe work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 23. 5. 2016Accepted: 10. 6. 2016.

[New Technologies for In Vivo Cancer Diagnostics]. / Nové technologie pouzívané pro in vivo diagnostiku nádoru.

BACKGROUND: The treatment of oncological diseases is based on the combination of surgery, representing the key step for the removal of the tumor tissue, radiotherapy, chemotherapy, and hormone therapy. However, the surgery is often accompanied by issue of determining the boundaries of the tumor. Prior the operation, the surgeon has information on preoperative findings, which indicate the location and extent of the tumor, but does not specify a clear boundary between the tumor and healthy tissue. This area cannot be recognized visually or by touch in most cases and when the tumor is not removed completely the patient has to undergo reoperation. AIM: Therefore, a number of research centers began to deal with the development of technology that would provide information about the state of the tissue in real time directly during surgery and would not require the collection or storage of tissue samples. These include MarginProbe, Spectropen tissue and spectroscopic scanner devices. Another group consists of imaging techniques using mass spectrometry approaches to determine the tissue specificity. Recently, the intraoperative mass spectrometry (REIMS) technique has undergone tremendous development. It uses an electronic scalpel using by the surgeon for cutting the tissue, when the resulting aerosol is discharged into the mass spectrometer that in tenths of seconds measures mass spectra of phospholipids, which are specific to the operated tissue (tumor or healthy). In the Czech Republic this technology has been already used for research purposes for the detection of drug deposited in the tumor and healthy tissue of mice suffering from melanoma. The obtained results show that with this apparatus it would be possible fundamentally affect the treatment and its efficacy in oncology as well. We will inform you about these new technologies and elucidate their principles and utilization.Key words: surgery - cancer - tumors - molecular diagnostics - mass spectrometry - databaseThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 17. 5. 2016Accepted: 6. 9. 2016.

p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53ß are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53ß, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53ß overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53ß expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases.

[What Can Study of Oligomerization of Proteins in the Process of Oncogenesis Bring Us?]. / Co muze prinést studium oligomerizace proteinu v procesu onkogeneze?

Many cellular proteins form oligomers. The equilibrium between monomeric and oligomeric states of these proteins is important for the regulation of protein activity. Modulation of the oligomerization equilibrium could be an interesting approach in the development of new therapeutic agents. This review summarizes information about protein oligomerization and modulation of this process, demonstrating the role of oligomerization in oncogenesis by tumor suppressor protein p53, which forms tetrameric structure. Today, many studies focus on finding compounds that stabilize its tetramers. Among the methods for studying oligomerization, we present hydrogen/ deuterium exchange method coupled with mass spectrometry which is suitable for the detection of protein-protein interaction and analysis of oligomerization dynamics.