[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.

[Rab Proteins, Intracellular Transport and Cancer]. / Proteiny Rab, vnitrobunecný transport a nádorová onemocnení.

BACKGROUND: Rab proteins are small monomeric enzymes which belong to the large Ras protein superfamily and allow hydrolysis of guanosine triphosphate (GTP) to guanosine (GDP). Up to now more than 60 proteins have been described that act primarily as regulators of intracellular transport. Rab GTPases are mostly located at the intracellular membranes, where they provide connections to motor proteins and to the cytoskeleton and control various steps of the traffic pathways including the formation and movement of vesicles or membrane fusion controlling secretion, endocytosis, recycling and degradation of proteins. Today, the deregulated expression of Rab protein is discussed in different types of malignancies. The number of identified diseases associated with mutations in Rab proteins or their cooperating partners increases and the evidence for the involvement of Rab to the human pathologies such as the immune failure, obesity and diabetes, Alzheimers disease or hereditary genetic diseases is growing. The malfunctions of Rab proteins caused by mutations or aberrant posttranslational modifications lead to changes in the protein and vesicle trafficking, which play a crucial role in the formation and development of cancer and the deregulation of Rab expression frequently influences the migration, invasion, proliferation and drug resistance of the tumor cells. AIMS: This article summarizes the main functions of Rab proteins in the cells, describes the mechanism of their activity and focuses on the current knowledge about the roles of these GTPases in carcinogenesis.Key words: Rab GTPases - protein transport - carcinogenesisThis 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. 5. 2016Accepted: 31. 5. 2016.

[Analysis of Phosphoproteome Changes in MDA MB 468 Cancer Cell Line in Response to Expression of p63 Isoforms Using Mass Spectrometry]. / Analýza zmen fosfoproteomu nádorové bunecné linie MDA MB 468 v odpovedi na expresi izoforem p63 pomocí hmotnostní spektrometrie.

Compared to normal cells, tumor cells can show different activity of kinases and phosphatases resulting in altered phosphorylation states of proteins affecting their activity within various signaling pathways. The detection of these alterations is essential for development of targeted therapy based on activation/ inhibition of specific signaling pathways. Various methods can be used for detection of protein phosphorylation; however, a comprehensive assessment of phosphoproteome is performed by mass spectrometry. The differences in phosphoproteome were studied using MDA  MB  468 cell line (with incorporated genes encoding isoforms of p63) derived from breast carcinoma. Cells with tetracycline-induced expression of the p63 isoforms were compared to control cells with wildtype expression. Denatured proteins from cell lysates were digested to peptides, enriched for phosphopeptides and subsequently separated using liquid chromatograph coupled with mass spectrometer Orbitrap Elite. Three different mass spectrometric methods were used for each sample analysis to find the most suitable conditions for the detection of phosphorylated peptides. Then phosphoproteins were identified and quantified. The number of identified phosphoproteins using all chosen mass spectrometric methods was similar; however, each method showed several unique phosphorylated proteins. Our analysis revealed that both p63 isoforms (TAp63α a Np63α) mainly affected phosphorylation of proteins associated with RNA splicing in MDA- MB- 468 cells.

[Mechanisms of drug resistance and cancer stem cells]. / Mechanizmy lékové rezistence a nádorové kmenové bunky.

Although the success of anticancer treatments has been increasing annually, drug resistance remains the dominant cause of death of cancer patients. Initial therapy often leaves residual dis-ease that leads to repeated tumor development or to loss of its sensitivity to available therapy. One reason of residual disease formation is the presence of cancer stem cells (CSCs). CSCs have been identified as a small population of cells that is capable of self  renewal and differentiation. It is supposed that these cells are responsible for cancer initiation, progression, metastasis, recurrence and drug resistance. Over the past years, much attention has been paid to development of CSCs related therapies and to identification of key molecules involved in controlling the specific properties of CSCs populations. This article reviews the basic mechanisms of drug resistance in relation to cancer stem cells.

P63 - an important player in epidermal and tumour development.

p63 is a transcription factor which plays an important role in epidermal development, differentiation and tumourigenesis. p63 belongs to the p53 protein family and at least six isoforms were identified to date. p63 isoforms play contrary roles during the development and formation of the epidermis as well as in cancer. p63 participates in epithelial development, where it affects proliferation and differentiation of epidermal cells. Inherited mutations in the TP63 gene generate different developmental defects and p63 knockout in mice results in the absence of epidermis. Another important role of p63 is the control of cell-cell adhesion, where it regulates desmosomes. The loss of proliferation and cell-cell adhesion control are important for tumourigenesis and overexpression of p63 can enhance tumour growth and inhibit apoptosis. This review briefly summarises the roles of p63 in epithelial development, cellular proliferation, adhesion and migration and reveals its share in tumourigenesis and metastasis.

A combined immunoprecipitation and mass spectrometric approach to determine deltaNp63-interacting partners.

Expression of p63 is essential for the formation of epidermis and other stratifying epithelia. Moreover p63 is highly expressed in several epithelial cancers and is involved in tumourigenesis and controlling chemo-sensitivity. The identification of p63 interacting partners is essential for understanding the complex network of gene regulation managing epithelial development and could also help to reveal signalling pathways participating in UV-damage response in human skin. We used a proteomic approach to identify proteins that interact with deltaNp63. Proteins were isolated by immunoprecipitation with deltaNp63 specific antibody and analysed by mass spectrometry. We identified 23 proteins as potential deltaNp63 binding partners that were not present in negative control samples. These results will be evaluated using other methods.

Identification of alphaB-crystallin, a biomarker of renal cell carcinoma by SELDI-TOF MS.

Spectrometric-based surface-enhanced laser desorption/ionization ProteinChip (SELDI-TOF) facilitates rapid and easy analysis of protein mixtures and is often exploited to define potential diagnostic markers from sera. However, SELDI- TOF is a relatively insensitive technique and unable to detect circulating proteins at low levels even if they are differentially expressed in cancer patients. Therefore, we applied this technology to study tissues from renal cell carcinomas (RCC) in comparison to healthy controls. We found that different biomarkers are identified from tissues than those previously identified in serum, and that serum markers are often not produced by the tumors themselves at detectable levels, reflecting the nonspecific nature of many circulating biomarkers. We detected and characterized áB-crystallin as an overexpressed protein in RCC tissues and showed differential expression by immunohistochemistry. We conclude that SELDI-TOF is more useful for the identification of biomarkers that are synthesized by diseased tissues than for the identification of serum biomarkers and identifies a separate set of markers. We suggest that SELDI-TOF should be used to screen human cancer tissues to identify potential tissue-specific proteins and simpler and more sensitive techniques can then be applied to determine their validity as biomarkers in biological fluids.