[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.

Prediction of ovarian cancer prognosis and response to chemotherapy by a serum-based multiparametric biomarker panel.

Currently, there are no effective biomarkers for ovarian cancer prognosis or prediction of therapeutic response. The objective of this study was to examine a panel of 10 serum biochemical parameters for their ability to predict response to chemotherapy, progression and survival of ovarian cancer patients. Sera from ovarian cancer patients were collected prior and during chemotherapy and were analysed by enzyme-linked immunosorbent assay for CA125, kallikreins 5, 6, 7, 8, 10 and 11, B7-H4, regenerating protein IV and Spondin-2. The odds ratio and hazard ratio and their 95% confidence interval (95% CI) were calculated. Time-dependent receiver-operating characteristic (ROC) curves were utilised to evaluate the prognostic performance of the biomarkers. The levels of several markers at baseline (c(0)), or after the first chemotherapy cycle (rc(1)), predicted chemotherapy response and overall or progression-free survival in univariate analysis. A multiparametric model (c(0) of CA125, KLK5, KLK7 and rc(1) of CA125) provided predictive accuracy with area under the ROC curve (AUC) of 0.82 (0.62 after correction for overfitting). Another marker combination (c(0) of KLK7, KLK10, B7-H4, Spondin-2) was useful in predicting short-term (1-year) survival with an AUC of 0.89 (0.74 after correction for overfitting). All markers examined, except KLK7 and regenerating protein IV, were powerful predictors of time to progression (TTP) among chemotherapy responders. Individual and panels of biomarkers from the kallikrein family (and other families) can predict response to chemotherapy, overall survival, short-term (1-year) survival, progression-free survival and TTP of ovarian cancer patients treated with chemotherapy.

[How external quality evaluation programs affect the quality of laboratory results in routine practice in the Czech Republic]. / Jak mohou programy externího hodnocení kvality ovlivnit kvalitu laboratorních výsledku v terénní praxi v Ceské republice.

Goals of our study were: 1. to assess quality of our analytical work and application of concepts of good laboratory practice using different EQA surveys, 2. to evaluate performance and efficiency of systems for internal quality control using EQA program that is independent on local subjective and objective influences and 3. to observe how single EQA programs differ and how they contribute the ability of laboratories to manage their overall quality. Using one-way ANOVA, we compared our 1999 results in the local EQA survey AKS (SEKK s r.o., Czech Republic) to our results in CN3 Comprehensive Chemistry Survey (College of American Pathologists, USA) for analyses provided in CN3 with the definitive method correlated target values, the design of which assures accuracy of the field methods on patient specimens. In 4 out of 9 methods, the material AKS predicted target values that were significantly different from that of CN3; calcium sodium, uric acid and cholesterol. The CN3 survey provided link of our results of internal quality control and national EQA to the largest proficiency testing available worldwide in clinical chemistry. Out data suggest that a possibility exists to improve the design of target values for materials used for small national EQA surveys as well as the program administration so that local characteristics of field method on patient specimens could be better monitored.

Quantitative determination of telomerase activity in breast cancer and benign breast diseases.

Telomerase plays an important role in maintaining the stability of chromosomes. This ribonucleoprotein prevents chromosome ends (telomeres) from gradual loss with each cell division. It enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Telomerase activity has been detected in the majority of tumor and germ cells and in immortalized cell lines. Quantitative telomerase PCR-ELISA (TeloTAGGG Telomerase PCR ELISA(PLUS)) was evaluated for distinguishing benign and malignant breast tissue. Activity of telomerase was determined in 27 samples of fibrocystic and dysplastic tissues, 28 fibroadenomas and phylloid tumors, and 154 breast cancer tissues; 59 specimens were analyzed retrospectively. Analytical precision and linearity of the assay was tested using breast carcinoma cell line ZR-75-1 and breast tumor tissue extracts. About 4% of tumor samples were excluded from analysis due to interferences in the PCR reaction. Relative telomerase activity differed significantly in the groups of dysplastic tissues, fibroadenomas and carcinomas. The highest activity was found in breast cancer tissue. This method can identify breast cancer tissue with 73% clinical sensitivity and 93% specificity as compared to benign breast tumors. We did not find a correlation between telomerase activity and the tissue levels of estrogen and progesterone receptors, HER-2/neu oncoprotein concentration, tumor size, and lymph node positivity. Probability of disease-free survival was significantly lower for patients with telomerase activity higher than median value. As the assay for telomerase activity has very high analytical sensitivity and high specificity for cancer cells, this routinely used method may prove useful for distinguishing malignant phenotype of breast tissues.

Significance of pre-treatment immunological parameters in colorectal cancer patients with unresectable metastases to the liver.

BACKGROUND/AIMS: In this study, we have compared the profiles of peripheral blood lymphocyte (PBL) subsets and serum cytokine levels of healthy individuals with those of patients with unresectable liver metastases from colorectal carcinoma before starting regional chemoimmunotherapy. Since the therapeutic responses are limited only to a subset of patients, we hypothesize that the initial status of immunity and individual immune response to a tumor might be significant to the therapeutic outcome. METHODOLOGY: Cellular and humoral immunological parameters were compared between 10 patients with colorectal cancer metastases to the liver responding and non-responding to regional intra-arterial chemo-immunotherapy, and 5 healty individuals. Analyses included a flow cytometric immunophenotyping of peripheral blood mononuclear cells (CD3, CD4, CD8, CD19, CD25, CD28, CD56, CD57, CD80 and HLA.DR), estimation of serum cytokine levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and other immunological parameters are soluble IL-2 receptor (sIL-2), carcinoembryonic antigen (CEA), gastrointestinal cancer-associated antigen (CA 19-9), and C-reactive acute phase protein (CRP). A significantly lower proportion of CD8 lymphocytes and a trend for decreased CD19, CD28 and CD80 was detected among colorectal cancer patients before liver-directed chemotherapy compared to healthy controls. RESULTS: The cancer patients showed a significantly increased population of peripheral NK cells as detected by both CD56+ and CD57+ phenotypes. Elevated serum levels of CRP, IL-4 and TNF-alpha, sIL-2R, but not IL-2, were also demonstrated in cancer patients as compared to controls. Activated CD25+ lymphocytes correlated negatively with CD28+ lymphocytes (r = -0.68, p < 0.01) and less significantly with CD4+ lymphocytes (r = -0.56, p < 0.05). The CD8+ cytotoxic cell subset might be negatively influenced by serum IL-4 (r = -0.57, p < 0.05). Positive correlation was found between sIL-2R and CRP (r = -0.78, p < 0.01), and between sIL-2R and TNF-alpha (r = 0.64, p < 0.05) serum levels in patients with progressive disease during the course of therapy, the initial proportions of CD4+, CD19+ and CD28+ lymphocytes were significantly lower than those among responders. Among humoral parameters, only sIL-2R showed a marginal correlation with therapeutic response, being more elevated among non-responding patients. Pre-treatment serum levels of CEA and CA 19-9 showed correlation with neither therapeutic response nor with any of the cellular or humoral immunological parameters analyzed. CONCLUSIONS: The results may serve as an initial guideline to open a discussion on the rationale of such a panel of tests, hopefully leading to standardized laboratory pre-selection and monitoring of patients treated with regional chemoimmunotherapy.

[Regional chemotherapy of liver tumors]. / Regionální chemoterapie nádoru jater.

Based on experience with regional intrahepatic chemotherapy in 67 patients with inoperable primary, and in particular secondary liver tumours, which comprises several hundred administered cycles of mostly continuous regional regimens, the authors summarize briefly the main principles and possibilities of this treatment. In a review of randomized studies they provide evidence for the advantage of regional chemotherapy, as compared with systemic treatment, in particular with regard to the higher percentage of therapeutic responses. In correctly indicated, and if possible early cases of hepatic tumourous affections, it is possible to potentiate the effect of regional chemotherapy by local destruction of tumourous foci by alcoholization, cryodestruction or resection. An integral part of this treatment is also monitoring of the effect by following up the dynamics of serum levels of tumour markers and by imaging methods. Because the most frequent cause of failure of this method are extrahepatic secondaries and secondary chemoresistance of the tumourous foci, improvement of results can be expected in particular from a combination of regional and systemic chemotherapy and the inclusion of cytokines into the therapeutic schemes.

Phylloid breast tumors and three steroid hormone receptors.

The concentrations of three steroid hormones (estrogen, progesteron and 1,25-dihydroxycholecalciferol) receptors (ER, PgR, DR) in tissue cytosol were analyzed in a group of 17 breast phylloid tumors. Comparison with breast carcinoma tissue samples (n = 37) did not reveal significant differences in average values of ER, PgR, and DR. Comparison with another control set of 30 samples of dysplastic tissue of the mammary gland showed significant differences only in PgR values. Only 18% of phylloid tumor samples contained levels above cut-of-line of all three receptors (ER, PgR, DR-5,10,10 resp. fmol/ mg protein). The most frequent combination was ER+PgR+DR-(41%). As far as we know, DR in phylloid breast tumors have never been examined before. In approximately 60% of our samples we found the expression of DR, in 36% the estimated values were above 10 fmol/mg protein. Cells of the tissue not expressing DR seem to belong to a special phenotype. We found no ER+PgR- or ER-PgR-combinations in them. The group which expresses DR is characterized by a higher dispersion of PgR values.

Biochemical analysis of breast cyst fluid as a possible predictor of breast carcinoma development.

The occurrence of breast cancer in patients with gross cystic disease is 2-5 times higher as compared to control group of women. During 3 years, 183 cyst fluid samples were analyzed in 129 females, in 30 patients of them the samples were analysed repeatedly. The distribution of the Na+/K+ ratio, considered as the measure of cancer risk, was found to be bimodal. In repeated analyses the type I cyst fluid markedly predominated (Na+/K+ < or = 4.0). A direct dependence on this ratio was found in the concentration of glucose, albumin, carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA) and its specific form, TPS; an indirect dependence was found for the level of uric acid, phosphates, alkaline phosphatase (ALP) and alpha-amylase (AMS). The predominance of apocrine metaplasia cells released into the cyst fluid is characteristic of type I cysts. A definitive assessment of significance of these parameters will be enabled by a long-term follow-up of the disease in the respective patients.

Early diagnosis of breast cancer dissemination by tumor markers follow-up and method of prediction.

A mathematical model of prediction of progression was tested in patients with breast cancer employing long-term monitoring of tumor markers CEA, CA 15-3, MCA and TPA, erythrocyte sedimentation rate (FW), and the enzymes gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and lactate dehydrogenase (LD) in serum. At the same time, specificity, sensitivity, lead time and positive predictive value were evaluated along with false positivity for all these parameters and their combinations. A model was proposed for the follow-up of patients with breast cancer after the completion of basic therapy.

The excretion of urinary enzymes, proteins and creatinine in patients receiving cisplatinum.

The sensitive parameters of tubular nephrotoxicity during 4-day chemotherapy with new combination schedule of cisplatinum and 5-fluorouracil were followed. The determinations of tubular enzymes beta-glucuronidase (GRS) and gamma-glutamyltransferase (GMT) in 24 hours urine, the excretion of creatinine and proteinuria were assayed before therapy and during 4 consecutive days of treatment. We recorded the significant increase of protein excretion and only slight increase of GMT and GRS activities after chemotherapy. The decrease of the creatinine excretion on the 3rd day of therapy was not statistically significant. Simultaneously followed serum creatinine and urea levels proved the elevation of creatinine only in 1 patient (the 1st grade according WHO classification). Our results suggest that the performed regime of chemotherapy produces only very low nephrotoxicity comparing to the 1-day administration of cisplatinum. These conclusions are confirmed also by the former examinations of GRS activities in cisplatinum therapy.

Phase I-II trial of aclacinomycin A given in a four-consecutive-day schedule to patients with solid tumours. A South-East European Oncology Group (SEEOG) Study.

Aclacinomycin A (ACM) in a daily dose of 30 mg/m2 was infused over 1 h on 4 consecutive days to 50 patients. Myelotoxicity was acceptable, nausea and vomiting was frequent, hair loss was mild. Grade 1-2 cardiac rhythm abnormalities were observed in 12% of the patients. Between days 1 and 4 the heart rate and the corrected Q-T interval increased while the amplitude of the T wave decreased significantly, cardiac contractility remained unchanged. In 24 evaluable breast cancer patients 1 complete remission (4%) and 2 partial remissions (8%) lasting for only 2-3 months were seen. None of the 8 patients suffering from ovarial cancer benefitted from ACM therapy.

Comparative evaluation of two administration schedules of cis-dichlorodiammineplatinum (DDP) in ovarian and head and neck cancers: a CMEA chemotherapy group study.

The therapeutic effectivity of two administration schedules of DDP were compared. The dose was either 100 mg/m2 infused for 4 h or 20 mg/m2 infused for 1 h on 5 consecutive days. The combined objective remission rate of the two regimens were 37/53 (23% CR ++ 44% PR) for ovarian cancer and 8/35 (9% CR + 14% PR) for head and neck cancer. WHO Grade 1-2 myelo- and nephrotoxicity was observed in 26% and 20%, respectively, out of the 105 cases evaluable for toxicity in the two groups. Nausea and vomiting was moderate to severe. Neither the remission rate nor the toxicity of the two schedules were significantly different.

Relation between dose, plasma concentration and toxicity in a phase I trial using high dose intermittent administration of an alkylating agent, diacetyldianhydrogalactitol (DADAG).

Diacetyldianhydrogalactitol (DADAG), a new alkylating sugar alcohol derivative, was administered as single, 30-min infusions in doses ranging from 390 to 1200 mg/m2. The dose-limiting toxicity was myelosuppression. The median times to WBC nadir and regeneration were 16 and 21 days, and to platelet nadir and recovery 20 and 27, respectively. Nausea and vomiting occurred frequently and were of moderate severity. For phase II studies 900 mg/m2 DADAG given every 4-6 weeks is recommended. The area under the plasma concentration time curve (AUC) for DADAG did not increase in proportion with dose escalation; it changed only from 235.5 +/- 70.7 to 262.4 +/- 71.5 micrograms h ml-1 between doses of 690 and 1050 mg/m2. No correlations between the dose administered and the nadir values for haemoglobin concentration, WBC and platelet counts, or the number of episodes of vomiting were demonstrable in this dose range. Such an association was revealed, however, when the above biological variables were related to the individual AUC for DADAG.