Research Profile

 

Welcome to the Department of cytokinetics. Our research group has been established several years ago in order to pursue a research program focused on the study of the effects of metabolites of arachidonic acid (AA), eicosanoids, in signal transduction of cytokines at both cellular and molecular levels. This program is based on our previous original data, which demonstrated that the balance between cyclooxygenase (COX) and lipoxygenase (LOX) metabolites plays a significant role in the regulation of proliferation of mouse hematopoietic cells - while LOX products play a positive role in hematopoiesis during post irradiation recovery in vivo, as well as in regulation of proliferation of human leukemic cells in vitro, COX products may produce an opposite effect.

The present research topics focus at a potential role of lipid membrane components and their derivatives in cell signaling after action of various environmental substances, with a particular emphasis being put on lipid nutrition components and xenobiotics. Our studies aim to understand the mechanisms modulating cytokinetics i.e. cell proliferation, differentiation and apoptosis and their role in carcinogenesis. Using both tumor and non-tumorigenic cells, new types of interactions of lipid compounds(short-chain and polyunsaturated fatty acids), drugs (non-steroidal anti-inflammatory drugs-NSAIDs, cytostatics) and selected environmental pollutants (polycyclic aromatic hydrocarbons, PCBs, dioxins) with physiological regulators of cytokinetics(cytokines, growth factors) are being investigated. A special attention is paid to relationships between effects of the substances investigated on biophysical properties of cell membranes, redox processes, cell communication and signaling (kinases, transcription factors, regulatory proteins of the cell cycle and apoptosis, etc.) and to consequences of these modulations on changes of the cytokinetics. The results contribute to understanding of the role of environmental components in carcinogenesis and to the search of new anticancer strategies. The group embodies considerable teaching activities.

 

Scientific interest:

Lipid dietary compounds in regulation of cytokinetics

Growth regulators in cancer cell signalling

Interaction of lipids and cytokines

Effects of anticancer drugs

Molecular and cellular mechanisms of toxicity of organic compounds

Link to our Methodology

 

 

Lipid dietary compounds in regulation of cytokinetics

Lipid nutritional compounds like polyunsaturated fatty acids (PUFAs) of omega-6 and omega-3 series have significant impact on cellular physiology, playing both structural and regulatory role. They can significantly affect maintenance of cell population homeostasis, and as such they are involved in development of various pathological disease states, including cancer. Cell lipid/phospholipid metabolism and membrane composition and properties depend on exogenous supplementation of fatty acids and there are significant differences between normal and transformed cells.

In the colon, PUFAs and other dietary components, like butyrate from fiber, may operate together and interact with endogenously produced factors (cytokines, growth factors). Thus, signals from nutritional compounds and endogenous factors regulating cell growth, differentiation and apoptosis are integrated within the cell and may have a substantial impact determining the final phenotype, metabolism and kinetics of colon epithelial cell population, and therefore are studied in our laboratory.

 

Growth factors in cancer cell signaling

Tissue microenvironment plays an important role in tumor initiation and progression. Growth factors - cytokines - play crucial role in cancer development and some of them belong to the significant autocrine/paracrine factors produced by various cell types in tumor microenvironment. Because of their important role during tumor progression, modulation of their signal transduction represent potential target for therapy. Research program in this direction is concentrated on the study of control and regulation of signaling in cancer with an initial focus on role of autocrine/paracrine produced growth factors. We are concerned with the mechanisms of possible modulation of expression of these factors (e.g. members of transforming growth factor beta family), and their role in regulation of cell migration, proliferation, differentiation and apoptosis. Special attention is given to the modulation of lipid metabolism and lipid membrane, as a potential strategy for regulation of the effects of autocrine/paracrine produced growth factors on tumor cells and in tumor microenvironment.

 

Interaction of lipids and cytokines

A particular attention is paid to the effects of selected types of omega-3 and omega-6 PUFAs, sodium butyrate (NaBt), their mutual interactions, and their interaction with endogenous regulators from tumor necrosis factor (TNF) family. We attempt to investigate in detail mechanisms of effects at various levels of cell organization (plasma membrane, cytosol, mitochondria, and nucleus), modifications of cellular lipid composition and function and their association with intracellular mechanisms influencing cell proliferation, differentiation and apoptosis, through comparing both normal and transformed colon epithelial cells. We focus on the changes of cellular lipid composition, membrane-associated events, oxidative metabolism, role of mitochondria, modulation of specific signaling pathways, activation of transcription factors and changes of cell cycle and apoptosis regulatory machinery. Moreover, we study relationships among differentiation and detachment-induced apoptosis (anoikis) and the role of adhesive molecule signal transduction during interaction of TNF family regulators, NaBt and specific inhibitors of arachidonic acid metabolic pathways. Our principal aim is to describe the role of lipid compounds in inter- and intracellular communication, which are involved in regulation of cytokinetics. Part of our research is also focused on the development of lipid nutrition preparations for prevention and supportive therapy of oncological diseases (disease-specific nutrition).

 

Effects of anticancer drugs

The research is focused on the interactions of phospho/lipids and their metabolism with specific drugs. 1) The mechanisms of action of non-steroidal anti-inflammatory drugs (NSAIDs), which act as inhibitors of arachidonic acid metabolism pathways, catalyzed by cyclooxygenases, lipoxygenases and cytochrome P450 monooxygenases, are investigated in both normal and transformed cells of various origins. Moreover, their interactions with inducers of differentiation and apoptosis (retinoic acid, vitamin D3, NaBt, TNF-alpha) are studied, with an aim to potentiate possible therapeutic effects.

2) The role of (phospho)lipid metabolism, most notably polyunsaturated fatty acids in effects of novel platinum cytostatics on a wide panel of cancer cell lines of different origin and resistance to cisplatin are being investigated. The main goals are to determine the growth suppressive effects of cytostatics and to gain a better insight into potential molecular mechanisms involved. Our attention is focused on the possible mechanisms of action, which are different of those primarily based on DNA damage. The signaling pathways involved in the control of cytokinetics are a primary subject of investigation. Particularly the role and balance at the level of a) regulatory proteins and receptors controlling apoptosis; b) molecules regulating cell cycle and proliferation, c) reactive oxygen species  and lipid peroxides are to be evaluated. The platinum drugs of new generation may overcome cell resistance, which may be further potentiated by modulating phospholipid metabolism. This might enable to treat the tumors that are hardly curable by currently-used cisplatin and its derivatives, and the higher efficiency of these new compounds may help to reduce doses, undesirable side effects and treatment costs.

 

Molecular and cellular mechanisms of toxicity of organic compounds

Organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs) or halogenated aromatic hydrocarbons (including dioxins, PCBs and other compounds) are widespread environmental contaminants, posing a serious threat to both environment and human health. The research program focuses on studies of their toxic modes of action in vitro. As these compounds may interact with a number of intracellular proteins, we are especially interested in their impact on transcriptional regulators, PAS and nuclear receptor family proteins, such as the aryl hydrocarbon receptor (AhR) and estrogen receptors (ER). We concentrate on the mechanisms related to deregulation of cell cycle, proliferation, apoptosis and cell-to-cell communication, which might be related to carcinogenic and endocrine-disrupting effects of these environmental pollutants. The principal aim is to identify target proteins and genes, which might play a key role in toxic effects of these compounds in vivo. PAHs are traditionally studied as genotoxins and mutagens. However, it is becoming increasingly evident that these compounds also exert nongenotoxic effects, such as disruption of cell proliferation/apoptosis control, expression and activity of xenobiotic-metabolizing enzymes, or inhibition of intercellular communication, which might significantly affect their ability to induce DNA mutations and aberrations that in turn give rise to cancer and other diseases. Therefore, we also aim to study interactions of genotoxic and nongenotoxic impact of these compounds on cells, using both individual PAHs and their environmental mixtures.

 

Methodology

Model transformed mammalian cell lines obtained mainly from European or American Cell Cultures Collections provide the system enabling dynamic studies of cytokinetics in these populations.

In selected time intervals after experimental treatment the cell proliferation, differentiation, death other cell parameter (see below) are detected simultaneously to obtain cytokinetic data. Flow cytometry is one of principal methods used, since it allows detection of several parameters, i. e. specific gene or intracellular molecule expression during the individual phases of the cell cycle or markers of differentiation and cell death. In some cases, detection of parameters is performed on a pre-selected population. Gene expression is also detected on the mRNA (QPCR) and protein level (Western Blotting). Specific intracellular localization pattern of particular proteins could be also visualized by means of fluorescent immunocytochemistry (ICC). Molecular aspects of DNA-protein and protein-protein interactions are studied by Electrophoretic Mobility Shift Assay (EMSA), Chromatin Immunoprecipitation (ChIP), and protein Immunoprecipitation (IP). RNA interference (RNAi) or knock-down of gene of interest can be used for determination of selected gene function. Change of activity or production of signalling molecules (i.e. secondary messengers, kinases, ROS etc.) is also possible to detect by different colorimetric, fluorimetric, and luminometric assays. In special cases, results obtained by in vitro experiments can be parallelly tested in in vivo models (mice).

 

Cytokinetic parameters

Proliferation parametersThese include absolute cell numbers, metabolic activity reflecting the number of viable cells, cell cycle detection and detection of DNA-replicating cells. Cell viability is detected using dye-exclusion assay.

Differentiation parametersThese include respiratory burst detection, detection of specific enzyme activities, expression of specific surface molecules and changes in cell morphology.

Apoptotic parametersThese include detection of DNA fragmentation on gel electrophoresis, detection of sub-G0/G1 peak or phosphatidylserine externalisation by flow cytometry, expression and cleavage of specific molecules (e.g. PARP, caspases, Bcl-2 family proteins), decrease of mitochondrial membrane potential, and detection of morphological changes.

Parameters of intercellular communication. These include detection of cell motility, localisation and expression pattern of adhesive molecules, and loss of cell-cell and/or cell-ECM interactions connected with the detachment-induced apoptosis (anoikis).

Signal transduction pathways parameters

Signal transduction pathways described in research profile are studied in detail by techniques mentioned above.

  

The laboratory contains top quality equipments for cell culture and detection of the cytokinetic parameters at the cellular and molecular levels:

 

a) Flow cytometer FACS CALIBUR  (Becton Dickinson) with sorting option. Two lasers enable parallel detection of 8 parameters.  It is possible to sort population parts of interest according to selected markers and to make further detail analyses of these cells. CellQuest Pro? and ModFit? softwares are used to analyse the data. Together with top high resolution cytometry and confocal microscopy (equipments shared by several groups in the Institute) it creates a complex methodology not only for analyses of cell structure and morphology, but also for analyzing large cell populations especially with regard to parameters of proliferation, differentiation and cell death.

 

b) Basic facilities for handling and cultivation of cells in vitro:

Laminar biohazard boxes (Jouan, Nuaire, Gelaire); COincubators (Jouan, Heraeus, Nuaire); sterilizators; centrifuges (Jouan, Boeco); Water bath (Julabo); Coulter Counter (models ZM, ZF); light microscopes (Zeiss, Jena, Olympus CK40), and fluorescent microscope IX-70 (Olympus) with image analysis software Analysis D and motorised stage (Marzhauser); ELISA reader (Asys Hitech); Absorbance and fluorescence reader Fluostar Galaxy (BMG); Ultralum (Ultralum Inc.)etc.

 

c) Facilities for methods of molecular biology:

Electroporator Biorad; Electrophoretic equipments (Biorad, Hoefer); Wet and semidry blotter (Hoefer) etc.

 

d) Special laboratory equipment for radioactive methods

 

e) Special laboratory for PCR-based and molecular cloning techniques

Thermocycler (MJ Research); Biohazard box (Faster), Dry incubator (Shel Lab).

 

A number of other eguipments are shared by several groups in the Institute of Biophysics - Molecular Dynamics SARASTRO 2000 confocal scanning laser microscope; chemiluminometer LKB Wallac; Molecular dynamics phosphor imager - Storm 860 system with ImageQuant software; ultracentrifuges; RotorGene 6000 (Corbett Research) etc.