Session I:

Structure of the nucleus
and regulation of gene expression

Maize in the maze: a rough guide to the puzzling epigenetics of paramutation

 

Jan Brzeski

 

Department of Education and Scientific Communication Copernicus Science Centre, Warsaw, Poland.

 

Epigenetic mechanisms maintain gene expression states through mitotic and sometimes meiotic cell divisions. Paramutation is an extreme example of epigenetic processes. Not only an established expression state is transmitted through meiosis to the following generations but also an information transfer occurs between alleles and leads to heritable changes in expression state. As a consequence the expression states can rapidly propagate in population, violating Mendelian genetics. Recent findings unraveled an essential role for siRNA-dependent processes in paramutation. Despite significant progress, the overall picture is still puzzling and many important questions remain to be answered.

 

FOLDED CHROMATIN DOMAIN INSTEAD OF AN ACTIVE CHROMATIN HUB: A MODEL BASED ON RECONSIDERED ESSENTIALS OF THE CHROMOSOME CONFORMATION CAPTURE PROCEDURE

 

Sergey V. Razin^1,2,3,  Ekaterina S. Gushchanskaya^1,2,3, Olga V. Iarovaia^1,3, Alexey A. Gavrilov¹

 

¹Institute of Gene Biology of the Russian Academy of Sciences, 119334 Moscow, Russia; ²Faculty of Biology, M.V. Lomonosov Moscow State University, 119992 Moscow, Russia; ³LIA 1066 French-Russian Joint Cancer Research Laboratory, Villejuif, France – Moscow, Russia.

 

Development of a panel of the so-called C-methods – experimental protocols that allow the study of the 3D organization of the eukaryotic genome – has permitted to make observations resulting in a new concept in molecular genetics. It became evident that the 3D genome organization constitutes a part of epigenetic mechanisms essential for maintaining the identity of differentiated cells. In this respect, the assembly of distant regulatory elements in common activatory complexes – active chromatin hubs – appears to be of primary importance. Here we show that one of the principal assumptions behind the C-methods is not correct. All C-methods are based on the “proximity ligation” which is preferential cross-ligation of interacting DNA fragments that remain joined by protein bridges after solubilization from formaldehyde-fixed nuclei. We show that the proximity ligation in the 3C procedure really occurs within non-lysed nuclei inside a cage formed by cross-linked chromatin fibers. This finding allows a new interpretation of the results of 3C analysis. Our data suggest that regulatory elements participating in formation of an active chromatin hub do not necessarily form a common complex stabilized by protein bridges, but rather are recruited to the same nuclear compartment where they retain a certain degree of mobility. This model is further supported by demonstration that in mouse embryonic liver cells treated according to the standard 3C protocol the number of cross-linked DNA fragments bearing promoters and upstream regulatory elements of the beta-globin locus does not exceed 1% of total number of these fragments.

 

An influence of chromatin topology on the accessibility of DNA

 

Sławomir Kumala¹, Yasmina Hadj-Sahraoui¹, Joanna Rzeszowska-Wolny²,
Ronald Hancock¹

 

¹Laval University Cancer Research Centre, Québec, Canada; ²Biosystems Group, Silesian University
of Technology, Gliwice, Poland.

 

The accessibility of DNA in chromatin is an essential factor in regulating its activities. We studied the accessibiiity of the DNA in a ~170 kb circular minichromosome to DNA cleaving reagents, using pulsed-field gel electrophoresis and fibre-FISH on combed DNA molecules. Only one of several potential sites in the minichromosome DNA was accessible to restriction enzymes in permeabilised cells, and in growing cells only a single site at an essentially random position was cut by poisoned topoisomerase II, neocarzinostatin, and γ-radiation which have multiple potential cleavage sites; further sites were then inaccessible in the linearised minichromosomes. Sequential exposure to combinations of these reagents also resulted in cleavage at only a single site. Minichromosome DNA containing single strand breaks created by a nicking endonuclease to relax any unconstrained superhelicity was also cut at only a single position by a restriction enzyme. Further sites became accessible after ≥95% of histones H2A and H2B, histone H1, and most nonhistone proteins were extracted.

These observations suggest that a global rearrangement of the three-dimensional packing and interactions of nucleosomes occurs when a circular minichromosome is linearised and results in its DNA becoming inaccessible to probes. Understanding this switch in DNA accessibility could be relevant to the accessibility of DNA in closed loops of genomic chromatin in vivo which are topologically analogous to a circular minichromosome.

 

New models of the nucleus and chromosomes

 

Ronald Hancock

 

Laval University Cancer Research Centre, Québec, Canada.

 

During the last five years there has been a revolution in our understanding of the structure of the cell nucleus and of metaphase chromosomes, mainly due to input of crucial ideas from polymer, colloid, and nanoscience. In the nucleus, chromosomes are confined with proteins, RNA, and DNA at concentrations of hundreds of mg/ml, and in these crowded conditions macromolecules and their interactions are influenced strongly by short-range forces termed entropic or depletion forces which are negligeable in dilute solutions. It has become clear that these strong but subtle forces are crucial determinants of many features of the nucleus and metaphase chromosomes, for example:

Formation of loops in chromatin is favoured by depletion forces, as shown by simulations of self-avoiding polymers. Chromosome territories are predicted to form due uniquely to the spontaneous segregation of mutually unentangled long polynucleosome chains. The assembly of nucleoli, PML bodies, and RNA pol II transcription factories appears to be driven by entropic forces. Diffusion in crowded conditions shows anomalies which increase the probability of finding a nearby target and consequently improve intermolecular propagation of signals.

In metaphase, the genome is surrounded by the cytoplasm where the measured concentration of diffusible macromolecules is ≥130 mg/ml. Metaphase chromosomes with well-conserved structure can be isolated by mimicking this crowded environment using inert crowding macromolecules but essentially no cations, suggesting that entropic forces due to crowding by cytoplasmic macromolecules play a significant role in the structure of metaphase chromosomes.

 

Session II:

Cellular responses to ionizing radiation
and stressing factors

(session co-organized by European Association for Cancer Research)

Microenvironment Matters:  Contributions to radiation carcinogenesis

 

Mary Helen Barcellos-Hoff

 

New York University School of Medicine, Department of Radiation Oncology, New York, NY 10016.

 

Our published and preliminary studies show that exposing mice to low doses of ionizing radiation prior to transplantation with oncogenically primed mammary epithelium accelerates mammary carcinogenesis in a TGFβ dependent manner and increases ER-negative, aggressive tumors (Nguyen et al. Cancer Cell, 2011). Here, we discuss how systemic radiation effects on host biology alter the course of carcinogenesis via the mammary microenvironment and stem cell regulation.  Together these data lead us to postulate that radiation primes the target epithelium for carcinogenesis by increasing stem cells and primes the host by recruitment and activation of macrophages to form pre-cancer niches, which in turn promote malignant progression. We hypothesize that epithelial cells undergoing neoplastic transformation in contact with activated host cells (e.g. macrophages and/or fibroblasts) form a pre-cancer niche, which mediates cancer development.  Notably, ionizing radiation acts on both components to accelerate the development of particular cancer subtypes. The identification of specific mechanisms by which microenvironment affects breast cancer subtype could provide avenues to prevent cancer in vulnerable populations. 

 

Research support from NASA Specialized Center of Research and DOE Low Dose Program.

 

Transmission of signals from irradiated rats to cage mates: an inter-animal bystander effect

 

Carmel Mothersill¹, Cristian Fernandez-Palomo¹, Elisabeth Schültke², Richard Smith¹, Elke Bräuer-Krisch³, Jean Laissue⁴, Christian Schroll², Jennifer Fazzari¹,
Colin Seymour¹

 

¹Medical Physics and Applied Radiation Sciences Department, McMaster University, Hamilton, Ontario, Canada; ²Stereotactic Neurosurgery and Laboratory for Molecular Neurosurgery, Freiburg University Medical Center, Freiburg, Germany; ³European Synchrotron Radiation Facility (ESRF), Grenoble, France; ⁴Institute
of Pathology, University of Bern, Switzerland.

 

Inter-animal signaling from irradiated to non-irradiated organisms has been demonstrated for whole body irradiated mice and also for fish. Endpoints studied include clastogenic  damage, and reproductive cell death. For fish, proteomic data has also been produced showing the induction of a protective proteome in both medaka and rainbow trout. The aim of the current study was to determine the effect of medical microbeam irradiation  using the ESRF synchrotron at Grenoble to the right brain hemisphere of the rat  to determine whether abscopal effects could be produced in the rest of the animal and also in cage mates housed with it. The results show strong signal production in the contra-lateral brain hemisphere and also in the distant bladder of the irradiated rats. Signal strength was almost identical in the cage mates housed for 48hrs with the irradiated rats. The proteomic  study showed  that the abscopal-associated proteins included A. an increase in the expression of NADH dehydrogenase, aconitase, ubiquinone and Glial fibrillary acid proteins, which are known to be present in gliomas, B. a reduction of the Prohibitin protein, which is thought to be a tumor suppressor in humans C. an increase on the expression of Heat shock cognate 71kDa protein, which is know to be involved in the disassembly of clathrin-coated vesicles and D. a decrease on the expression of Tubulin alpha-1A chain protein, which is known to be one of the major components  of microtubules.  All of these proteins would suggest a pro-carcinogenesis proteome. However in the cage mates, tubulin and aconitase  were present but in different isoforms, HSP 71 was also induced. Significantly, pyruvate kinase, fructose biphosphate and aldolase were also present  suggesting mobilization of energy reserves. This also happened in the bystander fish. One brain specific protein which was suppressed in the cage mate brain  is dihydropyrimidinase. This protein is prone to oxidation and  the oxidised molecule is associated with Alzheimer's disease. A reduction could be seen as beneficial in terms of reducing the overall extent of oxidased dihydropyrimidinase. On the other hand dihydropyrimidinase is a vital neurotransmitter so a reduction could also be seen as detrimental to cognitive brain function. Whatever way the induced changes in the cage mates influence the health of the animals, it is very clear that proximity to an irradiated animal induces proteomic changes in an unirradiated partner. The transmitting agent in fish and rats is thought to be urine. If similar signaling occurs between humans, the results could have implications for caregivers and hospital staff treating radiotherapy patients.

 

 

The First Cut is the Deepest

 

Colin Seymour

 

McMaster University, Canada

 

This was originally a song title by Cat Stevens in 1967. I will argue in this paper
that it should be regarded as the guiding principle for radiation and chemo therapy.
I shall demonstrate that the accepted principles of fractionation (equal effects per dose)
are wrong, and that possibly the most important treatment is the initial treatment. I shall use “in vitro” experiments performed in our laboratory to demonstrate the treatment rationale
we propose.

 

repair of oxidative DNA damage during development of colon cancer

 

Barbara Tudek^1,2, Elżbieta Speina¹, Alicja Winczura¹, Hubert Ludwiczak¹, Mateusz Chmielarczyk², Ryszard Oliński³

 

¹Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland; ²Institute of Genetics and Biotechnology, Warsaw University, Pawinskiego 5a, 02-106 Warsaw, Poland; ³Department of Clinical Biochemistry, Collegium Medicum, Nicolaus Copernicus University, Karłowicza 24, Bydgoszcz.

 

Inflammation, high fat, high red meat and low fibre consumption are the most important etiological factors of sporadic colorectal cancers (CRC). CRC originates from neoplastic transformation in a single epithelial cell and progresses from benign polyp to metastatic cancer. Mutational program in sporadic cancers involves APC gene, in which mutations occur most abundantly in the early phase of the process, and is followed by changes in RAS, TP53, and other genes. Progression of carcinogenic process is accompanied by augmentation of the oxidative stress, which manifests itself in the increased level of oxidatively damaged DNA bases in the colon epithelium, in blood leukocytes and urine, already at the earliest stages of disease development. Defence mechanisms are deregulated in CRC patients: (i) antioxidative vitamins level in blood plasma declines with the development of disease; (ii) mRNA level of base excision repair enzymes in blood leukocytes of CRC patients is significantly increased; however, excision rate is regulated separately, being increased for 8-oxoGua, while decreased for lipid peroxidation derived ethenoadducts, εAde and εCyt; (iii) excision rate of εAde and εCyt, as well as the level of poly(ADP-ribose) polymerase in colon tumors is significantly increased in comparison to asymptomatic colon margin, and ethenoadducts level is decreased. Such deregulation of repair processes may be caused by gene polymorphism, changes in transcription and translation rate, as well as direct effect of oxidative stress on repair proteins, and may be the driving force to colon carcinogenesis.

Session III:

Proteomics

(session co-organized by Polish Society of Proteomics)

High Resolution Mass Spectrometry Imaging – Comprehensive and Specific Histological Information At Cellular Resolution

 

Andreas Römpp, Nicolas Desbenoit, Katharina Huber, Bernhard Spengler

 

Justus Liebig University, Giessen, Germany.

andreas.roempp@anorg.chemie.uni-giessen.de

 

Mass spectrometry imaging (MS imaging) is the method of scanning a sample of interest and generating an image of the intensity distribution of a specific analyte ion. A full mass spectrum is acquired for each position sequentially. In contrast to most histological techniques, mass spectrometry imaging can differentiate (amino acid) modifications and does not require labeling of compounds. Our work is focused on further increasing the biologically relevant information that can be obtained by mass spectrometry imaging.

MS imaging experiments were performed with a high resolution atmospheric-pressure imaging source [1] attached to ‘LTQ Orbitrap’, ‘Exactive Orbitrap’ or ‘Q Exactive’ mass spectrometers (Thermo Scientific GmbH, Bremen). Mass accuracy was better than 2 ppm (root mean square) under imaging conditions. Tentative identification based on accurate mass was confirmed by on-tissue MS/MS experiments. MS images were generated with a bin size of ∆m/z = 0.01, which largely eliminates interferences from neighboring peaks in complex samples. 

Phospholipids, peptides and drug compounds were imaged in a wide range of murine and human tissue samples at a pixel size between 3 and 10 µm. Human tumor tissues were characterized in detail based on their phospholipid distribution. This provided structural features which were not directly visible in the histological staining experiments.  A drug compound (Imatinib) that was imaged in mouse kidney at 10 µm pixel size. These measurements revealed the detailed internal distribution of the compound within the mouse organ. Correlation with histological information based on the distribution of phospholipids (acquired simultaneously) allows for fast and easy interpretation of the drug compound distribution and areas of accumulation can be directly linked to certain tissue types. Phospholipids and smaller metabolites such as nucleic acids and cholesterol were also imaged in single cells. A full metabolic profile of was obtained from a single 7 µm pixel.

MS image analysis for all these experiments showed excellent agreement with histological staining evaluation. In addition it provided highly specific molecular information that can be used for biological interpretation. In many cases signals with very similar mass (∆m/z<0.1) showed distinctly different distributions, which demonstrates the need for high mass resolution in order to obtain reliable information from MS imaging experiments of biological samples.

Strategies for flexible data analysis on the basis of the data format imzML (www.imzml.org) will be discussed.

 

References:

[1] Römpp A. et al. (2010) Ang. Chem. Int. Ed. 49, 3834-3838.

Quantitative Study of Colorectal Cancer to a Depth of 10000 Proteins Using Laser Microdissected Formalin Fixed and Paraffin Embedded Tissue

 

Jacek R. Wiśniewski

 

Max-Planck-Institute for Biochemistry, Martinsried.

 

Development of sample preparation methods and their integration into LC-MS/MS workflows is the prerequisite for efficient exploration of proteomes. Filter aided sample preparation (FASP) method (1) allows protein-type-unbiased processing of microgram amounts of formalin fixed and paraffin embedded (FFPE) tissue (2,3) and a consecutive proteolytic digestion with multiple enzymes  increases depth of proteomic analysis (4). These developments combined with the newest generation of mass spectrometers (Q Exative) enable analyses of proteomes with unprecedented coverage.

Analysis of laser microdissected material from FFPE material of adenomatous polyps, colorectal cancer, and adjacent normally appearing enterocytes allowed identification 11,800 proteins. Expression levels of 2,200 proteins changed significantly between at least two types of sample. Tumor cells exhibit extensive alterations in the cells surface and nuclear proteomes.  The achieved results provide the most comprehensive description of proteomes of the native human cells published to date. They allow novel insights into the structural and functional organization of normal and diseased cells.

An important implication of this study is that proteomics does not require fresh or frozen material for studying human material for studying diseases. The archival formalin fixed and paraffin embedded material appears to be a valuable source of proteins, which can readily be compared between various stages of disorders and between different of tissues. Furthermore, the label free quantification approach used in this work offers a straightforward way to quantitatively analyze major proteomics features of clinical samples. It requires neither specific reagents for labeling of the peptides nor standards but enables determination of specific protein concentrations (e.g. mol/mg proteins or copy numer per cell). Clearly, these developments are now making proteomics readily applicable to the exploration of clinical samples in depth.

 

References:

[1] Wisniewski J. R., Zougman A., Nagaraj N., Mann M. (2009) Nature Met. 6, 359-362

[2] Wisniewski J. R., Ostasiewicz P., Dus K., Zielinska D. F., Gnad F., Mann M. (2012) Molec. Syst. Biol. 8, 611

[3] Wisniewski J. R., Ostasiewicz P., Mann M. (2011) J. Proteome Res. 10, 3040-3049

[4] Wisniewski J. R., Mann M. (2012) Analyt. Chem. 84, 2631-2637

PROTEOMICS THROUGH INTEGRATED MALDI AND ESI

 

M. Macht, A. Ingendoh, A. Asperger, A. Kiehne, D. Suckau, M. Becker, P. Hufnagel

 

Bruker Daltonik GmbH, Fahrenheitstrasse 4, 28359 Bremen, Germany.

 

Introduction:

The proteome is far more complex than it was ever expected at the genesis of the proteomics revolution. Dynamics in time, space and concentration as well as variability due to modifications and mutations require novel and complementary approaches to generate useful, reliable and complete information. As it turns out, there is no single platform able to unravel the proteome in its full complexity. To achieve comprehensive proteomic coverage – or even comprehensive sequence coverage of individual isolated proteins – different technologies have to be combined using individual strengthes.

Results and discussion:

Here we present a multi-tier approach to turn proteomic data into knowledge based on the combination of (LC-)MALDI-TOF and LC-ESI based technology for identification, quantification, characterization and localization of proteins combined with software tools for integration of different technology platforms allowing querying and reporting according to generally accepted guidelines.

The data presented covers top-down¹ as well as bottom-up analysis and shows ways to increase the proteome coverage in terms of identified peptides and their inferred proteins. Further information is obtained by the characterization of the proteins regarding their posttranslational modifications such as phosphorylation or glycosylation. We will also describe new tools for the comprehensive analysis of glycans and glycopeptides^2,3 parallel to unmodified peptides. We will demonstrate how quantitative information, using label-free as well as label-based approaches, can be obtained and turned into comparative data for in-depth sample analysis. As MALDI-TOF also allows for obtaining spatially resolved information directly from tissue⁴, we will show an example for a workflow where differences between different cancer types had been identified using MALDI-TOF imaging in combination with statistical analysis and subsequent identification of a protein cancer biomarker could be obtained using ETD ion trap tandem mass spectrometry⁵.

 

References:

[1] Macht M. (2009) Bioanalysis 1, 1131-48

[2] Ceroni A., Maass K., Geyer H., et al. (2008) J. Proteome Res. 7, 1650-9

[3] von der Lieth C. W., Freire A. A., Blank D., et al. (2011) Glycobiology 21, 493-502

[4] Schwamborn K., Caprioli R. M. (2010) Nat. Rev. Cancer 10, 639-46

[5] Rauser S., Marquardt C., Balluf B., et al. (2010) J. Proteome Res. 9, 1854-63

 

Rapid analysis of drugs of abuse as an initial step towards predictive toxicology

 

Marek Smoluch¹, Przemyslaw Mielczarek¹, Edward Reszke², Andrzej Ramsza³,
Jerzy Silberring^1,4

 

¹Department of Biochemistry and Neurobiology, AGH University of Science and Technology, Mickiewicza 30, 30-059 Krakow, Poland; ²ERTEC-Poland, Rogowska 146/5, 54-440 Wrocław, Poland; ³Institute of Applied Optics, Kamionkowska 18, 03-805 Warszawa, Poland; ⁴Centre of Polymer and Carbon Materials, Polish Academy of Sciences, Zabrze, Poland.

 

           The knowledge on the metabolic pathways and biotransformation of the most popular drugs, such as legal highs and homemade compounds, is crucial for elucidation of their possible toxicity and mechanism of action in human body. In vivo studies on metabolism are based on use of living animals. These methods need to be followed by extraction, isolation, and detection of metabolic products, which makes this technique time-consuming and technically demanding.

It was shown that simulation of the oxidative metabolism occurring in the liver and mainly caused by cytochrome P450, can be successfully mimicked with the electrochemical system (EC) combined with mass spectrometry detection. A novel, Flowing Atmospheric Pressure Afterglow (FAPA) plasma source for mass spectrometry has been developed and applied for this purpose. The source operating at ambient pressure can be used for direct analysis of organic compounds, such as drugs of abuse or counterfeit tablets, and can be considered as a soft ionization technique, as no or little fragmentation is observed. Sample application is possible in several ways, including direct analysis of solid compounds (e.g. tablets), deposition on a glass slide or on paper napkin (paper chromatography), and after nebulization. Moreover, the technique has been applied for on line coupling of LC system for separation of complex mixtures.

The analytical capabilities of the system were evaluated as an initial step towards predictive toxicology of drugs of abuse, their metabolites, and by-products. The presented technique can be complementary to other methods, and particularly useful for a large-scale, rapid evaluation of novel, unknown psychoactive substances where metabolites are frequently of great importance as potentially toxic.

 

This work was partially supported by the grants from the Ministry ofScience and Higher Education, No. NN 204 02 86 36 and NN 204 30 48 37.

 

Mass spectrometry based analysis of protein N-Homocysteinylation

 

Łukasz Marczak¹, Marta Sikora¹, Hieronim Jakubowski^1,2,3

 

¹Institute of Bioorganic Chemistry Polish Academy of Sciences, Noskowskiego 12/14, 61-707 Poznań; ²Department of Microbiology & Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark, NJ, USA; ³Department of Biochemistry and Biotechnology,University of Life Sciences, Poznań, Poland.

 

           Homocysteine (Hcy) is a sulfur-containing amino acid which is formed from methionine as side-product in the methylation process. Under standard conditions, Hcy released from cell can be further converted to cysteine by transsulphuration or back to methionine by remethylation process [1]. In the absence of cofactors of enzymes catalyzing the mentioned transformations (folic acid and vitamins B6 and B12) accumulation of Hcy in the blood plasma occurs. Accumulated Hcy in the presence of methionyl-tRNA synthetase may be converted into cyclic thiolactone of homocysteine which, due to high reactivity reacts specifically with ε-amino groups of lysine residues in protein chains. Such modifications of proteins have substantial impact on their physico-chemical properties, resulting in various pathological conditions, usually hazardous to human health [2].

           In the presented research we used mass spectrometry-based approach to localize N‑homocysteinylation sites of proteins. To evaluate the homocyteinylation sites of human albumin or fibrinogen, standard protein was modified with homocysteine thiolactone in vitro and control samples were prepared as well. All analyses of peptides derived from trypsin digestion were performed on mass spectrometers; both Maldi-ToF and LC-MS/MS systems were used for qualitative analysis and only LC-MS was applied for quantitation of modified peptides. Confirmation of N-homocysteinylation of predicted peptides was possible due to analysis of fragmentation obtained in tandem mass spectrometer (Q-ToF or Ion Trap MS). For our purposes albumin or fibrinogen was separated from human plasma samples and digested with trypsin. To fish out only N-homocysteinylated peptides affinity chromatography using aldehyde resin or resin with thiol groups was applied.

 

References:

[1] Jakubowski H (1997) J. Biol. Chem. 272, 1935-1942

[2] Jakubowski H (1999) FASEB J. 13, 2277-2283

 

Detection and quantification of MALDI ToF spectral peaks by using Gaussian mixture decomposition

 

M. Marczyk, A. Polanski, J. Polanska

 

Silesian University of Technology, Gliwice, Poland.

 

Computational approaches to processing proteomic mass spectra (MS) have been extensively developed in regards to applications of proteomics in medical diagnostics and molecular biology. Majority of algorithms for pre-processing of proteomic mass spectra include sequences of signal processing operations organized such that they lead to detection and quantification of spectral peaks. Important element of pre-processing of MS spectra is also alignment of spectral peaks between different spectra. When proteomic mass profiles are further analyzed and interpreted, spectral peaks are used as features of MS spectra; it is assumed that each spectral peak corresponds to a certain protein species, and the composition of mass spectrum carries direct information on composition of the analyzed samples.

Currently, there are already many algorithms implemented as publicly available software packages for proteomic MS spectral peak detection and quantification [3-20]. Different algorithms apply different procedures, different order and/or variants of signal processing operations. Algorithms can also differ with respect to types of proteomic MS spectra, which they can be specialized to (MALDI, SELDI, LCMS, MALDI-Tof/Tof, MSMS). In exhaustive evaluations concerning algorithms for analysis of proteomic mass spectra, based on both simulated and spike-in low and high resolution MALDI MS spectra, highest performance was achieved by algorithms based on computing continuous wavelet transform (CWT) of the spectral signal, with the “Mexican Hat” or Gaussian derivatives mother wavelet functions, and relating spectral peaks to either ridge lines or zero crossings in the parameter space. In comparisons it has shown high sensitivity for peak detection with quite low false discovery rate.

However, methods of CWT transforms are based on the assumption of disjoint components of spectral signal, which in practice is often not satisfied. Many components are overlapping which may lead to erroneous detection of peaks. In this paper we present a methodology of analysis of matrix-assisted laser desorption ionization time of flight mass spectra (MALDI-ToF MS) based on the Gaussian mixture decomposition. Gaussian mixture model is fitted to the data by maximizing the likelihood function by using a version of the expectation maximization (EM) algorithm. Initial values for EM iterations are computed by an algorithm where spectra are first decomposed into smaller parts and each or the part is approximated by a sum of Gaussian components. In the last step of the algorithm all smaller parts are aggregated into the full model of the spectrum.

Proposed methodology is applied to the simulated and spike in datasets and compared to previous algorithms. It is demonstrated that Gaussian model better describes overlapping components. Gaussian mixture decomposition is also applied as a methodology for construction of spectral classifiers. The method leads to differentiating components, which envelop regions, on the mass-to-charge (m/z) axis where there are most significant differences between cancer and control samples spectra. Computations lead to detecting reliable, differentiating components. Spectral classifiers based on Gaussian mixture decomposition are shown to outperform spectral classifiers based on spectral peaks.

 

Acknowledgement: The work was partially financed by NCN grant 2011/01/N/NZ2/04813.

 

Mass profiling of cancer serum proteome – does it provide any useful information?

 

Monika Pietrowska¹, Iwona Domińczyk¹, Agnieszka Gdowicz-Kłosok¹, Anna Walaszczyk¹, Magdalena Kalinowska-Herok¹, Małgorzata Roś¹, Karol Jelonek¹, Joanna Polańska², Michał Marczyk², Andrzej Polański², Piotr Widłak¹

 

¹Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-144 Gliwice, Poland; ²Silesian University of Technology, Gliwice, Poland.

 

MALDI-ToF mass spectrometry-based analyses of the low-molecular-weight fraction of serum proteome allow identifying profiles (signatures) that are potentially useful in detection and classification of a disease. However, features of serum proteome mostly reflect the general influence of disease development on patient’s organism and effects of the treatment. Hence, serum proteomics is an important tool for detection and characterization of changes related to progression of cancer and response to anticancer treatment. We compared serum proteome profiles of healthy donors and patients with three different types of cancer (head and neck squamous cell cancer, colorectal adenocarcinoma and non-small cell lung cancer) aiming to identify peptide signatures that were either common for all cancer samples or specific for cancer type. Classifiers built of selected spectral components allowed differentiation between healthy donors and cancer patients with about 70-80% sensitivity and specificity; the major differentiating components appeared to be associated with progression of cancer. Such components could be exemplified by those corresponding to fragments of serum amyloid A, the abundance of which apparently increased in blood of patients with more advanced cancer. We concluded that certain components of serum peptide signatures are common for different cancer signatures and putatively reflected general response of organism to the disease, yet other components of such signatures were more specific and most likely corresponded to clinical stage of the malignancy. Most interesting, features of serum proteome detected in blood of patients with low advanced disease have potential applicability in early detection/diagnosis of cancer. We also analyzed dynamics of changes in serum proteome profiles induced by anticancer treatment. Serum samples were collected before, during and after the end of radiotherapy in a group of patients with head and neck cancer. We found that numerous serum components significantly changed their abundance upon exposure to ionizing radiation, and that radiation-induced changes could be detected several months after the treatment. We also found that abundances of certain serum components associated with absorbed doses of radiation and intensity of radiation-induced reactions in irradiated tissues. Our data indicate that features of serum proteome could be a useful retrospective marker of exposure to ionizing radiation.

 

 

Session IV:

Selectively cytotoxic proteins

APOPTIN INDUCES CELL DEATH BY TARGETING VARIOUS TUMOR PROCESSES

 

Mathieu Noteborn, Henriette Lanz, Claude Backendorf

 

Molecular Genetics, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC Leiden, the Netherlands.

e-mail:m.noteborn@chem.leidenuniv.nl

 

Unbalances in cell proliferation, cell cycle regulation and/or cell death are underlying processes of cancer development. Many anticancer treatments fail because they are not accurate in targeting tumor-related processes and cause too high side effects. Viral and cellular proteins such as the avian virus-derived protein apoptin harbor a tumor-selective cell death activity potentiating the development of novel anticancer therapies and/or identifying essential processes leading to cancer formation.

           Bacterially produced recombinant ptd4-apoptin protein, applied on the skin of mice covering various xenografted tumors, showed the anti-tumor efficacy and safety aspects of apoptin. Combined treatment of the clinically applied chemotherapeutic agent dacarbazine and ptd4-apoptin protein resulted in an effective anticancer treatment with decreased side effects.

           Deregulation of the normal activity of protein phosphatase 2A (PP2A) is sufficient in triggering apoptin-induced cell death. Derailed PP2A activity is increasingly linked to oncogenic transformation including aberrant mitotic events.

           In cancer cells, apoptin is insensitive to proteasomal degradation, which still takes place in normal cells. This cancer-related loss of proteasomal susceptibility appears to be specific for apoptin protein as it is not found for the tumor suppressor protein p53.

In human cancer cells, apoptin becomes phosphorylated and is located in the nucleus, but not in normal cells. Biochemical characterization of the tumor-selective apoptin kinase activity points to a constitutive endogenous kinase located in both the nucleus and cytoplasm of cancer cells.

Weak inaccuracies in mitotic checkpoints are associated with aneuploidy and genetic instability, which are essential processes for cancer development. We have obtained evidence that apoptin likely senses mitotic inaccuracies in cancer cells, disrupting a cancerous process by inducing mitotic catastrophe leading to cell death.

Our results indicate the effectiveness and perspectives of apoptin for the development of novel therapeutic strategies for the treatment of human tumors.

Human Gyrovirus Apoptin shows a similar function to VP3/Apoptin

 

Mahvash Tavassoli, Jessica Bullenkamp, Joop Gaken

 

King's College London, Guy's Hospital Campus, LondonSE1 9RT, UK.

 

The chicken anemia virus-derived protein Apoptin/VP3 (CAV-Apoptin) has the important ability to induce tumor-selective apoptosis in a variety of human cancer cells. Recently the first human Gyrovirus (HGyV) was isolated from human skin swabs and blood. It shows significant structural and organizational resemblance toCAV and encodes a homologue of CAV-Apoptin/VP3. Using overlapping primers we constructed a synthetic human Gyrovirus Apoptin (HGyV-Apoptin) fused to green fluorescent protein in order to compare its apoptotic function in various human cancer cell lines to CAV-Apoptin. HGyV-Apoptin displayed a similar subcellular expression pattern as observed for CAV-Apoptin, marked by translocation to the nucleus of cancer cells, although it is predominantly located in the cytosol of normal human cells. Furthermore, expression of either HGyV-Apoptin orCAV-Apoptin in several cancer cell lines triggered apoptosis at comparable levels. We have previously shown that protein kinase C beta isoforms (PKC-beta I and II) phosphorylate CAV-Apoptin in some cancer cell types. Interestingly, HGyV-Apoptin also seems to interact with PKC-beta resulting in its translocation to the nucleus of human colon cancer and multiple myeloma cell lines.

Currently no association between HGyV and human disease has been identified, however the presence of a pro-apoptotic protein in the virus which is able to interacts with cellular survival and death mechanisms is intriguing and may lead to a potential therapeutic role for HGyV-Apoptin.

 

Inactivation of the TP53 gene or the p53 regulators Chk2/ATM predicts resistance to anthracyclinEs in breast cancer

 

Stian Knappskog

 

Section of Oncology, Institute of Medicine, University of Bergen, Norway; Mohn Cancer Research Laboratory, Haukeland University Hospital, Bergen, Norway.

 

Chemoresistance is the main obstacle to cure in most malignant diseases including breast cancer. While single parameter analysis and global gene expression profiles have provided much data on tumour biology, these strategies have failed to identify the molecular mechanisms causing in vivo resistance to chemotherapy. We have previously found TP53 mutations, in particular those affecting the L2/L3 domains, to be associated with anthracycline resistance. However, some tumors harboring wild-type TP53 are also therapy resistant.

Recently we have assessed the role of the two p53 activators Chk2 and ATM with respect to therapy resistance in cohorts of breast cancer patients treated with anthracycline monotherapy. Analyzing the ATM-chk2-p53 cascade in a cohort of 69 patients treated with doxorubicin or a mitomycin-containing regimen, low ATM levels (defined as the lower 5 - 50% percentiles) or mutations inactivating TP53 or CHK2 robustly predicted anthracycline resistance (p-values varying between 0.001 and 0.027 pending on the percentile used to define “low” ATM levels). These results were confirmed in an independent cohort of 109 patients treated with epirubicin monotherapy.

Our data indicate loss of function of the ATM-Chk2-p53 cascade to be strongly associated with resistance to anthracycline/mitomycin-containing chemotherapy in breast cancer in vivo.

 

MODELING OF INTERACTION BETWEEN BCR-ABL AND APOPTIN – NOVEL WAY FOR TARGETING THE DEREGULATED ABELSON KINASE ACTIVITY

 

Soumya Panigrahi¹, Jaganmohan Reddy Jangamreddy², Marek Los²

 

¹Department of Molecular Cardiology, Lerner Research Institute/NB-50, Cleveland, Ohio, USA; ²Department
of Clinical and Experimental Medicine (IKE) and Integrative Regenerative Medicine Center (IGEN), Linköping University, Linköping, Sweden.

 

Imatinib/Gleevec has been the first targeted anti-cancer therapeutic (it quite selectively targets the Bcr-Abl oncoprotein). However, when used as a monotherapy, it causes the rapid development of resistance, mainly due to mutations around the ATP/Gleevec-binding pocket. Thus, more effective alternative to Imatinib/Gleevec need to be developed. Our initial screening experiments have shown that a viral protein with cancer-selective toxicity, apoptin, directly interacts with and blocks Bcr-Abl. In the effort to develop apoptin-dervied Bcr-Abl inhibitors, we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its interaction with Bcr-Abl oncoprotein and its downstream signaling components. Furthermore, we validate some of the newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly (~95%) expressed in chronic myelogenous leukaemia (CML). CML is currently treated with an ATP-analogue Imatinib/Gleevec or their more advanced derivates, in combination with standard chemotherapeutics, as the above mentioned ATP-analogues when used alone, lead to rapid onsed or drug resistance. It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and Crk1 interact with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth and survival. Therefore, the oncogenic effect of Bcr-Abl could be inhibited by the interaction of small molecules with these domains. Apoptin is a viral protein with well-documented cancer-selective cytotoxicity. Apoptin attributes such as SH2-like sequence similarity with CrkL SH2 domain, unique SH3 domain binding sequence, presence of proline-rich segments, and its nuclear affinity render the molecule capable of interaction with Bcr-Abl. Despite almost two decades of research, the mode of apoptin’s action remains elusive because 3D structure of apoptin is unavailable. We performed in silico three-dimensional modeling of apoptin, molecular docking experiments between apoptin model and the known structure of Bcr-Abl, and the 3D structures of SH2 domains of Crkl and Bcr-Abl. We also biochemically validated some of the interactions that were first predicted in silico. This structure-property relationship of apoptin may help in unlocking its cancer-selective toxic properties. Moreover, such models will guide us in developing of a new class of potent apoptin-like molecules with greater selectivity and potency.

 

Session V:

Systems biology

Systems biology – introductory remarks

 

Mieczysław Chorąży

 

Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wyb. Armii Krajowej 15, 44-101 Gliwice, Poland.

 

In the second half of the last century the phenomena of life were often considered as an outcome of a simple linear relation between genotype and phenotype. Genes (DNA) were regarded as a sole information source, and primary causative factors determining all phenotypic traits. Gene was supposed to be the autonomous entity executing its deterministic power through coded protein which  acts as a final individual chemical molecule, responsible per se for physiological/pathological function.  This genocentric view was based on Crick-Watson “Central dogma”: DNA  makes RNA, RNA makes protein, and protein makes us. The typical, although a bit naïve and simplified thinking was that structural dysfunction of a gene, makes (via RNA) dysfunctional protein which is responsible for a disease. The hope that Human Genome Project will provide us with knowledge on gene structure and function essential for understanding  majority of physiological functions, and will enable us to recognize mechanisms of most diseases, and give us tools and methods to combat them  steadily faded.

At present we understand that in reality life phenomena are more complex than we thought some 20-30 years ago. In the living cell (organism) there are no autonomously acting molecules. The essence of  life lies not in the activity of individual chemical molecules, but rather is realized by the interaction between them. Millions of interacting macromolecules in a cell  form extremely complex systems (nets) having specific structure and properties. A prototype of such systems were metabolic maps (graphs) drawn some forty years ago. Graphs are used to depict internal structure of the system (net), where vertices (nodes) represent molecules (eg. protein, RNA, DNA-gene, enzyme, substrate) and edges (linkers) illustrate functional relation between them (transcription factor – binding to specific DNA regions, receptor and its interaction with other proteins in a signaling pathway, enzyme acting on substrate, etc.). Complex nets reveal various  motives, subnets, basic modules, feedback loops, hierarchical structures and are governed by specific rules. Processes within the networks are non-linear, but act according to the complex algorithmic rules, dynamic, and responsive to  the environment. These networks function in very variable, mostly unknown conditions, both on cellular and organism level and also are affected by external environment. We have only a very limited knowledge of their properties (structure, interaction dynamics, robustness, adaptation abilities, down-up and up-down regulation etc.). One expects that systems biology will be useful to understand the  nature of diseases and will provide new diagnostic and, hopefully, therapeutic possibilities.  

Systems biology, a new more holistic approach to study phenomena of life, is dealing with a study of structure, functions, activities, and laws governing of such complex networks and systems. Systems biology is trying to integrate into a functional “whole”: genetics, epigenetics, environmental factors and various  “-omics” (metabolomics, proteomics, transcriptomics, etc.). Systems biology posess a modest, yet continuously growing, set of tools and research methods (two-dimension electrophoresis, mass spectrometry, yeast two-hybrid method, various types of micromatrices, visualization in vivo of single protein molecules, and other). A major and potent new approach in systems biology is mathematical and computer modeling as well as informatics based on hundreds of data banks.

The presentation will be illustrated with some examples of systems biology approach applied to diseases and, in particular, to cancer.

Retrogenes – trash or treasure

 

Izabela Makałowska¹, Michał Kabza¹, Joanna Ciomborowska¹, Agnieszka Danek², Sebastian Deorowicz², Michał Szcześniak¹,Wojciech Rosikiewicz¹, Elżbieta Kaja¹, Andrzej Polański²

 

¹Laboratory of Bioinformatics, Faculty of Biology, Adam Mickiewicz University Poland; ²Institute
of Informatics, Silesian University of Technology, Gliwice, Poland.

 

One of the fundamental factors in the evolution of lineage-specific and species-specific traits is the birth of new genes. Retroposition is a major mechanism of gene duplications in which multi-exon genes give birth to single-exon copies that, in most cases, lack regulatory elements and are commonly believed to be pseudogenes. However, many of them are known to produce new genes and play very important role in shaping interspecies differences. They can also lead to regulatory RNAs or other regulatory elements. Retrogenes are considered to have little evolutionary significance. It is expected that retrocopies are selectively neutral, although, it is not universal for all retrogenes and mode of their evolution depends on the retrogene function. On the one hand, retrocopies, which replaced their parental gene are under strong purifying selection. On the other hand, many retrogenes demonstrate rapid changes in their structure.

Our knowledge of evolutionary history of retrogenes is still very limited, as most of the research has been conducted on few model organisms, including human, mouse and fruit fly. Even less is known about the recent history of retrogene formation at the population level. The availability of genomic sequences from dozens of animal species as well as genomes from multiple populations provided by the 1000 Genomes Project gave us the opportunity to perform large-scale analyses and shed some light on the subject. Our studies on 56 animal genomes clearly imply that the pattern of retrogenes’ evolution does not depend solely on their function. There are evident differences between various lineages in the number of retained retrogenes and the mode of their evolution. In addition, analyses of human genomes from ten different populations and RT PCR experiments on several transcriptomes from individuals belonging to the same population show that retrognes can be population-specific and individuals from the same group may differ in regard to retrogene functionality.

 

This work was supported by the National Science Centre grant No. 2011/01/B/ST6/06868
and grant No. 2011/01/N/NZ2/01701.

 

CROSS-TALK BETWEEN p53 AND Akt KINASE PATHWAYS

 

Marek Rusin

 

Center for Translational Research and Molecular Biology of Cancer, Maria Skłodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, 44-144 Gliwice, Poland

 

The cellular signaling pathways regulated by p53 and Akt proteins are intensely studied due to their importance in carcinogenesis and response of cancer cells to therapeutic agents. Both pathways are deregulated in significant percentage of tumors. For instance, recent comprehensive genomic characterization of squamous cell lung cancer revealed TP53 mutations in 81% of samples. The Akt kinase pathway was altered in 47% of cases. The p53 is a protein that inhibits cell growth or induces apoptosis when cells are subjected to various forms of stress including genotoxic stress induced by therapeutic agents. The Akt kinase, together with its upstream regulators and downstream effectors helps to maintain cell survival, growth and divisions, for instance, Akt phosphorylates and inactivates many pro-apoptotic proteins. The p53 and Akt signaling pathways are interconnected in opposite fashion. Akt phosphorylates and activates MDM2, which is a negative regulator of p53. On the other hand, p53 upregulates the expression of PTEN tumor suppressor gene, which is an indirect negative regulator of Akt. We study novel connections between p53 and Akt signaling pathways in cancer cells treated with anticancer drugs: actinomycin D, camptothecin and rapamycin.

microRNAs and their importance in laryngeal carcinoma

 

Joanna Janiszewska¹, Marcin Szaumkessel¹, Kinga Pelińska¹,
Magdalena Kostrzewska-Poczekaj¹, Małgorzata Jarmuż-Szymczak¹, Maciej Giefing¹, Krzysztof Szyfter^1,2

 

¹Institute of Human Genetics, Polish Academy of Sciences, Department of Environmental Mutagenesis, 60-479, Poznań, Poland; ²Department of Otolaryngology, K. Marcinkowski University of Medical Sciences, 60-355, Poznań, Poland.

 

The results of treatment of head and neck tumors remain poor for last decades. Head and neck tumors are relatively resistant to cytotoxic drug, therefore chemotherapy is not a proper choice. A little progress was noted for radiotherapy outcome only. Altogether, clinicians and researches expectations are focused on targeted therapy, where microRNAs seem to be the most promising target.

           miRNAs (microRNA, miRs) became a novelty in the late 90s as the new player on the scene of cancer science. Numerous extensive investigations have been performed with a hope of finding new tools for prognostic and diagnostic purposes and bridging them with a bright new way of understanding the molecular basis of carcinogenesis.

           We have analyzed 16 laryngeal squamous cell carcinoma and 5 primary larynx cancer cases by Agilent Human microarray miRNA expression 60K 1204 human and 144 human viral mature miRNAs according to miRbase 16.0 supplemented by updates.

           The analyses have revealed 27 induced and 9 repressed genes. Further, miRNAs were investigated according to literature background. Following the study, we have selected 3 miRNAs: miR-1246, miR-1290 and miR-4317 which revealed statistical significance in increase of relative expression level, as compared to control group (p<0.05). Moreover, they have not been reported previously in any head and neck cancer study.

Summarizing ‘the state of the art’ of knowledge about miRNA in head and neck cancers we can conclude that there is a delineated panel of miRNAs recurrently altered, which yield a relatively extensive information about HNSCC parameters and with potential prognostic value. miR-1246, miR-1290 and miR-4317 can be considered as potential new factors involved in carcinogenesis of  laryngeal carcinoma.

 

Variability and homeostasis in cycling cell populations

 

Marek Kimmel

 

Rice University, Houston.

 

A recognized source of variability in cell populations is asymmetry of assignment of biomolecules to progeny cells following cell division. It has been considered for past 50 years in diverse contexts. In conjunction with deterministic or stochastic cell growth regulation, unequal division has been mathematically proved to lead to stable distributions (homeostasis) in cell populations of cell characteristics such as cell mass and volume but also of discrete entities such as the numbers of membrane receptors, mitochondria and gene copies. New generation of data involving measurements of biomolecule level in individual cells, including distribution of the biomolecules between progeny cells following cell division, will allow new insights into generation and regulation of variability in cell population.

The talk reviews old and new mathematical models of dynamics of cell growth regulation, unequal division and death of cells and shows how new data fit in the framework of models. One of the examples involves tracing individual cells labeled with cell-cycle-phase- dependent FUCCI markers over a number or generations.

EXTENDED model of interaction between tumour cells

 

Andrzej Świerniak, Michał Krześlak

 

Silesian University of Technology, Gliwice, Poland.

 

Game theory has been applied in various branches of science, starting from economics where game theory was born, through behavioral and social science and ending on linguistic evolution, engineering and military.

One of the applications not mentioned before is biology. Everything started in 1970s when John Maynard Smith combined evolutionary biology and game theory [1]. Evolutionary game theory differs from standard game theory by deviating from rational approach of the competitive players, and strategies are treated as phenotypes of individuals acquired through the evolution.

Moreover, the players are members of a population containing individuals with different phenotypes (strategies), who can cooperate or compete for resources. As a result of different adaptations to the environment and following games through the time (generations) the population can tend to stabilize its structure at the same time gaining stable monomorphism or polymorphism of population’s phenotypes. Such state is called evolutionarily stable. Whereas evolutionary stable strategy (ESS) is defined as phenotype that, if adopted by the vast majority of a population, will not be displaced by any other phenotype. However opposite situation is very likely to occur.

Classical game problem studied by Maynard Smith was Hawk-Dove game and it assumed interaction between aggressive and giving up individuals among one population [1]. In dependency of costs and gains from winning different ratios of both strategies can occur in studied population.

Interactions between individuals may also happen among tumor cells. Evolutionary game is performed between cells with different phenotypes (both healthy and cancer cells). Main aim of these game theory models is to study the possibility of coexistence or even domination of newly formed tumor cells, which have acquired new strategies (phenotypes) by mutations. To our knowledge Tomlinson [2] first proposed a game theoretic model describing inter-cellular interactions. The models that followed described phenomena such as: avoidance of apoptosis [3], production of angiogenic growth factors, invasion and metastasis or radiation bystander effect. In this talk combination and by the same extension of two Tomlinson’s models [2], [3] is presented. The resulting model shows more complex population in terms of different phenotypes and stronger internal dependencies caused by parameters changing.

In [2] the author presents hypothesis that as a result of mutation a new phenotype, that gains benefits of harming neighboring cells, may occur and survive in population. The consequence of this phenomena is another phenotype that has acquired possibility to be resistant to harming substances.  These two strategies together with neutral one form one of the models presented in [2]. The implication of new features is defined by costs and benefits which concern phenotype’s fitness in population. The model in [3] considers growth factors production by tumor cell. These factors affect both the surrounding cells and the cells that produce them. As a result of such altruistic behavior only producers bear costs of growth factor performance.

The presented extended model considers four strategies (phenotypes) that can arise by mutations: cells that produce harming substances to gain an advantage, cells resistant to these substances, cells that produce growth factor which affects any other cell-kinds and neutral cells. Different equilibrium points, scenarios and possible extensions are also discussed.

 

References:

[1] Smith J. M. (1982) Evolution and the theory of games, Cambridge University Press.

[2] Tomlinson I. P. M. (1997) Game-theory models of interactions between tumour cells, Eur. J. Cancer 33, 1495-1500

[3] Tomlinson I. P. M., Bodmer W. F. (1997) Modeling the consequences of interactions between tumour cells, Brit. J. Cancer, 75, 157-160