1. Art and Diseño

CANCER EPIGENETICS
AND METABOLISM:
Connecting the Dots
UC Biotech
Cantanhede, Portugal
2-3 February 2015
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
Scientific Commission:
Paulo J. Oliveira, Ph.D. (CNC, Coimbra, Portugal)
Frederick Domann, Ph.D. (University of Iowa, Iowa City, IA, U.S.A.)
Maria T. Cunha-Oliveira, Ph.D. (CNC, Coimbra, Portugal)
Vilma A. Sardão, Ph.D. (CNC, Coimbra, Portugal)
Teresa L. Serafim, Ph.D. (CNC, Coimbra, Portugal)
Ignacio Vega-Naredo, Ph.D. (CNC, Coimbra, Portugal)
Organization:
Paulo J. Oliveira, Ph.D. (CNC, Coimbra, Portugal)
Maria T. Cunha-Oliveira (CNC, Coimbra, Portugal)
Luciana Ferreira (CNC, Coimbra, Portugal)
André Ferreira (CNC, Coimbra, Portugal)
Sandra Tomé (Biocant Park, Cantanhede, Portugal)
Leonor Jesus (CNC, Coimbra, Portugal)
Teresa L. Serafim (CNC, Coimbra, Portugal)
Vilma A. Sardão (CNC, Coimbra, Portugal)
Ignacio Vega-Naredo (CNC, Coimbra, Portugal)
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
TABLE OF CONTENTS
Program.................................................................................................3
Abstracts from invited speakers Day 1................................................5
Abstracts from invited speakers Day 2..............................................11
Abstracts from selected oral communications Day 1.......................16
Abstracts from selected oral communications Day 2.......................21
List of Posters......................................................................................26
Abstracts from Poster Communications............................................27
Sponsors..............................................................................................37
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Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
PROGRAM
Day 1, February 2
8:30 – 9:15
Registration
9:15 – 9:30
Opening of the meeting
9:30 – 10:30
Opening Plenary Lecture, Paolo Sassone-Corsi, University of California –
Irvine, USA
“Metabolism and epigenetics: the Clock link”
10:30 – 11:15
Valdemar Maximo, IPATIMUP, University of Porto, Portugal
“Mitochondrial regulation of epigenetics and its role in human diseases”
11:15 – 11:45 Coffee break, meet the sponsors
11:45 – 12:30
Gabriella Ficz, Barts Cancer Institute, University of London, UK
“Role of epimutations in cellular transformation”
12:30 - 13.00
Michael Rhodes, Nanostring Technologies, USA "Translational Research: from
Research to Diagnostics. The NanoString Digital Technology"
13:00 – 13:20
Selected short oral talk S1: Ana S. Rodrigues, CNC, Portugal,
“Pyruvate dehydrogenase and pluripotency: can we affect pluripotency
metabolically?”
13:20 - 15:00 Lunch and poster session
15:00 – 16.00
Plenary lecture, Carmen Jerónimo, Portuguese Oncology Institute-Porto (IPOPorto) & ICBAS-UP, Portugal
“Epigenetic deregulation in urological tumors: from biology to clinics”
16:00 – 16:45
Florian Pauler, Research Center for Molecular Medicine of the Austrian
Academy of Sciences, Austria
“Imprinted long non coding (lnc) RNAs as a paradigm for functional
lncRNAs in development and cancer”
16:45 – 17:15 Coffee break, meet the sponsors
17:15 – 17:35
Selected short oral talk S2: Ricardo P. Neves, CNC, Portugal and MRC
Molecular Haematology Unit, University of Oxford, UK
“Connecting the dots between cellular mitochondrial content and global
variability in gene expression and alternative splicing”
17:35 – 17:55
Selected short oral talk S3: Teresa L. Serafim, CNC, Portugal,
“Mitochondrial characterization of human breast cancer cell lines for in
vitro cancer research”
17:55 – 18:15
Selected short oral talk S4: Maria I. Almeida, INEB and Instituto de
Investigação e Inovação em Saúde, Portugal
“Extracellular vesicles derived from mesenchymal stem/stromal cells
contain microRNAs involved in osteoblast differentiation”
20:00 Meeting dinner
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
DAY 2, February 3
9:30 – 10:30
Plenary Lecture, Frederick Domann,
University of Iowa, USA
“Mitochondrial redox metabolism and its influence on the epigenetic
control of gene expression”
10:30 – 11:15
John G. Jones, CNC, University of Coimbra, Portugal
“Metabolic flux profiling of cancer cells using deuterated water and 13Ctracers”
11:15 – 11:45 Coffee break, meet the sponsors
11:45 – 12:30
Maarten Van Lohuizen,
Netherlands Cancer Institute, Amsterdam, The Netherlands
“Unexpected results of prolonged Ezh2 inhibition in an in vivo model for
Glioblastoma”
12:30 – 12:50
Selected short oral talk S5: Fátima Martel, Faculty of Medicine, University of
Porto, Portugal “The anticarcinogenic effect of the dietary compound
kaempferol in a human breast cancer cell line is dependent on inhibition of
glucose cellular uptake”
12:50 – 15:00 Lunch and poster session
15:00 – 15.45
João T. Barata,
Institute of Molecular Medicine, Lisbon, Portugal
“Signaling, Oncogenes and T-cell Leukemia”
15:45 – 16:45
Roundtable
“Cancer Epigenetics and Metabolism: Connecting the Dots?”
Moderated by: Frederick Domann, University of Iowa, USA
16:45 – 17:15 Coffee break, meet the sponsors
17:15 – 17:35
Selected short oral talk S6: Luciana Ferreira, CNC, Portugal, “Persistent effects in
mitochondrial genes transcription after incubation of H9c2 cells with the
anticancer drug Doxorubicin: an epigenetic origin?”
17:35 – 17:55
Selected short oral talk S7: Sofia Gomes, Faculty of Pharmacy, University of
Lisbon, Portugal “miR-143 and miR-145 overexpression in HCT116 colon cancer
cells enhances cetuximab-mediated antibody-dependent cellular cytotoxicity
leading to increased apoptosis”
17:55 – 18:15
Selected short oral talk S8: Marcelo Correia, CNC, Portugal, “Histone deacetylase
proteins: a possible glycolytic metabolic shifter in embryonic stem cells”
18:15 – 18:30 Closing session
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
ABSTRACTS FROM
INVITED SPEAKERS
DAY
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
OPENING PLENARY LECTURE
Paolo Sassone-Corsi
Center for Epigenetics and Metabolism,
University of California, Irvine, California 92697,
USA; [email protected]
“COMMON THREADS: EPIGENETICS, METABOLISM AND THE CLOCK”
Circadian rhythms govern a number of fundamental physiological functions in almost all
organisms, from prokaryotes to humans. The circadian clocks are intrinsic time-tracking
systems with which organisms can anticipate environmental changes and adapt to the
appropriate time of day. Disruption of these rhythms can have a profound influence to human
health and has been linked to depression, insomnia, jet lag, coronary heart disease,
neurodegenerative disorders and cancer. At the heart of circadian regulatory pathways is the
clock machinery, a remarkably coordinated transcription-translation system that utilizes also
dynamic changes in chromatin transitions and epigenetic control. Recent findings indicate that
regulation goes also the other way, since specific elements of the clock are able to sense
changes in the cellular metabolism. We have uncovered the role of the deacetylases SIRT1 and
SIRT6 in partitioning of the circadian epigenome, as well as the contribution of MLL1 as H3K4
methyltransferase. Notably, chromosome capture 4C studies have revealed that circadian genes
are organized in nuclear interactomes that dictate their coordinated expression,
Understanding in full detail the intimate links between cellular metabolism and the circadian
clock machinery will provide not only critical insights into system physiology and
endocrinology, but also novel avenues for pharmacological intervention towards metabolic
disorders.
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Cantanhede, Portugal 2-3 February 2015
Valdemar Maximo
Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), 4200465 Porto, Portugal; 2Department of Pathology and Oncology, Medical Faculty of the University
of Porto, 4200-319 Porto.
“MITOCHONDRIAL REGULATION OF EPIGENETICS AND ITS ROLE IN HUMAN
DISEASES”
Mitochondria have a central role in energy uptake and energy production, and as consequence
they play a key role in a wide variety of pathological conditions, such as cancer,
neurodegenerative diseases, diabetes and aging. Indeed, mitochondrial dysfunction, and
especially mitochondrial dysfunction caused by mutations in mtDNA have been implicated in a
wide range of age related pathologies. The mitochondrial activity is largely dependent on the
environmental availability of nutrients, namely carbohydrates, proteins and fats, and their
conversion, mainly by glycolysis and oxidative phosphorylation (OXPHOS), in energy rich
compounds, such as adenosine triphosphate (ATP), acetyl- Coenzyme A (acetyl-CoA), Sadenosyl-methionine (SAM), and nicotinamide adenine dinucleotide (NADH). ATP, acetyl-CoA,
SAM, and NAD+/NADH, in turn, are the high-energy substrates for protein (including histones)
phosphorylation, acetylation and deacetylation reactions, and DNA and protein methylation,
thus contributing to the regulation of several cell signalling pathways and cell epigenetic
landscape. Thus, the interrelationship between mtDNA, mitochondrial activity and cell
metabolism have an essential role in the maintenance of the levels of essential co-factors for
cell epigenetic mechanisms regulation, such as DNA methylation and protein acetylation.
The main goal of this study was to unveil how pathogenic mutations in mtDNA and/or
mitochondrial variants may lead to epigenetic changes, namely alterations in the pattern of
nuclear DNA methylation and/or acetylation of histones and other proteins.
Our data, showed that mitochondrial dysfunction, caused either by mtDNA mutations or
absence of mtDNA, as well as the presence of specific mitochondrial variants (different mtDNA
haplogroups) induce changes in cell metabolic profile, complex I activity, DNA methylation and
protein acetylation. Furthermore, we observed that mitochondrial dysfunction caused by
depletion of mtDNA, leads to the readjustment of several cellular processes through the
acetylation of several proteins, allowing the cell to survive and maintain its homeostasis.
Acknowledgements: This work was partially supported by the Portuguese Science and Technology Foundation
(FCT) through the project (PIC/IC/83037/2007). Further funding was obtained from the project ‘Microenvironment,
metabolism and cancer’ partially supported by Programa Operacional Regional do Norte (ON.2—O Novo Norte),
under the Quadro de Referência Estratégico Nacional (QREN), and through the Fundo Europeu de
Desenvolvimento Regional (FEDER). IPATIMUP is an associate laboratory of the Portuguese Ministry of Science,
Technology and Higher Education and is partially supported by the FCT.
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
Gabriella Ficz
Centre for Haemato-Oncology, Barts Cancer Institute, EC1M 6BQ, London, UK
“EPIGENETICS OF GROUND STATE PLURIPOTENCY”
DNA methylation is the last bottleneck in the reprogramming process and incomplete erasure
alters the differentiation potential of induced pluripotent cells. We have recently described the
mechanism of DNA methylation erasure in mouse embryonic stem cells (ESCs) through FGF
signalling inhibition (Erk1/2 and Gsk3b, called 2i) to achieve the blastocyst-like epigenetic
ground state pluripotency. We showed by whole-genome bisulphite sequencing that 2i induces
rapid and genome-wide demethylation on a scale and pattern similar to that in migratory
primordial germ cells (PGCs) and early embryos. Major satellites, intracisternal A particles
(IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves
oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired
maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and
Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region
in Dnmt3b that is highly responsive to signaling. These insights provide a framework for
understanding how signaling pathways regulate reprogramming to an epigenetic ground state
of pluripotency.
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
PLENARY LECTURE
Carmen Jerónimo
Cancer Biology & Epigenetics Group - Research Center; Portuguese Oncology Institute-Porto
(IPO-Porto), Porto, Portugal & & Abel Salazar Biomedical Sciences Institute (ICBAS), University
of Porto, Portugal
“EPIGENETIC DEREGULATION IN UROLOGICAL TUMORS: FROM BIOLOGY
TO CLINICS”
The most common genitourinary (GU) neoplasms (i.e., prostate, bladder, and kidney cancers),
represent the second most prevalent group of tumors only surpassed by lung cancer. All three
are generally clinically silent at their earliest stages, when curative treatment is most likely
successful. However, there are no consensual guidelines for GU cancer screening and available
methods are characterized by suboptimal sensitivity and specificity. Moreover, standard
clinical and pathological parameters meet with important limitations in the assessment of
prognosis in an individual basis. An increasing body of evidence is implicating several
epigenetic mechanisms as driving forces of neoplastic transformation. Among these, aberrant
DNA methylation is, by far, the most widely studied, followed by deregulated expression of
chromatin machinery proteins and microRNAs. Importantly, cancer-related epigenetic
alterations may provide novel cancer biomarkers, intended for early detection and diagnosis,
assessment of prognosis, and prediction of response to therapy. The importance of early
diagnosis in Oncology has been emphasized and although current methodologies (such as
cytology, histopathology, immunohistochemistry, etc) play a critical role, molecular markers
may complement or, eventually, replace them if more effective. Because epigenetic alterations
often precede the emergence of the malignant phenotype, they are advantageous for early
cancer detection. They might also be detected in clinical samples, including those obtained by
exfoliative cytology, endoscopic brush techniques, and biopsy, as well as urine, saliva, stool,
plasma and sputum samples, allowing for non- or minimally invasive molecular detection of
tumors. Moreover, early detection of cancer relapse before it manifests clinically (or on
imaging during routine patient follow-up) might be accomplished through epigenetic-based
biomarkers. Some epigenetic alterations have also been associated with tumor behavior and,
thus, may serve as biomarkers for prognosis.
In my presentation I will focus on the major findings obtained by our research team regarding
epigenetic mechanisms implicated in urological tumorigenesis as well as the usefulness of
epigenetic-based biomarkers for detection and prognosis in these cancers.
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
Florian Pauler
CeMM Research Center for Molecular Medicineof the Austrian Academy of Sciences,
Lazarettgasse 14, AKH BT 25.31090 Vienna, Austria
“IMPRINTED LONG NON CODING (LNC) RNAS AS A PARADIGM FOR
FUNCTIONAL LNCRNAS IN DEVELOPMENT AND CANCER”
Genomic imprinting is a developmentally important, epigenetic phenomenon that restricts
gene expression to one of the two parental alleles in mammals. Protein coding genes with
imprinted expression are therefore active on one parental allele and are silenced on the other.
Long non-protein coding (lnc) RNAs are long known to play a key role in this silencing process.
Therefore imprinted lncRNAs have been discovery models for the lncRNA mediated silencing.
For example the study of the imprinted lncRNA Airn in our lab has identified a novel silencing
mechanism that is independent of the lncRNA product and is only dependent on lncRNA
transcription. Recent technological advances revealed that lncRNAs are abundant transcripts
in mouse and human. Importantly lncRNA mapping studies found many novel lncRNAs, some
of which are promising tumor markers that might be directly involved in cancer related
processes like metastasis formation. However our work revealed that widely used
bioinformatics pipelines to identify lncRNAs might miss important transcripts like imprinted
lncRNAs that have an unusual RNA biology. After the association of lncRNAs with cancer and
disease it is now the next big challenge to identify the mode of action of these lncRNAs to
ultimately interfere with this function for therapeutic purposes. The findings and tools
established for and from the study of imprinted lncRNAs will help in this endeavor.
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ABSTRACTS FROM
INVITED SPEAKERS
DAY
2
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
PLENARY LECTURE
Frederick Domann
Department of Radiation Oncology, University of Iowa, Iowa City, IA 52246 USA
“MITOCHONDRIAL REDOX METABOLISM AND ITS INFLUENCE ON THE
EPIGENETIC CONTROL OF GENE EXPRESSION”
The earliest eukaryotic life forms simultaneously adopted mitochondria and chromatin, though
the molecular explanations for this and the mechanistic link between these apparently
simultaneous evolutionary events remains unknown. Cells respond to physiological and
environmental triggers through several key pathways including response to reactive oxygen
species, nutrient and ATP sensing, DNA damage response, and epigenetic alterations.
Mitochondria play a central role in these pathways not only through energetics and ATP
production but also through metabolites generated in the tricarboxylic acid cycle, alterations in
the balance of redox couples such as NAD+/NADH, and in mitochondria-nuclear signaling
related to mitochondria morphology, biogenesis, fission/fusion, mitophagy, apoptosis, and
epigenetic regulation. This presentation examines bidirectional interactions between
mitochondria and cellular pathways in response to physiological and environmental cues with
a focus on epigenetic regulation of biological processes that respond to endogenous and
exogenous signals through changes in homeostasis and altered mitochondrial function. When
the discovery that the enzymes responsible for initiating and perpetuating epigenetic events is
linked to metabolism by their cofactors a new paradigm for cellular retrograde signaling can
be created. Here, we summarize the foundation of such a paradigm on the origins of cancer, in
which metabolic alterations create an epigenetic progenitor that clonally expands to become
cancer. We suggest that metabolic alterations disrupt the production and availability of
cofactors such as S-adenosylmethionine, α-ketoglutarate, NAD(+), and acetyl-CoA to modify the
epigenotype of cells. We further speculate that redox biology can change epigenetic events
through oxidation of enzymes and alterations in metabolic cofactors that affect epigenetic
events such as DNA methylation. Combined, these metabolic and redox changes serve as the
foundation for altering the epigenotype of normal cells and creating the epigenetic progenitor
of cancer. A more comprehensive understanding of the retrograde signaling mechanisms
between the mitochondria and nucleus is central to elucidating the integration of
mitochondrial functions with other cellular responses with respect to disease. Recent studies
have highlighted the importance of mitochondrial functions in epigenetic regulation. Further
development and application of novel technologies, enhanced experimental models, and a
systems-type research approach will help to discern how mitochondrial dysfunction affects key
epigenetic pathways.
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
John G. Jones
CNC, University of Coimbra, Portugal
“METABOLIC FLUX PROFILING OF CANCER CELLS USING DEUTERATED
WATER AND 13C-TRACERS”
Cancer cells are programmed to be highly anabolic such that biosynthesis of lipid and
nucleotides are able to sustain high rates of proliferation. At the same time, they show a
marked dependence on glycolytic over mitochondrial ATP generation for energy needs. A
better understanding of the relationship between the biosynthetic carbon and energy demands
versus carbon and energy supply of cancer cells may identify critical metabolic bottlenecks
that may represent novel therapeutic targets for their selective demise. To help identify such
targets, it is important to develop metabolic tracer assays with high coverage of catabolic and
anabolic fluxes. To this end, the presentation will focus on how this may be achieved by the
integration of deuterated water (2H2O) - an universal tracer of biosynthetic pathways - with 13Ctracers, which monitor the fate of specific substrates into catabolic and anabolic pathways.
This will include methodological advances in NMR and MS measurements of metabolite 2H and
13
C-enrichment assays and novel metabolic activities of cancer cells that have been revealed by
such approaches.
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
Maarten Van Lohuizen
Division of Molecular Genetics, The Netherlands Cancer Institute, Amsterdam, The Netherlands
“UNEXPECTED RESULTS OF PROLONGED EZH2 INHIBITION IN AN IN VIVO
MODEL FOR GLIOBLASTOMA”
Repressive Polycomb-group (Pc-G) protein complexes and the counteracting Trithorax-group
(Trx-G) of nucleosome remodeling factors are involved in the dynamic maintenance of proper
gene expression patterns during development, acting at the level of chromatin structure. As
such, they are important controllers of cell fate and differentiation. When deregulated, these
master switches of gene expression are strongly implicated in formation of a diverse set of
cancers. Examples are the Pc-G gene Bmi1 and Ezh2 which are overexpressed in many cancers
including Glioblastoma. However, recently Ezh2 has also been found to be mutated/inactivated
in other forms of cancer, suggesting a highly context-dependent role as oncogene or tumor
suppressor. An outstanding question is how to identify among the many Pc-G bound genes the
cancer-relevant ones. hereto we have recently combined stringent ChIP-seq with custom
shRNAi library screening in in vivo models for glioblastoma. As Ezh2 is associated with poor
prognosis and metastasis in many cancer settings there is an increasing interest for
development of selective Ezh2 inhibitors as potential new ways for epigenetic cancer therapy.
We have developed conditional shRNAi inhibition in mouse models for aggressive Glioma to
study the effects of Ezh2 inhibition on tumor initiation and maintenance. Whereas initial
tumor regression upon Ezh2 inhibition was observed in vivo, prolonged Ezh2 inhibition caused
unexpected profound changes in tumor plasticity, differentiation status with important
consequences for tumor progression and treatment options, which will be discussed.
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Cantanhede, Portugal 2-3 February 2015
João T. Barata
Instituto de Medicina Molecular, Lisbon University Medical School, Portugal l
“SIGNALING, ONCOGENES AND T-CELL LEUKEMIA”
Interleukin 7 (IL-7) and its receptor (IL-7R), formed by IL-7Rα (encoded by IL7R) and γc, are
essential for normal T-cell development and homeostasis. However, IL-7/IL-7R-mediated
signaling can also have a “dark side”, promoting T-cell leukemia proliferation in vitro and
expansion in vivo. In addition to the evidence that IL-7 present in the tumor microenvironment
has a role in accelerating leukemia progression, we and others found that around 10% of T-cell
acute lymphoblastic leukemia (T-ALL) patients display oncogenic IL7R gain-of-function
mutations. The IL-7/IL-7R axis activates phosphoinositide 3-kinase/Akt/mammalian target of
rapamycin (PI3K/Akt/mTOR) pathway, thereby mediating viability, cell cycle progression and
growth of T-ALL cells. IL-7-mediated PI3K/Akt/mTOR-dependent survival of leukemia cells
relies, at least in part, on the upregulation of GLUT1 and increased glucose uptake. GLUT
activity appears to associate with IL-7-tiggered upregulation of reactive oxygen species, which,
surprisingly, are required for T-ALL cell viability. Interestingly, our RNA sequencing studies
indicate that the impact of IL-7 on the regulation of metabolism-related genes in T-ALL extends
beyond GLUT1 and suggest that IL-7/IL-7R-mediated signaling may promote a broad
transcriptional program with impact on metabolism. Moreover, we found that IL-7 regulates TALL cell viability not only by regulating apoptosis but also autophagy. Notably, IL-7 does so in a
complex manner that involves the activation of both anti-autophagic (PI3K/Akt/mTOR) and proautophagic (MEK/Erk) pathways, which ultimately allows IL-7 to promote leukemia cell
viability in opposite scenarios (e.g. nutrient-rich versus nutrient-poor conditions) by either
preventing or inducing autophagy.
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
ABSTRACTS FROM
SELECTED ORAL COMMUNICATIONS
DAY
1
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
SELECTED SHORT ORAL TALK S1
Ana S. Rodrigues1,2, Marcelo Correia1,2,3, Tânia Perestrelo1,2,3, Sandro L.
Pereira2, Maria I. Sousa2,3, Marcelo F. Ribeiro2,4, João Ramalho-Santos2,3,4
1
PhD Programme in Experimental Biology and Biomedicine (PDBEB), CNC - Center for
Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, 2Biology of
Reproduction and Stem Cell Group, CNC - Center for Neuroscience and Cell Biology, University
of Coimbra, Portugal, 3Institute for Interdisciplinary Research (IIIUC), University of Coimbra,
Portugal, 4Department of Life Sciences, Faculty of Sciences and Technology, University of
Coimbra, Coimbra, Portugal
“PYRUVATE DEHYDROGENASE AND PLURIPOTENCY: CAN WE AFFECT
PLURIPOTENCY METABOLICALLY?”
Similarly to Inner Cell Mass cells and to some types of tumours, embryonic stem cells (ESC) rely
mostly on glycolysis for energy supply. ESC present “silent” mitochondria with a low oxidative
phosphorylation activity; a high activity of hexokinase II (HK) to favour glucose use, and a
phosphorylated (inactive) pyruvate dehydrogenase (PDH) complex to steer pyruvate away from
the Krebs Cycle. The regulation of PDH activity thus plays an important role in the metabolic
flexibility. In order to better understand the metabolic profile of these cells we looked to the
regulation of the PDH complex and HK in pluripotent vs. differentiated mouse ESC, with the
ultimate goal of manipulating metabolism and possibly pluripotency. We set an experimental
design using PDHK inhibitor Dichloroacetate (DCA) and the HK inhibitor 3-Bromopyruvate
(3BP). Overall viability was not altered but proliferation was affected. Pluripotency was
negatively affected with both compounds elevating mitochondrial membrane potential and
ROS production causing alterations in the energetic status of these cells. Also DCA is promoting
a PDH more active, which is translated in a more oxidative state, the same was observed for
3BP though the effect was not as relevant. Interestingly we also observe a negative effect of
both compounds on glycolytic enzymes such as HK, Gapdh and PKM. To better understand the
possible metabolic shift we evaluated protein and mRNA levels for Hif 1alfa and p53. Both
compounds lead to a decrease in both Hif1 alfa and p53 protein levels.
Manipulating metabolism will cause a shift in pluripotency accompanied with mitochondrial
alterations and altered metabolic pathways. More importantly we established for the first time
that loss of pluripotency is accompanied by alterations on the PDH cycle independently were
we inhibit metabolism.
Acknowledgements: FCT for funding (grants PTDC/EBB-EBI/101114/2008, PTDC/EBB-EBI/120634/2010 and
PTDC/QUI-BIQ/120652/2010. QREN for funding (CENTRO-01-0762-FEDER-00204. CNC funding is supported by FCT
(Pest-C/SAU/LA000172011). PhD scholarships attributed to M.C. (SFRH/BD/51681/2011), T.P. (SFRH/BD/51684/2011)
and M.S. (SFRH/BD/86260/2012).
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
SELECTED SHORT ORAL TALK S2
Ricardo Pires das Neves1,2, Alberto Rastrojo3, Ana Lima1, Begoña Aguado3,
Raul Guantes4, Francisco J Iborra2,5
1
UC Biotech – Centre for Neurosciences and Cell Biology, Biocant Technology Park, 3060-197
Cantanhede, Portugal; 2MRC Molecular Haematology Unit, Weatherall Institute of Molecular
Medicine, John Radcliffe Hospital, University of Oxford Headington, Oxford, OX3 9DS, UK;
3
Centro Biología Molecular “Severo Ochoa”, CSIC-UAM, C/ Nicolás Cabrera 1; Campus de
Cantoblanco, 28049 Madrid, Spain; 4Department of Condensed Matter Physics, Materials
Science Institute ‘Nicolás Cabrera’ and Institute of Condensed Matter Physics (IFIMAC)
Universidad Autónoma de Madrid; Campus de Cantoblanco, 28049 Madrid, Spain; 5Centro
Nacional de Biotecnología, CSIC, Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain.
[email protected]
“CONNECTING THE DOTS BETWEEN CELLULAR MITOCHONDRIAL CONTENT
AND GLOBAL VARIABILITY IN GENE EXPRESSION AND ALTERNATIVE
SPLICING.”
Noise in gene expression is a main determinant of phenotypic variability. Increasing
experimental evidence suggests that genome-wide cellular constraints largely contribute to the
heterogeneity observed in gene products. It is still unclear, however, which global factors
mostly affect gene expression noise, and to which extent. We have previously shown that the
number of mitochondria transmitted to daughter cells at mitosis presents broad fluctuations
and this constitutes an important source of variability in transcription rates. The speed of
transcription elongation is directly linked to ATP concentration and to the levels of
mitochondria inside the cell. Using high content image analysis we have simultaneously
monitored mitochondrial content and the levels of different proteins or markers of gene
expression activity to quantify the contribution of mitochondria to the global variability in
gene expression. We found that changes in mitochondria content can account for ~50% of the
variability observed in protein levels. This is the combined result of the effect of mitochondria
dosage on transcription and translation apparatus content and activities. Moreover, we found
that mitochondrial levels have a large impact on alternative splicing, thus modulating both the
abundance and type of mRNAs. A simple mathematical model where mitochondrial content
simultaneously affects transcription rate and splicing site choice can explain the alternative
splicing data. The results of this study suggest that cells use mitochondrial content (and/or
probably function) to control mRNA abundance, translation and alternative splicing, which
may ultimately impact on cellular phenotype.
Acknowledgments: part of this work was co-funded by FEDER (QREN), through Programa Mais Centro under
projects CENTRO-07-ST24-FEDER-002008, and through Programa Operacional Factores de Competitividade COMPETE and National funds via FCT – Fundação para a Ciência e a Tecnologia under project(s) PestC/SAU/LA0001/2013-2014 and PTDC/SAU-ENB/113696/2009. Ana Lima has a FCT scholarship SFRH/BD/51942/2012.
18
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
SELECTED SHORT ORAL TALK S3
Teresa L. Serafim1, Teresa Cunha-Oliveira1, Vilma A. Sardão1, Cláudia Deus1,
Amy Greene2, Edward Perkins2 and Paulo J. Oliveira1.
1
Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 2Department of
Biomedical Sciences, Mercer University School of Medicine, Savannah Campus, Georgia, USA
“MITOCHONDRIAL CHARACTERIZATION OF HUMAN BREAST CANCER CELL
LINES FOR IN VITRO CANCER RESEARCH.”
Mitochondria are organelles present in all nucleated cells, being critical for many cellular
functions, such as ATP production by oxidative phosphorylation (OXPHOS), cell death
regulation, reactive oxygen species (ROS) generation and ionic homeostasis. Therefore,
mitochondria have been implicated in several diseases, including cancer. In fact,
comparatively to normal cells, it is suggested that cancer cells present differences in terms of
mitochondrial physiology that can contribute to the general metabolic remodeling observed in
cancer cells and which can contribute to different resistance to chemotherapeutics. In the
present work a set of commercial human breast cancer cell lines (MCF-7, HS578T and MDA-MB231) grown in high-glucose medium, were characterized regarding mitochondrial physiology
with the goal of detecting differences that can explain specific alterations in terms of behavior
and resistance to classic chemotherapeutics. Additionally, the transition to a glucose-free,
galactose/glutamine media, which is known to remodel metabolism to an OXPHOS-based, was
also evaluated. Specific alterations and cell behavior were compared with human normal
breast MCF-12A cell line. The results show that the mitochondrial membrane potential is
generally higher in tumor cell lines, especially in the MCF-7 cell line, which present condensed
mitochondria network. In galactose/glutamine, a remodeling of the mitochondrial network
occurred, with decreased membrane potential, elongated mitochondrial network of tumor cells
and increased mobility of HS578T cells. The tumor cell lines had also higher content of
mitochondrial complex subunits, confirmed by the increase of specific mitochondrial
transcripts for OXPHOS components and mitochondrial DNA copy number. When investigating
the effects of classic mitochondrial poisons, the MCF-7 cell line was more susceptible to the
compounds tested.
The data shows that the three commercial breast cancer cell lines tested presented alterations
in mitochondrial physiology, which may impact overall metabolism and resistance to
chemotherapeutic-induced cell death. The work presents a simple framework for the validity
of personal anti-cancer treatments based on distinct tumor physiology.
Acknowledgments: the present work was support by grants from the Portuguese Foundation for Science and
Technology (PTDC/QUI-QUI/101409/2008 to PJO and SFRH/BPD/75959/2011 to TLS).
19
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
SELECTED SHORT ORAL TALK S4
Maria I. Almeida1,2, Andreia M. Silva1,2,3, Susana G. Santos1,2, Mário A.
Barbosa1,2,3
1
INEB - Instituto de Engenharia Biomédica, Portugal, 2Faculdade de Engenharia, Universidade
do Porto, Portugal; 2Instituto de Investigação e Inovação em Saúde, Universidade do Porto,
Portugal; 3ICBAS – Instituto de Ciências Biomédica Abel Salazar, Universidade do Porto,
Portugal; [email protected]
“EXTRACELLULAR VESICLES DERIVED FROM MESENCHYMAL
STEM/STROMAL CELLS CONTAIN MICRORNAS INVOLVED IN OSTEOBLAST
DIFFERENTIATION.”
MicroRNAs are small non-coding RNAs involved in several biological processes important for
cancer pathogenesis and tissue regeneration, including cell differentiation. Mesenchymal
stem/stromal cells (MSC) have great therapeutic potential since they can differentiate into
osteoblasts, chondroblasts and adipocytes. MSC secrete abundant extracellular vesicles (EV),
which are known to contain and/or be associated with microRNAs and are able to protect them
from degradation.
In this study, we investigated the content of MSC-derived EV for microRNAs involved in
osteoblast differentiation, and consequently in bone repair. We also studied the
communication between MSC and others cell types present in the context of an injury, namely
macrophages, via EV.
We used primary MSC isolated from human bone marrow and a mouse preosteoblastic cell
line. EV produced by these cells were isolated by differential (ultra)-centrifugation. EV were
visualized by transmission electron microscopy (TEM) and their size was estimated by dynamic
light scattering (DLS). EV RNA was isolated and microRNAs expression was determined by
quantitative real-time PCR. For MSC-macrophages cross-talk, EV derived from MSC were
stained with PKH26 dye and added to macrophages.
MSC-derived EV displayed a cup-like morphology, as observed by TEM, and an average size
bellow 200nm, as detected by DLS. Several microRNAs involved in MSC osteogenic
differentiation were detected in EV derived from MSC, namely miR-29b, miR-29c and miR-20a.
Additionally, EV derived from MSC were found to be internalized by macrophages, as assessed
by confocal microcopy.
Taken together, these results suggest that MSC-derived EV might have a dual role in bone tissue
repair/regeneration, acting both in osteogenic pathways regulation and inflammatory
processes.
Authors thank Centro Hospitalar São João for donating buffy coats and bone marrow samples. Work funded by
Programa Operacional Factores de Competitividade–COMPETE, FCT–Fundação para a Ciência e a Tecnologia (PEstC/SAU/LA0002/2013; SFRH/BD/85968/2012; SFRH/BPD/91011/2012) and North Region Operational Program-ON.2
(‘‘Project on Biomedical Engineering for Regenerative Therapies; Cancer-NORTE-07-0124-FEDER-000005’’), through
QREN. M.I.Almeida would also like to thank to Boehringer Ingelheim Fonds.
20
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
ABSTRACTS FROM
SELECTED ORAL COMMUNICATIONS
DAY
2
21
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
SELECTED SHORT ORAL TALK S5
Cláudia Azevedo1, Ana Correia-Branco1, João R. Araújo1, João T.
Guimarães1,2, Elisa Keating1,3, Fátima Martel1
1
Department of Biochemistry (U-38 FCT), Faculty of Medicine, University of Porto, Portugal;
2
Department of Clinical Pathology, São João Hospital Center, Porto, Portugal; 3Center for
Biotechnology and Fine Chemistry, School of Biotechnology, Portuguese Catholic University,
Porto, Portugal
“THE ANTICARCINOGENIC EFFECT OF THE DIETARY COMPOUND
KAEMPFEROL IN A HUMAN BREAST CANCER CELL LINE IS DEPENDENT ON
INHIBITION OF GLUCOSE CELLULAR UPTAKE.”
Cancer cells present an altered metabolism, with an increased rate of glucose uptake and
lactate production instead of oxidative metabolism (the Warburg effect) [1]. Dietary
polyphenols are known to possess cancer preventive and anticancer effects [2]. Recently, our
group verified that the polyphenols quercetin and epigallocatechin-3-gallate inhibit glucose
uptake by the MCF-7 breast cancer cell line [3]. So, we decided to investigate if other
polyphenols could also interfere with glucose uptake by these cells.
Uptake of 3H-deoxy-D-glucose (3H-DG) by MCF-7 cells was time-dependent, saturable and
inhibited by cytochalasin B plus phloridzin. In the short-term (26 min), myricetin, chrysin,
genistein, resveratrol, kaempferol and xanthohumol (10-100 µM) inhibited 3H-DG uptake.
Kaempferol was found to be the most potent inhibitor of 3H-DG uptake (IC50 of 4 µM (1.6-9.8)),
behaving as a mixed-type inhibitor. In the long-term (24h), kaempferol was also able to inhibit
3
H-DG uptake, associated with a 40% decrease in GLUT1 mRNA levels. Interestingly enough,
kaempferol (100 µM) revealed antiproliferative (sulforhodamine B and 3H-thymidine
incorporation assays) and cytotoxic (extracellular lactate dehydrogenase activity
determination) properties, which were mimicked by low extracellular (1 mM) glucose
conditions and reversed by high extracellular (20 mM) glucose conditions. Finally, exposure of
cells to kaempferol induced an increase in extracellular lactate levels over time (to 731±32% of
control after a 24-h exposure), most probably related to inhibition of MCT1-mediated lactate
cellular uptake.
In conclusion, kaempferol potently inhibits glucose uptake by MCF-7 cells, apparently by
decreasing GLUT1-mediated glucose uptake. The antiproliferative and cytotoxic effect of
kaempferol in these cells appears to be dependent on this effect.
References:
[1] Cairns et al. (2011) Nature Rev Cancer 11:85-95
[2] Gupta and Prakash (2014) J Complement Integr Med 11:151-69
[3] Moreira et al. (2013) Exp Cell Res 319:1784-179
22
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
SELECTED SHORT ORAL TALK S6:
Luciana L. Ferreira1,2, Teresa Cunha-Oliveira1, Paulo J. Oliveira1
1
CNC, Center for Neuroscience and Cell Biology, University of Coimbra, UC-Biotech-Biocant
Park, 3060-197 Cantanhede, Portugal; 2Doctoral Programme in Medical Biochemistry and
Biophysics, University of Coimbra, Portugal
“PERSISTENT EFFECTS IN MITOCHONDRIAL GENES TRANSCRIPTION AFTER
INCUBATION OF H9C2 CELLS WITH THE ANTICANCER DRUG DOXORUBICIN:
AN EPIGENETIC ORIGIN?”
Doxorubicin (DOX) is a widely used and efficient chemotherapeutic agent prescribed for the
treatment of several types of tumors. However, despite its clinical potential, DOX treatment is
associated with a persistent, cumulative and dose-dependent cardiac toxicity. In fact, the
chronic cardiotoxicity can appear years or even decades after the end of the treatment, which
is particularly common in childhood tumor survivors. The exact mechanisms for this persistent
cardiotoxic memory remain under-explored and more studies are needed to elucidate the
causes of this delayed toxicity. In this context, the present work aims to investigate in vitro
persistent gene expression alterations, with special relevance on mitochondrial related-genes.
H9c2 cardiomyoblast cells were incubated with sub-lethal DOX concentrations (0.01 and 0.025
micromolar) during 24h, sub-cultured 72h after the end of the treatment and further subcultured for 72h after the first cell passage. RNA and DNA were isolated at four different timepoints and gene expression and mitochondrial DNA copy number were determined by realtime qPCR analysis. The affected genes included the mitochondrial mt-nd1, mt-cytb, mt-co1 and
mt-atp8 genes, whose expression significantly increased nine days after the end of the
treatment, confirming a DOX-dependent persistent effect. However, no statistically significant
alteration was observed in the expression of the mitochondrial transcription factor gene tfam
or in mitochondrial DNA copy number. In turn, dnmt1 expression consistently decreased in all
time-points, suggesting that alterations in DNA methylation may also occur and play a role in
the regulation of mtDNA transcription. In conclusion, the persistent alterations in
mitochondrial genes expression observed in the absence of the drug, confirm that DOX has
persistent effects in cardiac cells. More interestingly, despite complementary analysis is still
lacking, it is also possible that DOX might interfere with epigenetic regulation.
ACKNOWLEDGMENTS: This work was supported by the FCT (Fundação para a Ciência e a Tecnologia) grants
PTDC/DTP-FTO/1180/2012, PEst-C/SAU/LA0001/2013-2014 and co-funded by FEDER/Compete and National Budget,
CENTRO-07-ST24-FEDER-002008, as well as personal fellowships (SFRH/BD/52429/2013L.F.).
23
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
SELECTED SHORT ORAL TALK S7:
Sofia E. Gomes1, André E. S. Simões1, Diane M. Pereira1, Rui E. Castro1,
Alexandra Fernandes2, Catarina Rodrigues2, Pedro M Borralho1, Cecília M.
P. Rodrigues1
1
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,
Lisbon, Portugal; 2Faculty of Sciences and Technology, New University of Lisbon, Lisbon,
Portugal
“MIR-143 AND MIR-145 OVEREXPRESSION IN HCT116 COLON CANCER CELLS
ENHANCES CETUXIMAB-MEDIATED ANTIBODY-DEPENDENT CELLULAR
CYTOTOXICITY LEADING TO INCREASED APOPTOSIS”
MiRNAs have been widely studied in human cancers as they elicit strong effects on cell growth,
tumorigenesis and therapy response. miR-143 and miR-145 are tumor supressor miRNAs,
downregulated in colon cancer. We have shown that miR-143 overexpression reduces tumor
growth and proliferation, with increased apoptosis, while sensitizing to 5-fluoruracil. In this
study, we hypothesize that overexpression of miR-143 and miR-145 in colon cancer cells is able
to sensitize to cetuximab, promoting cetuximab-mediated antibody-dependent cellular
cytotoxicity (ADCC) through modulation of signalling pathways involved in effector cellmediated elimination of cancer cells.
We produced miR-143 or miR-145 overexpressing (and empty vector control) cell lines in KRAS
mutant HCT116 cells. We evaluated viability and proliferation of cells exposed to cetuximab.
Alterations in protein expression patterns of HCT116 cell lines exposed to cetuximab or vehicle
were identified by proteomic analysis of two-dimensional gel electrophoresis. Cetuximabmediated ADCC was evaluated using xCELLigence system and LDH assay, in cells exposed to
cetuximab, and/or peripheral blood mononuclear cells (PBMCs) isolated from human healthy
donors as effector cells.
Our results show that miR-143 and miR-145 overexpression increases the sensitivity of HCT116
cells to cetuximab, resulting in 40% reduction of cetuximab IC50, compared to control (p<0.01).
Importantly, overexpression of miR-143 and miR-145 triggered cetuximab-mediated ADCC by
increasing nuclear fragmentation and caspase-3/7 activity (p<0.05). Caspase inhibition reduced
the cytotoxic effect of PBMCs and cetuximab (p<0.05). In addition, inhibition of granzyme B, a
serine protease involved in effector cell-mediated granule secretory pathway, abrogated
cetuximab-mediated ADCC, reducing caspase-3/7 activity in HCT116 cells overexpressing miR143 or miR-145 (p<0.01). In addition, overexpression of miR-143 negatively regulated Bcl-2
protein levels, which was further reduced upon treatment with cetuximab (p<0.01). Bcl-2
silencing reduced HCT116 cell viability with a concomitant increase in cell death and
cetuximab-mediated ADCC, compared to control siRNA (p<0.05). Moreover, proteomic analysis
showed that miR-143 or miR-145 overexpression altered the expression of proteins involved in
several biological pathways/processes, including cell growth and proliferation, protein folding
and cell metabolism.
Collectively, restoration of miR-143 and/or miR-145 may provide a new approach for
developing a therapeutic strategy to re-sensitize colon cancer cells to cetuximab.
(PTDC/SAU-ORG/119842/2010, SFRH/BD/88619/2012, SFRH/BD/79356/2011, SFRH/BD/96517/2013, SPG)
24
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
SELECTED SHORT ORAL TALK S8:
Marcelo Correia1,2,3, Tânia Perestrelo1,2,3, Ana S. Rodrigues1,3, Sandro L.
Pereira3, Maria I. Sousa3,4, Marcelo F. Ribeiro3,4, João Ramalho-Santos3,4
1
PhD Programme in Experimental Biology and Biomedicine (PDBEB), CNC - Center for
Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Institute for
Interdisciplinary Research (IIIUC), University of Coimbra, Coimbra, Portugal; 3Biology of
Reproduction and Stem Cell Group, CNC - Center for Neuroscience and Cell Biology, University
of Coimbra, Coimbra, Portugal; 4Department of Life Sciences, Faculty of Sciences and
Technology, University of Coimbra, Coimbra, Portugal
“HISTONE DEACETYLASE PROTEINS: A POSSIBLE GLYCOLYTIC METABOLIC
SHIFTER IN EMBRYONIC STEM CELLS”
Embryonic stem cells (ESCs) are a promising field in regenerative medicine due to their
capacity of self-renewal and differentiation in all cell types of an adult individual. ESCs, like the
blastocyst’s inner cell mass and many cancer types, rely mostly in a glycolytic over oxidative
metabolism to supply their biosynthetic requirements to proliferate. When ESCs undergo
differentiation, a metabolic shift occurs from glycolysis to a predominant oxidative flux.
In this work, we have pharmacologically manipulated metabolism targeting different
metabolic pathways in E14Tg2.a murine ESCs, namely using Antimycin A – an electron
transport chain complex III inhibitor – and Dichloroacetic acid – an PDHK inhibitor. Overall
viability was not affected and both drugs are able to affect pluripotency positively and
negatively, respectively. Moreover, Antimycin A inhibits differentiation of ESCs into
dopaminergic neurons.
Although metabolic manipulation is shown here to be important to pluripotency and/or
differentiation, there are no clues about if epigenetic regulation is influencing the crosstalk
between pluripotency and metabolism. In order to address this question, a knockout murine
ESCs line for a Histone Deacetylase class protein was used to unveil possible effects of
epigenetic alterations in the metabolic profile of our cells. In fact, our results show that the
silenced Histone Deacetylase leads to reinforcement of the referenced metabolic profile of
ESCs, given that its absence induces a more pronounced glycolytic metabolism paralleled by a
decrease in oxidative metabolism.
Overall our results suggest metabolism as a powerful tool to induce alterations in
pluripotency/cellular fate in ESCs. Moreover, the results involving the epigenetic related
enzyme, suggests that epigenetics in ESCs could be an important mechanism how metabolism
could be regulated.
Acknowledgements: FCT for funding [grants PTDC/EBB-EBI/101114/2008, PTDC/EBB-EBI/120634/2010 and
PTDC/QUI-BIQ/120652/2010; PhD scholarships attributed to M.C. (SFRH/BD/51681/2011), T.P. (SFRH/BD/51684/2011)
and M.S. (SFRH/BD/86260/2012)]. QREN for funding (CENTRO-01-0762-FEDER-00204). CNC funding is supported by
FCT (Pest-C/SAU/LA000172011).
25
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
LIST OF POSTERS
Poster #
Presenter
Title
Cláudia M. Deus
Cytotoxic Effects of Resveratrol in Breast Cancer
Cells are Sirtuin 3-Independent
André M. Ferreira
Doxorubicin decreases global DNA methylation
levels, depletes mtDNA and causes downregulation of transcripts for metabolic end
epigenetic remodeling enzymes
P3
Nuno G. Machado
Influence of Exogenous Lactate on H9c2
Cardiomyoblast Gene Expression and
Proliferation
P4
Sílvia Magalhães
Novais
Cardiolipin Synthesis and Modifications During
P19 Embryonic Carcinoma Cell Differentiation
Vera M. Ribeiro
Lipidomics approach of B-cell chronic
lymphocytic leukemia: a study to identify new
biomarkers for disease diagnosis
P6
Vera F. MonteiroCardoso
Adaptation and survival of Caco-2 cells to glucose
deprivation involve and interconnected
remodeling of bioenergetics and lipid
metabolisms
P7
Albert A.
Rizvanov
Effect of Pluronic P85 Block Copolymer on
transcriptome of human tooth germ stem cells in
vitro
P8
Albert A.
Rizvanov
Application of anticancer drug based on
recombinant histone protein as inhibitor of
adenoviral infection in vitro
Vilena V. Ivanova
Serum of patients with auto-immune diseases
demonstrates higher affinity toward genomic
DNA, RNA and mitochondrial DNA from
immortalized HEK 293FT cells
P1
P2
P5
P9
26
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
ABSTRACTS FROM
POSTER
COMMUNICATIONS
27
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P1
Cláudia M. Deus1, Teresa L. Serafim1, Andreia Vilaça1,2, Susana M. Cardoso3,
Paulo J. Oliveira1
1
CNC- Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal;
Department of Life Sciences, University of Coimbra, Coimbra, Portugal; 3CERNAS- Department
of Environment, Agricultural College of Coimbra, Coimbra, Portugal
2
“CYTOTOXIC EFFECTS OF RESVERATROL IN BREAST CANCER CELLS ARE
SIRTUIN 3-INDEPENDENT”
Introduction: Resveratrol is a polyphenol, identified as chemopreventive or cytotoxic agent
against various types of cancer. Resveratrol has been described to stimulate the activity of
sirtuins, which have an important role in cancer progression and therapy. The objective of this
study was to investigate the cytotoxicity of resveratrol on human breast cancer cells and
identify whether this cytotoxicity is related with modulation of Sirtuins1 and 3 protein content.
Materials and methods: Two human breast cancer cell lines (MDA-MB-231 and MCF-7) were
cultured in high glucose and glucose-free/glutamine/pyruvate-containing media (OXPHOS
media). To silence SIRT1 and SIRT3, cells were transfected with small interfering RNA (siRNA)
specific for hSIRT1 and hSIRT3. Cells were treated with 10, 25 and 50 µM resveratrol during 24h
and 48h and viability, cell death, mitochondrial physiology, oxygen consumption and sirtuin
content were determined using standard methods.
Results: Cell proliferation studies showed that 50 μM resveratrol during 48h induced a decrease
in MDA-MB-231 and MCF-7 cell mass when cells were cultured in OXPHOS medium. Also,
resveratrol treatment led to morphological changes in the mitochondrial network and
chromatin condensation, suggesting apoptotic cell death. Furthermore, live/dead assay showed
that 25 and 50 μM resveratrol treatment during 24h promoted MDA-MB-231 and MCF-7 cell
death when cells were cultured in glucose or OXPHOS medium, respectively. Resveratrol
promoted differentiation of MDA-MB-231 cells. When incubated in the OXPHOS medium
increased ATP production was measured in MCF-7. However, resveratrol treatment decreased
and increased this parameter in MCF-7 and MDA-MB-231, respectively. Moreover, glycolytic
reserve and capacity in MCF-7 was decreased after resveratrol treatment. The two cell lines
cultured in glucose medium showed that Sirtuin1 content was increased after resveratrol
treatment, while the amount of Sirtuin3 was decreased in MCF-7. Transfected cells showed that
resveratrol effects in both breast cancer cell mass and cell death are independent of SIRT3 but
appear to be related with SIRT1.
Conclusions: Our data suggests that SIRT3 are not involved in resveratrol cytotoxic effects in
breast cancer cells and the modulation of Sirtuins content have different degrees of resveratrol
toxicity on distinct cell lines.
Acknowledgements: Supported by FCT-Portugal (PTDC/SAU-TOX/110952/2009, PEst-C/SAU/LA0001/2013-2014, cofunded by FEDER/ COMPETE/National Funds).
28
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P2
André M. Ferreira, Andreia Vilaça, Filipa S. Carvalho, Rita Garcia, Ana
Burgeiro, Teresa Cunha-Oliveira, Paulo J. Oliveira
CNC- Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal
“DOXORUBICIN DECREASES GLOBAL DNA METHYLATION LEVELS,
DEPLETES MTDNA AND CAUSES DOWN-REGULATION OF TRANSCRIPTS FOR
METABOLIC END EPIGENETIC REMODELING ENZYMES”
Doxorubicin (DOX), a potent and broad-spectrum antineoplastic agent, causes a cumulative
dose-dependent cardiomyopathy of late-onset that may ultimately lead to congestive heart
failure. The mechanisms responsible for the delayed nature of this pathology remain poorly
understood. Since DOX induces a persistent depression in cardiomyocyte gene expression
patterns, it is reasonable to hypothesize that a “toxic memory” is being formed by means of
epigenetic modulation. To test this hypothesis, eight week-old male Wistar rats (n = 6 per
group) were administered 7 weekly injections with DOX (2 mg/kg) or equivalent saline solution
and sacrificed one week after the last injection. We assessed gene expression patterns and
mtDNA copy number by qRT-PCR and global DNA methylation by ELISA in cardiac tissue. DOXtreated animals demonstrated a trend towards depressed transcripts for various functional
gene groups in cardiac tissue. Regarding genes related to mitochondrial biogenesis, we
observed a significant decrease in PGC-1α and TFAM transcripts, as well as a global trend
towards a decrease in the various oxidative phosphorylation subunits encoded by nDNA and
mtDNA. A concomitant decrease in mtDNA copy number was also observed. The master
regulator of lipid metabolism, PPARα, as well all assessed transcripts involved in lipid
metabolism (ACOX1, ACAA2, CACT, ACC, TAZ), were also found to be down-regulated. Regarding
epigenetic modulation, we observed a decrease in transcripts involved in histone acetylation
(CBP, EP300) and histone deacetylation (HDACs 3-10). Regarding DNA methylation, we observed
a global decrease in 5-mC content in DNA from cardiac tissue from DOX-treated animals.
Interestingly, this was accompanied by a decrease in the gene expression of MAT2A and
DNMT1. These results are consistent with the interplay between mitochondrial dysfunction
and epigenetic alterations in the persistent nature of DOX-induced cardiotoxicity. One
possibility is that DOX may modulate gene expression patterns via alterations to the epigenetic
landscape, which could be related to a disturbance in metabolite availability.
Acknowledgements: Supported by Foundation for Science and Technology, Portugal, grants PTDC/DTPFTO/1180/2012, PEst-C/SAU/LA0001/2013-2014 and co-funded by FEDER/Compete and National Budget, CENTRO-07ST24-FEDER-002008, as well as personal fellowships (SFRH/BD/64694/2009 to F.C.).
29
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P3
Machado NG1, Cunha-Oliveira T1, Burgeiro A1, Oliveira PJ1
1
CNC, Center for Neuroscience and Cell Biology, UC-Biotech, Biocant Park, University of
Coimbra, Portugal
“INFLUENCE OF EXOGENOUS LACTATE ON H9C2 CARDIOMYOBLAST GENE
EXPRESSION AND PROLIFERATION”
During physical activity, lactate is not a waste metabolite but a fuel source and “good” signaling
molecule instead, and was shown to up-regulate reactive oxygen species (ROS)-responsive
antioxidants genes, activating mitochondrial biogenesis and inhibiting cell proliferation. Thus,
the impact of lactate in muscle cells may be important for many physiological and pathological
conditions. In fact, physical activity prevents chemotherapy-induced cardiac toxicity, e.g. the
cardiovascular toxicity associated with anthracycline treatment. Since anthracycline toxicity is
higher in the differentiated H9C2 cardiomyoblast population, as previously demonstrated by
us, we are interested in finding out how lactate orchestrates the cell fate between proliferation
and differentiation states in that cell line and how that effect can explain the potential
protective role of physical exercise during cancer treatment-associated toxicity. We incubated
undifferentiated H9C2 cells (UNDIF) with several concentrations of lactate ([lactate]) during
48h to test the hypothesis that lactate regulates transcripts related to cell proliferation, lactate
metabolism, mitochondrial biogenesis, antioxidant defenses and sirtuins. By using the
sulphorodamine B method (SRB), treatment with exogenous lactate (10, 20 and 30 mM) for 48h
promoted cell growth inhibition in UNDIF, showing a small dose-dependent effect and being
toxic for supra-physiological levels (100 mM). Inhibition of H9c2 proliferation appeared to be
independent of medium acidification. qRT-PCR analysis showed that lactate treatment had little
effect on transcripts measured with exception for sirtuin 3 (SIRT3, p<0.05) and cardiac troponin
c (cTNT, p<0.01 and p<0.001).
We conclude that the inhibition seen on UNDIF proliferation appears to be independent of the
genes investigated here. Besides SIRT3 and cTNT, other inhibitory factors are probably
responsible for lactate-induced cellular adaptations.
Supported by Foundation for Science and Technology, Portugal, grants PTDC/DTP-FTO/1180/2012, PEstC/SAU/LA0001/2013-2014 and co-funded by FEDER/Compete and National Budget, CENTRO-07-ST24-FEDER-002008,
as well as personal fellowships (SFRH/BD/66178/2009- NGM)
30
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P4
Sílvia Magalhães Novais1,2; Maria Rosário Domingues3; Elisabete Maciel3;
Tânia Melo3; Ignacio Vega-Naredo2; Paulo J. Oliveira2
1
Department of Life Sciences, University of Coimbra, Coimbra, Portugal; 2CNC-Center for
Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Cantanhede,
Portugal; 3Mass Spectrometry Centre, Chemistry Department, University of Aveiro, Aveiro,
Portugal.
“CARDIOLIPIN SYNTHESIS AND MODIFICATIONS DURING P19 EMBRYONIC
CARCINOMA CELL DIFFERENTIATION”
The evolution of the carcinogenic process requires the knowledge of physiology and
metabolism of cancer stem cells (CSCs), which is crucial for the development of novel effective
therapies. Mitochondria are unequivocally a target for cancer therapy and crucial in cell death.
Given the relevance of mitochondrial metabolism for stemness maintenance and
mitochondrial control of apoptosis, as well as the importance of cardiolipin (CL) for
mitochondrial metabolism, we propose here the study CL alterations during P19 embryonal
carcinoma cells (CSCs) differentiation. Our results indicate that, although the total content of CL
did not significantly change, CL remodeling, possible mediated by increased of taffazin and
cardiolipin synthase proteins, may lead to a metabolic transformation towards the typical
oxidative metabolism that occurs during the differentiation of P19 CSCs. Differences in CL
molecular species were observed during differentiation of P19 CSCs. Moreover, the
differentiation of P19 CSCs increased the peroxidation of CL whereby the P19 CSCs present a
lower amount of non-oxidized CL. We conclude that the metabolic remodeling during P19 CSCs
differentiation probably also involves alterations in CL metabolism that probably are involved
in converting P19 differentiated cells more susceptible to mitochondrial-targeted antitumor
agents.
Acknowledgements: Supported by the Portuguese Foundation for Science and Technology (FCT), co-funded by
COMPETE/FEDER/National budget (PTDC/QUI-BIQ/101052/2008, PTDC-SAU-TOX-117912-2010, CENTRO-07-ST24FEDER-002008 and PEst-C/SAU/LA0001/2013-2014).
31
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P5
Vera M. Ribeiro1, Rosário P. Leite2 and Romeu A. Videira1
1
Chemistry Center – Vila Real (CQ-VR), University of Trás-os-Montes e Alto Douro (UTAD), Vila
Real, Portugal; 2Genetic Service, Cytogenetic Laboratory, Hospital Center of Trás-os-Montes and
Alto Douro, Vila Real, Portugal
“LIPIDOMICS APPROACH OF B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA: A
STUDY TO IDENTIFY NEW BIOMARKERS FOR DISEASE DIAGNOSIS”
B-cell chronic lymphocytic leukemia (B-CLL), the most common leukemic disorder in the
western hemisphere, is characterized by an abnormal accumulation of mature B lymphocytes
in the blood, bone marrow and secondary lymphoid organs. Although apoptosis dysregulation
connected with characteristics genetic alterations and with dynamic changes of membrane Bcell phenotypes are recognized as pathological hallmarks of disease, the molecular
mechanisms underlying B-CLL still uncertain in their nature and progression. Additionally,
new diagnostic tools are need since the traditional tests that combine fluorescence in situ
hybridization (FISH) with cytogenetic analysis are not able to identify at least 20% of B-CLL
cases. In the present work, a lipidomics approach were used to assess the blood lipid profiles of
six human patients diagnosed with B-CLL, three of which exhibiting abnormal karyotype and
positive result on FISH analysis and others three without the genetic abnormalities detected by
FISH and cytogenetic analyses, with the goal to detect new molecular markers with potential to
be used in the diagnosis of B-CLL. HPLC-MS/MS analyses of lipid extracts revealed significant
changes in phospholipid profile of blood samples from patients with normal karyotype and
negative FISH when compared with samples from patients with abnormal karyotype and
positive FISH. Increased levels of phosphatidylethanolamines (PE) associated with a decrease
of both phosphatidylinositol (PI) and cardiolipins (CL) content were detected in blood samples
of patients group with abnormal karyotype and positive FISH. Since the results were
normalized by the number of lymphocytes, the decreased CL levels indicate a lower
mitochondrial mass and/or activity suggesting that the bioenergetics metabolism of
lymphocytes is affected in B-CLL. Although the relative abundance of choline-containingphospholipids (SM, PC) is similar in both groups, significant differences are detected in the
relative abundance of the most representative molecular species of these phospholipid classes.
Despite preliminary, our data suggest that lipidomics strategy can be used to identify lipid
biomarkers to improve diagnosis of B-CLL.
Acknowledgments: This work was supported by Foundation for Science and Technology (FCT), and European
Union Funds (FEDER/COMPETE) [Project Grants: PEst-OE/QUI/UI0616/2014]. Romeu A. Videira was supported by
BPD/ENOEXEL/UTAD/269/2014.
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C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P6
Vera F. Monteiro-Cardoso1, Amélia M. Silva2, Maria M. Oliveira1, Francisco
Peixoto2 and Romeu A. Videira1
1
Chemistry Center – Vila Real (CQ-VR), University of Trás-os-Montes e Alto Douro (UTAD), Vila
Real, Portugal; 2Centre for the Research and Technology of Agro-Environmental and Biological
Sciences (CITAB), UTAD, Vila Real , Portugal
ADAPTATION AND SURVIVAL OF CACO-2 CELLS TO GLUCOSE DEPRIVATION
INVOLVE AND INTERCONNECTED REMODELING OF BIOENERGETICS AND
LIPID METABOLISMS
Cancer cells can adapt their metabolic activity under nutritional hostile conditions in order to
ensure both bioenergetics and biosynthetic requirements to survive. In this study, the effect of
glucose deprivation on Caco-2 cells bioenergetics activity and putative relationship with
membrane lipid changes were investigated. Glucose deprivation induces a metabolic
remodeling characterized at mitochondrial level by an increase of oxygen consumption, arising
from an improvement of complex II and complex IV activities and an inhibition of complex I
activity. This effect is accompanied by changes in cellular membrane phospholipid profile.
Caco-2 cells grown under glucose deprivation show higher phosphatidylethanolamine content
and decreased phosphatidic acid content. Considering fatty acid profile of all cell
phospholipids, glucose deprivation induces a decrease of monounsaturated fatty acid (MUFA)
and n-3 polyunsaturated fatty acids (PUFA) simultaneously with an increase of n-6 PUFA, with
consequent drop of n-3/n-6 ratio. Additionally, glucose deprivation affects significantly the fatty
acid profile of all individual phospholipid classes, reflected by an increase of peroxidability
index in zwitterionic phospholipids and a decrease in all anionic phospholipids, including
mitochondrial cardiolipin. These data indicate that Caco-2 cells metabolic remodeling induced
by glucose deprivation actively involves membrane lipid changes associated with a specific
bioenergetics profile which ensure cell survival.
Acknowledgments : This work was supported by Foundation for Science and Technology (FCT), and European
Union Funds (FEDER/COMPETE) [Grants: PTDC/SAU-NMC/115865/2009; FCOMP-01-0124-FEDER-022696:PEstC/AGR/UI4033/2011, PEst-OE/AGR/UI0772/ 2011 and PEst-C/QUI/UI0616/2011]. Vera F. Monteiro-Cardoso is
supported by FCT grant BI/PTDC/SAU NMC/115865/2009
33
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P7
Atousa Ataei1, Nataliya L. Blatt1, Mansour Poorebrahim2, Mehmet E.
Yalvaç3, Albert A. Rizvanov1
1
Kazan Federal University, Kazan, Russia; 2Tehran University of Medical Science, Tehran, Iran;
3
Children's Hospital Columbus, Center for Gene Therapy, Columbus, United States
“EFFECT OF PLURONIC P85 BLOCK COPOLYMER ON TRANSCRIPTOME OF
HUMAN TOOTH GERM STEM CELLS IN VITRO”
Pluronics blocking copolymers, such as P85, are currently considered as a drug delivery
carriers in various disorders and cancers. These compounds can impact many biological
processes but there are few reports explaining their molecular function in the cell. The
inhibiting effect of Pluronics on cancer cells depends on their chemical structure and
hydrophobic properties. Encapsulation of anti-cancer drugs in the Pluronic micelles alters their
distribution in vivo leading to higher rate of accumulation inside the tumor when compared to
free drugs. Although Pluronics are extensively studied using tumor cell lines, there is little
known about the effect of Pluronics on normal stem cells.
Here we present our data on genome-wide gene expression in human tooth stem cells (hTGSCs)
treated with Pluronic P85 Block Copolymer (P58). Illumina microarray method was used in this
study. Substantial changes in the 252 genes were detected in hTGSCs exposed to P85 treatment.
The gene enrichment approach was carried out using database for annotation, visualization
and integrated discovery (DAVID) and results were classified in several biologically meaningful
clusters. Finally, we constructed a global regulatory network of stem cell differentiation
pathways and multi-drug resistance (MDR) process using available bioinformatics databases to
demonstrate association of P85-modulated genes with stem cells differentiation and MDR
processes as well as cross-talk between stem cell differentiation pathways with MDR associated
genes. In conclusion, our results were in line with many of P85-mediated biological processes
published previously and can help us to gain better molecular perception of P85 biological
effects.
34
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P8
Valeriya V. Solovyeva, Albert A. Rizvanov
Kazan Federal University, Russia
“APPLICATION OF ANTICANCER DRUG BASED ON RECOMBINANT HISTONE
PROTEIN AS INHIBITOR OF ADENOVIRAL INFECTION IN VITRO”
Histones are a family of basic proteins that associate with DNA in the nucleus and help
condense it into chromatin. The balance of histone acetylation and deacetylation is an
epigenetic layer with a critical role in the regulation of gene expression. Anticancer drug
OncoHist based on recombinant histone H1.3 has strong anti-proliferative properties against
leukemic blast cells. OncoHist has been studied extensively as potential treatment for
hematologic malignancies, such as leukemias, lymphomas and myelomas. Here we present our
data on analysis of antiviral properties of the recombinant histone H1.3 in in vitro using
replication-deficient recombinant adenovirus serotype 5 encoding green fluorescent protein.
Recombinant adenovirus was generated using ViraPower Adenoviral Expression System
(Invitrogen). Recombinant adenovirus was pre-incubated with recombinant histone H1.3 to
assess its effect on adenoviral infection of HeLa cells. The efficiency of adenoviral infection was
determined by number of GFP+ cells using flow cytometry. To determine the effect of
recombinant histone H1.3 on the ability of adenoviral particles to form plaques we used
HEK239A cells (Invitrogen). To investigate the effect of recombinant histone H1.3 on adenoviral
infection we used its maximum non-toxic dose — 250 ug/ml. In our work we demonstrated for
the first time that recombinant histone H1.3 significantly reduces the infectivity of
recombinant adenovirus. Using of a mixture of recombinant histone H1.3 and recombinant
adenovirus lead to decreases the fluorescence intensity (2% GFP-positive cells) in contrast to
standard adenovirus infection (14% GFP-positive cells). Also, recombinant histone H1.3 exerts
an inhibitory effect on plaque formation on HEK293A cells, infected with recombinant
adenovirus. Adding of histone H1.3 to the recombinant adenovirus at a dilution 10-6 lead to a
reduction of plaque formation in 7,7 times, which confirms antiviral properties of histone H1.3
towards adenoviruses.
Further experiments are needed to determine mechanism of
interaction of recombinant adenoviral particles with histone H1.3. Our study provides new
direction for clinical application of recombinant of histone H1.3 as an antiviral drug for the
treatment of adenoviral infections.
35
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
P9
Vilena V. Ivanova1, Svetlana F. Khaiboullina1,2, Irina G. Starostina1, Albert
A. Rizvanov1
1
Kazan Federal University, Kazan, Russia; 2University of Nevada, Reno, NV, USA
“SERUM OF PATIENTS WITH AUTO-IMMUNE DISEASES DEMONSTRATES
HIGHER AFFINITY TOWARD GENOMIC DNA, RNA AND MITOCHONDRIAL
DNA FROM IMMORTALIZED HEK 293FT CELLS”
Systemic lupus erythematosus (SLE) is the auto-immune disease characterized by a plethora of
clinical presentation and cycles of remission and reactivation. Circulating anti-dsDNA
antibodies are hallmark features of SLE and detected in the serum as a reliable marker for SLE
diagnosis and are used as a key criterion for disease classification. Recently, potential
immunogenicity of peroxy-nitrite modified mitochondrial DNA (mtDNA) has been shown in
SLE patients. This suggests that circulating mtDNA can act as a trigger point for the
development of autoimmune disorders.
Our data demonstrate that the sera from SLE patients display higher immunoreactivity toward
total RNA and DNA from immortalized HEK 293FT cells when compared to healthy controls.
Additionally, significantly higher levels of immunoreactivity to mtDNA were observed for the
sera of SLE patients when compared to healthy controls. Finally, the sera from more than half
of all SLE cases (58.3%) demonstrated immunoreactivity to all types of nucleic acid
preparations as compared to only one serum sample (12.5%) from healthy donor.
Since a greater number of SLE patients displayed immunoreactivity toward mtDNA when
compared to healthy controls, we conclude that mtDNA from cells with high potential to
malignant transformation may play a role in pathogenesis of SLE. This assumption is
corroborated by the recent report of plasmacytoid dendritic cell activation by mtDNA leading
to the production of IFNα.
36
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Ca n t a n h e d e , P o r t u g a l 2 - 3 F e b r u a r y 2 0 1 5
SPONSORS
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37
C A N C E R E P I G E N E T I C S A N D M E TA B O L I S M : C o n n e c t i n g t h e D o t s
Cantanhede, Portugal 2-3 February 2015
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