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The frequency of selected polymorphisms, one in each gene coding for proteins with antioxidative properties (CAT(rs1001179), SOD2(rs4880), GPX1(rs1050450), and NQO1(rs689452)), was compared between patients suffering from pain-related temporomandibular disorders (TMDp ; n = 85) and control subjects (CTR ; n = 85). The same was evaluated when participants were divided with respect to oral behavioural habits frequency into high-frequency parafunction (HFP ; n = 98) and low- frequency parafunction (LFP ; n = 72) groups. Another aim was to investigate whether polymorphisms in these genes can be associated with participants’ psychological and psychosomatic characteristics. Polymorphisms were genotyped using the genomic DNA extracted from buccal mucosa swabs and real-time TaqMan genotyping assays. No differences in genotype distribution between TMDp patients and control subjects were found. Still, TMDp patients who were homozygous for minor allele A, related to the GPX1 polymorphism rs1050450, reported significantly more waking-state oral behaviours than GA + GG genotype carriers (score: 30 vs. 23, p = 0.019). The frequency of genotype AA for rs1050450 polymorphism was higher in HFP than in LFP participants (14.3% vs. 4.2%, p = 0.030). The most important predictors of waking- state oral behaviours were depression, anxiety, AA genotype (rs1050450), and female sex. The explored gene polymorphisms were not found to be significant risk factors for either TMDp or sleep- related oral behaviours. The association of waking-state oral behaviours with selected gene polymorphisms additionally supports previous assumptions that daytime bruxism is more closely linked to various stress manifestations, which might also be reflected through the variability related to the cellular antioxidative activity.

Aim: The aim of this study was to assess the association between single nucleotide polymorphisms (SNPs) in COMT (catechol-O- methyltransferase) and OPRPN (opiorphin) genes with temporomandibular disorders. Materials and Methods: The case-control study included 170 subjects: 85 patients diagnosed with pain-related temporomandibular disorders (TMDp) (according to Diagnostic Criteria for TMD) and 85 healthy controls (CTR). Characteristic Pain Intensity, assessed using the Graded Chronic Pain Scale, divided TMDp patients into high (HPI) and low intensity pain (LPI) groups. Genomic DNA was extracted from buccal mucosa swabs and used in the analysis of SNPs in COMT (rs4680, rs4818) and OPRPN (rs1387964) genes. SNP analysis was performed by qPCR using the TaqMan SNP Genotyping assays. Genotype distribution between studied groups was analyzed with respect to dominant and recessive genetic models. In both models, the minor allele represented the risk allele. The recessive model estimated the frequency of carriers of both minor alleles while the dominant model estimated the frequency of carriers of one or both minor alleles. Results: We didn’t find any significant difference in genotype frequencies of COMT rs4680 and rs4818 SNPs between TMDp and CTR groups. Two minor allele carriers (CC genotype) of rs1387964 polymorphism were significantly more frequent in TMDp than in CTR group (12.9% vs. 3.5%, p=0.025). When comparing HPI to LPI groups, we did not observe any significant difference in the genotype distribution of rs1387964 SNP. Two minor allele carriers (GG genotype) of rs4680 and rs4818 SNPs were significantly more represented in HPI compared to the LPI group (40% vs. 11.4% ; 24% vs. 7.5%, respectively). Conclusion: This study provides evidence that CC genotype of rs1387964 SNP in OPRPN gene might be associated with painful TMD, while GG genotypes of rs4680 and rs4818 SNPs in COMT gene appear to be associated with pain intensity. If confirmed in further research, determining the genetic predisposition for TMD could contribute to the development of a genetic test that could assess the risk of developing the disease

Aim: To investigate the potential impact of genetic background on treatment outcomes in patients with temporomandibular disorders (TMD). Materials and Methods: Sixty TMD patients diagnosed with painful TMD (according to DC/TMD) were treated with stabilization splints. Genomic DNA was extracted from buccal swabs and analyzed for polymorphisms: rs4818, rs6269 in the catechol-O-methyltransferase (COMT) gene, and rs1387964 in the opiorphin (OPRPN) gene. Treatment outcomes, assessed at baseline and after 6 months of treatment, included a range of mouth opening, level of jaw limitation, pain intensity (measured with Graded Chronic Pain Scale_GCPS and Visual Analogue Scale_VAS), anxiety, and depression. Mann-Whitney U test was used to compare changes in treatment outcomes between different genotypes. Results: Dominant and recessive genetic models were used in the assessment. In both models, the minor allele represented the risk allele. A recessive model estimated the effect of being homozygous with the minor allele and the dominant model estimated the effect of carrying one or both minor alleles.”Patients carrying two copies of the minor allele of rs1387964 (CC genotype) reported significantly less pain reduction than patients carrying the other two genotypes (GCPS: p=0.02 ; VAS: p=0.04). Patients carrying one or two copies of the minor allele of rs4818 (CG+GG) exhibited less reduction of jaw functional limitation (mastication, p=0.03) and less improvement of mouth opening (p

Objectives: Temporomandibular disorders (TMD) represent heterogeneous musculoskeletal conditions. Their complex etiology is yet to be thoroughly explored. The aim of the research presented is to investigate genetic predisposition as a risk factor for the TMD onset. Methods: Anamnestic and clinical data have been collected. Genomic DNA will be extracted from the buccal swabs obtained from TMD patients (DC/TMD) and control subjects, and used for the determination of selected polymorphisms by TaqMan SNP Genotyping Assays (Carlsbad, United States, Life Technologies). Gene polymorphisms clustering is expected to be related to the presence or absence of TMD. Whether specific clusters can be related to the source of pain and its intensity, presence of bone changes, psychological features, previous orthodontic therapy and treatment response to occlusal splint therapy will be explored. Results: We expect to show that a particular genotype may be a potential risk factor for the development of TMD. Conclusions: This innovative approach, focused on genetic predisposition for TMD has the potential for development of a commercial genetic test which would allow for risk estimation with respect to the TMD onset. Accordingly, it may be a potential tool to be used for decision making on early interventions and active avoidance of environmental risk factors.

Uvod: Usporediti pojavnost specifičnih polimorfizama (SNP) u genima povezanim s odgovorom na bol između oboljelih od kroničnih temporomandibularnih poremećaja (TMP) i zdravih ispitanika. Materijali i metode: Svim ispitanicima uzet je obrisak sluznice obraza (59 bolesnika i 49 zdravih ispitanika). Oboljeli od TMP-a su, prema ljestvici stupnjevanja kronične boli, razvrstani u skupinu s niskim (n= 26) i visokim (n=33) intenzitetom boli (CPI, od engl. Characteristic Pain Intensity ; raspon 0-100). Nakon izdvajanja DNA prethodno uspostavljenom metodom, genotipizirana su tri polimorfizma metodom TaqMan SNP Genotyping: a) u genu za opioidni µ-receptor (OPRM1, rs1799971), u genu za opiorfin (OPRPN, rs1387964) i u genu za percepciju boli (COMT, rs4646310). Podaci su analizirani χ2- testom, t- testom i analizom varijance. Rezultati: Genotipovi rs1799971: a) ukupno: A/A 75%, G/G 0%, A/G 25% ; b) TMP: A/A 71%, A/G 29% ; c) zdravi ispitanici: A/A 79%, A/G 21%. U skupini s visokim intenzitetom boli je značajno viša učestalost homozigota A/A, u odnosu na skupinu s niskim intenzitetom boli (81, 8% vs. 57, 7%, χ2-test: p=0, 042). Genotipovi rs1387964: a) ukupno T/T 61%, C/C 6, 5%, T/C 32, 5%, ; b) TMP: T/T 65, 5 %, C/C 7%, T/C 27, 5 % ; c) zdravi ispitanici: T/T 55, 1 %, C/C 6, 1%, T/C 38, 8%. Niže vrijednosti intenziteta boli pronađene su u skupini homozigota C/C (CPI=39, 5), u usporedbi s homozigotima T/T (CPI=53, 7) i heterozigotima T/C (CPI=50, 93), ali bez statistički značajne razlike među skupinama (F=1, 476, p=0, 238). Genotipovi rs4646310: a) ukupno: A/A 3, 7%, G/G 68, 9%, A/G 27, 4% ; b) TMP: A/A 1, 8%, G/G 71, 9%, A/G 26, 3% ; c) zdravi: A/A 6, 1%, G/G 65, 3%, A/G 28, 6%. Najviše vrijednosti intenziteta boli su u skupini homozigota A/A (CPI=86), a slijede homozigoti G/G (CPI=50, 05). Intenzitet boli je u ove dvije skupine značajno viši nego u heterozigota A/G (CPI=45, 26) (F=3, 941, p=0, 025). Zaključak: Usporedbom bolesnika s TMP i zdravih ispitanika nisu pronađene razlike u pojavnosti odabranih SNP, u genima povezanim s odgovorom na bol. Razlike u pojavnosti genskih polimorfizama unutar skupine TMP mogu se povezati s intenzitetom kronične boli, što ukazuje na potrebu za ovakvim razvrstavanjem, u daljnjem istraživanju. Potpora: Istraživački projekt Hrvatske zaklade za znanost IP‐2019‐04‐6211 (Glavni istraživač: Iva Alajbeg) i Projekt razvoja karijera mladih istraživača (DOK-2020-01) (student: Marko Zlendić).

Introduction: To establish a rapid and simple DNA isolation method from buccal swabs of patients with temporomandibular disorders (TMD) for the future analyses of specific polymorphisms in selected genes. Materials and methods: Buccal swabs were obtained from 40 subjects (22 TMD and 18 control patients) who agreed to participate in the study. Sample processing and DNA isolation were performed using a DNA isolation kit ChargeSwitch® PCR Clean-Up Kit(Carlsbad, United States, Life Technologies) by the research group at the Department of Removable Prosthodontics. The procedure of DNA isolation and purification relies on the use of magnetic beads. DNA quality was checked by 1% gel electrophoresis and polymerase chain reaction (PCR) amplification of exon 11 of the RET protooncogen, at the Rudjer Boskovic Institute. Results: A satisfying amount of DNA was obtained from all 40 buccal mucosal swabs. Samples will be further used for genotypization of polymorphism, which may be associated with the etiology of temporomandibular disorders, using the TaqMan™ SNP Genotyping Assay chemistry (Carlsbad, United States, Life Technologies). Conclusion: The presented method has proven to be a good option, which will be used in the future research. Support: Croatian Science Foundation Project IP- 2019-04-6211 (PI: Iva Alajbeg) and “Young Researchers’ Career Development Project - Training of Doctoral Students” (DOK-2020-01) (student: Marko Zlendić) Key words: polymorphisms, deoxyribonucleic acid, buccal swab, temporomandibular disorders, polymerase chain reaction

Depletion of glucose/glutamine is associated with a reduced level of Nicotinamide Adenine Dinucleotide Phosphate (NADPH), which is an important cofactor for replenishing cellular glutathione. Accordingly, starvation leads to increased levels of reactive oxygen species (ROS). For decreasing the ROS, cells activate detoxifying machinery based on the action of transcription factor Nuclear Factor Erythroid 2 Like 2 (NRF2). One of the NRF2 targets is an antioxidative enzyme, NAD(P)H: Quinone Dehydrogenase 1 (NQO1), which has a binding site for NRF2 in its promoter. The NRF2-NQO1 axis was explored in three head and neck-originating cancer cell lines: Cal 27, Detroit 562 and FaDu and in immortalized fetal lung fibroblasts: IMR-90 Four nutritional conditions were applied for 48 hours:a)NC1: high glucose (4, 5 g/L) + glutamine (0, 584 g/L) ; b)NC2: low glucose (1 g/L) + glutamine (0, 584 g/L) ; c)NC3: traces of glucose and glutamine provided by fetal bovine serum (FBS ; 0.69 mM glucose ; 0.05 mM glutamine) ; d)NC4: traces of glucose + glutamine (0, 584 g/L). Cellular viability, proliferation and ROS were measured. Quantification of NRF2 and NQO1 transcripts was combined with chromatin immunoprecipitation (ChIP) for discovering H3K27me3 enrichment and the amount of NRF2 bound to NQO1 promoter. Among the three cancer cell lines, FaDu was the most dependent on glucose for maintaining proliferation and glutamine had a minimal effect on restoring FaDu replicative potential. IMR-90 cells were entirely dependent on glucose. NRF2- NQO1 axis response to nutritional stress was cell type specific. Cal 27, Detroit 562 and IMR-90 upregulated NQO1 transcription under the NC3 condition.e There was no significant change in the NQO1 transcription rate in FaDu. ChIP analyses revealed that NC3 influences the amount of H3K27me3 ( a 3.50 and 1.25-fold increase in Cal 27 and FaDu, respectively) and NRF2 a(3-fold increase in Cal 27, 33-fold decrease in FaDu), bound to the NQO1 promoter.

Trimethylation of lysine 27 in histone H3 (H3K27me3) is a crucial epigenetic mark that is needed for gene silencing, maintaining stem cell pluripotency and proper cellular differentiation. The establishment of this epi-mark depends on the activity of methyltransferase Enhancer of Zeste Homolog 2 (EZH2), which acts as a catalytical subunit of the Polycomb Repressive Complex 2 (PRC2). This enzyme plays an important part in various types of cancers. Depending on the cancer type/specific cellular epi-network, EZH2 can function as an oncogene (prostate cancer) or as a tumor suppressor (myeloid malignancies). According to the GenBank database, there are five experimentally confirmed EZH2 transcript variants (TV1 – TV5). The longest transcript (TV1 ; NM_004456.4 ; 2724 bases) is composed of 20 exons. By comparison with this transcript, TV2-TV5 lack an in-frame segment that is composed of 15 nucleotides in exon 8 (ex8D15). In addition to ex8D15, TV2 also lacks exon 4, while both TV4 and TV5 lack an in-frame segment composed of 27 nucleotides in exon 3 (ex3D27). In addition to this, TV5 lacks exon 14. Here we report two novel transcript variants. One variant (TVexon427), lacking both exon 4 and 27 nucleotides in exon 3, is present in embryonic fibroblasts and three different cancer cell lines under resting conditions and after exposure to various types of stress. The second newly discovered variant, TVins126, is present in Detroit 562 cells grown in medium without glucose for 24 hours. This transcript includes an in-frame insertion of a part of intron 14 (N=126 bases), resulting in premature STOP codon. The importance of different EZH2 transcripts in such cellular processes as proliferation, differentiation, as well as cancer cell colony forming and cancer cell migration, has been only recently addressed. The biological significance of EZH2 transcript variants discovered by our research group is the focus of our ongoing research.

Understanding cancer biology in 21st century is still a very difficult task. While clinical medicine needs to find ways for dealing with this disease at many levels, molecular medicine has been mostly focused on mutational analyses extending to research related to transcriptome/proteome/ interactome studies. All these efforts have been concentrated on discovering early diagnostic markers and improving disease treatment. In 2011, two hallmarks of cancer: reprogramming of energy metabolism and evading immune destruction were added to the previous six hallmarks, which were proposed by Hanahan and Weinberg in 2000. Currently, the cancer’s cell metabolism and its “hiding” from the host’s immunological system, tend to be the basis for understanding the pathophysiology of seemingly unrelated signs of systemic disturbance which we diagnose as “cancer”. Several top-quality papers published in 2016/2017 strongly shed new light on an old hypothesis, officially suggested back in 1931, that cancer may represent a systemic disease. This new data must be considered in the setting of the human microbiome. Not understanding all its roles and functions, especially those related to the existence and/or absence of specific metabolites, may be an important gap in elucidating the most important aspects of cancer. Some of these issues, which should be viewed as “missing puzzles”, will be explained and carefully discussed.

Veliki depresivni poremećaj (MDD, od engl. Major Depressive Disorder) jedan je od najčešćih psihosomatskih poremećaja, sa snažnom tendencijom porasta broja oboljelih. Do 2020. godine mogao bi postati drugi najveći zdravstveni svjetski problem. Uzroci nastanaka bolesti, kao i klinička slika, vrlo su složeni. Ovakva složenost posljedica je aktivnosti velikog broja gena, od kojih svaki „pridonosi“ nastanku i izražaju bolesti s relativno malim udjelom. Sve veći broj rezultata mnogih studija upućuje na važnost epigenetičkih mehanizama regulacije aktivnosti gena, kao poveznicu bioloških i drugih čimbenika koji se povezuju s nastankom depresije. Većina istraživanja pokazuje uzročno-posljedičnu vezu između različitih bioloških i psihosocijalnih čimbenika, s jedne strane, te međuovisne promjene obrazaca metilacije/demetilacije molekule DNK i promjene koda histona, s druge strane. Sve je više podataka o značajnoj ulozi različitih nekodirajućih molekula RNK u nastanku depresivnog poremećaja. Konačno, pokazalo se da mnogi antidepresivi djeluju na epigenom. Ovaj učinak otvara potpuno novo poglavlje u razumijevanju patogeneze i epigenetičke podloge liječenja depresivnog poremećaja.

Green tea is the second most consumed beverage in the world. Its healthy effects are ascribed primarily to the following catechins, its polyphenolic components: (–)-epigallocatechin-3-O- gallate (EGCG), (–)-epigallocatechin (EGC), (-)- epicatechin-3-O-gallate (ECG) and (–)-epicatechin (EC). Herein are explored the effects of in vivo metabolic transformations of the green tea catechins on their absorption, distribution, metabolism, excretion and toxicity (ADME/Tox) by employing various computational approaches and tools. A comprehensive understanding of ADME/Tox characteristics of the compounds is essential for understanding their biological activities. The programs VolSurf+ [1] and ADMET PredictorTM [2] are used for generating molecular descriptors that are further applied in Principal Component Analysis (PCA) to distinguish these compounds and their main metabolites (Figure 1) regarding ADME/Tox features and to compare in silico predictions with experimental data available in literature. The effect of metabolic transformation on the distribution of catechins in the body is investigated by applying the program AutoDock4 [3] for non-covalent molecular docking into the extended Sudlow’s binding site I of human serum albumin (HSA). Figure 1 Green tea catechins and their main in vivo metabolites. References [1] Cruciani G, Crivori P, et al. (2000) Molecular fields in quantitative structure-permeation relationships: the VolSurf approach. J. Mol. Struct. 503:17-30 [2] ADMET PredictorTM 8.1, Simulations Plus, Inc., USA [3] Morris GM, Huey R, et al. (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem. 30: 2785- 91.

Many 1, 4-dihydropyridines (DHPs) possess redox properties. In this review DHPs are surveyed as protectors against oxidative stress (OS) and related disorders, considering the DHPs as specific group of potential antioxidants with bioprotective capacities. They have several peculiarities related to antioxidant activity (AOA). Several commercially available calcium antagonist, 1, 4-DHP drugs, their metabolites, and calcium agonists were shown to express AOA. Synthesis, hydrogen donor properties, AOA, and methods and approaches used to reveal biological activities of various groups of 1, 4-DHPs are presented. Examples of DHPs antioxidant activities and protective effects of DHPs against OS induced damage in low density lipoproteins (LDL), mitochondria, microsomes, isolated cells, and cell cultures are highlighted. Comparison of the AOA of different DHPs and other antioxidants is also given. According to the data presented, the DHPs might be considered as bellwether among synthetic compounds targeting OS and potential pharmacological model compounds targeting oxidative stress important for medicinal chemistry.

Primjena kurkumina u prevenciji zloćudne bolesti, kroz reprogramiranje metablizma stanice

Prikaz otkrića koja su dovela do uspostave personaliziranih pristupa u liječenju raka. Prikaz dobrih i loših strana personaliziranog liječanja u oboljelih od zloćudne bolesti

Uvod: Medularni karcinom štitne žlijezde čini samo pet posto svih zloćudnih tumora štitne žlijezde. U 75 % slučajeva se pojavljuje sporadično. Nasljedni se oblici javljaju u sklopu autosomno-dominantnog sindroma multiple multiple endokrine neoplazije tipa 2, MEN 2, u preostalih 25 % slučajeva. Razlog nastanka sindroma je prisustvo mutacije u protoonkogenu RET koji kodira nastanak receptora s tirozin kinaznom aktivnošću. Zbog učestalih mutacija ovog gena de novo (do 75 %), nasljedni je oblik tumora nerijetko teško prepoznati. Cilj: Odrediti nasljedne oblike medularnog karcinoma štitne žlijezde u 115 osoba, od 1995- 2015. godine, s klinički dijagnosticiranim sporadičnim medularnim karcinomom štitne žlijezde. Metode: Izdvajanje genomske DNA iz stanica pune krvi, umnažanje eksona 10, 11, 13, 15 i 16 metodom lančane reakcije polimerazom, pročišćavanje i sekvenciranje pročišćenih odsječaka. Očitavanje slijeda DNA i usporedba sa slijedom baza DNA (eng. referral DNA sequence) koje su pohranjene u Genskoj banci. Rezultati: Rezultati ovih analiza ukazuju na povećanu učestalost nasljednih mutacija u eksonu 13 (kodoni 790 i 791), u odnosu na susjedne zemlje. Rezultati dodatno omogućuju a) rano prepoznavanje nositelja mutiranog gena ; b) isključivanje prisustva mutacije, u srodnika oboljelih. Zaključak: Molekularno-genetička analiza protoonkogena RET trebala bi postati dio rutinske kliničke procedure u svih bolesnika s novodijagnosticiranim medularnim karcinomom štitne žlijezde.

Kronološki prikaz otkrića koja su dovela uvođenja personaliziranog liječenja raka u kliničku praksu.

Sveobuhvatni prikaz podataka i rezultata u području liječenja raka primjenom konvencionalnih citostatika u kombinaciji s kurkuminom.

Prikazani su najznačajniji epigenetički procesi uključeni u nastanak i napredovanje zloćudnih tumora glave i vrata.

Popularno napisan rad o zdravom zivotu u Hrvatskoj, s naglaskom na mediteranski način prehrane u sprečavanju nastanka raka.

It has been known for a long time that a healthy lifestyle, which includes a healthy diet, decreases the risk of cancer. Among many different spices which are scientifically proved to have antitumorous potential, curcumin is probably the best studied. Research being done in South-East Europe is in its earliest stages. We are convienced that curcumin influences the epigenome network of a cell, as the basis for its cancer preventive action From an epigenomic point of view, cancer is a disease of dysregulated genes functioning. This is a well- known consequence of several different cellular processes, of which aberrant DNA methylation and histone modifications play very important roles. Our particular scientific interest in cancer research focuses on specific histone modification, H3 Lysine-27 trimethylation, H3K27me3, which was shown to be present in the promoter regions of different tumor suppressor genes, leading to their silencing. Two enzymes are critical for creating and erasing this potent epigenomic mark. The histone methyltransferase, EZH2 (enhancer of zeste homologue) constitutes the catalytic component of the polycomb repressive complex-2 (PRC2) and is the key enzyme in Lysine-27 trimethylation process. Histone-27 demethylase JMJD3 acts in the opposite direction, through binding to PRC2 target genes promoters, where it regulates their H3K27me3 level and, consequentially, their transcriptional activity. We have explored the influence of different concentrations of curcumin on EZH2 gene expression/activity through measuring its expression by qRT-PCR, Western blot and immunocytochemistry in two different cancer cell lines. Additional experiments, performed on JMJD3 gene activity through RT-PCR, have shown how, upon treatment with curcumin, the balance between EZH2 and JMJD3 transcripts dramatically changes. In light of these discoveries, several possible scenarios for cancer prevention will be discussed.

The Importance of Aberrant DNA Methylation in Cancer

Curcumin and resveratrol influence the balance between EZH2 and JMJD3 in cancer cell lines in vitro

The constant interplay between DNA methylation, histone modifications and small interfering RNAs – including micro-RNA – provides the basis for establishing a cellular epigenomic network. Its occurrence depends on various endogenic and exogenic factors and is specific to a given type of cell and during a precise period of time. Thus, an epigenomic network is where phenomena known for a long time as »tissue and time specific gene expression« – both necessary for proper cell functioning – are manifested. A cancer cell is an excellent example of disturbed epigenomic network. Because of the reversible nature of the epigenomic process, the network can be partially or fully restored by the use of epigenomic drugs, to date, the inhibitors of DNA methylation and histone deacetylases. The possible intervention on the epigenome offers a very powerful tool in different fileds, including understanding disease pathophysiology, its treatment and, very significantly, its prevention.

The field of epigenomics has been officially introduced in Croatia in 2008, through establishment of Laboratory of Epigenomics (LE), at the Rudjer Boskovic Institute. While several research groups in Zagreb and Rijeka implement the approach related to epigenome in their research, the effort is mostly focused on exploring the methylome in cancer (especially as a valid link between inflammatory process and cancer) and in hereditary disease, particularly S-adenosylhomocysteine hydrolase (AHCY) deficiency. The LE members (one senior research associate, two research associates, one young PhD scientist, one PhD research assistant and, from very recently, one technition) perform their research in the field of cancer both, in vitro and in vivo. Currently, the Laboratory is funded through the National Grant (PI: Dr. Gall Troselj) only. The major focus of the group is: a) in vitro: treating different cancer cell lines with six different treatments that influence known basic biochemical mechanisms (e.g. 5-aza ; methylation processes ; PJ-34 and 3-AB: poly-ADP-ribosylation). Additional treatments include epigenome modifiers, yet not fully profiled as a specific modifiers of very certain cellular processes: curcumin, resveratrol and sirtinol. The potential consequences of their action on selected genes (IGF2, H19, CTCF, BORIS) is followed at the level of DNA, mRNA (quantitatively: qRT-PCR and qualitatively: RT-PCR with different primer combinations if splice variants are expected), proteins and protein/DNA interactions. In certain situations, the results obtained on this model generate and point out a specific process that may influence the biologic properties of the native cancer. Accordingly, in extenso work based on these preliminaries, translate on head and neck tumors (mostly laryngeal cancer). The collected samples are part of the Croatian Tumor Tissue Bank. Some recent data will be presented at the meeting.

The cation independent mannose 6-phosphate/insulin-like growth factor 2 (M6P/IGF2R) is a multifunctional receptor. It is included in a variety of cellular processes which become disregulated in cancer. Its tumor suppressor role was recognized a long time ago. However, due to its multifunctionality, it is not easy to understand the extent of its relevance to normal cellular physiology. Accordingly, it is even more difficult understanding its role in carcinogenesis. This review presents critical and focused highlights of data relating to M6P/IGF2R, obtained during more than 25 years of cancer research.

Liver is a unique mammalian organ with a great capacity of regeneration related to its function. After surgical resection or injury, hepatic cells, especially hepatocytes, can proliferate rapidly to repair the damage and to regenerate the structure without affecting the function of the liver. Loss of catalase activity during regeneration indicates that oxidative stress is present in the liver not only in pathological conditions but also as a “physiological” factor during regeneration. As we have shown in our previous work, liver stem cell- like cells treated with 4-hydroxynonenal (HNE), a cytotoxic and growth regulating lipid peroxidation product, recover in the presence of spleen cells. In the current study we characterized this novel cell line as liver-derived progenitor/oval-like cells, (LDP/OCs), i.e. functional liver stem-like cells. We showed that LDP/OC were OV6 positive, with abundant glycogen content in the cytoplasm and expressed α-fetoprotein, albumin, biliverdin reductase and γ-glutamyl transferase. Also, we compared their growth in vitro with the growth of cultured primary hepatocytes stressed with HNE and co-cultured with autologous spleen cells. The influence of spleen cells on HNE-treated primary hepatocytes and on LDP/OCs showed that spleen cells support in a similar manner the recovery of both types of liver cells indicating their important role in regeneration. Hence, LDP/OC cells may provide a valuable tool to study cell interactions and the role on HNE in liver regeneration.

The transcription of imprinted IGF2 is regulated by five promoters in a tissue-and age-specific manner. Its antisense transcript originates from the promoter located within a CpG island near IGF2 promoters P2-P4. The IGF-Bi fragment in this region has insulator properties and contains methylation-sensitive CTCF-binding sites. Our aim was to determine the influence of DNA methylation and ADP-ribosylation on the regulation of IGF2 promoter usage and IGF2AS transcription in two human tumor cell lines. They were chosen based on their different origin, Cal27 (tongue– mesodermal) and HT-29 (colon-endodermal) considering the activation of IGF2 by mesodermal or endodermal enhancers. Cells were treated with PARP-1 inhibitor, 3-aminobenzamide (3-AB), to inhibit poly(ADP-ribosyl)ation and promote DNA methylation, or 5-azacytidine (5-aza) to induce DNA demethylation and inhibit poly(ADP-ribosyl)ation. DNA methylation of IGF2AS promoter was analyzed by COBRA. Expression of IGF2AS was analyzed by real-time PCR. The IGF2 promoter usage was analyzed by RT-PCR. Both untreated cell lines had hypomethylated IGF2AS promoter to which CTCF could bind, but IGF2AS was expressed only in HT-29 cells. The IGF2 promoter usage differed: in HT-29 cells all five IGF2 promoters were active, while in Cal27 cells IGF2 originated only from promoters P3 and P4. Treatment with 5-aza downregulated IGF2AS expression in HT-29 cells by 60% and silenced IGF2 transcription from P2. Treatment with 3-AB downregulated IGF2AS expression in HT-29 cells by 30% concomitant with downregulation of IGF2 P2 transcription to undetectable level. Methylation status of IGF2AS promoter and transcription of IGF2 from other promoters was unchanged. Both treatments had no effect on IGF2AS promoter methylation, its expression or IGF2 promoter usage. Our results suggest that: 1) IGF2AS expression coincides with IGF2 expression from P1 and P0 ; 2) binding of CTCF to hypomethylated IGF2-Bi element prevents transcription from P1, P0 and P2 only in mesoderm-derived Cal27, but not endoderm-derived HT29 cells.

The maintenance of IGF2/H19 imprinting depends on the methylation of H19 ICR, the presence of CTCF and ADP-ribosylation. The transcription of IGF2 is regulated by five promoters in a tissue-and age-specific manner. The imprinted IGF2 has an antisense transcript originating from the promoter located within a CpG island near IGF2 promoters P2-P4. The IGF2- Bi fragment in this region has insulator properties and contains methylation-sensitive CTCF-binding sites. Our aim was to determine the influence of DNA methylation and ADP- ribosylation on the regulation of IGF2 promoter usage and IGF2AS transcription. Human tumor cell lines HT-29 and Cal-27 were chosen for this study based on different global DNA methylation (HT-29 being 2.2 x more methylated than Cal-27) and the ADP-ribosylating capacity (lower in HT-29). They were treated with PARP-1 inhibitor, 3-aminobenzamide (3-AB), to inhibit poly(ADP-ribosyl)ation and promote DNA methylation, or 5-azacytidine (5-azaC) to induce DNA demethylation. DNA methylation of DMR0 and IGF2AS promoter was analyzed by COBRA. ADP-ribosylating capacity was estimated based on the elevation of NAD level in the cells after 3-AB treatment. Expression of IGF2AS was analyzed by qPCR. The promoter specific transcripts of IGF2 were analyzed by RT-PCR. Both untreated cell lines had hypomethylated IGF2AS promoter to which CTCF could bind, but IGF2AS was expressed only in HT-29 cells with low ADP-ribosylating capacity. The IGF2 promoter usage differed: in HT-29 cells IGF2 transcription originated from promoters P1, P0, P3 and P4 but not from P2, although P2 transcripts including alternative exon 4b (P2/4b) were present. In Cal27 cells, only promoters P3 and P4 were active. The DMR0 was hemimethylated in HT-29 and hypomethylated in Cal-27 cell line. Treatment with 5-azaC, which causes DNA demethylation and inhibits poly(ADP- ribosyl)ation, downregulated IGF2AS expression in HT-29 cells by 60% most likely due to the inability of AP-1 to bind IGF2AS promoter in the complete lack of ADP-ribosylation. The same treatment had no effect on IGF2AS expression in Cal-27 cells but caused upregulation of IGF2 P2 and the appearance of P2/4b transcripts, likely due to the inhibitory effect of 5-azaC on ADP- ribosylation. The P1 and P0 promoters remained silent in Cal27 cells. No changes in methylation of IGF2AS promoter and DMR0 were observed in either cell line. Upon 3-AB treatment, the expression of IGF2AS was downregulated in HT-29 cells by 30% concomitant with emergence of very low level IGF2 P2 transcripts. Methylation status of IGF2AS promoter and transcription of IGF2 from other promoters has not changed, while DMR0 methylation has increased. The expression of IGF2AS was still undetectable in Cal27 cells upon 3-AB treatment. This treatment resulted in appearance of IGF2 P2 and P2/4b transcripts. No changes in methylation of IGF2AS promoter or DMR0 were detectd. Our results suggest that in tested cell lines: 1) IGF2AS and IGF2 P2 transcription are mutually exclusive ; 2) binding of CTCF to hypomethylated IGF2-Bi element prevents transcription from P1, P0 and P2/4b only in poly(ADP-ribosyl)ation-competent cells ; and 3) IGF2 transcription originates from P1 and P0 only in IGF2AS-expressing cells, regardless of DMR0 methylation.