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In the present study we examined a hypothesis that salinomycin, an antibiotic ionophore, might efficiently potentiate the cytotoxic effect of dichloroacetate (DCA), a metabolic inhibitor, on cancer cells. We used two human colorectal cancer derived cell lines - DLD-1 and HCT116. First, we performed series of dose response experiments in 2D cell culture in mono- and combination therapy and evaluated effects, based on Chou-Talalay method. This analysis revealed that salinomycin in combination with DCA acted synergistically in both cell lines. Secondly, in order to recapitulate the in vivo tumour architecture, we tested doses, selected from 2D experiments, in 3D multicellular spheroid culture in mono- and in combination therapy. In such conditions, the effect of salinomycin and DCA was additive in DLD-1 and synergistic in HCT116 cell lines. Further, we demonstrate that synergistic effect of compounds might be related with inhibitory effect of DCA on multidrug resistance proteins (MRPs) and with reduction of intracellular pH that in cancer cells is elevated. The activity of MRPs as well as potency of certain drugs is pH-dependent. Finally in order to disclose the rationale for sensitivity variations to combination therapy between two cell lines, we have investigated gene expression profiling of DLD-1 and HCT116 cell lines in 2D and 3D cell cultures and presume that decreased sensitivity of DLD1 cells to combination therapy could be attributed to increased stemness of DLD-1 cells in 3D cultures compared to HCT116 cells.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor with the highest incidence rates in Lithuania. CcRCC is characterized by the highest mortality rate of genitourinary cancers, therefore, new diagnostic and/or prognostic biomarkers are urgently needed. Considering this, the current study aimed to develop a gene-specific DNA methylation tool for non-invasive and early kidney cancer diagnosis and follow-up. Genome-wide DNA methylation profiling utilizing microarrays allowed the identification of a set of novel presumable ccRCC-specific DNA methylation biomarkers, having moderate to high diagnostic and/or prognostic potential. Moreover, identified DNA methylation alterations at the regulatory regions of selected genes appeared to be amenable for non-invasive detection in the urine samples of ccRCC patients, where they outperformed the diagnostic and prognostic value of previously described biomarkers and even some other parameters currently used in the clinical practice. Thus, the results showed the promising potential of the chosen genes as candidates for further development of non-invasive tools for kidney cancer patients testing. Although further comprehensive verification is mandatory to prove the clinical significance of the provided DNA methylation biomarkers for ccRCC, the obtained results seem to be rather promising and will definitely encourage these validation studies.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney tumor with the highest incidence rates in Lithuania. CcRCC is characterized by the highest mortality rate of genitourinary cancers, therefore, new diagnostic and/or prognostic biomarkers are urgently needed. Considering this, the current study aimed to develop a gene-specific DNA methylation tool for non-invasive and early kidney cancer diagnosis and follow-up. Genome-wide DNA methylation profiling utilizing microarrays allowed the identification of a set of novel presumable ccRCC-specific DNA methylation biomarkers, having moderate to high diagnostic and/or prognostic potential. Moreover, identified DNA methylation alterations at the regulatory regions of selected genes appeared to be amenable for non-invasive detection in the urine samples of ccRCC patients, where they outperformed the diagnostic and prognostic value of previously described biomarkers and even some other parameters currently used in the clinical practice. Thus, the results showed the promising potential of the chosen genes as candidates for further development of non-invasive tools for kidney cancer patients testing. Although further comprehensive verification is mandatory to prove the clinical significance of the provided DNA methylation biomarkers for ccRCC, the obtained results seem to be rather promising and will definitely encourage these validation studies.

Although local PCa treatment is quite effective and aggressive disease develops for a minority of patients, such PCa cases are associated with worse prognosis and shorter survival; also there is a lack of reliable biomarkers for the evaluation of response to treatment. Blood- and urine-circulating RNA can be used as lowly-invasive biomarkers for diagnostic and prognostic purposes in PCa. We found that miR-148a, -365, -375, and -429 are PCa-specific and have significant associations with clinical-pathological parameters of PCa. Also we demonstrated that the combination of urinary miR-148a and miR-375 is a sensitive molecular tool for noninvasive diagnosis of PCa, while the combination of miR-19b and miR-19a/b specifically predicts biochemical recurrence. Castration resistant prostate cancer (CRPC) remains incurable. Due to the heterogeneous nature of CRPC, approximately 30% of patients are already resistant to these new drugs. In patients treated with abiraterone acetate (AA), urinary miR-148 predicts progression-free survival, while the combination of miR-148a and miR-375 – overall survival. One of the resistance mechanisms could be AR splicing variants (AR-Vs) which lack a ligand binding domain and are constitutively active. We demonstrated that higher level of AR-FL and -V1 transcripts in AA-treated CRPC patients’ blood predicts progression-free survival and overall survival.

Although local PCa treatment is quite effective and aggressive disease develops for a minority of patients, such PCa cases are associated with worse prognosis and shorter survival; also there is a lack of reliable biomarkers for the evaluation of response to treatment. Blood- and urine-circulating RNA can be used as lowly-invasive biomarkers for diagnostic and prognostic purposes in PCa. We found that miR-148a, -365, -375, and -429 are PCa-specific and have significant associations with clinical-pathological parameters of PCa. Also we demonstrated that the combination of urinary miR-148a and miR-375 is a sensitive molecular tool for noninvasive diagnosis of PCa, while the combination of miR-19b and miR-19a/b specifically predicts biochemical recurrence. Castration resistant prostate cancer (CRPC) remains incurable. Due to the heterogeneous nature of CRPC, approximately 30% of patients are already resistant to these new drugs. In patients treated with abiraterone acetate (AA), urinary miR-148 predicts progression-free survival, while the combination of miR-148a and miR-375 – overall survival. One of the resistance mechanisms could be AR splicing variants (AR-Vs) which lack a ligand binding domain and are constitutively active. We demonstrated that higher level of AR-FL and -V1 transcripts in AA-treated CRPC patients’ blood predicts progression-free survival and overall survival.

Combination therapy is the cornerstone of cancer treatment. The simultaneous application of cytotoxic drugs potentiates their efficacy compared with monotherapy because it targets the key pathways in a synergistic or an additive manner. Such therapy is likely to diminish drug resistance, while simultaneously providing cytotoxic benefits, such as inhibition of tumor growth, decrease of cancer stem cell population, reduction of metastatic potential, and induction of apoptosis. In the present study, we investigated the effects of two drugs – dichloroacetate and salinomycin and the combination of both agents on the colorectal as well as lung cancer cell lines in vitro in 2D and 3D cell culture models. We also have tested therapeutic potential of this combination therapy in vivo in an allograft C57BL/6 mice model. In vitro studies were dedicated to investigate potential mechanism of salinomycin+dichloroacetate action and with in vivo studies we analyzed effects of this therapy on tumor growth, metastatic site formation, and the expression of tumor progression markers.

Early diagnosis of prostate cancer (PCa) and patients’ stratification into different risk groups are critical for successful management of the disease. DNA methylation of tumor suppressor genes (TSGs) is a potential source of diagnostic and prognostic biomarkers. The present study focused on identification and validation of novel DNA methylation biomarkers of PCa. During the first stage of experimental work, several TSGs were selected for methylation analysis in prostate tumors followed by evaluation of the most potential biomarkers for noninvasive screening in urine. Although the obtained results were promising, the relatively low specificity for PCa encouraged to search for biomarkers with excelling performance. The second stage of the study began with genome-wide DNA methylation profiling, which led to identification of a set of novel putative biomarkers with diagnostic and/ or prognostic value that might even surpass currently available tools. The prostate cancer dataset of The Cancer Genome Atlas project was utilized as an independent validation cohort. Promoter methylation of the newly identified genes was also demonstrated to be detectable in urine of PCa patients, which shows its potential application for noninvasive testing. Although additional validation is required, the obtained results seem to be rather promising. Protein-coding and miRNA host genes with newly identified methylation differences in PCa might be attractive targets for epigenetic therapeutics.

Early diagnosis of prostate cancer (PCa) and patients’ stratification into different risk groups are critical for successful management of the disease. DNA methylation of tumor suppressor genes (TSGs) is a potential source of diagnostic and prognostic biomarkers. The present study focused on identification and validation of novel DNA methylation biomarkers of PCa. During the first stage of experimental work, several TSGs were selected for methylation analysis in prostate tumors followed by evaluation of the most potential biomarkers for noninvasive screening in urine. Although the obtained results were promising, the relatively low specificity for PCa encouraged to search for biomarkers with excelling performance. The second stage of the study began with genome-wide DNA methylation profiling, which led to identification of a set of novel putative biomarkers with diagnostic and/ or prognostic value that might even surpass currently available tools. The prostate cancer dataset of The Cancer Genome Atlas project was utilized as an independent validation cohort. Promoter methylation of the newly identified genes was also demonstrated to be detectable in urine of PCa patients, which shows its potential application for noninvasive testing. Although additional validation is required, the obtained results seem to be rather promising. Protein-coding and miRNA host genes with newly identified methylation differences in PCa might be attractive targets for epigenetic therapeutics.

Prostate cancer (PCa) is the most common cancer in men and the second leading cause of male deaths in Europe. Several recent studies have suggested an association between promoter hypermethylation of hypoxia and angiogenesis-related genes and development of various types of cancer including PCa. Identification of new potential epigenetic biomarkers could greatly enhance our ability to diagnose PCa in the early stages and to predict the course of the disease after the surgery. The aim of our study - was to investigate with qualitative method methylation profile of ADAMTS12, FILIP1L and EPAS1 that were selected according to the DNA methylation microarrays analysis and evaluate the diagnostic and prognostic value of the selected epigenetic markers for PCa detection. Total epigenome analysis of prostate tumors using DNA methylation microarrays technology allowed the identification of genes associated with angiogenesis and hypoxia ADAMTS12, FILIP1L, and EPAS1 which are characterized by DNA methylation changes in tumors. Methylation status of regulatory and promoter sequences of selected genes was validated in 129 PCa and 30 benign prostate (NPT) samples by methylation-specific PCR (MSP). As an additional control group were used 17 benign prostate hyperplasia (BPH) samples. ADAMTS12 and FILIP1L promoter methylation was detected in 109 of 129 (84%) PCa samples for each gene. In NPT, methylation frequencies of ADAMTS12 and FILIP1L were 6% (2 of 35) and 11% (4 of 35), respectively and the difference was statistically significant versus PCa (P < 0.001) cases. No methylation changes were observed in BPH samples. EPAS1 gene was unmethylated in all analyzed PCa and NPT samples. Methylation of ADAMTS12 gene promoter was significantly associated with biochemical disease recurrence (P = 0.006) and was frequently observed in higher tumor stage (P = 0.071) and in higher Gleason score (P = 0.098) cases of evaluated tumors. No statistically significant correlations were observed between ADAMTS12 or FILIP1L methylation status and prostate-specific antigen (PSA) level, prostate mass, or patients’ age. Our study revealed a significant difference in methylation frequencies of the ADAMTS12 and FILIP1L genes between prostate tumor and non-tumor tissues and BPH, and potential prognostic significance of ADAMTS12 gene promoter‘s methylation. Quantitative genes’ expression analysis will allow a more accurate assessment of sensitivity and specificity of these genes as molecular markers in PCa.

Investigation of RNA and DNA function often requires sequence-specific incorporation of various reporter and affinity probes. This can be achieved using AdoMet-dependent methyltransferases (MTases) as they can be active with synthetic AdoMet analogues equipped with transferable chains larger than the methyl group. These chains usually carry reactive groups that can be further chemically appended with required reporters. For this, azide-alkyne 1,3-cycloaddition (AAC), also called “click”, reaction is particularly attractive. This work shows that the HhaI cytosine-5 DNA MTase (variant Q82A/Y254S/N204A) catalyzes efficient sequence-specific transfer of hex-2-ynyl side chains containing terminal alkyne or azide groups from synthetic cofactor analogues to DNA. Both the enzymatic transfer and subsequent “click” coupling of a fluorophore can be performed even in cell lysates. For RNA labeling, the activity of an archaeal RNA 2‘-O-MTase C/D ribonucleoprotein complex (RNP) with synthetic cofactors was investigated. It was shown that synthetically reprogrammed guide RNA sequences can be used to direct the C/D RNP-dependent transfer of a prop-2-ynyl group to predetermined nucleotides in substrate RNAs. Followed by AAC this can be used for programmable sequence-specific labeling of a variety of RNA substrates in vitro. These new possibilities for specific labeling of nucleic acids can be adopted in biochemistry, biomedical, nanotechnology, etc. research. Tiriant DNR ir RNR, neretai svarbu prijungti įvairius reporterinius ar giminingumo žymenis griežtai apibrėžtose (sekos) vietose – t.y. specifiškai. Tam galima pasitelkti fermentus metiltransferazes (MTazes). Natūraliai jos naudoja kofaktorių AdoMet, tačiau gali būti aktyvios ir su sintetiniais jo analogais, turinčiais ilgesnes nei metil- pernešamas grandines. Jei šios grandinės turi galines funkcines grupes, prie jų vėliau cheminių reakcijų pagalba galima prijungti norimus žymenis. Tam itin patogi azidų-alkinų cikloprijungimo (AAC), dar vadinama „click“, reakcija. Šiame darbe parodyta, kad DNR citozino-5 MTazė HhaI (variantas Q82A/Y254S/N204A) efektyviai katalizuoja sekai specifinę heks-2-inil- grandinių, turinčių galines alkinil- arba azido- grupes, pernašą nuo sintetinių kofaktorių ant DNR. Naudojant šią MTazės-kofaktorių sistemą bei AAC, visą specifinio DNR žymėjimo procesą galima atlikti netgi ląstelių lizate. RNR žymėjimui ištirtas archėjų RNR 2‘-O-MTazės C/D ribonukleoproteininio komplekso aktyvumas su sintetiniais kofaktoriais. Parodyta galimybė sintetiškai keičiant kreipiančiąją RNR, prop-2-inilgrupės pernašą nukreipti į norimas įvairių substratinių RNR sekos vietas ir po to AAC reakcijos pagalba prijungti fluoroforą. Taigi, sukurtas naujas molekulinis įrankis, leidžiantis be suvaržymų pasirinkti norimą pažymėti RNR seką. Šios naujos specifinio nukleorūgščių žymėjimo galimybės gali būti pritaikytos biochemijos, biomedicinos, nanotechnologijų ir kitose tyrimų srityse... [toliau žr. visą tekstą]

Tiriant DNR ir RNR, neretai svarbu prijungti įvairius reporterinius ar giminingumo žymenis griežtai apibrėžtose (sekos) vietose – t.y. specifiškai. Tam galima pasitelkti fermentus metiltransferazes (MTazes). Natūraliai jos naudoja kofaktorių AdoMet, tačiau gali būti aktyvios ir su sintetiniais jo analogais, turinčiais ilgesnes nei metil- pernešamas grandines. Jei šios grandinės turi galines funkcines grupes, prie jų vėliau cheminių reakcijų pagalba galima prijungti norimus žymenis. Tam itin patogi azidų-alkinų cikloprijungimo (AAC), dar vadinama „click“, reakcija. Šiame darbe parodyta, kad DNR citozino-5 MTazė HhaI (variantas Q82A/Y254S/N204A) efektyviai katalizuoja sekai specifinę heks-2-inil- grandinių, turinčių galines alkinil- arba azido- grupes, pernašą nuo sintetinių kofaktorių ant DNR. Naudojant šią MTazės-kofaktorių sistemą bei AAC, visą specifinio DNR žymėjimo procesą galima atlikti netgi ląstelių lizate. RNR žymėjimui ištirtas archėjų RNR 2‘-O-MTazės C/D ribonukleoproteininio komplekso aktyvumas su sintetiniais kofaktoriais. Parodyta galimybė sintetiškai keičiant kreipiančiąją RNR, prop-2-inilgrupės pernašą nukreipti į norimas įvairių substratinių RNR sekos vietas ir po to AAC reakcijos pagalba prijungti fluoroforą. Taigi, sukurtas naujas molekulinis įrankis, leidžiantis be suvaržymų pasirinkti norimą pažymėti RNR seką. Šios naujos specifinio nukleorūgščių žymėjimo galimybės gali būti pritaikytos biochemijos, biomedicinos, nanotechnologijų ir kitose tyrimų srityse... [toliau žr. visą tekstą] Investigation of RNA and DNA function often requires sequence-specific incorporation of various reporter and affinity probes. This can be achieved using AdoMet-dependent methyltransferases (MTases) as they can be active with synthetic AdoMet analogues equipped with transferable chains larger than the methyl group. These chains usually carry reactive groups that can be further chemically appended with required reporters. For this, azide-alkyne 1,3-cycloaddition (AAC), also called “click”, reaction is particularly attractive. This work shows that the HhaI cytosine-5 DNA MTase (variant Q82A/Y254S/N204A) catalyzes efficient sequence-specific transfer of hex-2-ynyl side chains containing terminal alkyne or azide groups from synthetic cofactor analogues to DNA. Both the enzymatic transfer and subsequent “click” coupling of a fluorophore can be performed even in cell lysates. For RNA labeling, the activity of an archaeal RNA 2‘-O-MTase C/D ribonucleoprotein complex (RNP) with synthetic cofactors was investigated. It was shown that synthetically reprogrammed guide RNA sequences can be used to direct the C/D RNP-dependent transfer of a prop-2-ynyl group to predetermined nucleotides in substrate RNAs. Followed by AAC this can be used for programmable sequence-specific labeling of a variety of RNA substrates in vitro. These new possibilities for specific labeling of nucleic acids can be adopted in biochemistry, biomedical, nanotechnology, etc. research.

Lung and head-neck tumours are malignancies with high rates of incidences and deaths in Lithuania and world-wide. The most important risk factor for these malignancies is tobacco smoke. Tobacco smoke contains a mixture of well known carcinogens and procarcinogens, which are shown to induce genetic and epigenetic alterations. In this study, we aimed to determine the frequency and profile of epigenetic (DNA methylation) changes in pathogenesis of tobacco smoke-related lung and head-neck tumours. Tumour supressor genes (TSGs) p16, p14, RARB, RASSF1, MGMT and DAPK were studied by methylation specific PCR and pyrosequencing in lung, head-neck and salivary gland carcinomas (n=212, n=31 and n=36, respectively). Also, scrapes of oral mucosa from subjects with no cancer indication (n=11) and histologically normal salivary gland tissues (n=19) were analysed. MGMT protein expression was analysed by immunohistochemistry in 287 of salivary gland speciments. In lung tumours, promoter hypermethylation was detected in 20.8% of p16, 31.1% of RARB, 28.9% of RASSF1, 15.1% of MGMT and 19.8% of DAPK1 gene. The prevalence of p16 hypemethylation was significantly higher in males than females (p=0.018), squamous cell carcinomas than adenocarcinomas (p=0.025). Hypermethylation of ≥1 gene promoter is associated with TP53 mutations (p=0.027). Methylation index and the frequency of p16 promoter methylation in neversmokers exposed to second-hand tobacco smoke is similar to indices observed in tumours from smokers. In head-neck tumour scrapings, promoter hypermethylation was detected in 23% of p16, 20% of p14, 19% of RASSF1, 20% of RARB, and 50% of MGMT gene. Hypermethylation in ≥1 gene promoter is higher in specimens obtained from smokers than neversmokers (p=0.047). In salivary gland carcinomas and histologically normal tissues promoter hypermethylation was detected in 27.8% and 15.8%, respectively. Epigenetic changes in gene MGMT promoter are associated with loss of MGMT protein expression (p=0.021). The loss of MGMT protein expression in salivary gland tumours is characteristic for patients with poor prognosis (high grade, certain histologic types, lymph node involved tumours). Our study contributes to understanding of tobacco smoke induced molecular alterations in lung and head-neck tumours. Hypermethylation of TSG promoters reflect the harmful effect of tobacco smoke or second-hand tobacco smoke exposure. Analysis of promoter hypermethylation of TSGs in oral scrapings from high risk subjects (smokers) may be a valuable tool for an early prediction of head-neck cancer risk. In salivary gland carcinomas, hypermethylation of gene MGMT promoter causes the loss of MGMT protein expression. This alteration is significant in pathogenesis of salivary gland tumours.

The 12th International St Gallen conference on breast cancer (2011) proposed patient categorization for systemic therapy, based on intrinsic breast cancer subtypes, defined by imunohistochemistry (IHC) test results. Since this classification is based on semi-quantitative expression of IHC biomarker expression, an issue of defining and applying cutoff values remains. Essential improvement in the IHC testing has become possible with digital image analysis tools enabling quantitative evaluation of the IHC data. This study explores data obtained by digital image analysis methods applied to evaluate a comprehensive biomarker dataset (p53, AR, p16, BCL2, SATB1, HIF1) along with well established (ER, PR, HER2, Ki67) biomarkers. Also, an extensive set of genetic and epigenetic biomarkers has been tested. For the first time, the dataset of 10 IHC biomarkers, evaluated by digital analysis was explored by the means of factor analysis to establish the intrinsic factors of biological variation and informative value of IHC biomarkers and their combinatiions. The results also provided insights into the significance and combinatorial effects of the established and relatively new biomarkers (p16, SATB1, HIF1, Ki67/BCL2, etc.).

XII tarptautinėje St Gallen krūties vėžio konferencijoje (2011) Ekspertų komisijos priimta nauja pacientų klasifikacija sisteminei terapijai atlikti, paremta biologiniais krūties vėžio subtipais, kurie apibrėžiami imunohistocheminiu tyrimu (IHC). Tačiau ši nauja navikų klasifikacija iš esmės pagrįsta pusiau kiekybiniu biožymenų raiškos vertinimu, todėl išlieka aktuali ribinių verčių nustatymo problematika. Esminiai pokyčiai IHC tyrimų srityje galimi atsiradus skaitmeninio vaizdinimo technologijoms, leidžiančiomis IHC tyrimų rezultatus analizuoti kiekybiniais parametrais. Darbe naudojant skaitmeninį vaizdo analizės metodą atliktas išsamus biologinių žymenų tyrimas leido palyginti svarbių, tačiau nepakankamai ištirtų (p53, AR, p16, BCL2, SATB1, HIF1) IHC žymenų informatyvumą su esamų prognozinių žymenų (ER, PR, HER2, Ki67) rodikliais. Ištirtas platus genetinių ir epigenetinių krūties vėžio žymenų spektras. Pirmą kartą 10 IHC žymenų rinkinio, įvertinto skaitmeninės analizės būdu, duomenys panaudoti faktorinės analizės metodu nustatyti jų variacijų vidinius veiksnius, atskleidžiančius biologinius dėsningumus ir IHC žymenų bei jų derinių informatyvumą. Šios analizės rezultatai leido naujai įvertinti publikuotų krūties vėžio IHC žymenų bei jų derinių (p16, SATB1, HIF1, Ki67/BCL2 ir kt.) informatyvumą. The 12th International St Gallen conference on breast cancer (2011) proposed patient categorization for systemic therapy, based on intrinsic breast cancer subtypes, defined by imunohistochemistry (IHC) test results. Since this classification is based on semi-quantitative expression of IHC biomarker expression, an issue of defining and applying cutoff values remains. Essential improvement in the IHC testing has become possible with digital image analysis tools enabling quantitative evaluation of the IHC data. This study explores data obtained by digital image analysis methods applied to evaluate a comprehensive biomarker dataset (p53, AR, p16, BCL2, SATB1, HIF1) along with well established (ER, PR, HER2, Ki67) biomarkers. Also, an extensive set of genetic and epigenetic biomarkers has been tested. For the first time, the dataset of 10 IHC biomarkers, evaluated by digital analysis was explored by the means of factor analysis to establish the intrinsic factors of biological variation and informative value of IHC biomarkers and their combinatiions. The results also provided insights into the significance and combinatorial effects of the established and relatively new biomarkers (p16, SATB1, HIF1, Ki67/BCL2, etc.).

Krūties vėžys yra dažniausia Lietuvos ir viso pasaulio moterų onkologinė liga. Ši liga pasižyminti nevienoda eiga, todėl molekulinė vėžio analizė yra labai svarbi. Šiuo metu ligos eiga prognozuojama, remiantis riboto informatyvumo klinikinių žymenų sistema, o gydymui tik pavieniais atvejais skiriami atrankūs vaistai, nukreipti į ligą sukėlusį genetinį pakitimą. Naviko molekulinė analizė padeda aptikti pažaidas vėžio genuose ar jų raiškos pakitimus ir informuoja apie ligos išsivystymo priežastis, padeda prognozuoti ligos progresavimo tikimybę, atskleidžia taikinius naujos kartos gydymo priemonėms. Siekiant įvertinti krūties vėžio epigenetinių biožymenų efektyvumą mes tyrėme reguliacinių genų, dalyvaujančių ląstelės ciklo kontrolėje, signalų perdavime, apoptozėje ir DNR reparacijoje, promotoriaus sekų hipermetilinimą. Tyrimui buvo atrinktos 76 pirminės ankstyvos stadijos (pT1-2) krūties karcinomos. Metilinimo pakitimai promotoriaus sekoje buvo tiriami septyniuose naviką slopinančiuose genuose (p14, p16, RARβ, RASSF1A, DAPK, GSTP1 ir MGMT), taikant metilinimui jautrią PGR. „Tikro laiko“ PGR metodas buvo įdiegtas epigenetinių pakitimų tyrimams cirkuliuojančioje vėžio DNR, išskirtoje iš ligonių kraujo plazmos. Didžioji dalis ankstyvos stadijos krūties karcinomų (63/76) turėjo bent vieno tirto geno hipermetilinimą. Bendras epigenetinių žymenų informatyvumas – 83%. Dažniausiai hipermetilinimas krūties karcinomose nustatytas gene RASSF1A (56/76). Statistinė analizė parodė... [toliau žr. visą tekstą] Breast cancer is the most prevalent malignancy of women in Lithuania and word-wide. The course of disease differ markedly among patients, therefore molecular characterisation of tumour is very important. However, current prognostic markers are mainly based on clinical parameters and do not enable a reliable selection of the patients with high risk of disease progression. Molecular characterisation of tumour through detection of changes in cancer-related genes can help to estimate the risk of cancer progression, detect the molecular targets for modern treatment strategies. In order to evaluate the suitability of epigenetic biomarkers for molecular characterisation of breast cancer we analysed promoter hypermethylation in a wide panel of regulatory genes involved in cell cycle control, signalling, apoptosis and DNA repair. 76 primary breast carcinomas of early stage (pT1-2) were selected for the study. Aberrant methylation in promoter regions of seven tumour suppressor genes (p16, p14, RARβ, RASSF1A, DAPK, GSTP1 and MGMT) was analysed by means of methylation-specific PCR. The “Real Time” PCR method was adapted for detection of epigenetic changes in circulating tumour DNA from plasma of cancer patients. Most of the early-stage breast tumours (63/76) exhibited hypermethylation in at least one gene involved in analysis. The overall sensitivity of the epigenetic biomarkers was 83%. Gene RASSF1A was the most frequently (56 of 76 cases) hypermethylated gene in breast tumours... [to full text]

Magistrinio darbo metu buvo optimizuotos sąlygos tirti trijų auglį slopinančių genų promotoriaus metilinimo pakitimus šviežiai šaldytos DNR ir iš parafininių audinių blokų išskirtos DNR mėginiuose metilinimui jautrios PGR (MSP) metodu. Ištirtos p16, RARβ ir RASSF1A genų DNR metilinimo pažaidos 43 nerūkančiųjų ir 60 rūkančiųjų nesmulkialąstelinės žmogaus plaučių karcinomos (PK) DNR mėginiuose. Šie ASG, kurių produktai dalyvauja ląstelės ciklo reguliacijoje, signalų perdavime ar diferenciacijoje, dažnai būna inaktyvuoti plaučių patogenezės metu kaip atsakas į tabako kancerogenų poveikį. Mūsų tyrimo metu nustatyti didesni hipermetilinimo dažniai p16 ir RASSF1A genų promotoriuose rūkančiųjų plaučių karcinomose (33 proc. ir 44 proc.) palyginus su nerūkančiaisiais (12 proc. ir 21 proc.). Be to, hipermetilinimas p16 geno promotoriuje buvo dažnesnis plokščialąstelinėse karcinomose (39 proc.) nei adenokarcinomose (18 proc.), bei geno inaktyvacija stebėta dažniau vyrų (36 proc.) negu moterų (9 proc.) vėžiniuose plaučių audiniuose. Nenustatyti statistiškai reikšmingi skirtumai tarp ASG metilinimo nerūkančiųjų, turėjusių ar neturėjusių kontakto su rūkalais namuose bei darbe, grupėse. Nustatyti dideli tirtų ASG metilinimo dažniai rūkančiųjų ir nerūkančiųjų grupėse rodo epigenetinių pažaidų svarbą plaučių vėžio patogenezėje. Epigenetinių pažaidų tyrimai ateityje gali tapti patikimu plaučių vėžio biožymeniu, o genų p16 ir RASSF1A metilinimo analizė gali pasitarnauti vėžio prevencijai... [toliau žr. visą tekstą] Aberrant methylation in the promoter region of tumour suppressor genes (TSGs) is one of the mechanisms responsible for gene silencing during carcinogenesis. In lung cancer, p16, RASSF1A and RAR genes are frequently altered by hypermethylation in promoter region as a consequence of exposure to tobacco smoke. The main aim of our study was to optimize conditions for methylation specific PCR (MSP) and to analyze aberrant methylation in p16, RASSF1A and RAR genes in primary non-small cell lung carcinomas (NSCLC) from smokers and never-smokers. Aberrant methylation was analysed in 43 NSCLC from non-smokers and in 60 NSCLC from smokers, and relations to smoking status, histology, age and sex were evaluated. We found hypermethylation of p16 and RASSF1A genes to be frequent events in tobacco smoking patients. The rate of epigenetic alterations in p16 gene promoter was 33%, while RASSF1A gene was hypermethylated in 44% of tumour samples. Hypermethylation of p16 was more prevalent in squamous cell carcinomas (39%) than adenocarcinomas (18%), and in males (36%) than females (9%). There was no relationship between promoter hypermethylation and age. No significant differences in frequencies of hypermethylation were detected in NSCLC from never-smokers with and without documented exposure to environmental tobacco smoke. Analysis of epigenetic inactivation of tumour suppressor genes, particularly p16 and RASSF1A, may provide a valuable biomarker for lung cancer prevention in... [to full text]

Aberrant methylation in the promoter region of tumour suppressor genes (TSGs) is one of the mechanisms responsible for gene silencing during carcinogenesis. In lung cancer, p16, RASSF1A and RAR genes are frequently altered by hypermethylation in promoter region as a consequence of exposure to tobacco smoke. The main aim of our study was to optimize conditions for methylation specific PCR (MSP) and to analyze aberrant methylation in p16, RASSF1A and RAR genes in primary non-small cell lung carcinomas (NSCLC) from smokers and never-smokers. Aberrant methylation was analysed in 43 NSCLC from non-smokers and in 60 NSCLC from smokers, and relations to smoking status, histology, age and sex were evaluated. We found hypermethylation of p16 and RASSF1A genes to be frequent events in tobacco smoking patients. The rate of epigenetic alterations in p16 gene promoter was 33%, while RASSF1A gene was hypermethylated in 44% of tumour samples. Hypermethylation of p16 was more prevalent in squamous cell carcinomas (39%) than adenocarcinomas (18%), and in males (36%) than females (9%). There was no relationship between promoter hypermethylation and age. No significant differences in frequencies of hypermethylation were detected in NSCLC from never-smokers with and without documented exposure to environmental tobacco smoke. Analysis of epigenetic inactivation of tumour suppressor genes, particularly p16 and RASSF1A, may provide a valuable biomarker for lung cancer prevention in high-risk groups.