- Email: [email protected]
A new epigenetic mechanism of temozolomide action in glioma cells
Abstracts / New Biotechnology 33S (2016) S1–S213
by heat and drought stresses. Studying grain development and the effects of environmental factors is a primary goal to improve grain quality. Hexaploid genome and relatively long life cycle of wheat makes gene expression studies difficult, therefore the diploid Brachypodium distachyon became a monocotyledonous model system for studying temperate cereals. The aim of this project is to study whether Brachypodium would be an ideal model for the analysis of storage proteins. Wheat prolamin ortholog genes were identified by sequence homology, annotations and EST databases. Five genes (BRADI1G50200, BRADI2G20910, BRADI2G38530, BRADI2G39940, BRADI3G17070) were used later in the gene expression studies. The expression profiles were studied during anthesis and grain filling by quantitative RT-PCR. The gene expression patterns of these genes were similar to the expression profile of wheat orthologs. Gene expression was also investigated under heat or drought stress conditions. Both factors appeared to shorten the period of grain development, although not in a linear fashion. When the expression profiles were compared there was a significant shift between the gene expression peaks of stressed and control plants. Our previous study in wheat indicated that not only the presence but the pattern of the cis-regulatory elements had impact on the expression profile of prolamins, therefore the promoter regions of these genes were analysed. The analysis of the expression profile of transcription factors involved in the accumulation of storage proteins is in progress. http://dx.doi.org/10.1016/j.nbt.2016.06.1297
P19-4 Changes in DNA methylation in maize under herbicide stress conditions Joanna Gracz 1,∗ , Agata Tyczewska 2 1 2
Institute of Bioorganic Chemistry, Poland PAS, Poland
DNA methylation and chromatin modifications have been shown to be involved in gene silencing at both transcriptional and posttranscriptional levels. DNA methylation (both asymmetric and symmetric) plays a crucial role in the regulation of gene expression, in the activity of transposable elements, in the defense against foreign DNA, and even in the inheritance of specific gene expression patterns. The link between stress exposure and sequence specific changes in DNA methylation was hypothetical until recently, when it was shown that stresses can induce changes in gene expression through hypomethylation or hypermethylation of DNA. Our initial experiments indicated that there are changes in global DNA methylation levels under herbicide stress conditions in maize. To reveal more detailed changes in DNA methylation in two maize lines displaying different susceptibility to RoundUp® we used methylation sensitive amplified polymorphism (MSAP). It is a technique where isoschizomers Hpa II and Msp I are used to determine the differences in DNA methylation due to enzyme’s differential sensitivity to DNA methylation. We observed differences in methylation profiles between the two tested lines under herbicide stress conditions. Differentiating DNA bands were eluted from PAA gels and sequenced. Analyses of 197 DNA fragments using Blastn and Maize GDB databases allowed us to divide them into several groups representing genes encoding for transporter proteins, transferases, methyltransferases, genes involved in stress responses but also transposons. For more detailed analyses (DNA methylation, chromatin modifications) we chose 4 genes encoding
S167
for proteins that could potentially be involved in herbicide stress response. http://dx.doi.org/10.1016/j.nbt.2016.06.1298
P19-5 Epigenetic of DNA Methylation Patterns in Lowland and Upland Malaysian Rice Cultivars under Induced Drought and Salinity Wai Sum Yap 1,∗ , Yang Lin Yeo 1 , Kok Song Lai 2 , Chien Yeong Wee 3 1
UCSI University, Kuala Lumpar, Malaysia Universiti Putra, Malaysia 3 MARDI, Malaysia 2
Rice, being a staple food in Malaysia, is planted widely across the region. Nevertheless, its yield is often halted by drought and high soil salinity. Being sessile in nature, plants have evolved strategies to cope with the changing environment. This study was aimed to evaluate DNA methylation patterns of lowland (MR219) and upland (Sambung) rice cultivars under induced drought (5, 10, 15, 20% PEG) and salt (50, 100, 150, 200 mM NaCl) by employing methylation sensitive amplified polymorphism (MSAP) analysis. Our data showed that the level of hemi-methylation in MR219 and Sambung under drought stress was ranged from 1.32% to 23.18% and 0.82% to 15.16%, respectively while the level of full methylation in MR219 and Sambung was ranged from 29.14% to 58.94% and 33.77% to 47.02%, respectively. Increased in salinity stress had decreased the percentage of hemi-methylation in Sambung, but increased the level of full methylation in both rice cultivars. The hemi-methylation level of MR219 increased and decreased gradually when exposed to increasing order of salt concentrations. In regard to drought stress, hemi-methylation of MR219 at 20% PEG was about 17.6-fold higher than control whereas hemi-methylation in Sambung was decreased with 18.5-fold, 2.6-fold, 7.4-fold and 1.3-fold at 5%, 10%, 15% and 20% PEG, respectively compared to control. This study reveals the effect of drought and salinity stresses on DNA methylation patterns in two contrasting rice cultivars, demonstrating the role of epigenetic mechanism in stress adaptation. Further study is carried out to determine the responsible genes involved in drought and salinity stresses. http://dx.doi.org/10.1016/j.nbt.2016.06.1299
Nucleic acid technologies P20-1 A new epigenetic mechanism of temozolomide action in glioma cells Dorota Gurda Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poland Glioblastoma constitute about 70% of all intracranial tumors with the highest morbidity and mortality rate. Current medical treatment based on surgical intervention followed by radioand chemotherapy fails to achieve patient’s long-term survival. Temozolomide (TMZ) is an alkylating agent used as a chemotherapeutic in brain tumor therapy. Although it is a first-line treatment for glioblastoma multiforme, progression and recurrence of the disease is still observed. The key to make TMZ therapy more effective is to fully understand mechanism of its action.
S168
Abstracts / New Biotechnology 33S (2016) S1–S213
To explore mechanism underlying TMZ action we analyzed changes in 5-methylcytosine (m5 C) in DNA of cancer versus normal cell lines after TMZ treatment (100–1000 M) for 3–48 h. We observed DNA hypermethylation followed by global demethylation. These changes may lead firstly to inhibition of genes related to DNA methyltransferases (DNMTs) activation. Subsequently observed demethylation is caused by oxidative stress to the cells. Removal of m5 C, being silencing results in progression of cancerogenesis through activation of pathologic protein genes. The influence of TMZ on cell cycle was evaluated by flow cytometry. TMZ treatment resulted in G2M phase arrest in a concentration dependent manner, while reduced the number of cells in G0/G1 phase. Our results suggest the new mechanism of TMZ action via modulation in m5 C level. High concentration in short time induced increase of global m5 C contents in DNA, whereas low concentration and longer time of exposure resulted in hypomethylation. Therefore administration with low doses of TMZ and short time should be considered as optimal therapy. http://dx.doi.org/10.1016/j.nbt.2016.06.1300
Macugen, an anti-VEGF aptamer for treatment of age-related macular degeneration, is the first and only FDA approved therapeutic aptamer. Also, many of therapeutic aptamers, now are in clinical trials phases. For example; ARC-1779, an aptamer against activated von-willebrand (vWF) in patients with purpura, thrombotic thrombocytopenic and vWF type 2, has finished phase 2 of clinical trials in 2008. Furthermore, Nox-E36, an anti MCP-1 aptamer, has completed phase 2 of clinical trials in patients with type 2 diabetes mellitus and albuminuria. In diagnosis, many aptamers that are in research procedure have designed against cancer cell surface biomarkers. Some of these cell surface biomarkers are: EpCAM, nucleolin and integrinavß3. Aptamers have different other applications. By conjugation with certain sensors, aptamers can be use for environmental toxicity studies and cell/tissue imaging. According to growing science and more consideration towards aptamers, it is expected that aptamers not only, will be therapeutics, but also will help to cancer diagnosis and environmental toxicity rescue in the next few years. http://dx.doi.org/10.1016/j.nbt.2016.06.1302
P20-2 P20-4 Metal ion triggers for reversible switching of DNA polymerase Hyun Gyu Park ∗ , Ki Soo Park, Chang Yeol Lee
Effect of kinetin riboside on proapoptotic activities in human cancer and normal cell lines
Korea Advanced Institute of Science and Technology (KAIST), Republic of Korea
Marta Orlicka-Płocka ∗ , Dorota Gurda, Agnieszka Fedoruk-Wyszomirska, Jan Barciszewski, Eliza Wyszko
We herein describe a new strategy that modulates DNA polymerase activity in a reversible and switchable manner. The method relies on the formation of specific metal ion-mediated base pairings (T-Hg2+ -T and C-Ag+ -C) within a DNA aptamer with an inhibitory capability against DNA polymerase. Through a detailed investigation, we verified that Hg2+ and Ag+ ions function as novel external stimuli (activators and inhibitors) to control DNA polymerase activity by stabilizing the respective T-T and C-C mismatching and consequently altering the secondary structure of the DNA aptamer. Finally, the reversible and switchable regulation of DNA polymerase activity was successfully achieved through successively applying metal ions and their binding ligands. This work will not only advance the artificial modulation of DNA-related biological reactions, for which little is known, but also expand the application of metal ion-mediated base pairs.
Instytut Chemii Bioorganicznej Polskiej Akademii Nauk, Poland
http://dx.doi.org/10.1016/j.nbt.2016.06.1301
P20-3 Aptamer: A new emerging tool for therapeutics and theranostics Gilar Gorji Bahri School of Pharmacy, Iran Aptamers are composed of DNA/RNA and termed chemical antibodies with 8–25 kDa molecular weight. They can be use for targeted drug deliver. They have several advantages over conventional antibodies. Some of them are: fast and efficient penetration to tissues, non-immunogenic properties, thermoresistant, low structural variation during synthesis and low production cost. In this review, we will discuss new studies on aptamers, their different applications and introduce some of the aptamers that are in clinical trials phases for different applications and targets.
Kinetin riboside (KR) is naturally occurring N6-substituted purine derivative and belongs to hormones family that play role in regulation of plant growth. KR has been recently reported to have a strong anticancer effect, however, the molecular mechanism has not been elucidated. There are findings that natural small molecular compounds may influence selectively various steps of intracellular signaling and molecular pathways that are crucial for cell growth, proliferation and apoptosis. We investigated kinetin riboside ability to induce cell apoptosis by flow cytometry and confocal microscopy analysis. Using flow cytometry, we estimated rate of KR-induced apoptosis in T98G (glioblastoma multiforme), HepG2 (hepatocellular carcinoma) and CCL-110 (skin fibroblasts) cell lines after 24 h and 48 h. We showed, that KR is a potent apoptosis-inducing agent in a concentration and time dependent manner. For instance, T98G cell line, treated with 1 M KR for 24 h, showed almost two-fold greater of apoptosis (30%) compared to control cells (18.5%). However, the apoptosis was completely prevented when cells were treated with 5-Iodotubericidin, an inhibitor of adenosine kinase, it indicated that the intracellular phosphorylation of KR is necessary for its cytotoxic effects to T98G, HepG2 and CCL-110 cells. To determine the proapoptotic activities of small molecular compounds to cancer cells, we also investigated adenosine, kinetin, trans-zeatin riboside and ortho-topolin riboside as controls. Small molecular compounds-based therapy seems to be very potent and perspective in civilization diseases treatment, including anti-cancer therapy. KR is a very good example of cell toxicity inducer, inhibiting cell division and initiating apoptosis. http://dx.doi.org/10.1016/j.nbt.2016.06.1303
by heat and drought stresses. Studying grain development and the effects of environmental factors is a primary goal to improve grain quality. Hexaploid genome and relatively long life cycle of wheat makes gene expression studies difficult, therefore the diploid Brachypodium distachyon became a monocotyledonous model system for studying temperate cereals. The aim of this project is to study whether Brachypodium would be an ideal model for the analysis of storage proteins. Wheat prolamin ortholog genes were identified by sequence homology, annotations and EST databases. Five genes (BRADI1G50200, BRADI2G20910, BRADI2G38530, BRADI2G39940, BRADI3G17070) were used later in the gene expression studies. The expression profiles were studied during anthesis and grain filling by quantitative RT-PCR. The gene expression patterns of these genes were similar to the expression profile of wheat orthologs. Gene expression was also investigated under heat or drought stress conditions. Both factors appeared to shorten the period of grain development, although not in a linear fashion. When the expression profiles were compared there was a significant shift between the gene expression peaks of stressed and control plants. Our previous study in wheat indicated that not only the presence but the pattern of the cis-regulatory elements had impact on the expression profile of prolamins, therefore the promoter regions of these genes were analysed. The analysis of the expression profile of transcription factors involved in the accumulation of storage proteins is in progress. http://dx.doi.org/10.1016/j.nbt.2016.06.1297
P19-4 Changes in DNA methylation in maize under herbicide stress conditions Joanna Gracz 1,∗ , Agata Tyczewska 2 1 2
Institute of Bioorganic Chemistry, Poland PAS, Poland
DNA methylation and chromatin modifications have been shown to be involved in gene silencing at both transcriptional and posttranscriptional levels. DNA methylation (both asymmetric and symmetric) plays a crucial role in the regulation of gene expression, in the activity of transposable elements, in the defense against foreign DNA, and even in the inheritance of specific gene expression patterns. The link between stress exposure and sequence specific changes in DNA methylation was hypothetical until recently, when it was shown that stresses can induce changes in gene expression through hypomethylation or hypermethylation of DNA. Our initial experiments indicated that there are changes in global DNA methylation levels under herbicide stress conditions in maize. To reveal more detailed changes in DNA methylation in two maize lines displaying different susceptibility to RoundUp® we used methylation sensitive amplified polymorphism (MSAP). It is a technique where isoschizomers Hpa II and Msp I are used to determine the differences in DNA methylation due to enzyme’s differential sensitivity to DNA methylation. We observed differences in methylation profiles between the two tested lines under herbicide stress conditions. Differentiating DNA bands were eluted from PAA gels and sequenced. Analyses of 197 DNA fragments using Blastn and Maize GDB databases allowed us to divide them into several groups representing genes encoding for transporter proteins, transferases, methyltransferases, genes involved in stress responses but also transposons. For more detailed analyses (DNA methylation, chromatin modifications) we chose 4 genes encoding
S167
for proteins that could potentially be involved in herbicide stress response. http://dx.doi.org/10.1016/j.nbt.2016.06.1298
P19-5 Epigenetic of DNA Methylation Patterns in Lowland and Upland Malaysian Rice Cultivars under Induced Drought and Salinity Wai Sum Yap 1,∗ , Yang Lin Yeo 1 , Kok Song Lai 2 , Chien Yeong Wee 3 1
UCSI University, Kuala Lumpar, Malaysia Universiti Putra, Malaysia 3 MARDI, Malaysia 2
Rice, being a staple food in Malaysia, is planted widely across the region. Nevertheless, its yield is often halted by drought and high soil salinity. Being sessile in nature, plants have evolved strategies to cope with the changing environment. This study was aimed to evaluate DNA methylation patterns of lowland (MR219) and upland (Sambung) rice cultivars under induced drought (5, 10, 15, 20% PEG) and salt (50, 100, 150, 200 mM NaCl) by employing methylation sensitive amplified polymorphism (MSAP) analysis. Our data showed that the level of hemi-methylation in MR219 and Sambung under drought stress was ranged from 1.32% to 23.18% and 0.82% to 15.16%, respectively while the level of full methylation in MR219 and Sambung was ranged from 29.14% to 58.94% and 33.77% to 47.02%, respectively. Increased in salinity stress had decreased the percentage of hemi-methylation in Sambung, but increased the level of full methylation in both rice cultivars. The hemi-methylation level of MR219 increased and decreased gradually when exposed to increasing order of salt concentrations. In regard to drought stress, hemi-methylation of MR219 at 20% PEG was about 17.6-fold higher than control whereas hemi-methylation in Sambung was decreased with 18.5-fold, 2.6-fold, 7.4-fold and 1.3-fold at 5%, 10%, 15% and 20% PEG, respectively compared to control. This study reveals the effect of drought and salinity stresses on DNA methylation patterns in two contrasting rice cultivars, demonstrating the role of epigenetic mechanism in stress adaptation. Further study is carried out to determine the responsible genes involved in drought and salinity stresses. http://dx.doi.org/10.1016/j.nbt.2016.06.1299
Nucleic acid technologies P20-1 A new epigenetic mechanism of temozolomide action in glioma cells Dorota Gurda Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poland Glioblastoma constitute about 70% of all intracranial tumors with the highest morbidity and mortality rate. Current medical treatment based on surgical intervention followed by radioand chemotherapy fails to achieve patient’s long-term survival. Temozolomide (TMZ) is an alkylating agent used as a chemotherapeutic in brain tumor therapy. Although it is a first-line treatment for glioblastoma multiforme, progression and recurrence of the disease is still observed. The key to make TMZ therapy more effective is to fully understand mechanism of its action.
S168
Abstracts / New Biotechnology 33S (2016) S1–S213
To explore mechanism underlying TMZ action we analyzed changes in 5-methylcytosine (m5 C) in DNA of cancer versus normal cell lines after TMZ treatment (100–1000 M) for 3–48 h. We observed DNA hypermethylation followed by global demethylation. These changes may lead firstly to inhibition of genes related to DNA methyltransferases (DNMTs) activation. Subsequently observed demethylation is caused by oxidative stress to the cells. Removal of m5 C, being silencing results in progression of cancerogenesis through activation of pathologic protein genes. The influence of TMZ on cell cycle was evaluated by flow cytometry. TMZ treatment resulted in G2M phase arrest in a concentration dependent manner, while reduced the number of cells in G0/G1 phase. Our results suggest the new mechanism of TMZ action via modulation in m5 C level. High concentration in short time induced increase of global m5 C contents in DNA, whereas low concentration and longer time of exposure resulted in hypomethylation. Therefore administration with low doses of TMZ and short time should be considered as optimal therapy. http://dx.doi.org/10.1016/j.nbt.2016.06.1300
Macugen, an anti-VEGF aptamer for treatment of age-related macular degeneration, is the first and only FDA approved therapeutic aptamer. Also, many of therapeutic aptamers, now are in clinical trials phases. For example; ARC-1779, an aptamer against activated von-willebrand (vWF) in patients with purpura, thrombotic thrombocytopenic and vWF type 2, has finished phase 2 of clinical trials in 2008. Furthermore, Nox-E36, an anti MCP-1 aptamer, has completed phase 2 of clinical trials in patients with type 2 diabetes mellitus and albuminuria. In diagnosis, many aptamers that are in research procedure have designed against cancer cell surface biomarkers. Some of these cell surface biomarkers are: EpCAM, nucleolin and integrinavß3. Aptamers have different other applications. By conjugation with certain sensors, aptamers can be use for environmental toxicity studies and cell/tissue imaging. According to growing science and more consideration towards aptamers, it is expected that aptamers not only, will be therapeutics, but also will help to cancer diagnosis and environmental toxicity rescue in the next few years. http://dx.doi.org/10.1016/j.nbt.2016.06.1302
P20-2 P20-4 Metal ion triggers for reversible switching of DNA polymerase Hyun Gyu Park ∗ , Ki Soo Park, Chang Yeol Lee
Effect of kinetin riboside on proapoptotic activities in human cancer and normal cell lines
Korea Advanced Institute of Science and Technology (KAIST), Republic of Korea
Marta Orlicka-Płocka ∗ , Dorota Gurda, Agnieszka Fedoruk-Wyszomirska, Jan Barciszewski, Eliza Wyszko
We herein describe a new strategy that modulates DNA polymerase activity in a reversible and switchable manner. The method relies on the formation of specific metal ion-mediated base pairings (T-Hg2+ -T and C-Ag+ -C) within a DNA aptamer with an inhibitory capability against DNA polymerase. Through a detailed investigation, we verified that Hg2+ and Ag+ ions function as novel external stimuli (activators and inhibitors) to control DNA polymerase activity by stabilizing the respective T-T and C-C mismatching and consequently altering the secondary structure of the DNA aptamer. Finally, the reversible and switchable regulation of DNA polymerase activity was successfully achieved through successively applying metal ions and their binding ligands. This work will not only advance the artificial modulation of DNA-related biological reactions, for which little is known, but also expand the application of metal ion-mediated base pairs.
Instytut Chemii Bioorganicznej Polskiej Akademii Nauk, Poland
http://dx.doi.org/10.1016/j.nbt.2016.06.1301
P20-3 Aptamer: A new emerging tool for therapeutics and theranostics Gilar Gorji Bahri School of Pharmacy, Iran Aptamers are composed of DNA/RNA and termed chemical antibodies with 8–25 kDa molecular weight. They can be use for targeted drug deliver. They have several advantages over conventional antibodies. Some of them are: fast and efficient penetration to tissues, non-immunogenic properties, thermoresistant, low structural variation during synthesis and low production cost. In this review, we will discuss new studies on aptamers, their different applications and introduce some of the aptamers that are in clinical trials phases for different applications and targets.
Kinetin riboside (KR) is naturally occurring N6-substituted purine derivative and belongs to hormones family that play role in regulation of plant growth. KR has been recently reported to have a strong anticancer effect, however, the molecular mechanism has not been elucidated. There are findings that natural small molecular compounds may influence selectively various steps of intracellular signaling and molecular pathways that are crucial for cell growth, proliferation and apoptosis. We investigated kinetin riboside ability to induce cell apoptosis by flow cytometry and confocal microscopy analysis. Using flow cytometry, we estimated rate of KR-induced apoptosis in T98G (glioblastoma multiforme), HepG2 (hepatocellular carcinoma) and CCL-110 (skin fibroblasts) cell lines after 24 h and 48 h. We showed, that KR is a potent apoptosis-inducing agent in a concentration and time dependent manner. For instance, T98G cell line, treated with 1 M KR for 24 h, showed almost two-fold greater of apoptosis (30%) compared to control cells (18.5%). However, the apoptosis was completely prevented when cells were treated with 5-Iodotubericidin, an inhibitor of adenosine kinase, it indicated that the intracellular phosphorylation of KR is necessary for its cytotoxic effects to T98G, HepG2 and CCL-110 cells. To determine the proapoptotic activities of small molecular compounds to cancer cells, we also investigated adenosine, kinetin, trans-zeatin riboside and ortho-topolin riboside as controls. Small molecular compounds-based therapy seems to be very potent and perspective in civilization diseases treatment, including anti-cancer therapy. KR is a very good example of cell toxicity inducer, inhibiting cell division and initiating apoptosis. http://dx.doi.org/10.1016/j.nbt.2016.06.1303