First we showed which the reduction in Topo I DNA relaxation activity was not a consequence of a decrease in the enzyme protein level; thus, contamination did not alter the expression of Topo I. influencing the anti-cancer capacity of Topo I antagonists. Introduction Mycoplasmas, which belong to the Mollicutes class, are the smallest self-replicating eubacteria, devoid of a cell wall and surrounded only by a plasma membrane. Their small genome size (ranging from 580 to 1380 kbp) results in limited metabolic capabilities and parasitism [1], [2]. Mycoplasmas can be found as parasites in a wide range of hosts including humans, animals, insects, plants, and cells produced in tissue culture. In humans, some Mycoplasma species are found as commensal inhabitants, while other were shown to be associated with infectious diseases and post-infection pathologies [3], [4]. Most of the known Mycoplasma species are found as membrane surface parasites, and recently, some were shown to enter the cells and become intracellular residents [5]. Mycoplasma may cause chronic infections due to sophisticated mechanisms for evasion from immune surveillance (i.e., molecular mimicry, a unique type of antigenic variance), up-regulating or down-regulating cytokine secretion, adhesion molecules expression, transcription factors expression, MAP kinases activity, apoptotic pathways, and more [2], [3]. Recently, many reports have strongly supported the ability of Mycoplasma to cause or promote oncogenic transformation [6]C[9], and the search for the link between Mycoplasma and malignancy is currently being explored [10]. The lipoproteins (LPMf) of was shown to inhibit the apoptosis process induced by tumor necrosis factor (TNF) [17], [18]. All these led to the assumption that contamination of tumor cells by Mycoplasma may impact the activity and expression of essential nuclear enzymes such as topoisomerases, which are the targets of several anti-cancer drugs and thus interfere with the anti-cancer efficacy of these drugs. DNA topoisomerases are a family of essential nuclear enzymes that are responsible for controlling the topological state of the DNA molecules. They participate in most DNA transactions such as replication, transcription, recombination, and chromatin remodeling [19]C[21]. DNA topoisomerases are classified as either type I (cleaves one strand of DNA) or type II (cleaves two strands of DNA). Both enzyme types are further categorized into subgroups according to structural and functional features. Users of each family of enzymes are unique in sequence, structure, and functions [22]. The catalytic activity of DNA topoisomerases entails the formation of transient covalent bridges of enzyme-DNA complexes. A tyrosyl group in the active site of the enzyme attacks a phosphodiester bond around the DNA backbone and remains covalently attached to one side of the break, leaving an opposite free hydroxyl (OH) end that allows the religation step, after DNA topology is usually resolved, by a second nucleophilic attack of the covalent enzyme-DNA phosphotyrosine bond, releasing the enzyme for the next catalytic cycle. The involvement of these enzymes in essential cellular processes tagged topoisomerases as important targets for anti-cancer treatments and for the development of potent, more effective, anticancer drugs [22], [23]. The cytotoxicity of Topoisomerases inhibitors such as Camptothecin (CPT) and its derivatives TPT and CPT-11 (which are approved for clinical use), stems from their ability to stabilize the cleavable complex of TopoCDNA, which introduces single and double strand breaks in the DNA [21], [24], [25]. Topoisomerase activity is usually influenced by several post-translational modifications, among them phosphorylation, poly-ADP-ribosylation, and ubiquitination. Recent work done in our laboratory exhibited the O-GlcNAcylation of Topo IB, which affects its activity [26]. The phosphorylation of DNA topoisomerase I by casein kinase II (CK II) and protein kinase C (PKC) up-regulate the enzyme DNA relaxation activity, whereas dephosphorylation by alkaline phosphatase inhibited this activity. In addition, poly-ADP ribosylation by poly-ADP ribose polymerase (PARP-1) of the enzyme protein was found to down-regulate its activity [20], [27]. PARP-1 is known to be activated by DNA breaks; however recently, it was reported that PARP-1 can be activated by phosphorylated ERK2 in the absence of stress conditions or DNA damage [28]. In recent studies Mycoplasma was demonstrated to be capable of activating numerous MAPKs, such as SAPK/JNK, p38, NF-kB, AP-1, and ERK 1/2 in response to Mycoplasma-derived membrane lipoproteins [11], [29]C[31]. Thus it is important to.t-test: *p<0.05, **p<0.01, ***p<0.005 (TIF) Click here for additional data file.(1.2M, tif) Acknowledgments We thank Liraz Platinum, Fabian Afergan, Elena Robinstein, Orly Sagi, and Frida Inghel from your mycoplasma laboratory, Soroka University Medical Center, Beer-Sheva, Israel, for technical assistance; and Refael Peleg for helpful remarks. Funding Statement Funding was provided by the Seed Research fund, Ben-Gurion University or college. results of this study suggest that modification of Topo I activity by may alter cellular gene expression and the response of tumor cells to Topo I inhibitors, influencing the anti-cancer capacity of Topo I antagonists. Introduction Mycoplasmas, which belong to the Mollicutes class, are the smallest self-replicating eubacteria, devoid of a cell wall and surrounded only by a plasma membrane. Their small genome size (ranging from 580 to 1380 kbp) results in limited metabolic capabilities and parasitism [1], [2]. Mycoplasmas can be found as parasites in a wide range of hosts including humans, animals, insects, plants, and cells grown in tissue culture. In humans, some Mycoplasma species are found as commensal inhabitants, while other were shown to be associated with infectious diseases and post-infection pathologies [3], [4]. Most of the known Mycoplasma species are found as membrane surface parasites, and recently, some were shown to enter the cells and become intracellular residents [5]. Mycoplasma may cause chronic infections due to sophisticated mechanisms for evasion from immune surveillance (i.e., molecular mimicry, a unique type of antigenic variation), up-regulating or down-regulating cytokine secretion, adhesion molecules expression, transcription factors expression, MAP kinases activity, apoptotic pathways, and more [2], [3]. Recently, many reports have strongly supported the ability of Mycoplasma to cause or promote oncogenic transformation [6]C[9], and the search for the link between Mycoplasma and cancer is currently being explored [10]. The lipoproteins (LPMf) of was shown to inhibit the apoptosis process induced by tumor necrosis factor (TNF) [17], [18]. All these led to the assumption that infection of tumor cells by Mycoplasma may affect the activity and expression of essential nuclear enzymes such as topoisomerases, which are the targets of several anti-cancer drugs and thus interfere with the anti-cancer efficacy of these drugs. DNA topoisomerases are a family of essential nuclear enzymes that are Vardenafil responsible for controlling the topological state of the DNA molecules. They participate in most DNA transactions such as replication, transcription, recombination, and chromatin remodeling [19]C[21]. DNA topoisomerases are classified as either type I (cleaves one strand of DNA) or type II (cleaves two strands of DNA). Both enzyme types are further categorized into subgroups according to structural and functional features. Members of each family of enzymes are distinct in sequence, structure, and functions [22]. The catalytic activity of DNA topoisomerases involves the formation of transient covalent bridges of enzyme-DNA complexes. A tyrosyl group in the active site of the enzyme attacks a phosphodiester bond on the DNA backbone and remains covalently attached to one side of the break, leaving an opposite free hydroxyl (OH) end that Vardenafil allows the religation step, after DNA topology is resolved, by a second nucleophilic attack of the covalent enzyme-DNA phosphotyrosine bond, releasing the enzyme for the next catalytic cycle. The involvement of these enzymes in essential cellular processes tagged topoisomerases as important targets for anti-cancer treatments and for the development of potent, more effective, anticancer drugs [22], [23]. The cytotoxicity of Topoisomerases inhibitors such as Camptothecin (CPT) and its derivatives TPT and CPT-11 (which are approved for clinical use), stems from their ability to stabilize the cleavable complex of TopoCDNA, which introduces single and double strand breaks in the DNA [21], [24], [25]. Topoisomerase activity is influenced by several post-translational modifications, among them phosphorylation, poly-ADP-ribosylation, and ubiquitination. Recent work done in our laboratory demonstrated the O-GlcNAcylation of Topo IB, which affects its activity [26]. The phosphorylation of DNA topoisomerase I by casein kinase II (CK II) and protein kinase C (PKC) up-regulate the enzyme DNA relaxation activity, whereas dephosphorylation by alkaline phosphatase inhibited this activity. In addition, poly-ADP ribosylation by poly-ADP ribose polymerase (PARP-1) from the enzyme proteins was discovered to down-regulate its activity [20], [27]. PARP-1 may be triggered by DNA breaks; nevertheless recently, it had been reported that PARP-1 could be triggered by phosphorylated ERK2 in the lack of tension circumstances or DNA harm [28]. In latest research Mycoplasma was proven with the capacity of activating different MAPKs, such as for example SAPK/JNK, p38, NF-kB, AP-1, and ERK 1/2 in response to Mycoplasma-derived membrane lipoproteins [11], [29]C[31]. Therefore it’s important to investigate the chance that the mobile Topo I as well as the effectiveness of CPTs as anti-cancer real estate agents might be suffering from Mycoplasma infection. Components and Strategies Cells Human breasts tumor cell lines -MCF7 (American Type Tradition Collection, HTB-22) and.The co-cultures were incubated for five hrs at 37C, 5% CO2. changes of Topo I activity by may alter mobile gene expression as well as the response of tumor cells to Topo I inhibitors, influencing the anti-cancer capability of Topo I antagonists. Intro Mycoplasmas, which participate in the Mollicutes course, will be the smallest self-replicating eubacteria, without a cell wall structure and surrounded just with a plasma membrane. Their little genome size (which range from 580 to 1380 kbp) leads to limited metabolic features and parasitism [1], [2]. Mycoplasmas are available as parasites in an array of hosts including human beings, animals, insects, vegetation, and cells cultivated in tissue tradition. In human beings, some Mycoplasma varieties are located as commensal inhabitants, while additional were been shown to be connected with infectious illnesses and post-infection pathologies [3], [4]. A lot of the known Mycoplasma varieties are located as membrane surface area parasites, and lately, some were proven to get into the cells and be intracellular occupants [5]. Mycoplasma could cause chronic attacks due to advanced systems for evasion from immune system monitoring (i.e., molecular mimicry, a distinctive kind of antigenic variant), up-regulating or down-regulating cytokine secretion, adhesion substances expression, transcription elements manifestation, MAP kinases activity, apoptotic pathways, and even more [2], [3]. Lately, many reports possess strongly supported the power of Mycoplasma to trigger or promote oncogenic change [6]C[9], as well as the search for the hyperlink between Mycoplasma and tumor is currently becoming explored [10]. The lipoproteins (LPMf) of was proven to inhibit the apoptosis procedure induced by tumor necrosis element (TNF) [17], [18]. Each one of these resulted in the assumption that disease of tumor cells by Mycoplasma may influence the experience and manifestation of important nuclear enzymes such as for example topoisomerases, which will be the focuses on of many anti-cancer drugs and therefore hinder the anti-cancer effectiveness of these medicines. DNA topoisomerases certainly are a family of important nuclear enzymes that are in charge of managing the topological condition from the DNA substances. They take part in most DNA transactions such as for example replication, transcription, recombination, and chromatin redesigning [19]C[21]. DNA topoisomerases are categorized as either type I (cleaves one strand of DNA) or EP type II (cleaves two strands of DNA). Both enzyme types are additional classified into subgroups relating to structural and practical features. Members of every category of enzymes are specific in sequence, framework, and features [22]. The catalytic activity of DNA topoisomerases requires the forming of transient covalent bridges of enzyme-DNA complexes. A tyrosyl group in the energetic site from the enzyme episodes a phosphodiester relationship for the DNA backbone and continues to be covalently mounted on one side from the break, departing an opposite free of charge hydroxyl (OH) end which allows the religation stage, after DNA topology can be resolved, by another nucleophilic attack from the covalent enzyme-DNA phosphotyrosine relationship, liberating the enzyme for another catalytic routine. The involvement of the enzymes in important mobile procedures tagged topoisomerases as essential focuses on for anti-cancer remedies and for the introduction of potent, far better, anticancer medications [22], [23]. The cytotoxicity of Topoisomerases inhibitors such as for example Camptothecin (CPT) and its own derivatives TPT and CPT-11 (that are accepted for clinical make use of), is due to their capability to stabilize the cleavable complicated of TopoCDNA, which presents single and dual strand breaks in the DNA [21], [24], [25]. Topoisomerase activity is normally influenced by many post-translational modifications, included in this phosphorylation, poly-ADP-ribosylation, and ubiquitination. Latest work done inside our lab showed the O-GlcNAcylation of Topo IB, which impacts its activity [26]. The phosphorylation of DNA topoisomerase I by casein kinase II (CK II) and proteins kinase C (PKC) up-regulate the enzyme DNA rest activity, whereas dephosphorylation by alkaline phosphatase inhibited this activity. Furthermore, poly-ADP ribosylation by poly-ADP ribose polymerase (PARP-1) from the enzyme proteins was discovered to down-regulate its activity [20], [27]. PARP-1 may be turned on by DNA breaks; nevertheless recently, it had been reported that PARP-1 could be turned on by phosphorylated ERK2 in the lack of tension circumstances or DNA harm [28]. In latest research Mycoplasma was proven with the capacity of activating several MAPKs, such as for example SAPK/JNK, p38, NF-kB, AP-1, and.A lot of the research regarding and MAPKs were performed using mycoplasmal items or high temperature inactivated Mycoplasma (HIM). response of tumor cells to Topo I inhibitors, influencing the anti-cancer capability of Topo I antagonists. Launch Mycoplasmas, which participate in the Mollicutes course, will be the smallest self-replicating eubacteria, without a cell wall structure and surrounded just with a plasma membrane. Their little genome size (which range from 580 to 1380 kbp) leads to limited metabolic features and parasitism [1], [2]. Mycoplasmas are available as parasites in an array of hosts including human beings, animals, insects, plant life, and cells harvested in tissue lifestyle. In human beings, some Mycoplasma types are located as commensal inhabitants, while various other were been shown to be connected with infectious illnesses and post-infection pathologies [3], [4]. A lot of the known Mycoplasma types are located as membrane surface area parasites, and lately, some were proven to get into the cells and be intracellular citizens [5]. Mycoplasma could cause chronic attacks due to advanced systems for evasion from immune system security (i.e., molecular mimicry, a distinctive kind of antigenic deviation), up-regulating or down-regulating cytokine secretion, adhesion substances expression, transcription elements appearance, MAP kinases activity, apoptotic pathways, and even more [2], [3]. Lately, many reports have got strongly supported the power of Mycoplasma to trigger or promote oncogenic change [6]C[9], as well as the search for the hyperlink between Mycoplasma and cancers is currently getting explored [10]. The lipoproteins (LPMf) of was proven to inhibit the apoptosis procedure induced by tumor necrosis aspect (TNF) [17], [18]. Each one of these resulted in the assumption that an infection of tumor cells by Mycoplasma may have an effect on the experience and appearance of important nuclear enzymes such as for example topoisomerases, Vardenafil which will be the goals of many anti-cancer drugs and therefore hinder the anti-cancer efficiency of these medications. DNA topoisomerases certainly are a family of important nuclear enzymes that are in charge of managing the topological condition from the DNA substances. They take part in most DNA transactions such as for example replication, transcription, recombination, and chromatin redecorating [19]C[21]. DNA topoisomerases are categorized as either type I (cleaves one strand of DNA) or type II (cleaves two strands of DNA). Both enzyme types are additional grouped into subgroups regarding to structural and useful features. Members of every category of enzymes are distinctive in sequence, framework, and features [22]. The catalytic activity of DNA topoisomerases consists of the forming of transient covalent bridges of enzyme-DNA complexes. A tyrosyl group in the energetic site from the enzyme episodes a phosphodiester connection over the DNA backbone and continues to be covalently mounted on one side from the break, departing an opposite free of charge hydroxyl (OH) end which allows the religation stage, after DNA topology is normally resolved, by another nucleophilic attack from the covalent enzyme-DNA phosphotyrosine connection, launching the enzyme for another catalytic routine. The involvement of the enzymes in important mobile procedures tagged topoisomerases as essential goals for anti-cancer remedies and for the introduction of potent, far better, anticancer medications [22], [23]. The cytotoxicity of Topoisomerases inhibitors such as for example Camptothecin (CPT) and its own derivatives TPT and CPT-11 (that are accepted for clinical make use of), is due to their capability to stabilize the cleavable complicated of TopoCDNA, which presents single and dual strand breaks in the DNA [21], [24], [25]. Topoisomerase activity is certainly influenced by many post-translational modifications, included in this phosphorylation, poly-ADP-ribosylation, and ubiquitination. Latest work done inside our lab confirmed the O-GlcNAcylation of Topo IB, which impacts its activity [26]. The phosphorylation of DNA topoisomerase I by casein kinase II (CK II) and proteins kinase C (PKC) up-regulate the enzyme DNA rest activity, whereas dephosphorylation by alkaline phosphatase inhibited this activity. Furthermore, poly-ADP ribosylation by poly-ADP ribose polymerase (PARP-1) from the enzyme.This reduction was also observed when non-live (sonicated) Mycoplasma was used rather than live Mycoplasma, recommending that the result on cellular Topo I is certainly mediated by Mycoplasma surface area proteins/glycoproteins probably. towards the Mollicutes course, will be the smallest self-replicating eubacteria, without a cell wall structure and surrounded just with a plasma membrane. Their little genome size (which range from 580 to 1380 kbp) leads to limited metabolic features and parasitism [1], [2]. Mycoplasmas are available as parasites in an array of hosts including human beings, animals, insects, plant life, and cells expanded in tissue lifestyle. In human beings, some Mycoplasma types are located as commensal inhabitants, while various other were been shown to be connected with infectious illnesses and post-infection pathologies [3], [4]. A lot of the known Mycoplasma types are located as membrane surface area parasites, and lately, some were proven to get into the cells and be intracellular citizens [5]. Mycoplasma could cause chronic attacks due to advanced systems for evasion from immune system security (i.e., molecular mimicry, a distinctive kind of antigenic variant), up-regulating or down-regulating cytokine secretion, adhesion substances expression, transcription elements appearance, MAP kinases activity, apoptotic pathways, and even more [2], [3]. Lately, many reports have got strongly supported the power of Mycoplasma to trigger or promote oncogenic change [6]C[9], as well as the search for the hyperlink between Mycoplasma and tumor is currently getting explored [10]. The lipoproteins (LPMf) of was proven to inhibit the apoptosis procedure induced by tumor necrosis aspect (TNF) [17], [18]. Each one of these resulted in the assumption that infections of tumor cells by Mycoplasma may influence the experience and appearance of important nuclear enzymes such as for example topoisomerases, which will be the goals of many anti-cancer drugs and therefore hinder the anti-cancer efficiency of these medications. DNA topoisomerases certainly are a family of important nuclear enzymes that are in charge of managing the topological condition from the DNA substances. They take part in most DNA transactions such as for example replication, transcription, recombination, and chromatin redecorating [19]C[21]. DNA topoisomerases are categorized as either type I (cleaves one strand of DNA) or type II (cleaves two strands of DNA). Both enzyme types are additional grouped Vardenafil into subgroups regarding to structural and useful features. Members of every category of enzymes are specific in sequence, framework, and features [22]. The catalytic activity of DNA topoisomerases requires the forming of transient covalent bridges of enzyme-DNA complexes. A tyrosyl group in the energetic site from the enzyme episodes a phosphodiester connection in the DNA backbone and continues to be covalently attached to one side of the break, leaving an opposite free hydroxyl (OH) end that allows the religation step, after DNA topology is resolved, by a second nucleophilic attack of the covalent enzyme-DNA phosphotyrosine bond, releasing the enzyme for the next catalytic cycle. The involvement of these enzymes in essential cellular processes tagged topoisomerases as important targets for anti-cancer treatments and for the development of potent, more effective, anticancer drugs [22], [23]. The cytotoxicity of Topoisomerases inhibitors such as Camptothecin (CPT) and its derivatives TPT and CPT-11 (which are approved for clinical use), stems from their ability to stabilize the cleavable complex of TopoCDNA, which introduces single and double strand breaks in the DNA [21], [24], [25]. Topoisomerase activity is influenced by several post-translational modifications, among them phosphorylation, poly-ADP-ribosylation, and ubiquitination. Recent work done in our laboratory demonstrated the O-GlcNAcylation of Topo IB, which affects its.
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