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Effects of partial silencing of genes coding for enzymes involved in glycolysis and tricarboxylic acid cycle on the enterance of human fibroblasts to the S phase.

Konieczna A, Szczepańska A, Sawiuk K, Węgrzyn G, Łyżeń R - BMC Cell Biol. (2015)

Bottom Line: We found that silencing of certain genes resulted in either less efficient or delayed enterance to the S phase.Decreased levels of expression of HK2, GADPH, CS1, ACO2, FH and MDH2 caused also a substantial impairment in DNA synthesis efficiency.The presented results illustrate the complexity of the influence of genes coding for enzymes of glycolysis and the tricarboxylic acid cycle on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308, Gdańsk, Poland. a.konieczna@biol.ug.edu.pl.

ABSTRACT

Background: Previously published reports indicated that some enzymes of the central carbon metabolism (CCM), particularly those involved in glycolysis and the tricarboxylic acid cycle, may contribute to regulation of DNA replication. However, vast majority of such works was performed with the use of cancer cells, in the light of carcinogenesis. On the other hand, recent experiments conducted on bacterial models provided evidence for the direct genetic link between CCM and DNA replication. Therefore, we asked if silencing of genes coding for glycolytic and/or Krebs cycle enzymes may affect the control of DNA replication in normal human fibroblasts.

Results: Particular genes coding for these enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of certain genes resulted in either less efficient or delayed enterance to the S phase. This concerned following genes: HK2, PFKM, TPI, GAPDH, ENO1, LDHA, CS1, ACO2, SUCLG2, SDHA, FH and MDH2. Decreased levels of expression of HK2, GADPH, CS1, ACO2, FH and MDH2 caused also a substantial impairment in DNA synthesis efficiency.

Conclusions: The presented results illustrate the complexity of the influence of genes coding for enzymes of glycolysis and the tricarboxylic acid cycle on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process.

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Related in: MedlinePlus

Effects of siRNA-mediated silencing of glycolityc genes on enterance of cells in S phase. Cells were seeded on Petri dishes, transfected with siRNA specific for indicated gene (□) and synchronized. Analogous experiments without siRNA were treated as controls (■). After cell cycle releasing, the cells were collected every two hours, starting from 14 h, and analyzed by flow cytometry. Presented results are mean values from at least three independent experiments, with error bars indicating SD. Statistically significant differences relative to the control are indicated by asterisks
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Fig3: Effects of siRNA-mediated silencing of glycolityc genes on enterance of cells in S phase. Cells were seeded on Petri dishes, transfected with siRNA specific for indicated gene (□) and synchronized. Analogous experiments without siRNA were treated as controls (■). After cell cycle releasing, the cells were collected every two hours, starting from 14 h, and analyzed by flow cytometry. Presented results are mean values from at least three independent experiments, with error bars indicating SD. Statistically significant differences relative to the control are indicated by asterisks

Mentions: The time and efficiency of the enterance of the cells to the S phase following silencing of expression of particular genes were estimated. Two types of effects were observed in cells treated with siRNAs impairing expression of some genes, less efficient or delayed enterance to S phase. When genes coding for enzymes catalyzing reactions of glycolysis were silenced, the less efficient enterance in the S phase, as measured by the percentage of cells in this phase, was observed for fibroblasts with impaired expression of HK2, PFKM, TPI, GAPDH and LDHA, with the most pronounced effect in the case of GAPDH (Fig. 3). Delayed enterance in the S phase, with a similar fraction of cells entering this phase, was observed in fibroblasts with silenced the ENO1 gene (Fig. 3). Analysis of other phases of the cell cycle under these conditions is presented as additional data [Additional file 1 and 2].Fig. 3


Effects of partial silencing of genes coding for enzymes involved in glycolysis and tricarboxylic acid cycle on the enterance of human fibroblasts to the S phase.

Konieczna A, Szczepańska A, Sawiuk K, Węgrzyn G, Łyżeń R - BMC Cell Biol. (2015)

Effects of siRNA-mediated silencing of glycolityc genes on enterance of cells in S phase. Cells were seeded on Petri dishes, transfected with siRNA specific for indicated gene (□) and synchronized. Analogous experiments without siRNA were treated as controls (■). After cell cycle releasing, the cells were collected every two hours, starting from 14 h, and analyzed by flow cytometry. Presented results are mean values from at least three independent experiments, with error bars indicating SD. Statistically significant differences relative to the control are indicated by asterisks
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4446904&req=5

Fig3: Effects of siRNA-mediated silencing of glycolityc genes on enterance of cells in S phase. Cells were seeded on Petri dishes, transfected with siRNA specific for indicated gene (□) and synchronized. Analogous experiments without siRNA were treated as controls (■). After cell cycle releasing, the cells were collected every two hours, starting from 14 h, and analyzed by flow cytometry. Presented results are mean values from at least three independent experiments, with error bars indicating SD. Statistically significant differences relative to the control are indicated by asterisks
Mentions: The time and efficiency of the enterance of the cells to the S phase following silencing of expression of particular genes were estimated. Two types of effects were observed in cells treated with siRNAs impairing expression of some genes, less efficient or delayed enterance to S phase. When genes coding for enzymes catalyzing reactions of glycolysis were silenced, the less efficient enterance in the S phase, as measured by the percentage of cells in this phase, was observed for fibroblasts with impaired expression of HK2, PFKM, TPI, GAPDH and LDHA, with the most pronounced effect in the case of GAPDH (Fig. 3). Delayed enterance in the S phase, with a similar fraction of cells entering this phase, was observed in fibroblasts with silenced the ENO1 gene (Fig. 3). Analysis of other phases of the cell cycle under these conditions is presented as additional data [Additional file 1 and 2].Fig. 3

Bottom Line: We found that silencing of certain genes resulted in either less efficient or delayed enterance to the S phase.Decreased levels of expression of HK2, GADPH, CS1, ACO2, FH and MDH2 caused also a substantial impairment in DNA synthesis efficiency.The presented results illustrate the complexity of the influence of genes coding for enzymes of glycolysis and the tricarboxylic acid cycle on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308, Gdańsk, Poland. a.konieczna@biol.ug.edu.pl.

ABSTRACT

Background: Previously published reports indicated that some enzymes of the central carbon metabolism (CCM), particularly those involved in glycolysis and the tricarboxylic acid cycle, may contribute to regulation of DNA replication. However, vast majority of such works was performed with the use of cancer cells, in the light of carcinogenesis. On the other hand, recent experiments conducted on bacterial models provided evidence for the direct genetic link between CCM and DNA replication. Therefore, we asked if silencing of genes coding for glycolytic and/or Krebs cycle enzymes may affect the control of DNA replication in normal human fibroblasts.

Results: Particular genes coding for these enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of certain genes resulted in either less efficient or delayed enterance to the S phase. This concerned following genes: HK2, PFKM, TPI, GAPDH, ENO1, LDHA, CS1, ACO2, SUCLG2, SDHA, FH and MDH2. Decreased levels of expression of HK2, GADPH, CS1, ACO2, FH and MDH2 caused also a substantial impairment in DNA synthesis efficiency.

Conclusions: The presented results illustrate the complexity of the influence of genes coding for enzymes of glycolysis and the tricarboxylic acid cycle on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process.

Show MeSH
Related in: MedlinePlus