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Toxins vapC and pasB from prokaryotic TA modules remain active in mammalian cancer cells.

Wieteska Ł, Skulimowski A, Cybula M, Szemraj J - Toxins (Basel) (2014)

Bottom Line: However, research concerning the adoption of toxins from these systems shows their great potential as a tool for molecular biology and medicine.As for the toxin PasB, observed changes were more subtle than for the VapC.The level of expression for both the genes was monitored by QPCR and did not reveal statistically significant differences within the same cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Medical University of Lodz, ul. Mazowiecka 6/8, 92-215 Lodz, Poland. l.wieteska@gmail.com.

ABSTRACT
Among the great number of addictive modules which have been discovered, only a few have been characterized. However, research concerning the adoption of toxins from these systems shows their great potential as a tool for molecular biology and medicine. In our study, we tested two different toxins derived from class II addictive modules, pasAB from plasmid pTF-FC2 (Thiobacillus ferrooxidans) and vapBC 2829Rv (Mycobacterium tuberculosis), in terms of their usefulness as growth inhibitors of human cancer cell lines, namely KYSE 30, MCF-7 and HCT 116. Transfection of the pasB and vapC genes into the cells was conducted with the use of two different expression systems. Cellular effects, such as apoptosis, necrosis and changes in the cell cycle, were tested by applying flow cytometry with immunofluorescence staining. Our findings demonstrated that toxins VapC and PasB demonstrate proapoptotic activity in the human cancer cells, regardless of the expression system used. As for the toxin PasB, observed changes were more subtle than for the VapC. The level of expression for both the genes was monitored by QPCR and did not reveal statistically significant differences within the same cell line.

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Cell cycle analysis of cells transfected with constructs: pEGFP-PasB, pEGFP-VapC and control pEGFP. Histograms show propidium iodide signal strength (A) and population range (B) taken into analysis. Visible increase of Sub-G1 population, mostly for cells transfected with pEGFP-VapC. (C) histograms showing number (%) of cells in Sub-G1 phase (visible as light green on plots in section (C)).
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toxins-06-02948-f005: Cell cycle analysis of cells transfected with constructs: pEGFP-PasB, pEGFP-VapC and control pEGFP. Histograms show propidium iodide signal strength (A) and population range (B) taken into analysis. Visible increase of Sub-G1 population, mostly for cells transfected with pEGFP-VapC. (C) histograms showing number (%) of cells in Sub-G1 phase (visible as light green on plots in section (C)).

Mentions: Due to the fact that both PasB and VapC act as specific RNAses, their expression should not have affect the cell cycle. In addition, the sub-G1 population responsible for apoptotic cells was expected to be expanded. To confirm that hypothesis, we analyzed the cell cycle of KYSE 30 and MCF-7 cell lines 72 h after transfection with pEGFP-PasB, pEGFP-VapC and pEGFP as the control vector (Figure 5). After transfection with pEGFP-VapC, the sub G1 population increased remarkably for KYSE 30 and MCF-7 cells (p < 0.002 and p < 0.0001 respectively). There was also a statistically significant increase in the sub-G1 population for MCF-7 cells transfected with pEGFP-PasB (p < 0.02). That effect, however, was approximately two times weaker compared to pEGFP-VapC and not detectable in the KYSE 30 cell line. Among the tested lines, KYSE 30 appeared to be the most susceptible to VapC expression. That line behaved most stably and the reproducible results were easier to obtain. In similarly designed research, susceptibility variations among cell lines were also observed [28]. As for the apoptosis induction potency of the PasB from plasmid pTF-FC2, according to our results, two out of three experiments performed produced a statistically significant drop in viable cells and apoptosis induction, but induced changes were more subtle than those observed for VapC. Importantly, changes in the potency were not caused by the different levels of expression which were monitored with the QPCR method and did not reveal significant disparity. The RelE toxin cleaves ribosomal A-site of the mRNA in prokaryotes and eukaryotes and the mRNA cleavage pattern is conserved within family; however, the sequence alignment with PasB shows major changes within the active site of the toxin; thus, more research into PasB biology is needed. It is worth noticing that a toxin whose activity depends on the interaction not only with the mRNA but also with the ribosome, remains active in eukaryotic cells. This effect was also observed for the RelE toxin from Escherichia coli and it was demonstrated that it occurs by the identical mechanism of action [29]. Authors claim that this observation supports the view that the structural organization of the A site of the small ribosomal subunit is very similar in bacteria and mammals [29].


Toxins vapC and pasB from prokaryotic TA modules remain active in mammalian cancer cells.

Wieteska Ł, Skulimowski A, Cybula M, Szemraj J - Toxins (Basel) (2014)

Cell cycle analysis of cells transfected with constructs: pEGFP-PasB, pEGFP-VapC and control pEGFP. Histograms show propidium iodide signal strength (A) and population range (B) taken into analysis. Visible increase of Sub-G1 population, mostly for cells transfected with pEGFP-VapC. (C) histograms showing number (%) of cells in Sub-G1 phase (visible as light green on plots in section (C)).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4210878&req=5

toxins-06-02948-f005: Cell cycle analysis of cells transfected with constructs: pEGFP-PasB, pEGFP-VapC and control pEGFP. Histograms show propidium iodide signal strength (A) and population range (B) taken into analysis. Visible increase of Sub-G1 population, mostly for cells transfected with pEGFP-VapC. (C) histograms showing number (%) of cells in Sub-G1 phase (visible as light green on plots in section (C)).
Mentions: Due to the fact that both PasB and VapC act as specific RNAses, their expression should not have affect the cell cycle. In addition, the sub-G1 population responsible for apoptotic cells was expected to be expanded. To confirm that hypothesis, we analyzed the cell cycle of KYSE 30 and MCF-7 cell lines 72 h after transfection with pEGFP-PasB, pEGFP-VapC and pEGFP as the control vector (Figure 5). After transfection with pEGFP-VapC, the sub G1 population increased remarkably for KYSE 30 and MCF-7 cells (p < 0.002 and p < 0.0001 respectively). There was also a statistically significant increase in the sub-G1 population for MCF-7 cells transfected with pEGFP-PasB (p < 0.02). That effect, however, was approximately two times weaker compared to pEGFP-VapC and not detectable in the KYSE 30 cell line. Among the tested lines, KYSE 30 appeared to be the most susceptible to VapC expression. That line behaved most stably and the reproducible results were easier to obtain. In similarly designed research, susceptibility variations among cell lines were also observed [28]. As for the apoptosis induction potency of the PasB from plasmid pTF-FC2, according to our results, two out of three experiments performed produced a statistically significant drop in viable cells and apoptosis induction, but induced changes were more subtle than those observed for VapC. Importantly, changes in the potency were not caused by the different levels of expression which were monitored with the QPCR method and did not reveal significant disparity. The RelE toxin cleaves ribosomal A-site of the mRNA in prokaryotes and eukaryotes and the mRNA cleavage pattern is conserved within family; however, the sequence alignment with PasB shows major changes within the active site of the toxin; thus, more research into PasB biology is needed. It is worth noticing that a toxin whose activity depends on the interaction not only with the mRNA but also with the ribosome, remains active in eukaryotic cells. This effect was also observed for the RelE toxin from Escherichia coli and it was demonstrated that it occurs by the identical mechanism of action [29]. Authors claim that this observation supports the view that the structural organization of the A site of the small ribosomal subunit is very similar in bacteria and mammals [29].

Bottom Line: However, research concerning the adoption of toxins from these systems shows their great potential as a tool for molecular biology and medicine.As for the toxin PasB, observed changes were more subtle than for the VapC.The level of expression for both the genes was monitored by QPCR and did not reveal statistically significant differences within the same cell line.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Medical University of Lodz, ul. Mazowiecka 6/8, 92-215 Lodz, Poland. l.wieteska@gmail.com.

ABSTRACT
Among the great number of addictive modules which have been discovered, only a few have been characterized. However, research concerning the adoption of toxins from these systems shows their great potential as a tool for molecular biology and medicine. In our study, we tested two different toxins derived from class II addictive modules, pasAB from plasmid pTF-FC2 (Thiobacillus ferrooxidans) and vapBC 2829Rv (Mycobacterium tuberculosis), in terms of their usefulness as growth inhibitors of human cancer cell lines, namely KYSE 30, MCF-7 and HCT 116. Transfection of the pasB and vapC genes into the cells was conducted with the use of two different expression systems. Cellular effects, such as apoptosis, necrosis and changes in the cell cycle, were tested by applying flow cytometry with immunofluorescence staining. Our findings demonstrated that toxins VapC and PasB demonstrate proapoptotic activity in the human cancer cells, regardless of the expression system used. As for the toxin PasB, observed changes were more subtle than for the VapC. The level of expression for both the genes was monitored by QPCR and did not reveal statistically significant differences within the same cell line.

Show MeSH
Related in: MedlinePlus