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Characterization of ectonucleotidases in human medulloblastoma cell lines: ecto-5'NT/CD73 in metastasis as potential prognostic factor.

Cappellari AR, Rockenbach L, Dietrich F, Clarimundo V, Glaser T, Braganhol E, Abujamra AL, Roesler R, Ulrich H, Battastini AM, Battastini AM - PLoS ONE (2012)

Bottom Line: On the other hand, D283 cell line did not hydrolyze AMP.It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity.In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas e da Saúde, UFRGS, Porto Alegre, Rio Grande do Sol, Brasil.

ABSTRACT
Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

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Analysis of ectonucleotidases expression in MB cell lines.Total RNA isolated from cell cultures. RT-PCR and real time-PCR reactions were performed as described in Materials and Methods. (A) RT-PCR analysis of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA (B) Quantitative analysis of the relative expression of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA in MB cell lines were performed by real time-PCR where GAPDH expression was used as internal control for normalization of expression levels. Data were analyzed by the One-Way ANOVA test followed by the Tukey post hoc test. The experiments were performed three times in triplicate with (* p<0,01) and (*** p<0.001) indicating statistical relevant difference compared to control data.
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pone-0047468-g002: Analysis of ectonucleotidases expression in MB cell lines.Total RNA isolated from cell cultures. RT-PCR and real time-PCR reactions were performed as described in Materials and Methods. (A) RT-PCR analysis of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA (B) Quantitative analysis of the relative expression of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA in MB cell lines were performed by real time-PCR where GAPDH expression was used as internal control for normalization of expression levels. Data were analyzed by the One-Way ANOVA test followed by the Tukey post hoc test. The experiments were performed three times in triplicate with (* p<0,01) and (*** p<0.001) indicating statistical relevant difference compared to control data.

Mentions: ATP transduces a variety of signals into adjacent cells, and these effects are controlled by ectonucleotidase activities [15]. Considering that MB cell lines secrete ATP into the extracellular medium, mRNA transcription of ectonucleotidases in these cell lines was verified by RT-PCR (Figure 2A) and real time-PCR (Figure 2B). We observed that all MB cell lines expressed only NTPDase5 mRNA, but NTPDase1, 2 and 3 gene expression could not be detected (Figure 2A). Real time PCR analysis showed prominent expression of NTPDase5 in D283 cells when compared to other cell lines (Figure 2B). Furthermore, the D283 cell line revealed E-NPP1, 2 and 3 amplicons with expected sizes; real-time PCR confirmed that these expression profiles were significantly higher than those in other MB cell lines. The Daoy and ONS76 MB cell lines expressed all three types of E-NPPs, but in very low proportions, not differing significantly between them.


Characterization of ectonucleotidases in human medulloblastoma cell lines: ecto-5'NT/CD73 in metastasis as potential prognostic factor.

Cappellari AR, Rockenbach L, Dietrich F, Clarimundo V, Glaser T, Braganhol E, Abujamra AL, Roesler R, Ulrich H, Battastini AM, Battastini AM - PLoS ONE (2012)

Analysis of ectonucleotidases expression in MB cell lines.Total RNA isolated from cell cultures. RT-PCR and real time-PCR reactions were performed as described in Materials and Methods. (A) RT-PCR analysis of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA (B) Quantitative analysis of the relative expression of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA in MB cell lines were performed by real time-PCR where GAPDH expression was used as internal control for normalization of expression levels. Data were analyzed by the One-Way ANOVA test followed by the Tukey post hoc test. The experiments were performed three times in triplicate with (* p<0,01) and (*** p<0.001) indicating statistical relevant difference compared to control data.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475694&req=5

pone-0047468-g002: Analysis of ectonucleotidases expression in MB cell lines.Total RNA isolated from cell cultures. RT-PCR and real time-PCR reactions were performed as described in Materials and Methods. (A) RT-PCR analysis of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA (B) Quantitative analysis of the relative expression of E-NPPs, NTPDases, Ecto-5′-NT/CD73, ALP and ADA in MB cell lines were performed by real time-PCR where GAPDH expression was used as internal control for normalization of expression levels. Data were analyzed by the One-Way ANOVA test followed by the Tukey post hoc test. The experiments were performed three times in triplicate with (* p<0,01) and (*** p<0.001) indicating statistical relevant difference compared to control data.
Mentions: ATP transduces a variety of signals into adjacent cells, and these effects are controlled by ectonucleotidase activities [15]. Considering that MB cell lines secrete ATP into the extracellular medium, mRNA transcription of ectonucleotidases in these cell lines was verified by RT-PCR (Figure 2A) and real time-PCR (Figure 2B). We observed that all MB cell lines expressed only NTPDase5 mRNA, but NTPDase1, 2 and 3 gene expression could not be detected (Figure 2A). Real time PCR analysis showed prominent expression of NTPDase5 in D283 cells when compared to other cell lines (Figure 2B). Furthermore, the D283 cell line revealed E-NPP1, 2 and 3 amplicons with expected sizes; real-time PCR confirmed that these expression profiles were significantly higher than those in other MB cell lines. The Daoy and ONS76 MB cell lines expressed all three types of E-NPPs, but in very low proportions, not differing significantly between them.

Bottom Line: On the other hand, D283 cell line did not hydrolyze AMP.It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity.In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Ciências Básicas e da Saúde, UFRGS, Porto Alegre, Rio Grande do Sol, Brasil.

ABSTRACT
Medulloblastoma (MB) is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy) and inosine accumulation (ONS76). On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

Show MeSH
Related in: MedlinePlus