Limits...
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.

Show MeSH

Related in: MedlinePlus

Basal nucleotide secretion by MB cell lines.MB cell lines were incubated for 90 minutes with incubation medium in the absence of substrate, as described in Materials and Methods. Extracellular nucleotide/nucleoside concentrations (ATP, ADP, AMP, ADO and INO) were measured by HPLC and concentrations were expressed as µmol product/mg protein (mean±S.D.). Experiments were performed three times in triplicate and the data obtained were analyzed for statistical relevance using a Two-Way ANOVA test followed by the Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475694&req=5

pone-0047468-g001: Basal nucleotide secretion by MB cell lines.MB cell lines were incubated for 90 minutes with incubation medium in the absence of substrate, as described in Materials and Methods. Extracellular nucleotide/nucleoside concentrations (ATP, ADP, AMP, ADO and INO) were measured by HPLC and concentrations were expressed as µmol product/mg protein (mean±S.D.). Experiments were performed three times in triplicate and the data obtained were analyzed for statistical relevance using a Two-Way ANOVA test followed by the Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.

Mentions: MB cells were cultured and used for determination of adenine nucleotide/nucleoside secretion. All three analyzed cell lines predominantly secrete ATP to the extracellular medium (Figure 1). The Daoy cell line presented a higher secretion (100.04 µMol ±14.99) than the ONS76 and D283 MB cell lines (55.61 µMol ±0.145 and 26.48 µMol ±10.54, respectively). It is important to note that the three MB cell lines secreted ADP and ADO to the extracellular medium, although in much lower concentrations than secreted ATP. Differences in secretion between MB cell lines were not statistically significant. The detected INO probably resulted the deamination of ADO by ADA present in these cells.


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)

Basal nucleotide secretion by MB cell lines.MB cell lines were incubated for 90 minutes with incubation medium in the absence of substrate, as described in Materials and Methods. Extracellular nucleotide/nucleoside concentrations (ATP, ADP, AMP, ADO and INO) were measured by HPLC and concentrations were expressed as µmol product/mg protein (mean±S.D.). Experiments were performed three times in triplicate and the data obtained were analyzed for statistical relevance using a Two-Way ANOVA test followed by the Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475694&req=5

pone-0047468-g001: Basal nucleotide secretion by MB cell lines.MB cell lines were incubated for 90 minutes with incubation medium in the absence of substrate, as described in Materials and Methods. Extracellular nucleotide/nucleoside concentrations (ATP, ADP, AMP, ADO and INO) were measured by HPLC and concentrations were expressed as µmol product/mg protein (mean±S.D.). Experiments were performed three times in triplicate and the data obtained were analyzed for statistical relevance using a Two-Way ANOVA test followed by the Bonferroni post hoc test. (***) p<0.001 and (*) p<0.05 compared to control data.
Mentions: MB cells were cultured and used for determination of adenine nucleotide/nucleoside secretion. All three analyzed cell lines predominantly secrete ATP to the extracellular medium (Figure 1). The Daoy cell line presented a higher secretion (100.04 µMol ±14.99) than the ONS76 and D283 MB cell lines (55.61 µMol ±0.145 and 26.48 µMol ±10.54, respectively). It is important to note that the three MB cell lines secreted ADP and ADO to the extracellular medium, although in much lower concentrations than secreted ATP. Differences in secretion between MB cell lines were not statistically significant. The detected INO probably resulted the deamination of ADO by ADA present in these cells.

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