Differential expression of nucleotide pyrophosphatase/phosphodiesterases by Walker 256 mammary cancer cells in solid tumors and malignant ascites.
OBJECTIVE
Expression of ectoenzymes responsible for nucleotide phosphohydrolysis to form adenosine may represent a mechanism that facilitates the proliferation and spread of malignancy. In this study, we have identified and characterized the ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) family members expressed during the subcutaneous tumor growth and in the ascitic form of Walker 256 mammary tumor cells.


METHODS
The biochemical characteristics in ascitic forms and expression of NPP 1, 2, and 3 in both solid and ascitic forms of Walker 256 tumor were investigated using RT-PCR and real-time PCR.


RESULTS
Walker 256 tumor cells demonstrate E-NPP activities that are associated with extracellular hydrolysis of p-Nph-5'-TMP, and define the biochemical characteristics. The K(m) and maximal velocity for the hydrolysis of p-Nph-5'-TMP in the ascitic tumor cells were in accordance with the NPP reaction. The mRNA expression in the cells of the ascitic form of Walker 256 tumor revealed transcripts for NPP2 and NPP3, whereas elevated expression of NPP3 was observed in solid tumor, after 6, 10, and 15days of inoculation. The dominant gene expressed in both forms of the tumor was the NPP3 enzyme. However, this enzyme was expressed more during tumor development in vivo, when compared with the ascitic cells.


CONCLUSIONS
We have previously demonstrated that Walker 256 tumor cells express mRNA for ecto-5'-nucleotidase and E-NTPDases. Thus, coexistence with NPP3 suggests an ectonucleotidase "enzyme chain" that is responsible for the sequential hydrolysis of ATP to adenosine, which may be an important therapeutic target in anticancer therapy.
Buffon
Andréia
A
Departamento de Análises, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil. andreia.buffon@ufrgs.br
Casali
Emerson A
EA
Cardoso
Valesca V
VV
Zerbini
Luiz F
LF
Robson
Simon C
SC
Sarkis
João J F
JJ
Wink
Márcia R
MR
D000818:Animals; D001201:Ascites / Q000201:enzymology; D002279:Carcinoma 256, Walker / Q000201:enzymology*; D002412:Cations / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology*; D007700:Kinetics; D009363:Neoplasm Proteins / Q000378:metabolism; D009368:Neoplasm Transplantation; D009705:Nucleosides / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D011755:Pyrophosphatases / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
D002412:Cations; D009363:Neoplasm Proteins; D009705:Nucleosides; D009711:Nucleotides; D012333:RNA, Messenger; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases