Inhibition of glutaminase expression increases Sp1 phosphorylation and Sp1/Sp3 transcriptional activity in Ehrlich tumor cells.
Tumor cells expressing antisense glutaminase RNA show a drastic inhibition of glutaminase activity and they acquire a more differentiated phenotype. We have studied the expression of Sp1 and Sp3 transcription factors in both Ehrlich tumor cells and their derivative 0.28AS-2 antisense glutaminase expressing cells. The expression of phosphorylated Sp1 in 0.28AS-2 cells was 3-fold the expression in EATC. Full length Sp3 was also incremented in 0.28AS-2 cells. Sp1 and Sp3 binding to a consensus Sp1 probe was higher in 0.28AS-2 nuclear extracts, as determined by supershift assays. Sp1-DNA binding was inhibited by phosphatase treatment, demonstrating that phosphorylation of Sp1 is critical for its DNA binding capacity. The Sp1 and Sp3 DNA binding found in 0.28AS-2 cells was also correlated with an increased Sp1 activity, as shown in transient transfections assays carried out with a luciferase reporter plasmid. Incubation of Ehrlich tumor cells with the differentiation agent PMA could not totally reproduce the Sp1/Sp3 changes observed in 0.28AS-2 cells. However, it was demonstrated that the intracellular concentration of glutamine, but not glutamate or aspartate, is increased in 0.28AS-2 cells. In conclusion, the antisense inhibition of glutaminase leads to an increased expression of phosphorylated Sp1 and that correlates with an increase in Sp1 activity.
Segura
Juan Antonio
JA
Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Campus de Teatinos, Universidad de Málaga, 29071 Málaga, Spain. jsegura@uma.es
Donadio
Ana Carolina
AC
Lobo
Carolina
C
Matés
José Manuel
JM
Márquez
Javier
J
Alonso
Francisco José
FJ
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000235:genetics* / Q000473:pathology*; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D005972:Glutaminase / Q000096:biosynthesis*; D010766:Phosphorylation; D016372:RNA, Antisense; D016329:Sp1 Transcription Factor / Q000096:biosynthesis* / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000096:biosynthesis* / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D016372:RNA, Antisense; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D005972:Glutaminase