Identification of genes downregulated in tumor cells expressing antisense glutaminase mRNA by differential display.
Ehrlich ascites tumor cells (EATC) is a highly proliferative malignant cell line derived from mouse mammary epithelia, whereas their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, show a less transformed phenotype and loss of their tumorigenic capacity in vivo correlated with an inhibition of glutaminase expression. The mRNA differential display technique was applied to these two cell lines for the identification and isolation of genes whose transcription was altered. Side-by-side comparisons of cDNA patterns among relevant RNA samples revealed four genes significantly downregulated in 0.28AS-2 cells: high-mobility group Hmga2 protein, Fmnl3 or formin-like protein 3, Nedd-4 ubiquitin-protein ligase, and ubiquitin carboxyl-terminal hydrolase Usp-15. These positives were confirmed by Northern analysis. The four targeted genes have relevant functions in cell growth and proliferation. Our results show the validity of mRNA differential display technique to get insights into the molecular mechanisms underlying the acquisition of a more differentiated phenotype by tumor cells after inhibition of glutaminase expression.
Martín-Rufián
Mercedes
M
Departamento de Biología Molecular y Bioquímica, Laboratorio de Química de Proteínas, Facultad de Ciencias, Universidad de Málaga, Málaga, Spain.
Segura
Juan A
JA
Lobo
Carolina
C
Matés
José M
JM
Márquez
Javier
J
Alonso
Francisco J
FJ
D000818:Animals; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D018076:DNA, Complementary / Q000235:genetics; D015536:Down-Regulation; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005972:Glutaminase / Q000235:genetics / Q000502:physiology*; D051379:Mice; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats
D018076:DNA, Complementary; D016372:RNA, Antisense; D012333:RNA, Messenger; D005972:Glutaminase