Identification of functional genes during Fas-mediated apoptosis using a randomly fragmented cDNA library.
We describe a general strategy for the identification of functional genes that, when downregulated, result in a selectable phenotype. This strategy is based on expression selection of cDNA fragments that counteract their cognate genes. A cDNA library containing random fragments expressed in human HepG2, A375 and CLS-354 cells was used to identify functional genes whose inhibition conferred resistance to Fas-induced apoptosis. Thirty-five clones were isolated, 28 of which were derived from unknown genes, that tagged 19 individual genes and 7 of which referred to known genes that tagged the apoptosis-related protein (APR)-1, -2 and indoleamine-pyrrole 2,3,-dioxygenase (IDO). The ability of APR-1-, -2- and IDO-derived antisense RNAs to induce resistance to Fas in HepG2, A375 and CLS-354 cells suggested that APR-1, -2 and IDO genes are involved in the machinery of Fas-mediated apoptosis. Our gene discovery strategy provides a generally applicable procedure to identify functional genes that interfere with apoptosis, and may therefore be clinically relevant for tumor therapy.
Hassan
M
M
Institute of Pathology, Heinrich-Heine-University, Moorenstr. 5, 40225, Düsseldorf, Germany.
Mirmohammadsadegh
A
A
Selimovic
D
D
Nambiar
S
S
Tannapfel
A
A
Hengge
U R
UR
D000906:Antibodies / Q000494:pharmacology; D017209:Apoptosis / Q000235:genetics*; D002478:Cells, Cultured; D015870:Gene Expression; D015723:Gene Library; D006801:Humans; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D007371:Interferon-gamma / Q000494:pharmacology; D010641:Phenotype; D016372:RNA, Antisense / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014366:Tryptophan Oxygenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000187:drug effects / Q000276:immunology
D000906:Antibodies; D050503:Indoleamine-Pyrrole 2,3,-Dioxygenase; D016372:RNA, Antisense; D019014:fas Receptor; D007371:Interferon-gamma; D014366:Tryptophan Oxygenase