Design of artificial transcription factors to selectively regulate the pro-apoptotic bax gene.
The tumor suppressor p53 is the most commonly mutated gene in human cancers. Active p53 is able to stimulate the transcription of a variety of genes including the pro-apoptotic gene bax, as well as p21, a cell cycle regulator. In this study we produced novel zinc finger transcription factors that would selectively increase the expression of bax, but not of other p53 targets. Reporter gene assays in p53-negative Saos-2 cells showed that the novel zinc finger proteins stimulated transcription driven by a minimal bax promoter, but not that driven by a minimal p21 promoter. Moreover, electromobility shift assays demonstrated that the novel transcription factors could bind the bax promoter sequence with high affinity and selectivity. Expression of a five zinc finger protein (5ZFAV) in COS-7 cells resulted in an increase in Bax protein levels, indicating that this novel transcription factor could act on endogenous gene expression. Expression of 5ZFAV also drastically reduced Saos-2 cell survival; this effect could be reversed by the general caspase inhibitor B-D-FMK. These data suggest that 5ZFAV is able to induce apoptosis through increased Bax expression. Further, while expression of 5ZFAV in p53-deficient Saos-2 cells reduced cell survival, there was little effect on U-2 OS cells which have wild-type p53. Thus the selective induction of the pro-apoptotic bax gene may be a valuable adjunct to cancer chemotherapy by diminishing survival of p53-deficient tumor cells.
Falke
D
D
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA. dfalke@med.unc.edu
Fisher
M
M
Ye
D
D
Juliano
R L
RL
D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D020169:Caspases / Q000502:physiology; D006801:Humans; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D019253:Proto-Oncogene Proteins c-bcl-2; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000502:physiology; D016335:Zinc Fingers; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D020169:Caspases