A novel method for the determination of basal gene expression of tissue-specific promoters: an analysis of prostate-specific promoters.
Because the toxicity of suicide gene therapeutics is directly related to basal promoter activity, we developed an assay to test for promoter "leakiness" using a diphtheria toxin mutant. Sequences of 15 prostate-specific gene promoter constructs were cloned in an expression plasmid (pBK; Stratagene, La Jolla, CA) backbone driving expression of an attenuated mutant of diphtheria toxin A (tox176). Low expression levels of the DT-tox176 result in significant protein synthesis inhibition reflected by a decreased expression of the luciferase activity of a simultaneously transfected CMV luciferase construct. ID50 (dose of plasmid with 50% luciferase inhibition) was calculated for each promoter construct in different cell lines. Highest transactivational activity (ID50 <75 ng) was found for the CMV promoter in all cell lines, which is in agreement with the dual luciferase assay findings. Unlike the dual luciferase findings, however, the DT-tox176 assay showed protein inhibition of CN65 (PSA promoter/enhancer) and PSE-hK2 (PSA enhancer and basal human kallikrein 2 promoter) in HEK293 and DLD cells indicating "leakiness" of these promoter constructs. Low basal promoter activity in nonprostate cell lines was found for the minimal PSA promoter, hK2, DD3, and OC promoters. The DT-tox176 assay can better predict basal promoter activity compared to less sensitive dual luciferase assay.
van der Poel
H G
HG
Brady Urologic Institute, Johns Hopkins Medical School, Baltimore, Maryland 21287, USA.
McCadden
J
J
Verhaegh
G W
GW
Kruszewski
M
M
Ferrer
F
F
Schalken
J A
JA
Carducci
M
M
Rodriguez
R
R
D001681:Biological Assay / Q000379:methods*; D004167:Diphtheria Toxin; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008297:Male; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011467:Prostate; D011471:Prostatic Neoplasms / Q000235:genetics / Q000628:therapy; D014407:Tumor Cells, Cultured
D004167:Diphtheria Toxin