Expression of the Drosophila melanogaster S3 ribosomal/repair protein in T24 human bladder cells.
Repair of 8-oxo-7,8-dihydroguanine (8-oxoG) is inefficient in human cells due to the poor catalytic properties of hOGG1, the major DNA glycosylase involved in the removal of this oxidized base. The S3 ribosomal/repair protein from Drosophila melanogaster (dS3) is endowed with a potent 8-oxoG glycolytic activity coupled with a beta, delta-AP lyase. In vitro repair experiments have shown that pure GST-tagged dS3 can stimulate a > 40-fold increase in the rate of 8-oxoG repair by human cell extracts. In this study, we expressed dS3 fused to the Enhanced Green Fluorescent Protein (EGFP) in T24 human bladder cells in order to accelerate the repair of 8-oxoG in vivo. Limiting dilution and Fluorescence-Activated Cell Sorting (FACS) were used in an effort to isolate cells with elevated EGFP-dS3 expression; however, the cells that were isolated invariably had severe growth impairment. Curiously, EGFP-dS3 expression was slightly increased after recovering cells from liquid nitrogen, but it was not possible under those conditions to achieve a significant acceleration of 8-oxoG repair. The data confirm and extend our previous results obtained with Chinese hamster CHO cells and indicate that elevated expression of dS3 may be toxic to at least some types of mammalian cells, thus limiting its use in vivo as a protective factor against oxidative DNA damage.
Ropolo
Monica
M
Mutagenesis Laboratory, Istituto Nazionale Ricerca Cancro, Largo Rosanna Benzi n. 10, 16132 Genova, Italy.
Geroldi
Alessandro
A
Rossi
Ottavio
O
Degan
Paolo
P
Zupo
Simona
S
Poggi
Alessandro
A
Frosina
Guido
G
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D045744:Cell Line, Tumor; D004260:DNA Repair / Q000502:physiology*; D029721:Drosophila Proteins / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012269:Ribosomal Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D001749:Urinary Bladder Neoplasms / Q000235:genetics / Q000378:metabolism*
D029721:Drosophila Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C485192:green fluorescent protein, Aequorea victoria; C044152:ribosomal protein S3; D049452:Green Fluorescent Proteins; C024829:8-hydroxyguanine; D006147:Guanine