Ectopic expression of human topoisomerase IIalpha fragments and etoposide resistance in mammalian cells.
Cellular resistance to etoposide has been correlated both with reduced levels and an aberrant cytoplasmic accumulation of the drug's target, topoisomerase IIalpha (topo IIalpha). It is not known, however, whether a cytoplasmic pool of topo IIalpha is sufficient to confer drug resistance on cultured mammalian cells. In our study, we have transfected mouse fibroblasts and human 293 cells with truncated forms of human topo IIalpha fused to GFP and have examined the transformants for the subcellular localization of topo IIalpha and their resistance to etoposide. Transient transfection resulted in high-level expression of all GFP-topo IIalpha fusions tested, whereas in stably transfected cells the levels varied significantly. Transfectants expressing a central or a carboxy-terminal topo IIalpha domain (aa 428-1504, 639-1028 or 1028-1504) accumulated high levels of the fusion proteins, while only very low amounts of GFP-topo IIalpha proteins were observed in cell lines expressing constructs that retain the amino-terminus of the enzyme (aa 1-1214, aa 1-939, aa 1-611). Our results suggest that the topo IIalpha amino-terminus affects the stability of truncated forms of the enzyme in mammalian cells, perhaps due to targeted degradation. Assays that screen for cell vitality and DNA synthesis reveal no significant changes in etoposide sensitivity in transfected cells expressing high levels of cytoplasmic or nuclear localized topo II fusion proteins. Retroviral expression of a cytoplasmically anchored domain of human topo IIalpha also failed to confer drug resistance. These results suggest that a cytoplasmic pool of topo IIalpha is not sufficient to render cultured mammalian cells drug resistant.
Ernst
A I
AI
Division of Oncology, Department of Internal Medicine, University Hospital, Zürich, Switzerland.
Soltermann
A
A
Sigrist
J A
JA
Widmer
L
L
Gasser
S M
SM
Stahel
R A
RA
D016475:3T3 Cells / Q000187:drug effects / Q000201:enzymology; D000818:Animals; D000951:Antigens, Neoplasm; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004250:DNA Topoisomerases, Type II / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins; D004351:Drug Resistance; D005047:Etoposide / Q000494:pharmacology*; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005047:Etoposide; D004250:DNA Topoisomerases, Type II