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Ectopic expression of human topoisomerase IIα fragments and etoposide resistance in mammalian cells

✍ Scribed by Angelika I. Ernst; Alex Soltermann; Jürg A. Sigrist; Lukas Widmer; Susan M. Gasser; Rolf A. Stahel

Publisher
John Wiley and Sons
Year
2000
Tongue
French
Weight
596 KB
Volume
88
Category
Article
ISSN
0020-7136

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✦ Synopsis

Cellular resistance to etoposide has been correlated both with reduced levels and an aberrant cytoplasmic accumulation of the drug's target, topoisomerase II␣ (topo II␣). It is not known, however, whether a cytoplasmic pool of topo II␣ 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 II␣ fused to GFP and have examined the transformants for the subcellular localization of topo II␣ and their resistance to etoposide. Transient transfection resulted in high-level expression of all GFP-topo II␣ fusions tested, whereas in stably transfected cells the levels varied significantly. Transfectants expressing a central or a carboxy-terminal topo II␣ domain (aa 428-1504, 639-1028 or 1028-1504) accumulated high levels of the fusion proteins, while only very low amounts of GFP-topo II␣ 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 II␣ 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 II␣ also failed to confer drug resistance. These results suggest that a cytoplasmic pool of topo II␣ is not sufficient to render cultured mammalian cells drug resistant.

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