摘要：

Stable variants resistant to pyrazofurin (PF) and 6-azauridine (AZUrd) were serially selected in increasing drug concentrations from an MC3T3-E1 nontumorigenic murine osteoblastic cell line. Monophosphates of both AZUrd and PF competitively inhibit orotidine-5′-monophosphate decarboxylase (ODCase) activity of the UMP synthase multifunctional enzyme. When compared to the wild type cells, the AZUrd r and PF r lines were 3000- and 10 000-fold more resistant, respectively. Flow cytometry indicated tetraploidy in wild type cells and a reduction of DNA content in both resistant cell lines. DNA dot blot analysis showed no amplification of the gene coding for UMP synthase in either AZUrd r or PF r cells. Measurements of UMP synthase showed a 6-fold higher activity in AZUrd r cells and no significant difference in PF r cells as compared to wild type. Sensitivity to 5-fluorouracil was increased in the AZUrd r line as opposed to PF r and normal cell lines, indicating an increased orotate phosphoribosyltransferase activity in the AZUrd r cells. In comparison to wild type cells, PF r cells were 100-fold resistant to 6-methylmercaptopurine riboside, suggesting a lack of adenosine kinase activity. The control and AZUrd r cells showed equal sensitivity to 5-fluorouridine, thus indicating unchanged uridine kinase levels. While PF r cells were not cross-resistant to AZUrd, the AZUrd r cells were cross-resistant to PF. These results indicate the possibility of an altered ODCase active site. Although amplification of unrelated sequences cannot be excluded, our findings show that bone tetraploid, nontumorigenic cells acquire drug resistance through mechanisms other than the amplification of a target gene and that this resistance is accompanied by the partial loss of a chromosomal complement.

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