Phenotypic resistance to methotrexate and N-phosphonacetyl L-aspartate is induced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA)
✍ Scribed by Anne R. Kinsella; Margaret Fox
- Publisher
- John Wiley and Sons
- Year
- 1988
- Tongue
- French
- Weight
- 763 KB
- Volume
- 42
- Category
- Article
- ISSN
- 0020-7136
No coin nor oath required. For personal study only.
✦ Synopsis
Three different 3T6 mouse fibroblast cell clones with in-1984) a single dose of MTX was used, whereas in the expertrinsically different sensitivities to methotrexate (MTX) have iments described by others ; Barsoum and been isolated from an OriginallY heterogeneous PoPulation- ) cells were exposed to several equal doses When these 3 different clones were exposed to M T X in the during the selection period by renewing the drug-containing of methotrexate resistant (MTXR) colonies was greatest in the most sensitive clone. M ~x ~ colonies isolated and cultured erations, thus raising the possibility that amplification was unin the presence and absence of M T X and TPA were analysed stable and had been lost prior to analysis. for dihydrofolic acid reductase (dhfr) levels by flow cytometry In the present study we have measured dhfr content by use after binding of fluorescent methotrexate. None Of the 58 of fluorescent MTX, dhfr COPY number in DNA, and levels of clones showed major changes in dhfr levels. Dot-blot analysis dhfr m~~~ in putative MTXR clones isolated after singleor mRNA levels consistent with gene amplification. Southern analysis of 6 further clones indicated that only I clone isolated and absence Of MTX TPA* In addition, since we susby multi-step selection had amplified dhfr sequences. TPA-pected that the cells may not amplify. readily at the dhfr !oCUS, enhanced mouse 3T6 N-phorphonacetyl-L-apartate (PALA)-we analysed PALAR Clones for amphficatlon Of the multlfuncresistant colony recovery and mouse 3T3 MTXR colony recov-tional CAD gene. ery were also shown, by dot-blot analysis, not to be due to gene amplification. The data indicate that TPA can have a profound effect on drug-resistant colony recovery by mechanirms other than induction of gene amplification. Presence of the tllmour promoter 12-Otetradecanoylphor-medium at 3-to 4&y intervals. Also, we routinely cultured bo1-13-acetate (TpA) the degree Of enhancement Of putative MTXR cells in the absence of MTX for several gen-Of I2 clones indicated no increaser in dhfr gene copy number and multi-dose selection protocols and cultured in the presence