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Thymidylate Synthetase Overproduction in 5-Fluorodeoxyuridine-Resistant Mouse Fibroblasts CINDY ROSSANA, LAKSHMI GOLLAKOTA RAO, and LEE F

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Thymidylate Synthetase Overproduction in 5-Fluorodeoxyuridine-Resistant Mouse Fibroblasts CINDY ROSSANA, LAKSHMI GOLLAKOTA RAO, and LEE F MOLECULAR AND CELLULAR BIOLOGY, Sept. 1982, p. 1118-1125 Vol. 2, No. 9 0270-7306/82/091118-08$02.00/0 Copyright 0 1982, American Society for Microbiology Thymidylate Synthetase Overproduction in 5-Fluorodeoxyuridine-Resistant Mouse Fibroblasts CINDY ROSSANA, LAKSHMI GOLLAKOTA RAO, AND LEE F. JOHNSON* Department ofBiochemistry, The Ohio State University, Columbus, Ohio 43210 Received 9 February 1982/Accepted 29 April 1982 We describe the isolation and characterization of a series of 5-fluorodeoxyuri- dine (FdUrd)-resistant mouse 3T6 cell lines that overproduce thymidylate synthe- tase (TS) by up to 50-fold compared with the parental cells. The resistant cells were selected by growing 3T6 cells or a methotrexate-resistant 3T6 cell line (M50L3, isolated previously in our laboratory) in gradua$ increasing concentra- tions of FdUrd. Uridine and cytidine were included in the culture medium to reduce toxicity from metabolic products of FdUrd. Cells that were resistant to the drug by virtue of loss of thymidine kinase activity were eliminated by selection in medium containing hypoxanthine, methotrexate, and thymidine. M5OL3 cells were found to adapt to FdUrd more readily than 3T6 cells. A number of clones were isolated that were able to grow in the presence of 3 ,uM (M50L3 derived) or 0.3 ,uM (3T6 derived) FdUrd. Several were found to overproduce TS by 10 to 50- fold compared with normal 3T6 cells. All were found to have thymidine kinase activity, although the enzyme level was significantly reduced in some clones. The overproduced TS was inactivated by 5-fluorodeoxyuridylic acid at the same concentration as the enzyme from 3T6 cells. TS was purified from the LU3-7 clone (50-fold overproducer) by affinity chromatography on methotrexate-poly- acrylamide. The monomer molecular weight was about 38,000, which was the same as the molecular weight of the monomer in 3T6 cells. The overproduction trait was gradually lost (half-life, 3 weeks) when LU3-7 cells were grown in the absence of FdUrd. The overproducing cells will provide an abundant supply ofTS and (very likely) its mRNA and may serve as a convenient model system for detailed studies of the regulation of TS gene expression during the cell cycle. Thymidylate synthetase (TS) (thymidylate Proliferating cells are able to develop resist- synthase, EC 2.1.1.45) is the enzyme that cata- ance to FdUrd by a number of mechanisms. If lyzes the final reaction in the de novo synthesis cells growing in FdUrd are also provided with of thymidylic acid. This reaction involves the thymidine, they are able to synthesize TMP by reductive transfer of the methylene group from using the salvage enzyme TK (which is not N5,N"0-methylene tetrahydrofolate to the 5 po- inhibited by the drug). In the absence of thymi- sition of deoxyuridylic acid to form thymidylic dine, cells have been found to develop resist- acid (reviewed in reference 32). TS is inhibited ance to FdUrd by losing the ability to convert it in a stoichiometric manner by the substrate to FdUMP as a result of a loss of TK activity (4, analog 5-fluorodeoxyuridylic acid (FdUMP), 30). Cells might also be able to develop resist- which forms a covalent linkage with the enzyme ance by decreasing the rate of transport of the at the active site (27). The enzyme can be analog into the cell, by increasing the rate of inactivated in vivo by exposing cells to the catabolism of the drug, or by synthesizing an nucleoside form of the inhibitor, 5-fluorodeoxy- altered TS with a decreased affinity for FdUMP. uridine (FdUrd), which is transported into the Another possible mechanism for the develop- cell and then converted to FdUMP by the en- ment of resistance to FdUrd is the overproduc- zyme thymidine kinase (TK) (11). Inhibition of tion of TS. Previous studies have shown that TS activity in rapidly proliferating cells leads to cells are able to develop resistance to other starvation for thymidylic acid, inhibition of specific enzyme inhibitors by overproducing the DNA synthesis, and cell death. For this reason, target enzymes. For example, cells are able to FdUrd or similar analogs have been widely used develop resistance to high concentrations of as antineoplastic drugs (reviewed in references 6 methotrexate (MTX) or N-phosphonacetyl-L-as- and 8). partate by overproducing the enzymes dihydro- 1118 VOL. 2, 1982 THYMIDYLATE SYNTHETASE OVERPRODUCTION 1119 folate reductase (DHFR) (2, 10) or aspartate saline solution and stained with hematoxylin. The transcarbamylase (16), respectively. The mecha- number of clones per dish was normalized to the nism for overproduction of these enzymes has number of clones that were observed when the cells been studied extensively (reviewed in reference were plated in the same medium without FdUrd to give the relative cloning efficiency. Each clone con- 28). tained at least 30, and usually several hundred cells. In this paper we show that cultured mouse Determination of TS, DHFR, and TK activity. Cells 3T6 fibroblasts are able to develop resistance to were grown in the absence of FdUrd for 10 to 13 days high FdUrd concentrations by overproducing before enzyme assay. TS activity was determined by TS by up to 50-fold over the level found in the procedure of Roberts (24) as described previously normal 3T6 cells. The overproduced enzyme is (21). This procedure measures the release of inactivated by FdUMP at the same concentra- [3H]water from [5-3H]dUMP during the formation of tion as the enzyme in 3T6 cells and appears to TMP. DHFR activity was determined by the have the same molecular weight as that enzyme. [3H]MTX-binding assay (13, 14). TK activity was assayed by the procedure of Ives et al. (12) as de- scnrbed previously (13a). Enzyme activities were nor- MATERIALS AND METHODS malized to the amount of protein in the extract (20) to give the specific activity. Development of FdUrd-redstant cell lines. Cultures Purification of TS. MTX-polyacrylamide was pre- of cells were maintained in plastic petri dishes in the pared according to the procedure of Newbold and Dulbecco-Vogt modification of Eagle medium Harding (22). TS was purified from roller-bottle cul- (GIBCO) supplemented with 10%o dialyzed calf serum tures of LU3-7 cells by a modification of the proce- (Colorado Serum). A line of cells resistant to 0.3 ,uM dures of Dolnick and Cheng (9) and Rode et al. (26). FdUrd was developed from mouse 3T6 fibroblasts (29) Briefly, cell extracts were subjected to streptomycin in the following manner. Cells were plated in medium sulfate fractionation, ammonium sulfate precipitation, supplemented with 0.06 F.M FdUrd, 1 mM uridine, and and finally affinity chromatography on MTX- 1 mM cytidine. After the surviving cells had adapted polyacrylamide. TS was bound to the matrix, washed to the drug and were growing rapidly (which took extensively with buffers containing dUMP, and then several weeks), they were grown in a modification of eluted from the column with buffer lacking dUMP. The HAT medium (10 ,uM hypoxanthine, 16 ,uM thymi- fractions with the highest TS activity were pooled, and dine, 10 F.M MTX) to select for TK+ cells (18). The the purity of the enzyme was determined by sodium HAT-selected cells were grown in HT medium (10 ,uM dodecyl sulfate (SDS)-polyacrylamide gel electropho- hypoxanthine, 16 ,uM thymidine) for several cell dou- resis according to the procedure of Laemmli (17). blings to allow them to recover from the toxic effects TS-FdUMP complex formation. A modification of of MTX. The cells were then adapted to gradually the procedure of Lockshin et al. (19) was used to form increasing concentrations of FdUrd (0.1, 0.15, 0.2, and the covalent complex between TS and [3H]FdUMP (18 finally 0.3 ,uM) in medium containing 1 mM uridine Ci/mmol, Moravek Biochemicals). The amount of and 1 mM cytidine and then subjected to HAT selec- complex formed was determined by measuring radio- tion again. The surviving cells were plated at low activity in the trichloroacetic acid-insoluble fraction. density in medium lacking drug, and a series of clones The molecular weight of the complex was determined were picked with stainless steel cloning rings. Clones by electrophoresis on an SDS-7.5% polyacrylamide that grew well in medium containing 0.3 pLM FdUrd, 1 slab gel according to the procedure of Laemmli (17). mM uridine, and 1 mM cytidine and that had high Protein bands were visualized by staining with Coo- levels of TS activity were studied further. massie brilliant blue. Radioactive bands were detected In a similar manner, a line of cells resistant to 3 F.M by fluorography with En3Hance (New England Nuclear FdUrd was developed from M50L3 cells, a line of 3T6 Corp.) by a procedure described by the manufacturer. cells resistant to 50 ,uM MTX, which we isolated The impregnated gel was dried on Whatman no. 1 filter previously (33). M50L3 cells were grown in the ab- and sence of MTX for 2 weeks and then exposed to paper exposed to X-ray film at -80°C. medium supplemented with 0.04 ,uM FdUrd, 1 mM uridine, and 1 mM cytidine. After the cells had adapt- RESULTS ed to the drug level, the concentration of FdUrd was increased, and the process was repeated. The cells Isolation of FdUrd-resistant cells. Cells resis- were adapted to FdUrd at the following concentra- tant to high concentrations of FdUrd were iso- tions: 0.08, 0.2, 0.5, 1, 2, and finally 3 pLM FdUrd, in lated by serial selection in gradually increasing medium containing 1 mM uridine and 1 mM cytidine. concentrations of the drug. Uridine and cytidine Cells that had adapted to 3 ,uM FdUrd were selected in were included in the selection media to prevent HAT medium containing 300 ,uM MTX and then toxicity from possible metabolic products of cloned in medium containing 3 ,uM FdUrd, 1 mM FdUrd.
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