5-fluorouracil modulation of dihydrofolate reductase RNA levels in methotrexate-resistant KB cells

Cytotoxicity and growth inhibition by 5-fluorouracil in methotrexate-resistant dihydrofolate reductase gene-amplified KB cells in the presence of 30 microM thymidine correlates with incorporation of this fluorinated pyrimidine into RNA. Growth of these cells over several generations in the presence of inhibitory concentrations of 5-fluorouracil does not depress the steady state levels of either 18 or 28 S RNA but actually causes an increase. Similarly the rates of RNA and protein synthesis in 5-fluorouracil-treated cells are not decreased. The level of dihydrofolate reductase RNA from 5-fluorouracil-treated cells increases in a dose-dependent manner correlated with 5-fluorouracil incorporation into RNA. The qualitative size distribution of the dihydrofolate reductase RNA species is unaffected when examined by the Northern blotting technique indicating an RNA processing lesion is not induced by 5-fluorouracil incorporation into RNA. As the dose of dihydrofolate reductase RNA increases, there is no change in the level of dihydrofolate reductase specific activity, but the level of enzyme activity per cell increases. The relevance of these phenomena to the mechanism of 5-fluorouracil effect on RNA and relevance to combination chemotherapy with methotrexate are discussed.     

5-Fluorouracil inhibits dihydrofolate reductase precursor mRNA processing and/or nuclear mRNA stability in methotrexate-resistant KB cells

This laboratory previously reported that 5-fluorouracil (FUra) increases dihydrofolate reductase (DHFR) precursor mRNA (pre-mRNA) levels relative to DHFR mRNA levels in a methotrexate-resistant KB cell line; these data suggested that incorporation of FUra into RNA may, in part, lead to cell death through the inhibition of mRNA processing (Will, C. L., and Dolnick, B.J. (1987) J. Biol. Chem. 262, 5433-5436). Utilizing a methotrexate-resistant KB cell line designated 1BT, we now report the kinetic basis for altered levels of DHFR RNA observed in FUra-treated cells. Long-term exposure to FUra had no effect on the steady-state level of DHFR pre-mRNA containing intron V or I. However, steady-state levels of total DHFR mRNA decreased 2.0-fold on a per cell basis in cells exposed to 1.0 microM FUra. No significant change in the half-life of total DHFR mRNA or pre-mRNA was observed in cells exposed to FUra (t1/2 = approximately 11.5 h and 50 min, respectively). Nuclear/cytoplasmic RNA labeling experiments demonstrated that the rate of nuclear DHFR RNA conversion to cytoplasmic DHFR mRNA decreased approximately 1.8-fold in FUra-treated cells. These results provide further evidence the FUra may inhibit processing of mRNA precursors and/or affect the stability of nuclear DHFR mRNA.     

Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells.

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levls of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W.F., Davidson, S.V., and Siminovitch, L. (1976) Somatic Cell Genet.2,245-261). By selecting for increased resistance form these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In the report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.     

Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells.

We isolated overlapping recombinant cosmids that represent the equivalent of two complete dihydrofolate reductase amplicon types from the methotrexate-resistant CHO cell line CHOC400. The type I amplicons are 260 kilobases long, are arranged in head-to-tail fashion, and represent 10 to 15% of the amplicons in the CHOC400 genome. The type II amplicons are 220 kilobases long, are arranged in head-to-head and tail-to-tail configurations, and constituted the majority of the remaining amplicons in CHOC400 cells. The type II amplicon sequences are represented entirely within the type I unit. These are the first complete amplicons to be cloned from a mammalian cell line.     

Reduction of oxidised folates by dihydrofolate reductase from methotrexate-resistant Lactobacillus casei.

The use of alternative substrates by dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was investigated as a possible mechanism for the resistance of Lactobacillus casei to the cytotoxic drug methotrexate. The reduction of folic acid and 10-formylfolic acid by homogeneous enzyme was compared to that of the normal substrate, dihydrofolic acid. The three substrates have different pH optima and Km values. In addition, it was found that the reduction of 10-formylfolic acid was markedly stimulated by the presence of ions. Although the reduction was sensitive to methotrexate in all cases, the ion activation may be of importance in partially inhibited systems.     

Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts.

As a model cell culture system for studying polyoma-mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble portein, corresponding to an increase of approximately 100-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA. In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a 2-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin E1, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.     

Overproduction of dihydrofolate reductase and gene amplification in methotrexate-resistant Chinese hamster ovary cells.

Stable isolates of Chinese hamster ovary cells that are highly resistant to methotrexate have been selected in a multistep selection process. Quantitative immunoprecipitations have indicated that these isolates synthesize dihydrofolate reductase at an elevated rate over its synthesis in sensitive cells. Restriction enzyme and Southern blot analyses with a murine reductase cDNA probe indicate that the highly resistant isolates contain amplifications of the dihydrofolate reductase gene number. Depending upon the parenteral line used to select these resistant cells, they overproduce either a wild-type enzyme or a structurally altered enzyme. Karyotype analysis shows that some of these isolates contain chromosomes with homogeneously staining regions whereas others do not contain such chromosomes.     

Transfection of DHFR- and DHFR+ mammalian cells using methotrexate-resistant mutants of mouse dihydrofolate reductase

Site-directed mutagenesis was used to generate mutants of mouse dihydrofolate reductase more resistant to methotrexate than the wild type enzyme. The mutant genes were used to transfect either DHFR- or DHFR+ cell lines. These mutants, as well as the wild type gene, were able to confer methotrexate resistance to DHFR- CHO cells. The number of selected colonies decreased with increased concentrations of methotrexate. The number of colonies observed at 10 microM methotrexate is correlated with the Ki(MTX) of the enzyme: the higher the Ki, the higher the number of colonies for the corresponding mutant. In contrast, the transfection of DHFR+ cells gave a few numbers of colonies not different for the wild type and the mutants.     

Dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli: dihydrofolate monooxygenase activity

Dihydrofolate reductase from strain MB 1428 of Escherichia coli was shown to catalyze the oxidative cleavage of dihydrofolate at the C(9)N(10) bond. One of the products of the reaction was identified as 7,8-dihydropterin-6-carboxaldehyde through its proton magnetic resonance spectrum. The maximal enzymatic rate was 0.05 moles dihydrofolate cleaved per minute per mole enzyme at 25° and pH 7.2, and the K_M for dihydrofolate was 17.5 ± 2.5 μM. The enzymatic reaction was fully inhibitable with methotrexate. The mechanism of enzyme action was proposed to be an apparent “acidification” of dihydrofolate upon binding to the enzyme. Folate underwent an analogous oxidative cleavage by enzyme with a turnover number of 0.0014, which produced pterin-6-carboxaldehyde. Methotrexate was also slowly degraded by the enzyme.     

Acute effect of 5-fluorouracil on cytoplasmic and nuclear dihydrofolate reductase messenger RNA metabolism

Studies were completed in C3-L5178Y cells, in which the DNA-coding region for dihydrofolate reductase messenger RNA (DHFR-mRNA) is amplified, to determine the acute effect of 5-fluorouracil (FUra) on DHFR-mRNA metabolism. There was minimal to no effect of 100 microM FUra on total cytoplasmic DHFR-mRNA levels by 6 and 12 h and only a 25% reduction by 24 h. These results contrasted with the nuclear DHFR-mRNA levels which by 6 h following exposure to FUra increased by 80% in a dose-dependent manner. Furthermore, some of the increased nuclear DHFR-mRNA was found to be in a non-polyadenylated form. Under conditions to examine only RNA synthesized during the drug exposure, FUra was found to markedly enhance the level of newly synthesized nuclear DHFR-mRNA in a dose-dependent manner, while also producing an apparent dose-dependent reduction in the cytoplasmic DHFR-mRNA. RNA fractionated by 1.5% agarose-urea gel electrophoresis revealed two major cytoplasmic DHFR-mRNA species approximately 1.8 and 0.8 kilobases in size. Following a 24-h FUra exposure, a dose-dependent loss of the 0.8-kilobase DHFR-mRNA was observed. The combined results of these experiments indicate that FUra treatment reduces the ability of nascent DHFR-mRNA to relocate to the cytoplasm, suggesting either an inhibition of mRNA processing or nuclear-cytoplasmic transport.     

Nucleotide sequence of the dihydrofolate reductase gene of methotrexate-resistant Lactobacillus casei

The nucleotide sequence of the dihydrofolate reductase (DHFR) gene of a methotrexate-resistant strain of Lactobacillus casei, which is the source of DHFR for nuclear magnetic resonance (NMR) studies, has been determined. The derived amino acid sequence differs from that obtained by protein sequencing by the presence of aspartic acid instead of asparagine at position 8 and proline instead of leucine at position 90. The nucleotide sequences of 320-bp 5' and 335-bp 3' flanking regions of this gene have also been determined.