Inhibition of dihydrofolate reductase gene expression following serum withdrawal or db-cAMP addition in methotrexate-resistant mouse fibroblasts

We have used a methotrexate (MTX)-resistant mouse 3T6 cell line (M50L3), that overproduces dihydrofolate reductase (DHFR) and its mRNA by a factor of 300, to study the mechanism for turning off DHFR gene expression following withdrawal of serum factors or elevation of the intracellular level of cAMP. When resting (G0) M50L3 cells are serum-stimulated to reenter the cell cycle, the level of DHFR activity begins to increase at about the same time the cells begin synthesizing DNA. The increase in enzyme activity is preceded by increases in the synthesis rate of the enzyme, and the content and production rate of DHFR mRNA. These increases, as well as entry into S phase, are blocked when the cells are serum-stimulated in the presence of dibutyryl cyclic AMP (db-cAMP) and theophylline. In this study, we found that when these drugs were added, or the serum stimulus was withdrawn during S phase (20 h following stimulation), the subsequent increase in DHFR level was blocked. Immunoprecipitation of DHFR from pulse-labelled cells showed that both treatments led to a rapid decrease in synthesis rate of the enzyme. The effect on total protein synthesis was much less than on DHFR synthesis. In DNA-excess filter hybridization experiments, we found that the content of cytoplasmic DHFR mRNA decreased in parallel with the synthesis rate of the enzyme. This was due in part to a decrease in the production rate of DHFR mRNA relative to total mRNA. In addition, drug addition or serum withdrawal led to a significant destabilization of DHFR (as well as total) mRNA. About 85% of poly(A)(+) DHFR mRNA was associated with polysomes in resting, growing, or cAMP-treated cells, suggesting that DHFR gene expression was not controlled at the translational level under these conditions.     

Early changes in the synthesis of acidic nuclear proteins in human diploid fibroblasts stimulated to synthesize DNA by changing the medium

When resting WI-38 cells in a confluent monolayer were stimulated to proliferate by changing the medium, the incorporation of leucine-³H into nuclear acidic proteins was promptly stimulated, although its incorporation into total cellular proteins was unchanged or even decreased. Three fractions, all acidic by aminoacid analysis, were extracted from the nuclei: (1) ribonucleoproteins (RNP); (2) a fraction extractable with 0.15 M NaC1; and (3) a fraction tenaciously bound to the insoluble residue (residual fraction). A first increase occurred between one and three hours after stimulation in all three fractions. The synthesis of NaCl-soluble proteins then returned to control levels, while the synthesis of residual and RNP proteins remained high between 6 and 12 hours and increased even further at 18 hours, the peak of DNA synthesis. Pulse chase experiments indicated that the proteins synthesized in the first hour after stimulation have a turnover time of less than four hours, while the same fractions in non-proliferating cells were stable for at least 12 hours. 2-mercapto-1-(β-4-pyridethyl) benzimidazole, when added at the same time as the fresh medium, produced an inhibition of the increase in nuclear protein synthesis at one hour, but, if added at five hours after stimulation, it did not inhibit the increase in nuclear protein synthesis occurring at six hours. Actinomycin D (0.01 μg/ml) inhibited both the stimulation of DNA synthesis and the increases in nuclear acidic protein synthesis occurring at one and six hours after stimulation. These results seem to indicate that the serum factors responsible for the stimulation of WI-38 cells, after binding to cells, induce an early synthesis of acidic nuclear proteins which is sensitive to very low doses of actinomycin D. In turn, the newly synthesized proteins could in some way activate in the nuclei the genes that control DNA synthesis and cell division.     

Murine erythroleukemia cell variants: isolation of cells that have amplified the dihydrofolate reductase gene and retained the ability to be induced to differentiate

A series of murine erythroleukemia cell (MELC) variants was generated by selection for the ability to grow in increasing concentrations of the folate antagonist methotrexate (MTX). Growth of the parental MELC strain DS-19 was completely inhibited by 0.1 microM MTX. We isolated cells able to grow in 5, 40, 200, 400, and 800 microM MTX. Growth rates and yields were essentially the same in the presence or absence of the selective dose of MTX for all variants. MTX resistance was not the result of a transport defect. Dihydrofolate reductase (DHFR) from our variants and DS-19 was inhibited to the same extent by MTX. Variants had increased dihydrofolate reductase activities. The specific activity of DHFR was proportional to the selective concentration of MTX employed to isolate a given variant. DNA dot blotting established that the cloned variant (MR400-3) had a 160-fold increase in DHFR gene copy number relative to the parental strain (DS-19). Hybridization studies performed in situ established the presence of amplified DHFR genes on the chromosomes of the MTX-resistant but not the MTX-sensitive (parental) cells. Quantitation of DHFR mRNA by cytoplasmic dot blotting established that the amplified DHFR gene expression was proportional to gene copy number. Thus, MTX resistance was due to amplification of the DHFR gene. The variants retained the ability to be induced to differentiate in response to dimethyl sulfoxide and hexamethylenebis(acetamide) as evaluated by the criteria of globin mRNA accumulation, hemoglobin accumulation, cell volume decreases, and terminal cell division.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide changes in oxidatively stressed lymphocytes

Similar to HIV-1-induced suppression of thymus-derived lymphocytes (T cells), oxidatively stressed T cells show inhibited DNA synthesis and proliferation. The influence of oxidative stress on nucleotide pools was explored using ³H-uridine addition to OKT3-stimulated peripheral blood lymphocytes. The cells were preincubated and stimulated in the presence of 1 mM buthionine sulfoximine to inhibit GSH synthesis. This treatment gave rise to a significant reduction in dUDP and TTP biosynthesis following 18–32 hours stimulation, indicating possible impairment of ribonucleotide reductase activity.     

DNA synthesis in the nuclei of resting and proliferating cells of the NIH 3T3 line in monokaryons, homo- and heterodikaryons

Serum-deprived (0.5%) resting NIH 3T3 mouse fibroblasts were fused with stimulated cells taken at 2 hour intervals after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in monokaryons, homodikaryons, and heterodikaryous using radioautography with double-labeling technique. The presence of the resting nucleus in the common cytoplasm has an inhibitory effect on the entry of the stimulated nucleus into the S period in the medium containing either 0.5 or 10% serum, but DNA synthesis continues. After a 24 hour stay in the common cytoplasm with resting nuclei the stimulated nuclei return into the state of rest. When resting cells are stimulated by 10% serum, their inhibitory effect on stimulated nuclei in heterodikaryons still persists for at least 2 hours following stimulation. Preincubation of resting cells with cycloheximide for 4 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei. The data suggest that the resting cells produce an endogenous inhibitor of cell proliferation whose formation depends upon the synthesis of protein(s). When stimulated, cell can proliferate only upon decreasing the level of this inhibitor. The obtained results are consistent with the idea of a negative control of cell proliferation.     

Differences in the cytoplasmic distribution of newly synthesized poly (A) in serum-stimulated and resting cultures of BALB/c 3T3 cells.

Density-inhibited, serum-stimulated, and SV40 virus-transformed BALB/c 3T3 cultures were compared with respect to the rates of accumulation of cytoplasmic RNA molecules and with respect to the distribution of newly synthesized messenger RNA (mRNA) between polyribosomes and the post-ribosomal cell fraction. mRNA was isolated and quantitated by virtue of its association with radioactive polyadenylate (poly(A))-synthesized during a 90 min exposure of the cultures to ³H-adenosine. The rate of accumulation of cytoplasmic poly(A) rose slowly after serum stimulation and reached a value of 1.8 times that of resting cultures at 12 h after serum stimulation, which was also the time of onset of DNA synthesis. A change in the cytoplasmic distribution of newly synthesized poly(A) occurred more rapidly than the change in the rate of its synthesis, however. Resting cultures contained 37% of newly synthesized cytoplasmic poly(A)-containing RNA large enough to be mRNA in the post-ribosomal cell fraction, whereas virtually all of this material was found in polyribosomes at 3, 6 and 12 h after stimulation and in transformed cultures. The relatively infrequently translated mRNA of resting cultures was shown to be functional by cycloheximide treatment. (All BALB/c 3T3 cultures, resting or stimulated, contained about 20% of newly synthesized cytoplasmic poly(A) as nearly pure poly(A) in molecules of 4–6 Svedbergs in size, presumably too small to be mRNA.) We conclude that serum stimulation of density-inhibited cultures resulted in a more efficient use of the protein-synthesizing ability of the cell, and that the change in efficiency preceded increases in numbers of ribosomes and mRNA molecules.     

Plasmid amplification-promoting sequences from the origin region of Chinese hamster dihydrofolate reductase gene do not promote position-independent chromosomal gene amplification

Initiation of DNA synthesis occurs with high frequency at oriß, a region of DNA from the amplified dihydrofolate reductase (DHFR) domain of Chinese hamster CHOC 400 cells that contains an origin of bidirectional DNA replication (OBR). Recently, sequences from DHFR oriß/OBR were shown to stimulate amplification of cis-linked plasmid DNA when transfected into murine cells. To test the role of oriß/OBR in chromosomal gene amplification, linearized plasmids containing these sequences linked to a DHFR expression cassette were introduced into DHFR- CHO DUKX cells. After selection for expression of DHFR, cell lines that contain a single integrated, unrearranged copy of the linearized expression plasmid were identified and exposed to low levels of the folate analog, methotrexate (MTX). Of seven clonal cell lines containing the vector control, three gained resistance to MTX by 5 to 15-fold amplification of the integrated marker gene. Of 16 clonal cell lines that contained oriß/OBR linked to a DHFR mini-gene, only 6 gained resistance to MTX by gene amplification. Hence, sequences from the DHFR origin region that stimulate plasmid DNA amplification do not promote amplification of an integrated marker gene in all chromosomal contexts. In addition to showing that chromosomal position has a strong influence on the frequency of gene amplification, these studies suggest that the mechanism that mediates the experiment of episomal plasmid DNA does not contribute to the early steps of chromosomal gene amplification.     

The effect of folate and folate analogs upon dihydrofolate reductase and DNA synthesis in kidneys of normal mice

A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of ³H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity $\text{in}$$\text{vivo}$ were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity $\text{in}$$\text{vivo}$ was inhibited by 90%.³H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of ³H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase $\text{in}$$\text{vitro}$. Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner.The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA.     

BIOPTERIN V. DE NOVO SYNTHESIS OF DIHYDROBIOPTERIN: EVIDENCE FOR ITS QUINONOID STRUCTURE AND LACK OF DEPENDENCE OF ITS REDUCTION TO TETRAHYDROBIOPTERIN ON DIHYDROFOLATE REDUCTASE

Samples of quinonoid-l -erythrodihydrobiopterin (q-BH₂) and quinonoid-6-methyl-dihydro-pterin (q-6-MPH₂) were prepared by oxidation of l -erythro-5,6,7,8-tetrahydrobiopterin (BH₄) and 5,6,7,8-tetrahydro-6-methylpterin (6-MPH₄) and separated from ^D-erythro-7,8-dihydrobiopterin (7,8-BH₂) and 6-methyl-7,8-dihydropterin (7,8-6-MPH₂) as well as from the tetrahydropterins on phosphocellulose column by high-pressure liquid chromatography. The quinonoid dihydropterins were identified and quantitated by scan of their ultraviolet absorption and fluorescence emission spectra through their rearrangement to their 7,8-tautomer and also by gas chromatography of their rapidly synthesized trimethylsilyl derivative. Identification was also achieved by the enzymatic reduction of [³H]q-BH²to [³H]BH₄ by dihydrofolate reductase (DHFR). Direct proof for the enzymatic synthesis of the q-BH² from GTP or from 2-amino-6-(5′-triphosphoribosyl)-amino-5- or -6-formamido-6-hydroxypyrimi-dine (FPyd-P₃) was obtained by isolation of the compound which was identical in all respects to the q-BH₂ obtained by chemical synthesis from BH₄. The reduction of enzymatically synthesized q-BH² by dihydropteridine reductase (DHPR) to BH⁴ was not inhibited by methotrexate (MTX). When the enzymatically synthesized q-BH² was converted to 7,8-BH₂, it was reduced only by DHFR. This reduction, however, was inhibited by MTX. On the biosynthetic pathway from GTP to dihydrobiopterin, the enzyme responsible for the appearance of the quinonoid structure is the d -erythro-dihydroneopterin triphosphate synthetase, the product of which (quinonoid d -erythro-dihydroneopterin triphosphate) is converted to quinonoid dihydrobiopterin by l -erythro-dihydrobiopterin synthetase. Experiments in vivo established that DHFR does not participate in the reduction of dihydrobiopterin to tetra-hydrobiopterin when the former is synthesized from GTP de novo. MTX at 5 × 10^?6M exerted no inhibition on the reduction of the biosynthetic dihydrobiopterin to tetrahydrobiopterin in vivo, yet completely inhibited the reduction of intraventricularly injected tritiated dihydrofolate ([³H]FH₂) to tritiated tetrahydrofolate ([³H]FH₄).     

Effect of cyclic AMP on chromatin-bound protein kinases in WI-38 fibroblasts stimulated to proliferate.

When resting confluent monolayers of WI-38 fibroblasts are stimulated to proliferate by serum, DNA synthesis begins to increase between 15-18 h after stimulation. Chromatin-bound protein kinase activity increases in stimulated cells within 1 h after the nutritional change, concomitant with an increase in the template activity of nuclear chromatin. Addition of dibutyryl 3' : 5'-cyclic adenosine monophosphate (dibutyryl cyclic) AMP to the stimulating medium inhibits the entrance of cells into S phase, but only if dibutyryl cyclic AMP (5-10(-4) M) is added before the onset of DNA synthesis. The increases in chromatin template activity and in the chromatin-bound kinase activity are not inhibited by dibutyryl cyclic AMP in the early hours after stimulation, but are completely inhibited after the 5th hour from the nutritional change. This seems to indicate that in stimulated WI-38 cells, dibutyryl cyclic AMP exerts its inhibitory action somewhere between 5 and 12 h after stimulation. A number of protein kinase activities were extracted from chromatin with 0.3 M NaCl and partially resolved on a phosphocellulose column. Two distinct peaks of protein kinase activity appeared to be markedly increased in WI-38 cells 6 h after serum stimulation. Both peaks of increased activity were inhibited by dibutyryl cyclic AMP in vivo. Adenosine, sodium butyrate and adenosine 5'-monophosphate (AMP) do not inhibit the increase in DNA synthesis nor the increase in protein kinase activity. The results suggest that stimulation of cell proliferation in confluent monolayers of WI-38 cells causes an increase (or the new appearance) of certain chromatin-bound protein kinases, and that this increase is inhibited by cyclic AMP in vivo.     

DNA synthesis in the nuclei of stimulated NIH 3T3 cells in heterodikaryons obtained by the fusion of these cells with resting cells treated with cycloheximide

Serum-deprived (0.2%) resting NIH 3T3 mouse fibroblasts preincubated with cycloheximide (7.5 micrograms/ml) were fused with stimulated cells taken 10 hours after changing the medium to the one containing 10% serum, and DNA synthesis was investigated in nuclei of heterodikaryons, homodikaryons, and monokaryons, using radioautography with double-labeling technique. Preincubation of resting cells with the inhibitor of protein synthesis cycloheximide for 4, 3, 2, but not for 1 or 0.5 hours abolishes their ability to suppress DNA synthesis in stimulated nuclei in heterodikaryons. Three hours after the removal of cycloheximide from the media, the resting cells acquire once again the inhibitory effect towards the entry of stimulated nuclei into the S-period. The data suggest that the resting cells may produce a labile endogenous inhibitor of cell proliferation, and support the idea on the active metabolic processes occurring in the resting cells.     

Rapid increase in poly(A) (+) mRNA content following inhibition of protein synthesis

When resting 3T6 cells undergo a serum-induced transition to the growing state, the cytoplasmic content of ribosomal, transfer and messenger RNA increase as the cells prepare for DNA synthesis. The normal linear increase in mRNA content occurs even when the production of ribosomes is blocked. In this paper we determine the effect of inhibiting protein synthesis on the increase in poly(A) (+) mRNA content. Resting cells were serum stimulated in the presence of cycloheximide or puromycin at levels which inhibit protein synthesis by greater than 95%. Cytoplasmic poly(A) (+) mRNA content was determined at various times thereafter. We found that mRNA content increased five to ten times more rapidly in drug treated cells than in control cells stimulated in the absence of inhibitors. mRNA content increased 50–70% by one hour, and 60–90% by two hours following stimulation in the presence of inhibitor, and remained more or less constant thereafter. In contrast, mRNA content increased linearly in control stimulated cultures and did not double until about 15 hours after stimulation. The rapid increase in mRNA content is most likely the result of inhibition of protein synthesis rather than a secondary effect of the drug since the same observations were made in growth stimulated cells if protein synthesis was blocked with either puromycin or cycloheximide. A similar effect was also observed with resting 3T6, exponentially growing 3T6 and growing HeLa cells following exposure to cycloheximide, although the magnitude of the increase was less than that observed with growth stimulated cells. Puromycin had negligible effect on mRNA content in resting or exponentially growing cells. The rapid increase in cytoplasmic poly(A) (+) mRNA content was not due to rapid unbalanced export of nuclear poly(A) (+) RNA into the cytoplasm since there was no decrease in nuclear poly(A) content following serum stimulation in the presence of cycloheximide.     

Rapid changes in poly (A)(+) mRNA content in growth stimulated fibroblasts following perturbations in protein synthesis.

We have previously shown that when resting 3T6 cells are serum stimulated in the presence of inhibitors of protein synthesis, poly(A)(+) mRNA content increases extremely rapidly relative to cells stimulated in the absence of drug. Poly(A)(+) mRNA content nearly doubles within two hours, but then remains constant for at least ten hours (Johnson and Meister, '77). In this report we show that continuous exposure to both serum and cycloheximide are required to maintain this elevated mRNA level. Removal of either leads to an equally rapid decrease in poly(A)(+) mRNA content. If cycloheximide is withdrawn at either two or ten hours following serum stimulation in the presence of the drug, allowing the rapid (< 30 minutes) restoration of the rate of protein synthesis, we observe that poly(A)(+) mRNA content decreases within two hours to a level nearly equal to that found in resting cells prior to stimulation. If the drug is withdrawn but the serum stimulus is not, the rapid decrease in poly(A)(+) mRNA content is followed by an increase which is parallel to that which occurs in cultures stimulated in the absence of drug, but displaced from the latter by an interval approximately equal to the length of exposure of the drug. These results show that the mammalian cell is able to decrease as well as increase its content of poly(A)(+) mRNA in response to drug induced perturbations in the rate of protein synthesis. The changes in poly(A)(+) mRNA content occur extremely rapidly and may represent an attempt by the cell to correct the perturbation.     

Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth-stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5-fluorodeoxyuridine (5-FdUrd)-resistant cell line (LU3-7) that overproduces TS and its mRNA about 50-100-fold. In this paper we show that the LU3-7 cells were able to rest in the G0 state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10%, the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulse-labeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8-9-fold by 25 hr following serum stimulation. The half-life of TS in growing LU3-7 cells was measured in a pulse-chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3-7 cells and in the parental 3T6 cells, the LU3-7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.     

Ionic changes associated with lead stimulation of DNA synthesis in Balb/c3T3 cells

Lead at slightly subtoxic concentrations markedly stimulated the rate of DNA synthesis in cultured animal cells. This stimulation was closely correlated with formation of a precipitate that was adsorbed and taken up by the cells under certain medium conditions. Data suggest that a precipitate-induced perturbation of the surface membrane leads to intracellular changes responsible for stimulation of DNA synthesis. Maximum stimulation of³H-thymidine incorporation by optimum concentrations of lead is delayed about 8 h compared to that in serum stimulation. In cells stimulated significantly by lead, but not in unstimu-lated cells, a reproducible rise of about 13% in intracellular magnesium occurred over a 24 h period, with an 8 h lag in the increase compared to that observed in serum stimulation. In view of the increases in intracellular magnesium consistently associated with and preceding stimulation of DNA synthesis by several different mitogens including serum and insulin, the present time-coordinated positive correlation between magnesium and DNA synthesis provides evidence for the primary involvement of this divalent cation in growth stimulation produced by lead.