Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0038187 (starvation)
 24,951 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.
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PMID:Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene. 302 46

A clinically isolated strain of Escherichia coli, resistant to more than 1000 mg/l of trimethoprim, expressed chromosomal dihydrofolate reductase to a level 200-fold higher than that of drug sensitive E. coli K-12 strains, and this high cellular enzyme activity was found to increase further when the cells were cultured in the presence of trimethoprim. The induced increase in enzyme activity was dependent on the drug concentration. The increase was six-fold at 100 mg/l of trimethoprim. The aberrantly regulated dihydrofolate reductase gene mediating trimethoprim resistance could be transduced into E. coli K-12 or moved by recombination into an F' factor and then transferred into trans position in relation to the corresponding chromosomal gene. In either of these positions, the synthesis of dihydrofolate reductase could be induced to increase by adding trimethoprim to the culture medium. The observed induction was dependent on protein synthesis, since it could be abolished by chloramphenicol. No other folic acid analogue was found to induce increased expression of the dihydrofolate reductase gene. Also thymine starvation had no effect. Two further clinical isolates of E. coli, highly resistant to trimethoprim, were shown to produce drug resistant, plasmid-mediated dihydrofolate reductases, which were distinct from the earlier known enzyme types I and II.
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PMID:New observations regarding evolution of trimethoprim resistance. 380 98

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.
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PMID:Relationship between the expression of estrogen-regulated genes and estrogen-stimulated proliferation of MCF-7 mammary tumor cells. 398 99

We have studied the metabolism of dihydrofolate reductase (DHFR) RNA in cells synchronized in the G1 phase of the cell cycle by starvation for isoleucine and glutamine. The relative content and stability of DHFR mRNA and the relative rate of transcription of the DHFR gene are similar in starved and exponentially growing cells. However, the relative rate of labeling of DHFR mRNA is about three times lower in starved cells than in exponentially growing cells. When the starved cells are stimulated to reenter the cell cycle by feeding them with complete medium, the relative rate of labeling of DHFR mRNA increases about fourfold within 6 h. However, the relative rate of transcription of the DHFR gene changes very little during this period. Continuous labeling experiments show that starved cells convert DHFR heterogeneous nuclear RNA into cytoplasmic DHFR mRNA much more slowly than serum-limited or exponentially growing cells. Pulse-chase experiments show that DHFR mRNA sequences contained in DHFR heterogeneous nuclear RNA appear to be conserved in starved cells. In addition, the content of DHFR RNA sequences in the nuclei of starved cells is about three times greater than that in exponentially growing cells. Delayed processing of DHFR heterogeneous nuclear RNA is also observed when exponentially growing cells are treated with inhibitors of protein synthesis. Our results suggest that, although delayed processing leads to a decrease in the initial labeling rate of DHFR mRNA, it does not result in a decrease in the actual rate of production of the message.
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PMID:Delayed processing of dihydrofolate reductase heterogeneous nuclear RNA in amino acid-starved mouse fibroblasts. 664 25

Thymidine auxotrophic mutants were selectively isolated from mutagenized mouse FM3A cells by resistance to methotrexate in the presence of thymidine and 5-methyl-tetrahydrofolate with a frequency of 10(-5)-10(-6). In most of the thymidine auxotrophs the activity of thymidylate synthase was very low or undetectable, but dihydrofolate reductase activity was normal. Upon starvation of thymidine, the mutant cells immediately stopped growing and started to lyse within one day. In the presence of thymidine, the mutant cells grew quite normally. This phenotype behaved recessively in cell-cell hybrids, and the segregation profile of its marker indicated that the lesions in the mutants are not linked to the X chromosome. Prototrophic revertants could be isolated from these mutants, and they showed almost the normal level of thymidylate synthase activity. The selection method described here should be useful for isolating large numbers of thymidylate synthase-negative mutants from various mammalian cell lines.
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PMID:Selection of mammalian thymidine auxotrophic cell mutants defective in thymidylate synthase by their reduced sensitivity to methotrexate. 729 55

Hyperproduction of the type IV plasmid-encoded dihydrofolate reductase was studied in Escherichia coli J62-2 (pUK1123). Hyperproduction of the enzyme was shown to occur not simply as a response to a given concentration of trimethoprim but also to the presence of thymidine in the medium. Before hyperproduction occurred the bacteria began to elongate and die, thus showing the symptoms of thymine starvation. Hyperproduction also required the presence of L-methionine, adenine and glycine, suggesting that the elevated production of the enzyme was a response to the ability of trimethoprim to starve the cell of thymine metabolites.
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PMID:The role of thymine starvation in the expression of type IV plasmid-encoded trimethoprim-resistant dihydrofolate reductase. 847 16

Cells often acquire resistance to the antiproliferative agents methotrexate (MTX) or N-phosphonacetyl-L-aspartate (PALA) through amplification of genes encoding the target enzymes dihydrofolate reductase or carbamylphosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD), respectively. We showed previously that Syrian hamster BHK cells resistant to selective concentrations of PALA (approximately 3 x ID50) arise at a rate of approximately 10(-4) per cell per generation and contain amplifications of the CAD gene as ladder-like structures on one of the two B9 chromosomes, where CAD is normally located. We now find that BHK cells resistant to high concentrations of PALA (approximately 15 x ID50) appear only after prior exposure to selective concentrations of PALA for approximately 72 h. Furthermore, in contrast to untreated cells, BHK cells pretreated with selective concentrations of MTX give colonies in high concentrations of PALA, and cells pretreated with selective concentrations of PALA give colonies in high concentrations of MTX or 5-fluorouracil. As judged by measuring numbers of cells and metaphase cell pairs, BHK cells do not arrest completely when starved for pyrimidine nucleotides by treatment with selective concentrations of PALA for up to 72 h. We propose that DNA damage, caused when cells fail to stop DNA synthesis promptly under conditions of dNTP starvation, stimulates amplification throughout the genome by mechanisms--such as bridge-breakage-fusion cycles--that are triggered by broken DNA. Amplified CAD genes were analyzed by fluorescence in situ hybridization both in cells where amplification was induced by PALA pretreatment and in cells in which the amplification occurred spontaneously, before selection with PALA. The ladder-like structures that result from bridge-breakage-fusion cycles were observed in both cases.
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PMID:Inefficient growth arrest in response to dNTP starvation stimulates gene amplification through bridge-breakage-fusion cycles. 886 64

To understand the regulation and expression of pyrimidine biosynthesis in plants, we have examined the effect of the metabolic inhibitor 5-fluoroorotic acid (FOA) on uridine-5'-monophosphate synthase (UMPSase) expression in cell cultures of Nicotiana plumbaginifolia. UMPSase is the rate-limiting step of pyrimidine biosynthesis in plants. Addition of FOA causes an up-regulation of UMPSase enzyme activity in cell cultures after a lag phase of several days. Western-blot analysis demonstrated that the up-regulation in enzyme activity was caused by increased expression of the UMPSase protein. Northern-blot analysis demonstrated a higher level of UMPSase mRNA in the FOA-induced tissues than in control tissues. Run-on transcriptional assays showed that the UMPSase gene was transcriptionally activated after FOA treatment. The mechanism of toxicity of FOA is through thymine starvation. We found that addition of thymine abrogated the FOA-mediated up-regulation of UMPSase. In addition, methotrexate and aminopterin, which affect thymine levels by inhibiting dihydrofolate reductase, also up-regulate UMPSase in N. plumbaginifolia cells.
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PMID:Uridine 5'-Monophosphate Synthase Is Transcriptionally Regulated by Pyrimidine Levels in Nicotiana plumbaginifolia 949 Jul 73

Preimplantation mammalian embryos develop with a high degree of autonomy. To date, there have been no unequivocal demonstrations of a requirement for vitamins in preimplantation embryo development. Reduced folic acid acts as an important methyl donor in many reactions including the synthesis of thymidine. Thymidine does not accumulate in cells so it might be expected that significant amounts of reduced folate would be required to support the exponential increase in DNA synthesis that occurs during early embryo development. The reduction of folate is catalysed by dihydrofolate reductase (EC 1.5.1.3) which is selectively inhibited by the anti-cancer drug methotrexate. Methotrexate caused a dose-dependent inhibition of cell division in 1-cell, 2-cell and 8-cell mouse embryos with 50% inhibition of division occurring at concentrations of 1-10 microM. At a concentration of 0.1 microM only minimal inhibition of the initial cell division occurred, but continuous culture in this concentration of methotrexate completely inhibited further cell divisions. This suggests that most of the exogenous store of reduced folates was used in the first round of cell division. The effects of methotrexate were apparently primarily due to thymidine starvation, since a 10-fold excess of thymidine over methotrexate in culture media reversed the inhibition of development. Supplementing media with folic acid had no beneficial effect on the rate at which zygotes produced by in-vitro fertilization developed to the blastocyst stage. It is concluded that the development of the early embryo has an absolute requirement for reduced folate for thymidine synthesis which is met entirely by endogenous sources.
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PMID:Endogenous folic acid is essential for normal development of preimplantation embryos. 964 65

In mammalian cells reiterated binding sites for Sp1 and two overlapping and inverted E2F sites at the transcription start site regulate the dhfr promoter during the cell growth cycle. Here we have examined the contributions of the dhfr Sp1 and E2F sites in the repression of dhfr gene expression. In serum-starved cells or during serum stimulation, the Chinese hamster dhfr gene was not derepressed by trichostatin A (TSA), an inhibitor of histone deacetylases (HDAC). Immunoprecipitation experiments showed that HDAC1 and hypophosphorylated retinoblastoma protein (pRb) are associated with Sp1 in serum-starved CHOC400 cells. In transfection experiments, reporter plasmids containing the reiterated dhfr Sp1 sites were stimulated 10-fold by TSA, while a promoter containing four dhfr E2F sites and a TATA box was responsive to E2F but was completely unaffected by TSA. HDAC1 did not coprecipitate with p130-E2F DNA binding complexes, the predominant E2F binding activity in cell extracts after serum starvation, suggesting that p130 imposes a TSA-insensitive state on the dhfr promoter. In support of this notion, recruitment of GAL4-p130 to a dihydrofolate reductase-GAL4 reporter rendered the promoter insensitive to TSA, while repression by GAL4-pRb was sensitive to TSA. Upon phosphorylation of pRb and p130 after serum stimulation, the Sp1-pRb and p130-E2F interactions were lost while the Sp1-HDAC1 interaction persisted into S phase. Together these studies suggest a dynamic model for the cooperation of pRb and p130 in repression of dhfr gene expression during withdrawal from the cell cycle. We propose that, during initial phases of cell cycle withdrawal, the binding of dephosphorylated pRb to Sp1-HDAC1 complexes and complexes of E2F-1 -to -3 with DP results in transient, HDAC-dependent suppression of dhfr transcription. Upon withdrawal of cells into G(0), recruitment of p130 to E2F-4-DP-1 complexes at the transcription start site results in a TSA-insensitive complex that cooperates with Sp1-HDAC-pRb complexes to stably repress dhfr promoter activity in quiescent cells.
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PMID:Cooperation of E2F-p130 and Sp1-pRb complexes in repression of the Chinese hamster dhfr gene. 1115 99


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