Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.5.1.3 (dihydrofolate reductase)
5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the construction and characterization of retroviral vectors and packaging plasmids that produce helper-free retrovirus with titers of 1 X 10(6) to 5 X 10(6) within 48 h. These vectors contain the immediate early region of the human cytomegalovirus enhancer-promoter fused to the Moloney murine leukemia virus long terminal repeat at the TATA box in the 5' U3 region, yielding the pCL promoter. By selecting vectors designed to express genes from one of four promoters (dihydrofolate reductase, Rous sarcoma virus, long terminal repeat, or cytomegalovirus), the pCL system permits the investigator to control the level of gene expression in target cells over a 100-fold range, while maintaining uniformly high titers of virus from transiently transfected producer cells. The pCL packaging plasmids lack a packaging signal (delta-psi) and include an added safety modification that renders them self-inactivating through the deletion of the 3' U3 enhancer. Ecotropic, amphotropic (4070A), and amphotropic-mink cell focus-forming hybrid (10A1) envelope constructions have been prepared and tested, permitting flexible selection of vector pseudotype in accordance with experimental needs. Vector supernatants are free of helper virus and are of sufficiently high titer within 2 days of transient transfection in 293 cells to permit infection of more than 50% of randomly cycling target cells in culture. We demonstrated the efficacy of these vectors by using them to transfer three potent cell cycle control genes (the p16(INK4A), p53, and Rb1 genes) into human glioblastoma cells.
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PMID:The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. 876 92

The retinoblastoma tumor suppressor gene product (pRb) is involved in controlling cell cycle progression from G1 into S. pRb functions, in part, by regulating the activities of several transcription factors, making pRb involved in the transcriptional control of cellular genes. Transient-transfection assays have implicated pRb in the transcription of several genes, including c-fos, the interleukin-6 gene, c-myc, cdc-2, c-neu, and the transforming growth factor beta2 gene. However, these assays place the promoter in an artificial context and exclude the effects of far 5' upstream regions and chromosomal architecture on gene transcription. In these experiments, we have studied the role of pRb in the control of cell cycle-related genes within a chromosomal context and within the context of the G1 phase of the cell cycle. We have used adenovirus vectors to overexpress pRb in human osteosarcoma cells and breast cells synchronized in early G1. By RNase protection assays, we have assayed the effects of this virus-produced pRb on gene expression in these cells. These results indicate that pRb is involved in the transcriptional downregulation of the E2F-1, E2F-2, dihydrofolate reductase, thymidine kinase, c-myc, proliferating-cell nuclear antigen, p107, and p21/Cip1 genes. However, it has no effect on the transcription of the E2F-3, E2F-4, E2F-5, DP-1, DP-2, or p16/Ink4 genes. The results are consistent with the notion that pRb controls the transcription of genes involved in S-phase promotion. They also suggest that pRb negatively regulates the transcription of two of the transcription factors whose activity it also represses, E2F-1 and E2F-2, and that it plays a role in downregulating the immediate-early gene response to serum stimulation.
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PMID:Regulation of cellular genes in a chromosomal context by the retinoblastoma tumor suppressor protein. 967 66

The downstream effects of p15 and p16 gene deletions and loss of transcripts on dihydrofolate reductase (DHFR) were examined in 63 B-precursor (BP) acute lymphoblastic leukaemia (ALL) samples. p15 and/or p16 gene deletions were seen in 6% and 8%, respectively, of BP-ALL samples; however, losses of p15 and/or p16 transcripts were seen in 26 out of 63 (41%) samples. Loss of p15 transcripts (36.5%) exceeded that for p16 (17.5%). For the 26 BP-ALLs that lacked p15 and/or p16 transcripts, only six (23%) exhibited low levels of DHFR by flow cytometry assay with Pt430, a fluorescent anti-folate. Conversely, 18 out of 37 (49%) BP-ALL samples with intact p15 and/or p16 genes and transcripts showed low levels of DHFR (P = 0.04). In p15- and p16-null K562 cells transfected with a tetracycline-inducible p15 cDNA construct, induction of p15 transcripts and protein was accompanied by decreased growth rates, decreased S-phase fraction, decreased retinoblastoma protein phosphorylation, and markedly reduced levels of DHFR transcripts and protein. Collectively, our results suggest that losses of p15 and/or p16 gene expression result in elevated levels of DHFR in BP-ALL in children. However, additional downstream factors undoubtedly also contribute to elevated levels of this enzyme target.
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PMID:Relationship of p15 and p16 gene alterations to elevated dihydrofolate reductase in childhood acute lymphoblastic leukaemia. 1138 Apr 66

The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G(1) cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G(1) cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G(0) quiescent cells and becomes hypophosphorylated (approximately 2 mol of PO(4) to 1 mol of pRB) in early G(1) and hyperphosphorylated (approximately 10 mol of PO(4) to 1 mol of pRB) in late G(1) phase. Here, we report that hypophosphorylated pRB, present in early G(1), represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G(0) and hyperphosphorylated pRB in late G(1) fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT-dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G(1) and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G(1). Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16(INK4a) gene, contained hypophosphorylated pRB that was bound to E2Fs in early G(1) and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G(1) cyclin-Cdk complexes.
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PMID:Differential regulation of retinoblastoma tumor suppressor protein by G(1) cyclin-dependent kinase complexes in vivo. 1141 52

We analyzed the role of methylthioadenosine phosphorylase (MTAP) for chemoselective treatment of T-cell acute lymphoblastic leukemia (T-ALL). MTAP converts methylthioadenosine into adenine which serves as an alternative purine source, if de novo purine biosynthesis is inhibited by antimetabolites (i.e., methotrexate). The idea of the chemoselectivity concept is that tumors with MTAP deletion at chromosome 9p21 are more susceptible to antimetabolites than normal cells without such a deletion. First, we screened 13 T-ALL lines for 9p21 deletions by comparative genomic hybridization. Five cell lines revealed deletions at the short arm of chromosome 9, dim(9p21pter). Further analyses were performed with CEM cells in which the 9p21 deletion was corroborated by fluorescence in situ hybridization. CEM cells were transfected with an MTAP expression vector. A green fluorescent protein (GFP) plasmid was cotransfected, to monitor the transfection efficacy by flow cytometry. The response of MTAP-transfected cells to the antimetabolites methotrexate (MTX), trimetrexate (TMX), and L-alanosine (ALA) was decreased compared to mock control transfectants using growth inhibition assays. The activity of doxorubicin (DOX) which is not involved in DNA biosynthesis was not changed in MTAP transfectants. As the p16(INK4a) tumor suppressor gene resides also at 9p21, we transfected CEM cells with a p16(INK4a) expression vector. These transfectant cells were more resistant to all four drugs indicating that p16(INK4a) did not specifically affect antimetabolites. The chemoselective effect of antimetabolites in MTAP-deleted tumor cells may, however, be compensated by the development of drug resistance. To prove this possibility, we analyzed an MTX-resistant subline, CEM/MTX1500LV, in which the MTX-resistance conferring dihydrofolate reductase (DHFR) gene was amplified. While TMX exhibited considerable cross-resistance in CEM/MTX1500LV cells, ALA did not. Thus, ALA could exhibit chemoselectivity in 9p21/MTAP-deleted cells, even if DHFR amplification occurs. We conclude that ALA may be more suitable than MTX or TMX for MTAP-mediated chemoselective treatment of T-ALL. Pretherapeutical detection of 9p21 and MTAP deletion may be helpful in developing a predictive molecular chemosensitivity test for T-ALL.
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PMID:Methylthioadenosine phosphorylase as target for chemoselective treatment of T-cell acute lymphoblastic leukemic cells. 1198 41

Pemetrexed is a novel multitargeted antifolate that inhibits > or = 3 enzymes involved in folate metabolism and purine and pyrimidine synthesis. These enzymes are thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase. This agent has broad antitumor activity in phase II trials in a wide variety of solid tumors, and is approved in combination with cisplatin for the therapy of malignant mesothelioma. In a recent phase III trial, pemetrexed demonstrated equivalent efficacy to docetaxel, but with significantly less toxicity, in second-line treatment of non-small-cell lung cancer. The most common and serious toxicities of pemetrexed--myelosuppression and mucositis--have been significantly ameliorated by folic acid and vitamin B12 supplementation. More important, vitamin supplementation has not been shown to adversely affect efficacy in some tumor types. Tumors with codeletion of the methylthioadenosine phosphorylase gene, as a consequence of p16 deletions, may be particularly sensitive to pemetrexed. In this review, the biochemistry and mechanism of action of pemetrexed are discussed.
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PMID:Pharmacology and mechanism of action of pemetrexed. 1511 25

This study investigated associations between CpG island methylator phenotype (CIMP) colon cancer and genetic polymorphisms relevant to one-carbon metabolism and thus, potentially the provision of methyl groups and risk of colon cancer. Data from a large, population-based case-control study (916 incident colon cancer cases and 1,972 matched controls) were used. Candidate polymorphisms in methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS), transcobalamin II (TCNII), methionine synthase (MTR), reduced folate carrier (RFC), methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), dihydrofolate reductase (DHFR) and alcohol dehydrogenase 3 (ADH3) were evaluated. CIMP- or CIMP+ phenotype was based on five CpG island markers: MINT1, MINT2, MINT31, p16 and MLH1. The influence of specific dietary factors (folate, methionine, vitamin B(12) and alcohol) on these associations was also analyzed. We hypothesized that polymorphisms involved in the provision of methyl groups would be associated with CIMP+ tumors (two or more of five markers methylated), potentially modified by diet. Few associations specific to CIMP+ tumors were observed overall, which does not support the hypothesis that the provision of methyl groups is important in defining a methylator phenotype. However, our data suggest that genetic polymorphisms in MTHFR 1,298A > C, interacting with diet, may be involved in the development of highly CpG-methylated colon cancers. AC and CC genotypes in conjunction with a high-risk dietary pattern (low folate and methionine intake and high alcohol use) were associated with CIMP+ (OR = 2.1, 95% CI = 1.3-3.4 versus AA/high risk; P-interaction = 0.03). These results provide only limited support for a role of polymorphisms in one-carbon metabolism in the etiology of CIMP colon cancer.
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PMID:Genetic polymorphisms in one-carbon metabolism: associations with CpG island methylator phenotype (CIMP) in colon cancer and the modifying effects of diet. 1744 6

We demonstrate that the tea polyphenol, epigallocatechin-3-gallate, is an efficient inhibitor of human dihydrofolate reductase. Like other antifolate compounds, epigallocatechin-3-gallate acts by disturbing folic acid metabolism in cells, causing the inhibition of DNA and RNA synthesis and altering DNA methylation. Epigallocatechin-3-gallate was seen to inhibit the growth of a human colon carcinoma cell line in a concentration and time dependent manner. Rescue experiments using leucovorin and hypoxanthine-thymine medium were the first indication that epigallocatechin-3-gallate could disturb the folate metabolism within cells. Epigallocatechin-3-gallate increased the uptake of [(3)H]-thymidine and showed synergy with 5-fluorouracil, while its inhibitory action was strengthened after treatment with hypoxanthine, which indicates that epigallocatechin-3-gallate decreases the cellular production of nucleotides, thus, disturbing DNA and RNA synthesis. In addition to its effects on nucleotide biosynthesis, antifolate treatment has been linked to a decrease in cellular methylation. Here, we observed that epigallocatechin-3-gallate altered the p16 methylation pattern from methylated to unmethylated as a result of folic acid deprivation. Finally, we demonstrate that epigallocatechin-3-gallate causes adenosine to be released from the cells because it disrupts the purine metabolism. By binding to its specific receptors, adenosine can modulate different signalling pathways. This proposed mechanism should help us to understand most of the molecular and cellular effects described for this tea polyphenol.
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PMID:Effects of folate cycle disruption by the green tea polyphenol epigallocatechin-3-gallate. 1768 69

Luminescence resonance energy transfer (LRET) offers many advantages for accurate measurements of distances between specific sites in living cells, but progress in developing a methodology for implementing this technique has been limited. We report here the design, expression, and characterization of a test protein for development of a LRET methodology. The protein, which we call DAL, contains the following domains (from the N-terminus): Escherichia coli dihydrofolate reductase (DHFR), the third and fourth ankyrin repeats of p16(INK4a), a lanthanide-binding tag (LBT), and a hexahistidine tag. LBT binds Tb(3+) with a submicromolar dissociation constant. LRET was measured from the Tb(3+) site on LBT to transition metals bound to the hexa-His tag and to fluorescein methotrexate bound to DHFR. The measured distances were consistent with a molecular model constructed from the known crystal structures of the constituent domains of DAL. The results indicate that the two C-terminal ankyrin domains of p16(INK4a) are stably folded when combined with other protein domains. We found that Tb(3+) binds to DAL in the cytoplasm of live E. coli cells, and thus, DAL is useful as an indicator for studies of metal transport. We also used DAL to measure LRET from Tb(3+) to Cu(2+) in the cytoplasm of live E. coli cells. The rates of Tb(3+) and Cu(2+) transport were not affected by a proton uncoupler or an ATP synthase inhibitor. Reversal of the membrane potential had a small inhibitory effect, and removal of lipopolysaccharide had a small accelerating effect on transport. Changing the external pH from 7 to 5 strongly inhibited the Tb(3+) signal, suggesting that the Tb(3+)-LBT interaction is useful as a cytoplasmic pH indicator in the range of approximately pH 5-6.
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PMID:Luminescence resonance energy transfer in the cytoplasm of live Escherichia coli cells. 2173 54

Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase (eNOS) and NO production in endothelial cells. Tetrahydrobiopterin (BH4) is an important cofactor in regulating the balance between NO (eNOS coupling) and superoxide production (eNOS uncoupling). However, whether the decreased eNOS and NO production in CRIF1-deficient cells is associated with relative BH4 deficiency-induced eNOS uncoupling remains completely unknown. Our results showed that CRIF1 deficiency increased eNOS uncoupling and depleted levels of total biopterin and BH4 by reducing the enzymes of BH4 biosynthesis (GCH-1, PTS, SPR, and DHFR) in vivo and vitro, respectively. Supplementation of CRIF1-deficient cells with BH4 significantly increased the recovery of Akt and eNOS phosphorylation and NO synthesis. In addition, scavenging ROS with MitoTEMPO treatment replenished BH4 levels by elevating levels of GCH-1, PTS, and SPR, but with no effect on the level of DHFR. Downregulation of DHFR synthesis regulators p16 or p21 in CRIF1-deficient cells partially recovered the DHFR expression. In summary, CRIF1 deficiency inhibited BH4 biosynthesis and exacerbated eNOS uncoupling. This resulted in reduced NO production and increased oxidative stress, which contributes to endothelial dysfunction and is involved in the pathogenesis of cardiovascular diseases.
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PMID:CR6-interacting factor 1 deficiency reduces endothelial nitric oxide synthase activity by inhibiting biosynthesis of tetrahydrobiopterin. 3196 86


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