Disease relevance of Dhfr

• A plasmid, pQS4 , expressing large amount of Dhfr in both E. coli and B. subtilis was isolated through a two steps selection with two substrate analogues, trimethoprim followed by methotrexate [1].
• Dihydrofolate reductase deficiency causing megaloblastic anemia in two families [2].
• Friend murine erythroleukemia (F-MEL) cells were transfected with a plasmid bearing tandemly arranged mouse c-myc antisense and dihydrofolate reductase transcription units [3].
• The amino acid sequence of dihydrofolate reductase from the mouse lymphoma L1210 [4].
• Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells [5].

High impact information on Dhfr

• A search of published DNA sequences identified a mouse sequence, with about 77% similarity to the pseudogene sequence, in the negative strand of an intron in a mouse dihydrofolate reductase gene [6].
• To investigate autoregulation in vivo, transcription from the SV40 early promoter was quantitated in COS7 monkey cells transfected with plasmid vectors carrying the mouse dihydrofolate reductase gene (SV-dhfr vectors) [7].
• Growth of these methotrexate-resistant populations at higher methotrexate concentrations results in the emergence of cells expressing high levels of dihydrofolate reductase with a reduced methotrexate affinity [8].
• Unstable amplification of an altered dihydrofolate reductase gene associated with double-minute chromosomes [8].
• Initially, drug resistance results from amplification of the gene encoding the normal dihydrofolate reductase [8].

Chemical compound and disease context of Dhfr

• We have used a microtiter assay to study the toxicity of various folate analogs in a series of cultured human cell lines that exhibit different degrees of resistance to methotrexate, an inhibitor of dihydrofolate reductase [9].
• Trimetrexate inhibits progression of the murine 32Dp210 model of chronic myeloid leukemia in animals expressing drug-resistant dihydrofolate reductase [10].
• Chromosomal aberrations and dihydrofolate reductase gene amplification are observed in L5178Y mouse lymphoma cells after treatment with hydroxyurea [11].
• 5-Formyltetrahydrofolate promotes the net dissociation of methotrexate bound to dihydrofolate reductase in the Ehrlich ascites tumor (L. H. Matherly et al., Cancer Res., 43: 2694-2699, 1983) [12].
• Rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after cessation of dihydrofolate reductase activity in stationary versus log phase L1210 leukemia cells [13].

Biological context of Dhfr

• Analysis of the mouse Dhfr/Rep-3 major promoter region by using linker-scanning and internal deletion mutations and DNase I footprinting [14].
• The mouse dihydrofolate reductase (Dhfr) promoter region is buried within a CpG island (a region rich in unmethylated CpG dinucleotides), has a high G+C content, and lacks CAAT and TATA elements [14].
• Using single-strand conformation polymorphism analysis, Crtl1 was assigned to mouse chromosome 13, tightly linked to Dhfr [15].
• Analysis of the nucleotide sequence of this cDNA clone allows us to predict that the altered dihydrofolate reductase differs from the wild-type enzyme at a single amino acid, reflecting the substitution of an arginine for a leucine residue in a region of the polypeptide thought to form a hydrophobic pocket essential for inhibitor binding [16].
• We have constructed a cDNA library from a murine cell line expressing high levels of a dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) that displays an abnormally low affinity for methotrexate [16].

Anatomical context of Dhfr

• We demonstrate that this altered gene can be employed as a dominant selectable marker in cultured cells expressing normal levels of wild-type dihydrofolate reductase [16].
• Microcells prepared from diploid mouse fibroblasts were fused with mutant hamster cells lacking the dihydrofolate reductase gene (Dhfr), and DHFR+ microcell hybrids were selected in medium lacking purines and pyrimidines [17].
• Liver-cell homogenate from one patient had a previously undetectable level of dihydrofolate reductase restored to normal by high cation concentration in the assay [2].
• Through analysis of transiently and stably transfected NIH 3T3 cells, we have now demonstrated that DHFR promoter sequences extending from -270 to +20 are sufficient to confer similar regulation on a reporter gene [18].
• The ability of the tyr22-DHFR transgenic marrow to confer MTX-resistance to bone marrow transplant (BMT) recipients did not correlate with the level of mRNA expression or the number of transgene copies [19].

Associations of Dhfr with chemical compounds

• Functional analysis of the E2F1 core domain demonstrated that replacement of phenylalanine residues 413, 425, and 429 with alanine reduces both transcriptional activation of the dhfr promoter and protein-protein interactions with CBP, transcription factor (TF) IIH, and TATA-binding protein (TBP) [20].
• Aminopterin, an inhibitor of dihydrofolate reductase, has been demonstrated to allow HGPRT(-) cells to be negatively selected [21].
• Such plasmids specify a protein that has enzymatic properties, immunological reactivity and molecular size characteristic of the mouse dihydrofolate reductase, and render host cells resistant to the antimetabolic drug trimethoprim [22].
• The most active of these drugs, MGT-6a, was three orders of magnitude more effective than distamycin and inhibited complexes between E2F1 and the dihydrofolate reductase promoter by 50% at 0.00085 microM [23].
• These boron-containing compounds were shown to inhibit DNA synthesis; such inhibition was caused primarily by reducing de novo purine biosynthesis via inhibition of PRPP amidotransferase, IMP dehydrogenase and dihydrofolate reductase activities [24].

Physical interactions of Dhfr

• Activation depends on the E2F binding site in the DHFR promoter, known to mediate its activation at the G1/S transition in vivo [25].
• A mechanism of resistance to methotrexate. NADPH but not NADH stimulation of methotrexate binding to dihydrofolate reductase [26].

Regulatory relationships of Dhfr

• Therefore, we suggest that recruitment of CBP is one mechanism by which E2F activates the dhfr promoter [20].
• Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification [27].
• Effect of direct suppression of thymidylate synthase at the 5,10-methylenetetrahydrofolate binding site on the interconversion of tetrahydrofolate cofactors to dihydrofolate by antifolates. Influence of degree of dihydrofolate reductase inhibition [28].

Other interactions of Dhfr

• Assignment of genes encoding dihydrofolate reductase and hexosaminidase B to Mus musculus chromosome 13 [17].
• In particular, cyclin D1 overexpression leads to the activation of the dihydrofolate reductase (DHFR) gene promoter [25].
• Furthermore, C/EBPalpha can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines [29].
• We confirmed that our purification enriched for EEs that carry genes by fluorescent in situ hybridization of EEs (FISH-EEs), and we probed non-enriched and immunopurified EEs with a dihydrofolate reductase (DHFR) cDNA probe that is known to detect extrachromosomal amplification in Myc-activated cells [30].
• MEFs lacking both Rb alleles (Rb-/-) entered S phase in the presence of the dihydrofolate reductase inhibitor methotrexate [31].

Analytical, diagnostic and therapeutic context of Dhfr

• We investigated the effect of substrate binding on the mechanical stability of mouse dihydrofolate reductase using single-molecule force spectroscopy by atomic force microscopy [32].
• Transgenic mice expressing the tyr22 variant of murine DHFR: protection of transgenic marrow transplant recipients from lethal doses of methotrexate [19].
• These results indicate that tyr22-DHFR is likely to be superior to arg22-DHFR in conferring MTX-resistance in BMT recipients, illustrating its usefulness for chemoprotection during MTX chemotherapy and also potentially for in vivo selection of transduced cells in gene therapy trials [19].
• Molecular cloning, mapping, and DNA sequencing techniques have been used to characterize the functional human dihydrofolate reductase (DHFR) gene [33].
• The L5178YR cell line synthesizes approximately 10-11% of its total soluble cell protein as DHFR regardless of growth phase, as measured by direct immunoprecipitation with a monospecific antiserum [34].

References