• We have constructed retroviruses containing human ADA cDNA and a dominant selectable marker, a mutated dihydrofolate reductase gene (DHFR*) encoding methotrexate resistance [1].
• To begin this study, we constructed a plasmid containing a neomycin resistance gene, a human beta globin gene, and a wild-type DHFR cDNA, and transfected it into mouse erythroleukemia cells [2].
• The TS-DHFR from Plasmodium falciparum has also been cloned and has recently been expressed in Escherichia coli, albeit in small amounts [3].
• This study analyzed pretreatment dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes and treatment outcomes in a double-blind, placebo-controlled trial of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment for uncomplicated P. falciparum malaria [4].
• Protection studies with 3 in the FaDu squamous cell carcinoma cell line indicated that inhibition was completely reversed by leucovorin [(6R,S-5-formyltetrahydrofolate] or by a combination of thymidine and hypoxanthine, suggesting an antifolate effect directed at DHFR [5].

Psychiatry related information on DHFR

• Molecular cloning of human cDNA with a sequence highly similar to that of the dihydrofolate reductase gene in brain libraries derived from Alzheimer's disease patients [6].
• The decision-making process is discussed for a set of G-protein coupled receptor antagonists and validated on a literature example for dihydrofolate reductase inhibition [7].

Disease relevance of DHFR

• Although the DHFR 19-bp deletion polymorphism was not associated with overall breast cancer risk, we observed a borderline significant additive interaction (P = 0.06) between the DHFR genotype and multivitamin use [8].

High impact information on DHFR

• The fundamental issues related to this linkage are probed in the context of two enzymes that catalyze hydride transfer, namely dihydrofolate reductase and liver alcohol dehydrogenase [9].
• Use of multivitamin supplements containing folic acid diminished the adverse effects of dihydrofolate reductase inhibitors, but not that of antiepileptic drugs [10].
• We have examined DNA repair in the dihydrofolate reductase (DHFR) gene in cultured human cells treated with 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) using a newly developed assay for interstrand DNA cross-linking in defined genomic sequences [11].
• Selective removal of transcription-blocking DNA damage from the transcribed strand of the mammalian DHFR gene [12].
• Furthermore, in the 5' flanking region of the human DHFR gene, selective rapid repair occurs in the opposite DNA strand relative to the transcribed strand of the DHFR gene [12].

Chemical compound and disease context of DHFR

• The resulting expression plasmid conferred trimethoprim resistance to E. coli DH5 alpha and complemented the TS deficiency in chi 2913recA cells indicating the presence of active DHFR and TS [13].
• Co-expression of mutant DHFR and TS enzymes has additive effects in conferring resistance to pemetrexed-induced toxicity [14].
• In this study, we examined the sensitivity of the DHFR F/S-TS G52S fusion protein to the multitargeted antifolate, pemetrexed (LY231514, Alimta), which targets both DHFR and TS and is currently in phase III trials for the treatment of solid tumors and in combination with cisplatin has been shown to be an advance in the treatment of mesothelioma [14].
• Powerful new DHFR and TS directed agents are in advanced levels of clinical evaluation, and purine analogues directed against adenosine deaminase are newly available for treatment of indolent lymphomas [15].
• Sequence variations in the genes encoding dihydropteroate synthase and dihydrofolate reductase and clinical response to sulfadoxine-pyrimethamine in patients with acute uncomplicated falciparum malaria [16].

Biological context of DHFR

• Using in situ hybridization, we have now localized the functional DHFR gene to the region q11.1-q13.3 on chromosome 5 [17].
• Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression [18].
• There are no intervening sequences within the coding region for TS-DHFR, and the G+C content is high, with a strong bias for codons possessing guanine or cytosine in the third base [19].
• The human dihydrofolate reductase (DHFR) gene family comprises one functional gene and at least four intronless processed pseudogenes [17].
• Furthermore, a synthetic minimal promoter of DHFR, consisting of a single binding site for Sp1 and a binding site for the HIP1 initiator cloned into a bacterial vector sequence, required RNAP II with an intact CTD for activity in vitro [20].

Anatomical context of DHFR

• The upstream initiating transcripts present in all three cell lines are increased in amount in 6A3 cells as compared with the other cell lines, in about the same proportion as the three identified DHFR mRNAs [21].
• To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle [22].
• 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 [23].
• Phytohemagglutinin-stimulated peripheral blood lymphocytes from 6 patients with increased levels of DHFR showed no evidence of MTX resistance in vitro [24].
• The CHO cells transformed with the human ERCC-1 gene repaired the active DHFR gene much more efficiently than the non-transcribed sequences, a pattern similar to that seen in wild type CHO cells [25].

Associations of DHFR with chemical compounds

• The associated increase in DHFR expression resulted in increased resistance to methotrexate but had no effect on other classes of anticancer agents [26].
• Studies with the recombinant enzyme show that trimethoprim is a very poor inhibitor of P. carinii DHFR and, in fact, is a more potent inhibitor of human DHFR [27].
• Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells [28].
• At least six other minor 5' ends have been mapped to nucleotide positions -449 to -480 upstream of the DHFR gene and pertain to approximately 1% of the DHFR-specific polysomal polyadenylic acid-containing RNA [21].
• Interestingly, pyrimethamine-resistant strains of P. falciparum all have a common point mutation in the DHFR coding sequence (Thr/Ser 108 to Asn), which causes decreased binding of the folate analog [3].
• Cells with the mutant 3' UTR had a 2-fold increase in DHFR mRNA half-life, expressed higher DHFR mRNA and DHFR protein, and were 4-fold more resistant to methotrexate as compared with WT cells [29].

Physical interactions of DHFR

• Rb interacts with the family of transcription factors called E2F reducing transcription of genes that contain E2F binding sites in the promoter regions e.g. DHFR [30].
• The DHFR-TS nucleic acid sequence contains no introns and the single 1563-bp open reading frame encodes a 179 residue N-terminal DHFR domain connected by a 55 amino acid junction peptide to a 287 residue C-terminal TS domain [31].
• Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH [32].
• In all recrudescent parasites, however, the presence of Ile 51/Asn 108 dhfr mutations was coupled with the dhps Ala 436 [33].
• Insertion of a seven amino acid sequence from a structurally equivalent 'beta-blowout' sequence from human DHFR destabilizes E. coli DHFR by 3.6 kcal/mol and decreases catalytic efficiency (kcat/K(m)) 34-fold [34].

Regulatory relationships of DHFR

• Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells [22].
• Specifically, TS ligand induced domain-domain communication involving DHFR activation is observed only in the L. major enzyme and, whereas both DHFR activities involve a rate-limiting conformational change, the change occurs at different positions along the kinetic pathway [35].
• Only Tom20 inhibited the import of a fusion protein of the leader of aldehyde dehydrogenase attached to dihydrofolate reductase [36].
• By high resolution two-dimensional polyacrylamide gradient electrophoresis, human DHFR was shown to be selectively overproduced in VB2a-100 MTX-resistant cells whereas mouse DHFR protein "spots" present in MTX-sensitive parental hybrid were absent in these cells exhibiting 100 microM MTX resistance [37].
• Recombinant BHK-21 cells producing hIL-6 under the control of the CMV promoter were contransfected with the ras oncogene and dihydrofolate reductase gene, then selected with 50 nM methotrexate to coamplify the ras oncogene [38].

Other interactions of DHFR

• Transfectants with increased cyclin D1 expression also had increased DHFR mRNA and protein expression [26].
• Each reaction resulted in a rapid, time-dependent loss of TS activity and no effect on DHFR activity [39].
• DHFR/MSH3 amplification in methotrexate-resistant cells alters the hMutSalpha/hMutSbeta ratio and reduces the efficiency of base-base mismatch repair [40].
• We show that HCMV activates the DHFR promoter and that products of the HCMV major immediate-early gene region mediate the activation of the promoter specifically through the E2F site [41].
• These data indicate higher FPGS and lower DHFR levels as potential mechanisms contributing to greater MTXPG accumulation and cytotoxicity in B-lineage lymphoblasts [42].

Analytical, diagnostic and therapeutic context of DHFR

• Dihydrofolate reductase (DHFR) mRNA and protein levels were measured by northern blot and western blot analyses, respectively [26].
• Most of the recognized elements of secondary structure of other DHFRs, as ascertained by x-ray crystallography, are found in the predicted structure of the DHFR domain of the bifunctional protein [19].
• Consistent with the recent conclusion that P. carinii is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase [27].
• We used gel mobility shift assays to search for potential molecular mechanisms for this activation and found an "infection-specific" multimeric complex that bound to the E2F sites in the DHFR and E2 promoters in extracts from HCMV-infected cells but not in extracts from uninfected cells [41].
• Southern blot analysis with a human DHFR cDNA probe confirmed this increase in the gene copy number, and demonstrated a similar restriction pattern with Eco R1, Hind III, and Pst 1 as seen with a highly amplified human leukemia cell line, K562 [43].

References