Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases.

Melera, Peter W.; Davide, Joseph P.; Hession, Catherine; Scotto, Kathleen W.

doi:10.1128/mcb.4.1.38

Cited by 68 publications

(30 citation statements)

References 47 publications

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“…To date, five MTX-resistant cell lines, developed in vitro, have been identified and found to contain an altered DHFR with reduced affinity for antifolates (9,(25)(26)(27)(28). In each of these cases, sequencing of the DHFR gene has revealed a single nucleotide base change that causes a single amino acid replacement for the protein products.…”

Section: Discussionmentioning

confidence: 99%

Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation.

Dicker

Waltham

Volkenandt

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied expressed an altered dihydrofolate reductase (DEHFR) 5,6,7,8-tetrahydrofolate, an essential carrier of one-carbon units in the biosynthesis of thymidylate, purine nucleotides, and methyl compounds. DHFR has been the subject of intense study for >40 years as it is the target enzyme for several important drugs, including methotrexate (MTX; 4-amino-4-deoxy-10-methylfolic acid), trimethoprim [TMP; 2,4-diamino-5-(3,4,5-trimethoxybenzyl) repeated fixed maximally tolerated doses of MTX. To this end, a series of L1210 leukemic cells resistant to MTX were developed in vivo through drug dosage schedules analogous to clinical schedules (7). Twenty independently derived sublines were selected for maximum resistance to MTX and all displayed an elevated DHFR activity. Ten of these sublines had only this phenotype, while seven sublines also demonstrated a reduced influx for MTX (7). The remaining three sublines (designated L1210/MTX-1, -2, and -3) were found to have a DHFR with reduced affinity for MTX on the basis of preliminary DHFR activity titration experiments with MTX using crude tumor lysates (8). Here we describe the basis for this decreased antifolate binding. MATERIALS AND METHODSMaterials. General molecular biology reagents (including items for mRNA isolation and cDNA synthesis) and enzyme purification materials were from sources as indicated (9, 10

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Section: Discussionmentioning

confidence: 99%

Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation.

Dicker

Waltham

Volkenandt

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

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“…The dhfr cDNA sequence from exon 2 to exon 6 was subcloned by the insertion of a EcoRI-PstI fragment from the cDNA clone pA35 (46) into EcoRI-PstI-cleaved pBR322. This intermediate construct was cleaved at the exon 2-pBR322 junction 5' region aligned with DNA sequences of the mouse and human dhfr 5' regions.…”

Section: Methodsmentioning

confidence: 99%

“…Housekeeping genes do show physiological and tissue-specific variations in expression. The development of cell lines with amplified dhfr genes has greatly facilitated the cloning and characterization of several mammalian dhfr cDNAs and their genomic regions (7,9,14,46,47,51,78). These amplified cell lines provide a unique opportunity to study the transcription and processing of an otherwise low-copy S-phase-specific mRNA.…”

mentioning

confidence: 99%

Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

Mitchell¹,

Carothers²,

Han³

et al. 1986

Mol. Cell. Biol.

164

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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 occwrs 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. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312: [409][410][411][412][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.The mammalian dihydrofolate reductase gene product (DHFR) is one of a group of S-phase-responsive enzymes important for DNA replication. Like other enzymes involved in DNA synthesis, DHFR activity is greater in proliferating cells than in quiescent cells (34). DHFR catalyzes the NADPH-dependent reduction of folate to dihydrofolate and then to tetrahydrofolate. Reduced folates are essential cofactors in the biosynthesis of glycine, purine nucleotides, and thymidylic acid. The de novo synthesis of the DNA precursor thymidylic acid during S phase is the major tetrahydrofolate-consuming reaction, and the cellular requirement for DHFR is highest at this time (34). However, even in rapidly proliferating cells, sufficient DHFR catalytic activity can be maintained by as few as 10 to 20 copies of mRNA per cell (32).Genes coding for low-abundance mRNAs represent the major portion of RNA polymerase II-specific genes that are expressed in any given cell type. Our understanding of the regulation of such genes is only just beginning. Some of the proteins encoded by low-copy mRNAs are tissue-specific enzymes and regulatory proteins involved in metabolic activities unique to a particular cellular phenotype. Others are proteins expressed to some extent in all cells and are commonly known as housekeeping gene products. The dihydrofolate reductase (dhfr) gene is referred to as a house-* Corresponding author. keeping gene because DHFR plays a role in several fundamental biosynthetic reactions, but the ternlinology is not meant to indicate that dhfr gene expression is uniform in all cells or at all times in a given cell. Housekeeping genes do show physiological and tissue-specific variations in expression. The development of cell lines with amplified dhfr genes has greatly facilitated the cloning and characterization of several...

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“…Among the mutated sites identified was leu22Arg in the murine gene located at the MTXbinding site (Simonsen and Levinson, 1983). Mutations were identified in MTX-resistant hamster lung cancer cell lines including a Leu22Phe substitution (Melera et al, 1984;Melera et al, 1988). A variety of human cell lines have been selected for resistance to MTX due to mutations in DHFR.…”

Section: Alterations In Dhfrmentioning

confidence: 99%

Resistance to antifolates

2003

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Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases.

Cited by 68 publications

References 47 publications

Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation.

Methotrexate resistance in an in vivo mouse tumor due to a non-active-site dihydrofolate reductase mutation.

Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

Resistance to antifolates

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