Association of deletions and translocation of the reduced folate carrier gene with profound loss of gene expression in methotrexate-resistant K562 human erythroleukemia cells

• Published in: Biochemical pharmacology Vol. 61; no. 6; pp. 665 - 675
• Main Authors: Ching Ding, Bee, Witt, Teah L., Hukku, Bharati, Heng, Henry, Zhang, Long, Matherly, Larry H.
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 15.03.2001; Elsevier Science
• Subjects: 5' Untranslated Regions - genetics; Antimetabolites, Antineoplastic - pharmacokinetics; Antimetabolites, Antineoplastic - pharmacology; Antineoplastic agents; Azacitidine - analogs & derivatives; Azacitidine - pharmacology; Biological and medical sciences; Biological Transport; Carrier Proteins - biosynthesis; Carrier Proteins - genetics; Chromosome 21; Decitabine; DNA Methylation; Drug Resistance, Neoplasm - genetics; Gene Deletion; Gene Expression Regulation, Neoplastic; General aspects; Genes, Reporter; Histones - metabolism; Humans; Hydroxamic Acids - pharmacology; In Situ Hybridization, Fluorescence; K562 Cells; Karyotyping; Leukemia, Erythroblastic, Acute - genetics; Medical sciences; Membrane Proteins; Membrane transport; Membrane Transport Proteins; Methotrexate; Methotrexate - pharmacokinetics; Methotrexate - pharmacology; Pharmacology. Drug treatments; Promoter Regions, Genetic - physiology; Reduced folate carrier; Reduced Folate Carrier Protein; Resistance; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - biosynthesis; Translocation, Genetic; Resistance; Reduced folate carrier; Methotrexate; Membrane transport; Chromosome 21; Chromosomal aberration; Antineoplastic agent; Human; Transcription; Leukemia; Biological transport; Chromosome G21; Malignant hemopathy; In vitro; Folate; Established cell line; Deletion; Abnormal chromosome; Mutation; Tumor cell; Chromosome translocation
• ISSN: 0006-2952; 1873-2968
• DOI: 10.1016/S0006-2952(01)00535-4

Severe impairment of methotrexate membrane transport in methotrexate-resistant K562 (K500E) cells was characterized by a nearly complete loss of reduced folate carrier (RFC) transcripts and RFC protein. As determined by 5′-rapid amplification of cDNA ends (5′-RACE), ∼93% of the RFC transcripts in wild-type cells contained the KS43 5′-untranslated region transcribed from the RFC-B promoter. KS43 transcripts decreased > 90% in K500E cells. The basal and full-length RFC-B promoters were more active (3- and 2-fold, respectively) in directing transcription of a luciferase reporter gene in K500E than in wild-type cells. Treatment with a demethylating agent, 5-aza-2′-deoxycytidine, or with a histone deacetylase inhibitor, trichostatin A, did not increase the levels of RFC transcripts in K500E cells. No differences in RFC gene structure were detected between the lines on Southern blots; however, the RFC signals were decreased approximately 60% in K500E cells. DNA sequences were identical between the lines for the RFC coding region and the two 5′-non-coding exons and their respective promoters. Spectral karyotype analysis and fluorescence in situ hybridization in wild-type cells showed two normal chromosome 21 copies and one or two marker chromosomes, each with an RFC signal. In K500E cells, the RFC gene locus was no longer localized to a normal chromosome 21 (at 21q22.2), and a single RFC signal was associated with a small metacentric chromosome, characterized by a 21/22 translocation. Our results suggest that loss of RFC transcripts in K500E cells is unrelated to changes in the levels of critical transcription factors, or to differences in the extent of RFC promoter methylation or core histone deacetylation. Rather, this phenotype is due to the loss of one or more RFC alleles, and to a translocation of the remaining RFC allele with the formation of a 21/22 fusion chromosome.