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

Severe impairment of methotrexate membrane transport in methotrexate-resist ant K562 (K500E) cells was characterized by a nearly complete loss of reduc ed folate carrier (RFC) transcripts and RFC protein. As determined by 5'-ra pid amplification of cDNA ends (5'-RACE), similar to 93% of the RFC transcr ipts in wild-type cells contained the KS43 5'-untranslated region transcrib ed 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 differen ces 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 ka ryotype analysis and fluorescence in situ hybridization in wild-type cells showed two normal chromosome 21 copies and one or two marker chromosomes, e ach with an RFC signal. In K500E cells, the RFC gene locus was no longer lo calized 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 t ranslocation. Our results suggest that loss of RFC transcripts in K500E cel ls is unrelated to changes in the levels of critical transcription factors, or to differences in the extent of RFC promoter methylation or core histon e deacetylation. Rather, this phenotype is due to the loss of one or more R FC alleles, and to a translocation of the remaining RFC allele with the for mation of a 21/22 fusion chromosome. (C) 2001 Elsevier Science Inc. All rig hts reserved.