The chronic myelocytic cell line K562 contains a breakpoint in bcr and produces a chimeric bcr/c-abl transcript.

Abstract

In the DNAs of all Ph1-positive chronic myelocytic leukemia patients studied to date, a breakpoint on chromosome 22 (the Ph1 chromosome) can be demonstrated with a probe from the bcr (breakpoint cluster region). Although the K562 cell line was established from cells of a chronic myelocytic leukemia patient, we have been unable to detect the Ph1 chromosome by cytogenetic means. Employing a probe from the 5' region of bcr, we have cloned an amplified Ph1 breakpoint fragment from K562. This demonstrates that K562 contains multiple remnants of a Ph1 chromosome with a breakpoint within bcr and thus may serve as a model system for the study of Ph1-positive chronic myelocytic leukemia at a molecular level. The isolation of bcr cDNA sequences shows that parts of bcr encode a protein. Employing K562, we demonstrate the presence of an abnormally sized mRNA species hybridizing to c-abl and to a bcr cDNA probe, indicating the possible consequence of the Ph1 translocation on a transcriptional level in chronic myelocytic leukemia. The isolation and sequencing of a cDNA containing the breakpoint area of this mRNA provide further evidence for its chimeric structure. Cloning of large stretches of chromosomal DNA flanking bcr and c-abl sequences in K562 and identification of the exons participating in the formation of the chimeric mRNA shows that a splice of at least 99 kilobases is made to fuse the 3' bcr exon to the 5' c-abl exon. Furthermore two chimeric cDNAs were isolated containing chromosome 9 sequences that map 43.5 kilobases downstream from the K562 breakpoint. These chromosome 9 sequences neither hybridize to the 8.5-kilobase chimeric c-abl mRNA nor to normal c-abl mRNAs in Hela cells and probably represent incorrect splicing products present in the K562 cell line.

Evidence that the phl gene encodes a 160,000-dalton phosphoprotein with associated kinase activity.

“…Indeed, an abnormally sized abl mRNA of 8.5 kilobases (2, 3, and 7) and an abl protein of 210,000 molecular weight (P210) have been demonstrated in leukemic cells of CML patients (15k. In subsequent experiments, the mRNA was found to be chimeric, consisting of 5' phl exons fused to 3' c-abl sequences (9,21,23). Apart from the fact that it is specifically involved in the Ph' translocation, little is known regarding the phl gene and its translational product.…”

“…A chromosomal breakpoint was previously localized within the c-abl gene in one CML patient (12). Since the genomic sequences of the phl and abl genes are fused in a head-to-tail fashion, we hypothesized that an abnormal c-abl gene product could be expressed in Ph'-positive leukemia cells (9). Indeed, an abnormally sized abl mRNA of 8.5 kilobases (2, 3, and 7) and an abl protein of 210,000 molecular weight (P210) have been demonstrated in leukemic cells of CML patients (15k.…”

“…1A). These latter sequences are absent from the chimeric mRNA detected in the leukemic cells of CML patients and in the CML cell line K562 (9,23). Both segments were inserted into appropriate trpE expression vectors (22), and trpE fusion proteins were synthesized in Escherichia coli.…”

A novel BCR-ABL rearrangement in a Philadelphia chromosome-positive chronic myelogenous leukaemia variant with thrombocythaemia

“…8 A combined reverse transcription (RT) and polymerase chain reaction (PCR) method was used 9 followed by a further PCR using a nested primer set. Outer primers were based on published sequences 10,11 and were: (I) 5′-GAAGTGTTTCAGAAGCTTCTC-3′; (II) 5′-CTGGCCA-CAAATCATACAGTGC-3′. Inner primers and amplification conditions were as described.…”

Cell type dependent regulation of multidrug resistance-1 gene expression by AML1-ETO

“…Despite its malignant origin, K562 cells retain some capacity for differentiation into multiple cell types, a characteristic of the normal multipotent hematopoietic progenitor cell [35][36][37][38]. Because these cells express BCR-ABL, we examined whether this was the reason for the activation of the MDR1 promoter observed in the presence of AML1-ETO.…”