Characterization of Double BCR-ABL–positive Cell Lines Established from a Patient with Blasic Crisis of Chronic Myeloid Leukemia Who Showed Clinical Resistant to Imatinib

Pervious in vitro studies have shown that molecular alterations of BCR-ABL-positive leukemia cells such as amplification of BCR-ABL gene and/or mutation(s) of abl kinase domain cause resistant to imatinib. However recent study showed that alterations of imatinib bioavailability might be a important factor to cause clinical resistant in BCR-ABL-positive leukemia patients, showing a differences between in vivo and in vitro sensitivity to imatinib of BCR-ABL-positive cells. To analyze mechanism(s) of clinical resistance to imatinib and to overcome the resistance, we have sequentially established and characterized two leukemia cell lines from a patient with myeloid blastic crisis of chronic myeloid leukemia (CML) who showed progressively resistant to imatinib. Case report and establishment of cell lines: a 59-years-old women developed blastic crisis preceded by four years of chronic phase of CML. Increased blasts in crisis was positive for CD13, 33 and showed double Ph-chromosome in addition to complexed chromosomal alterations such as, add(3)(p13), add(3)(q11), add(5)(q11), der(19)(3;19) (p21;q13). After repeated courses of combination chemotherapy including, 600mg of imatinib was administered orally in combination with chemotherapeutic drugs. For a brief period Imatinib showed clinical effects and slowed the increase of BCR-ABL-positive cells, however myeloblast progressively increased in peripheral blood in spite of daily administration of imatinib and she died four months treatment with imatinib. Two myeloid leukemia cell lines, NS-1 and NS-2 were established, after obtaining informed consent, from peripheral blood at day 65 and day 95 after initiation of imatinib administration, respectively. Cell surface phenotype and karyotype of these cell lines were identical to original blasts. NS-1 and NS-2 cell lines were characterized compared with BCR/ABL-positive K562 erythroleukemia cell line as a control Quantitative analysis by real-time polymerase chain reaction showed that copy number of BCR-ABL transcript were 2.2 × 10⁵ and 1.6 × 10 ⁵/μg RNA in NS-1 and NS-2 respectively, showing slightly lower than those (5.8 × 10⁵) in K562 cell line. Although nucleotide sequence analysis showed that a point mutation in abl kinase domain resulted in amino acid substitution pro310ser in NS-1 cell line, no additional mutation was found in NS-2 cell line. Western blot analysis showed levels of both 210 KD BCR-ABL protein and BCR-ABL phosphorylation were similar in NS-1, NS-2 and K562 cells. Although two hours incubation with 10 mM imatinibin vitro did not show any detectable difference in levels of phosphorylation of BCR-ABL protein between NS-1 and NS-2 cell lines, sensitivity to imatinib measured by MTT assay showed that IC₅₀ was 0.1 mM, 0.5 mM and 1.0mMin NS-1, NS-2 and K562 cell lines respectively. The measured IC₅₀ of both NH-1 and NH-2 cell lines were much lower than reported plasma concentrations achieved by oral administration of 600 mg of imatinib (above 10 μM). The present results suggest

1. difference between in vivo and in vitro sensitivity to imatinib indicate that alteration of bioavailability of imatinib possibly involved in clinical resistance to this drug,

2. accumulations of BCR-ABL gene amplification and/or mutation are not necessarily a major reason of progressive clinical resistance to imatinib in BCR-ABL positive leukemia.