[Growth inhibition and differentiation of imatinib-resistant chronic myeloid leukemia cell induced by cell differentiation agent in vitro].
OBJECTIVE
To investigate the effects of uroacitide (CDA-2), a cell differentiation agent, on the growth inhibition and differentiation of imatinib-(IM) resistant chronic myeloid leukemia (CML) cells.


METHODS
IM resistant CML cell line K562R was established from the line K562. K562 and K562R CML cells were cultured with CDA-2 of different concentrations. MTI method was used to detect the survival rates. Bone marrow cells of IM-resistant and non-IM-resistant CML patients were collected and co-incubated with K562 and K562R cells. MTT and colony-forming assays were used to evaluate the efficacy of CDA-2 treatment for cell growth in K562 and K562R cell lines, and IM-resistant or non-IM-resistant bone marrow cells of the CML patients; Annexin-V staining was employed to detect the apoptosis. Cell differentiation was assessed by flow cytometry analysis with CD11b/CD14 markers, reverse transcriptase PCR (RT-PCR) for mRNA levels of NCF-1 and ORM-1 genes and Giemsa staining for the observation in morphology. Cell cycle distribution was detected by stained with propidium iodide and then analyzed by flow cytometer. RT-PCR also was employed for the expression of DNA methyltransferase.


RESULTS
Significant cell growth inhibition was found at a dose-dependent manner in the IM-resistant K562R cell line and IM-resistant bone marrow cells of the CML patients compared with the non-resistant K562 cell line and bone marrow cells of the CML patients following 7 days exposure to CDA-2. Although CDA-2 could significantly induce the apoptosis of K562R (15.38%) compared with K562 (5.28%) (P < 0.05), the major reason for the cell growth inhibition of K562R is CDA-2-induced cell differentiation, including the increase of expression of differentiation-related antigens CD11b/CD14, mRNA expression of NCF-1 and ORM-1, and cell cycle arrest in G1-phase at a dose-dependent manner. Because CDA-2 could significantly activate the p21 and p27 gene expression, downregulate the expression of cyclin D1, and down-regulate the expressions of DNMT1 and DNMT(3B) at mRNA level, CDA-2 might be a DNMT inhibitor for restoring some gene function that involved in cell cycle control by demethylation.


CONCLUSIONS
Inhibiting the growth and inducing the differentiation of K562R cells, CDA-2 is very likely to be a potential agent for the treatment of IM resistance CML patients.
Wang
Xiao-dan
XD
Institute of Hematology & Oncology, Harbin 150010, China.
Qiu
Lin
L
Lu
Run-zhang
RZ
Chen
Li-jun
LJ
Zhan
Zhao-min
ZM
Han
Bing-hong
BH
Zhang
Bo-long
BL
Ma
Jun
J
D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000652:urine*; D017209:Apoptosis / Q000187:drug effects; D001549:Benzamides; D015153:Blotting, Western; D039481:CD11b Antigen / Q000032:analysis; D049109:Cell Proliferation / Q000187:drug effects*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D005434:Flow Cytometry; D006801:Humans; D000068877:Imatinib Mesylate; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D019255:NADPH Oxidases / Q000235:genetics; D010879:Piperazines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000970:Antineoplastic Agents; D001549:Benzamides; D039481:CD11b Antigen; D018950:Lipopolysaccharide Receptors; D010879:Piperazines; D011743:Pyrimidines; D012333:RNA, Messenger; D000068877:Imatinib Mesylate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1