Inhibition of Overexpression of c-myb Protooncogene in K562 Cells Using c-myb Antisense Oligodeoxynucleotides.
- Author:
Pyoung Han HWANG
1
;
Ho Keun YI
;
Kyeong Mee LEE
;
Jung Soo KIM
;
Moo Kyung KIM
Author Information
1. Department of Pediatrics, Chonbuk National University Medical School, Chonju, Chonbuk, Korea. hwaph@moak.chonbuk.ac.kr
- Publication Type:Original Article
- Keywords:
c-myb;
Antisense oligodeoxynucleotides;
CML
- MeSH:
Bone Marrow Cells;
Bone Marrow Purging;
Cell Count;
Cell Line;
Cell Proliferation;
Cell Survival;
Cellulose;
Clone Cells;
Cloning, Organism;
Down-Regulation;
Granulocyte-Macrophage Colony-Stimulating Factor;
Humans;
Interleukin-3;
K562 Cells*;
Leukemia;
Leukemia, Myelogenous, Chronic, BCR-ABL Positive;
Leukemia, Myeloid, Acute;
Methylcellulose;
Nucleotides;
Oligodeoxyribonucleotides*;
Precursor Cell Lymphoblastic Leukemia-Lymphoma;
RNA;
RNA, Messenger
- From:Korean Journal of Pediatric Hematology-Oncology
1999;6(2):275-285
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
PURPOSE: The c-myb protooncogene encodes MYB protein that is critical for normal and leukemic hematopoietic cell proliferation and development. It is known that c-myb plays an important role in leukemogenesis as well. Aberrant expression of c-myb is seen in acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), and chronic myeloid leukemia (CML). We reasoned that down regulation of c-myb expression using synthetic antisense oligomers targeted to c-myb mRNA might prove useful antileukemic agents if leukemia cells were more dependent on c-myb function for proliferation than their normal counterparts. To investigate the applying possibility of c-myb antisense oligodeoxynucleotides as the useful antileukemic agents, we examined the cell viability, cloning efficiency and the expression of c-myb mRNA and MYB protein after the exposure of c-myb oligomers on normal bone marrow cells and leukemia cells. MATERIALS AND METHODS: We maintained in short-term suspension culture for 4 days to analyze the effect of c-myb oligomers on normal bone marrow cells and chronic myelocytic leukemia cell line K562. During suspension culture, cell counts and viability were periodically determined, and immediately seeded into duplicate methylcellulose cultures containing recombinanat human interleukin 3 and GM-CSF. Cells placed in semisolid cultures were allowed to grow for an additional 10~12 days. We counted the colonies, and then RNA and protein was extracted from cells cloned in liquified methyl- cellulose cultures. We detected the c-myb mRNA expression by RT-PCR and Southern hybridization analysis and MYB expression by Western hybridization analysis. RESULTS: c-myb sense oligomers had negligible effects on K562 cells growth in short- term suspension culture, while exposure to c-myb antisense oligomers resulted in a daily decline in cell number. In contrast, normal bone marow cell viability and numbers were unaffected by c-myb sense or antisense oligomers exposure. c-myb antisense oligodeoxy nucleotides strongly inhibited or completely abolished growth and colony formation of K562 cells. In contrast, c-myb sense oligomers did not affect. At antisense dose that inhibited leukemic cell growth, normal bone marrow cells survived. Thus, normal and leukemic cells showed the differential sensitivity to the toxic effect of c-myb antisense oligomers. RT-PCR, Southern hybridization analysis and Western hybridization analysis of c-myb antisense-treated K562 cells revealed a complete absence of c-myb mRNA expression and MYB expression. CONCLUSION: Results obtained from these studies suggest that inhibition of c-myb function with antisense oligodeoxynucleotides might eventually form the basis for a molecular biologic approach to leukemia therapy, perhaps most immediately as ex vivo bone marrow purging agent.
