Characterization of two novel retinoic acid-resistant cell lines derived from HL-60 cells following long-term culture with all-trans-retinoic acid.
Either all-trans-retinoic acid (RA) or vitamin D3 (VD) induces differentiation of the myeloid leukemia cell line HL-60. RA is available for the treatment of acute promyeloleukemia, although the development of resistance to the agent is a serious problem for differentiation-inducing therapy. To approach the mechanisms of resistance to RA, we developed two novel cell lines, HL-60-R2 and R9, which were subcloned after exposure to increasing concentrations of RA. The growth rate of HL-60-R2 cells was significantly increased by RA treatment, whereas the growth rate of HL-60-R9 was not affected. RA induces apoptosis in the parental HL-60 cells. The number of apoptotic cells, however, was not increased and nitroblue tetrazolium (NBT) reduction was not altered by 1 microM RA in either of the cloned cell lines. Treatment with VD induced monocytic differentiation and increased the expression of CD11b in HL-60 and HL-60-R9 cells, but not in HL-60-R2 cells. Flow cytometric and G-banding analysis demonstrated that R2 cells were near-triploid. The sequencing analysis revealed a deletion of three nucleotides in the sequence of the RAR alpha gene in HL-60-R9 cells, resulting in deletion of codon 286. No mutation was found in HL-60-R2 cells. Taken together, these data indicate that the resistance to RA is caused by the mutation in RAR alpha of HL-60-R9, but by other factor(s), which also affect the VD-response pathways, in HL-60-R2. The abnormal response to VD may be associated with the abnormal ploidy of the R2 cells.
Mori
J
J
Department of Aging Medicine and Geriatrics, Shinshu University School of Medicine, Nagano.
Suzuki
S
S
Hara
M
M
Kaneko
A
A
Yamashita
K
K
Kumagai
M
M
Sakuma
T
T
Kakizawa
T
T
Yamazaki
M
M
Takeda
T
T
Miyamoto
T
T
Ichikawa
K
K
Hashizume
K
K
D000970:Antineoplastic Agents / Q000494:pharmacology*; D002453:Cell Cycle; D002455:Cell Division / Q000187:drug effects; D002869:Chromosome Aberrations; D019008:Drug Resistance, Neoplasm; D018922:HL-60 Cells; D006801:Humans; D016177:Macrophage-1 Antigen / Q000032:analysis; D018168:Receptors, Retinoic Acid / Q000032:analysis; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000032:analysis; D014212:Tretinoin / Q000494:pharmacology*; D014807:Vitamin D / Q000494:pharmacology
D000970:Antineoplastic Agents; D016177:Macrophage-1 Antigen; C000607071:RARA protein, human; D018168:Receptors, Retinoic Acid; D000072482:Retinoic Acid Receptor alpha; D047488:Retinoid X Receptors; D014157:Transcription Factors; D014807:Vitamin D; D014212:Tretinoin