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 μM 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 RARα 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 RARα 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.