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HL-60 cells differentiate into granulocyte-like cells by all-trans retinoic acid (ATRA) treatment, and the cellular proliferation is markedly reduced during the differentiation. To elucidate the molecular mechanisms of the growth arrest during the cellular differentiation, we examined the regulated expression of the thymidylate synthase (TS) gene. Northern blot analysis revealed that the expression of the TS gene was almost suppressed in the differentiated HL-60 cells. The change in the levels of nuclear factors, NF-TS2 and NF-TS3, that bind to the 5'-terminal regulatory region of the human TS gene was examined during the differentiation of the HL-60 cells. The amount of NF-TS2 did not change significantly during the differentiation, whereas that of NF-TS3 clearly increased as the cells differentiated. We previously reported that NF-TS2 and NF-TS3 bind to the sequence around the initiation codon ATG of the human TS gene. Further analyses revealed that the DNA sequences of NF-TS2 and NF-TS3 are very similar, and the first and second positions of the ATG triplet codon are important for the formation of rigid DNA-protein complexes. The present findings concerning the binding site and changes during the differentiation induced by ATRA treatment are very similar to those previously reported on the differentiation induced by 1,25-dihydroxyvitamin D3 treatment. These findings suggest that NF-TS3 is involved in regulating the expression of the human TS gene during the differentiation of HL-60 cells, regardless of the terminal cell type: macrophage-like cells or granulocyte-like cells.

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Characterization of Nuclear Factors that Bind to the Human Thymidylate Synthase Gene in HL-60 Cells Differentiated by All-trans Retinoic Acid Treatnent.