IDENTIFICATION OF TRANSCRIPTION FACTORS EXPRESSED DURING ATRA-INDUCEDNEUTROPHIL DIFFERENTIATION OF HL60 CELLS

A recent clinical therapeutic initiative has been the use of chemical agents which induce the leukaemic cells to overcome their block in dif ferentiation. In order to understand this block the cascade of molecul ar events needs to be characterized. Haemopoietic differentiation is u ltimately controlled at the level of gene transcription which is media ted by an array of transcription factors. Many transcription factors c ontain similar structural protein sequences, and we have used an RT-PC R-based approach to isolate sequences, from transcription factor gene families which share similar domains. Degenerate primers corresponding to the TFIIIA zinc-finger consensus amino acid sequences and to the P OU-homeodomain and POU-specific domain were used to amplify genes on t he basis that they contained similarities in structural motifs shared within these families of transcription factors. A serum-independent HL 60 cell line was induced towards the neutrophil lineage by treatment w ith all-trans retinoic acid (ATRA) for 24 h, CD38(+) cells committed t owards this lineage were enriched and a population of these cells trea ted with dihydroxyvitamin D-3 to induce neutrophil maturation, RNA ext racted from uninduced, ATRA-induced CD38(+) cells, and vitamin D-3 tre ated maturing cell cultures were amplified using the degenerate primer s. PCR fragments were cloned, sequenced, clustered into homologous gro ups, and the group sequences searched on the GenBank database. The Oct 1 transcription factor, and a very close homologue, KIAA0144, was ide ntified using the POU family primers. The zinc-finger primers identifi ed three zinc-finger genes. The pattern of gene expression was suggest ed from the number of clones in each group at neutrophil commitment an d maturation. The differential expression of the genes in the zinc fin ger and POU families will lead to a better understanding of the cascad e of gene expression which occurs following ATRA-induced differentiati on.