HMG-box containing protein 1 (HBP1) is a recently described member of the high mobility group of transcription factors. We showed that HBP1 is expressed at low levels in immature myeloid cell lines, and at greatly increased levels following induction of differentiation. We also showed that HBP1 protein interacts physically and functionally with the retinoblastoma (RB) protein. We demonstrated that in human myeloid cell lines which express the myeloperoxidase (MPO) gene, HBP1 enhances the activity of the MPO promoter by binding to promoter DNA. However, its role in regulating myeloid development and differentiation remains to be elucidated. To begin to explore this question, we stably transfected the myeloid cell line, K562, with plasmid constructs derived from pBudCE4.1 (Invitrogen), which overexpress HBP1. K562 cells are a myeloid cell line which can be induced to differentiate (by appropriate chemicals) along erythroid or megakaryocytic pathways. Five sets of bulk cultures and multiple clones derived from each of them were obtained: 1) cell only; 2) CAT control; 3) HBP1 driven by CMV promoter (low level overexpression); 4) HBP1 driven by EF1 promoter (high level overexpression); and 5) HBP1 driven by CMV promoter plus RB driven by EF1 promoter. Overexpression of HBP1 with or without co-overexpression of RB resulted in slower cell growth rates compared with control cells. Cell cycle analysis revealed that HBP1 transfectants displayed marked retardation of S-phase progression by a 5-bromo-2-deoxyuridine (BrdU) incorporation method. Real-time RT-PCR showed that expression of cyclin D1 and cyclin D3 mRNAs significantly decreased in HBP1 transfectants. The D-type cyclins (cyclin D1, D2, and D3) promote cell cycle progression from G1 to S phase by binding to and activating the cyclin dependent kinases Cdk4 and Cdk6. HBP1 transfectants underwent increased apoptosis as demonstrated by intracellular DNA fragmentation, and the binding of annexin V to the cell surface. Consistent with this, substantial increases in Fas ligand mRNA levels were noted in HBP1 transfectants suggesting that apoptosis may be occurring by the Fas/Fas ligand pathway. HBP1 overexpression induced terminal, irreversible erythroid differentiation of K562 cells in the absence of hemin and enhanced hemin-induced erythroid differentiation, as evidenced by hemoglobin production and increased expression of glycophorin A on the K562 cell membrane. During this erythroid differentiation, c-Myc and c-Myb transcripts were downregulated whereas JunB transcription was upregulated. HBP1 overexpression increased TPA-induced induction of megakaryocytic markers such as CD41a and increased cell size as determined by flow cytometry. Similar effects were seen in clones derived from bulk cultures of transfected cells. In summary, overexpression of HBP1 in the myeloid cell line K562 resulted in reduced cell growth rates, increased rates of apoptosis, and increased differentiation of cells toward erythroid or megakaryocytic lineages. Real-time RT-PCR studies suggested that these changes resulted, at least in part, from changes in transcription of critical genes which control cell growth, apoptosis, and differentiation in myeloid cells. Our results support the hypothesis that HBP1 is intimately involved in the control of cell growth, apoptosis, and terminal differentiation in developing myeloid cells.