gp91phox is a subunit of the phagocyte respiratory burst oxidase catalytic unit. Transcription of CYBB, the gene encoding gp91 phox , is restricted to terminally differentiated phagocytic cells. An element in the proximal CYBB promoter binds a protein complex, referred to as hematopoiesis-associated factor (HAF1), that is necessary for interferon-␥ (IFN␥)-induced gp91 phox expression. In these investigations, we determined that HAF1 was a multiprotein complex, cross-immunoreactive with the transcription factors PU.1, interferon regulatory factor 1 (IRF-1), and interferon consensus sequencebinding protein (ICSBP). In electrophoretic mobility shift assay, the

The DNA binding affinity of HoxA10 is increased by partnering with Pbx proteins. A consensus sequence for Pbx1-HoxA10 DNA binding has been derived, but genuine target genes have not been identified. We noted that the derived Pbx-HoxA10 DNA-binding consensus is similar to a repressor element in the CYBB promoter. The CYBB gene, which encodes the respiratory burst oxidase component gp91phox , is expressed only in myeloid cells that have differentiated beyond the promyelocyte stage. In these studies, we demonstrate that interferon ␥ (IFN-␥)-induced differentiation of myeloid cell lines abolishes in vitro Pbx-HoxA10 binding to either the derived consensus or the similar CYBB sequence. We also demonstrate that HoxA10, overexpressed in myeloid cell lines, represses reporter gene expression from artificial promoter constructs with Pbx-HoxA10 binding sites. We determine that HoxA10 has endogenous repression domains that are not functionally altered by IFN-␥ treatment. However, IFN-␥-induced differentiation of myeloid cell lines leads to HoxA10 tyrosine phosphorylation, which decreases in vitro DNA binding to PbxHoxA10 binding sites. Therefore, these investigations identify the CYBB gene as a potential target for HoxA10 and define repression of genes expressed in mature myeloid cells as a novel role for HoxA10 during myeloid differentiation.

MATERIALS AND METHODS Monoclon~t antibody ?O c-myc protein 19El0) [8] was

Previous studies have shown that the tumour-promoting phorbol ester 12-O-tetradecanoyl phorbol-13 acetate (TPA) induces both morphological and functional differentiation in SH-SY5Y human neuroblastoma cells (Påhlman et al., 1981). In order to investigate the role of protein kinase C (PKC) in TPA-induced maturation of SH-SY5Y cells, we have used staurosporine, which is a potent inhibitor of protein kinases including PKC. Treatment of SH-SY5Y cells with 25 nM staurosporine for 72 hours caused an appearance of long, neuritelike processes with varicosities, terminated by growth cones. The morphological differentiation was accompanied by a cessation of DNA synthesis, induction of growth associated protein 43 (GAP-43), and neuropeptide Y (NPY) mRNA. These effects of staurosporine were comparable to those elicited by TPA. Staurosporine further induced a time-dependent increase in the expression of tyrosine hydroxylase protein and a 30-fold increase in the concentration of noradrenaline. TPA only induced a marginal increase in tyrosine hydroxylase expression. Both TPA and staurosporine induced an appearance of voltage-gated Ca2+ channels in SH-SY5Y cells detected with single-cell fluorescent measurements using fura-2. The Ca2+ channels were found almost exclusively in growth cones and varicosities. Staurosporine inhibited both basal and a TPA-induced phosphorylation of an endogenous 80kDa PKC substrate (p80), and also blocked c-fos proto-oncogene mRNA expression induced by the phorbol ester. Bryostatin 1, a potent activator of PKC, has failed to induce morphological or functional differentiation in SH-SY5Y cells (Jalava et al., 1990). Incubation of SH-SY5Y cells in the presence of 100 nM bryostatin 1 for 24 hours caused a complete disappearance of all immunoreactive alpha-, beta-, and zeta-PKC. The level of epsilon-PKC decreased by 70%. Staurosporine induced a partial translocation of the epsilon-isoenzyme but it failed to cause down-regulation of epsilon-PKC. Bryostatin 1-treatment did not interfere in the ability of staurosporine to induce morphological differentiation, cessation of DNA synthesis, and GAP-43 and NPY mRNA expression. The ability of staurosporine to stimulate tyrosine hydroxylase expression and to increase cellular content of noradrenaline was also unaffected. Taken together the results of this study show that staurosporine induces a mature neuronal noradrenergic phenotype in SH-SY5Y cells through an alpha-, beta-, and zeta-PKC-independent pathway.

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