Regulation of oct-1 gene transcription is different in lymphoid and non-lymphoid cells.

Transcription factor Oct-1 is expressed in all eukaryotic cells acting as a positive or negative regulator of gene transcription and DNA replication. Being a ubiquitous nuclear protein, Oct-1 also takes part in the regulation of tissue-specific gene expression. In this paper, we have found that human oct-1 gene is regulated by two promoters, located in OTF-1 locus upstream of 1U and 1L exons, respectively. The DNA region preceding U exon has a pattern typical of the constitutive gene promoters. The 5'-region upstream of 1L-exon is AT-rich, contains no TATA box, but has two octamer sequences targeted by Oct-1 and Oct-2 proteins. Analysis of promoter activity is carried out by transfection of recombinant plasmids in non-lymphoid HEK293 and lymphoid Raji cells. In non-lymphoid cells, efficiency of transcription from the 1U promoter several times exceeded that from the 1L promoter. The 1U promoter activity is little increased in the presence of an external enhancer. A different expression pattern was observed if the same constructs were transfected into lymphoid Raji cells. In this case, the level of transcription from the 1L promoter (the L-2 fragment, containing a proximal octamer site) in the presence of the enhancer was significantly higher than that of any fragments containing 1U promoter. It was shown that distal regions of both 1U and 1L were capable of silencing activity. In Raji cells, the enhancer completely overcomes the activity of U silencer, but only partly overcomes that of L silencer. Our data on tissue-specific features of 1L promoter and interaction of both oct-1 promoters with enhancer and silencers in different cell types point to a fine tissue-specific regulation of the oct-1 gene expression, especially in lymphoid cells.

Antisense CD11b integrin inhibits the development of a differentiated monocyte/macrophage phenotype in human leukemia cells.

Macrophage-like development of myeloid leukemia cells which can be induced by agents such as phorbol esters (TPA) is accompanied by integrin expression and cell adhesion. Thus, in differentiating myeloid leukemia cells CD11b is predominantly expressed which can associate with CD18 to form the functional heterodimeric integrin Mac-1. To elucidate the role of cell adhesion during macrophage-like differentiation, we transfected human U937 myeloid leukemia cells with a vector containing the CD11b gene in antisense orientation. Expression of the CD11b antisense gene in stably transfected U937 cells (as-CD11b cells) resulted in an attenuated response to TPA. As-CD11b cells demonstrated poor adhesion to solid substrate upon TPA treatment in contrast to U937 control cells. Constitutive expression of c-myc in as-CD11b transfectants was higher than in control cells and failed to be repressed by TPA treatment. Moreover, unlike control cells, antisense transfectants failed to induce expression of early response genes such as c-jun and the redox factor ref-1 upon TPA stimulation. Consequently, the induction of monocytic differentiation markers such as the activity of alpha-naphthyl acetate esterase, the capacity to reduce nitroblue tetrazolium and the expression of the vimentin gene was much lower in antisense transfectants than in control U937 cells. According to the failure to undergo a monocytic differentiation program, TPA treatment of as-CD11b cells resulted in a progressively increasing amount of apoptotic cells whereas the differentiated population of U937 control cells remained alive. Taken together, these data suggest that the integrin-mediated (particularly CD11b-mediated) adhesion of myeloid leukemia cells in the course of induced monocytic differentiation is crucial for cell attachment, development of a monocytic phenotype and subsequent survival.