ABSTRACT
Tracheobronchial epithelial (TBE) cells that normally do not express the squamous cell differentiation marker gene, SPR1, can be induced to produce it by 12-O-tetradecanoylphorbol-13-acetate (TPA). The regulation of SPR1 gene expression by TPA occurs, in part, at the transcriptional level in primary human and monkey TBE cells. Using a transient transfection assay, we observed that TPA stimulates the activity of the reporter gene, chloramphenicol acetyltransferase, by 2-4-fold in transfected TBE cells. However, this chloramphenicol acetyltransferase activity is cell type-specific with significantly less activity in transformed epithelial cell lines and no activity in non-epithelial cell types. TPA-dependent stimulation can also be demonstrated by co-transfection with plasmid DNAs that overexpress the JUN family of proteins, especially c-JUN. Overexpression of c-JUN and TPA treatment synergistically stimulate the SPR1 promoter activity by more than 40-fold. Deletion analysis of the promoter region demonstrates that the DNA fragment of the first 98 base pairs of the 5'-flanking region contains the basal promoter activity, while the region between -162 and -96 contains the cis-enhancer elements for both the basal and TPA/c-JUN-stimulating promoter activities. This observation is supported by in vivo genomic footprinting studies that reveal persistent protections in the following motifs of this region: -141 TRE, -131 GT, -123 ETS-like, and -111 TRE-like motifs and in the enhanced protections in -141 TRE and -111 TRE-like motifs in cells after the TPA treatment. Site-directed mutagenesis in this region demonstrates the involvement of both -141 TRE and -111 TRE-like motifs in TPA/c-JUN-dependent stimulation as well as enhanced basal transcriptional activity. However, it is primarily the -111 TRE-like motif that is involved in the mediation of the enhanced basal promoter activity of the human SPR1 gene. These results are further supported by gel mobility shift assays that demonstrate the involvement of c-JUN and these TRE motifs in the formation of the DNA-protein complex.
Subject(s)
Bronchi/metabolism , Cell Differentiation , Gene Expression , Promoter Regions, Genetic , Protein Biosynthesis , Proteins , Proto-Oncogene Proteins c-jun/metabolism , Trachea/metabolism , Transcription, Genetic , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Biomarkers , Cell Nucleus/metabolism , Cells, Cultured , Chloramphenicol O-Acetyltransferase/biosynthesis , Consensus Sequence , Cornified Envelope Proline-Rich Proteins , Epithelium/metabolism , Gene Expression/drug effects , Haplorhini , Humans , Membrane Proteins , Molecular Sequence Data , Oligodeoxyribonucleotides , Polymerase Chain Reaction , Proto-Oncogene Proteins c-jun/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Sequence Deletion , Tetradecanoylphorbol Acetate/pharmacology , TransfectionABSTRACT
Sixty-one patients (82 eyes) were studied after argon laser trabeculoplasty (ALT) to determine the lasting efficacy of such treatment. This investigation, now in its fourth year, was prospective, and the information derived was analysed with the aid of a computer. Success was defined as intraocular pressure (IOP) below baseline (22 mmHg). The mean follow-up time was 24.5 months, when the success rate was 74% compared with 75% at three months. Success declined to 45% at 42 months. No significant difference was noted when (a) first lasered eyes of all patients and those fellow eyes treated were analysed separately, (b) when right and left eyes were analysed separately, nor (c) when patients were divided into two treatment groups, (I) 100 burns at 1 W, and (II) 65 burns at 850 mW. Eight of 11 eyes showed progressive postlaser field loss despite below-baseline intraocular pressures. ALT is an alternative to carbonic anhydrase inhibitor therapy, with a success rate of 66.7% at two years. However, repeat ALT was successful in only 25% of patients seven months after treatment.
