Human 15-lipoxygenase-1 and telomerase gene expression in cancer and inflammation

Sammanfattning: Human 15-lipoxygenase type 1 (15-LOX-1) is an enzyme that catalyzes the oxygenation of free or membrane-bond polyunsaturated fatty acids containing at least one bis-allylic methylene moiety. The enzyme has been proposed to be involved in various physiological and pathophysiological activities including inflammation, apoptosis, cell maturation and tumorigenesis. However, its expression and function has not been well studied yet. Telomerase, a ribonucleoprotein reverse transcriptase, synthesizes TTAGGG telomeric repeats which are essential for the stability and integrity of chromosomes and is involved in cell transformation and lymphocyte activation during the processes of tumorigenesis and immune response, respectively. In this thesis, the studies aimed at defining the transcriptional regulation of 15-LOX-1 and telomerase. We also delineated the effect of 15-LOX-1 upregulation in lung epithelial cells on chemokine production. In paper I, we demonstrated that hyper- and hypomethylation of CpG islands in the 15- LOX-1 promoter region are intimately associated with the transcriptional repression and activation of the 15-LOX-1 gene, respectively. Inhibition of DNA methylation in the 15- LOX-1 negative Hodgkin lymphoma (HL) cell line L428 restores the inducibility of 15- LOX-1 by IL-4. Our results suggest that demethylation of the 15-LOX-1 promoter is a prerequisite for gene transactivation, which contributes to tissue- and cell-type-specific regulation of 15-LOX-1 expression. In paper II, we identified that three functional STAT6 binding sites are required for full activation of the 15-LOX-1 promoter in the 15- LOX-1 positive HL cell line L1236. We found that this region was occupied by STAT6 in L1236 cells but not in L428 cells. Furthermore, DNA hypomethylation and histone hyperacetylation were observed within the core promoter region of 15-LOX-1 only in L1236 cells. Our data indicate that STAT6 activation and chromatin remodeling by DNA demethylation and histone acetylation are crucial for transcriptional activation of 15- LOX-1 in cultured HL cells. In paper III, we ectopically expressed 15-LOX-1 in the human lung epithelial cell line A549. We found that over-expression of 15-LOX-1 in A549 cells leads to increased release of the chemokines Mip-1alpha, RANTES and IP-10, and thereby increases the recruitment of immature dendritic cells, mast cells and activated T cells. Our data suggest that an increased expression of 15-LOX-1 in lung epithelial cells is a pro-inflammatory event in the pathogenesis of asthma and other inflammatory lung disorders. In paper IV, we showed that SET and MYND domaincontaining protein 3 (SMYD3) directly trans-activates the human telomerase reverse transcriptase (hTERT) gene. We found that SMYD3 occupies its binding motifs in the hTERT promoter and is required for maintenance of histone H3-K4 trimethylation in this region. Knocking down SMYD3 in some tumor cell lines abolished trimethylation of H3-K4, attenuated the occupancy by the transactivators c-Myc and Sp1, and led to diminished histone H3 acetylation in the hTERT promoter region, which was coupled with down-regulation of hTERT mRNA and telomerase activity. These results suggest that SMYD3-mediated trimethylation of H3-K4 functions as a licensing element for subsequent transcription factors binding to the hTERT promoter.