Molecular characterization of a cancer-related single nucleotide polymorphism in the pro-inflammatory interleukin-1B gene.

Interleukin-1ß is a key pro-inflammatory cytokine that has been associated with chronic inflammation and inflammation-related cancer initiation and progression. There are inter-individual differences in IL1B expression which may be due to single nucleotide polymorphisms (SNPs) in the regulatory regions of the gene. We have previously shown that a SNP located in the promoter of the IL1B gene (the IL1B T-31C SNP) was associated with lung cancer risk. Interestingly, the presence of the C allele was also associated with reduced IL1B expression in normal lung tissue of lung cancer patients. In the present study, we found that differential binding patterns of nuclear proteins to oligonucleotide probes containing the IL1B -31C allele compared to those with the T allele were due to specific binding of the transcription factor Yin Yang 1 (YY1). We further found evidence that specific recruitment of YY1 to the -31C region of the IL1B promoter regulated IL1B gene expression using siRNA directed towards YY1. The results indicate that the presence of a C allele at the -31 position may lead to decreased expression of the IL1B gene due to a specific binding of YY1 in lung epithelial cells. Our study provides functional significance of allelic variation at a single locus in the IL1B promoter and contributes to understanding the regulation of IL1B in inflammation-related carcinogenesis.

3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells.

3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ((32)P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of IkappaB-alpha (suggesting activation of NF-kappaB) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-kappaB play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system.

The B[a]P-increased intercellular communication via translocation of connexin-43 into gap junctions reduces apoptosis.

Gap junctions are channels in plasma membrane composed of proteins called connexins. These channels are organized in special domains between cells, and provide for direct gap junctional intercellular communication (GJIC), allowing diffusion of signalling molecules <1 kD. GJIC regulates cell homeostasis and notably the balance between proliferation, cell cycle arrest, cell survival and apoptosis. Here, we have investigated benzo[a]pyrene (B[a]P) effects on GJIC and on the subcellular localization of the major protein of gap junction: connexin-43 (Cx43). Our results showed that B[a]P increased GJIC between mouse hepatoma Hepa1c1c7 cells via translocation of Cx43 from Golgi apparatus and lipid rafts into gap junction plaques. Interestingly, inhibition of GJIC by chlordane or small interference RNA directed against Cx43 enhanced B[a]P-induced apoptosis in Hepa1c1c7 cells. The increased apoptosis caused by inhibition of GJIC appeared to be mediated by ERK/MAPK pathway. It is suggested that B[a]P could induce transfer of cell survival signal or dilute cell death signal via regulation of ERK/MAPK through GJIC.