Nina E. Landvik

The TERT-CLPTM1L lung cancer susceptibility variant associates with higher DNA adduct formation in the lung

Genome-wide association studies have provided evidence that common variation at 5p15.33 [telomerase reverse transcriptase (TERT)-cleft lip and palate transmembrane 1-like (CLPTM1L)], 6p21.33 and 15q25.1 (CHRNA5-CHRNA3) influences lung cancer risk and cancer types with strong environmental risk factors. To independently validate these associations, we compared 5p15.33 (rs402710, rs401681), 6p21.33 (rs4324798) and 15q25.1 (rs1051730, rs16969968 and rs8034191) genotypes in 365 non-small cell lung cancer cases and 440 controls. Consistent with published data, variant genotypes of 5p15 (rs402710), 6p21 and 15q25 showed dose-dependent associations with lung cancer risk. To examine if variants influence the impact of environmental risk factors on lung carcinogenesis, we studied the relationship between genotype and levels of bulky aromatic/hydrophobic DNA adducts in lung tissue adjacent to tumor from 204 lung cancer cases. The risk allele of rs402710 (TERT-CLPTM1L locus) was associated with significantly higher levels of bulky aromatic/hydrophobic DNA adducts (P = 0.02). These data demonstrate a potential association between the TERT-CLPTM1L variant and levels of bulky DNA adducts measured by (32)P-postlabeling and hence a basis for susceptibility to the development of lung cancer.

A specific interleukin-1B haplotype correlates with high levels of IL1B mRNA in the lung and increased risk of non-small cell lung cancer

Epidemiological evidence suggests a relationship between chronic inflammation and lung cancer. Inflammation in the lung may be modulated by host genetic factors such as polymorphisms in inflammatory genes. Identification of polymorphisms in inflammatory genes may help understanding interindividual differences in susceptibility to lung cancer. We have investigated single-nucleotide polymorphisms (SNPs) and their haplotypes in the regulatory region of the IL1B gene in association to non-small cell lung cancer (NSCLC) risk. Our previous work showed that two promoter SNPs C-511T and T-31C modulated NSCLC risk. In the present study, we show that G-3893A and G-1464C located in the enhancer region of the IL1B gene may also affect this risk, with odds for developing NSCLC being 0.69 [95% confidence interval (CI), 0.52-0.92] for -3893 A-allele and 0.63 (95% CI, 0.47 - 0.83) for -1464 C-allele. The associations were particularly prominent in patients with TP53 mutations in the tumor. Inference of the haplotype structures showed that -3893 G, -1464 G, -511 C and -31 T formed a specific haplotype (GGCT) with near complete linkage disequilibrium in lung cancer patients but not in controls. Furthermore, the risk haplotype (GGCT) was present in 65% of cases compared with 36% of controls. Quantitative analysis of RNA in normal lung tissue of the patients showed that the risk haplotype was correlated with significantly higher IL1B messenger RNA (mRNA) levels compared with the non-risk haplotype (ACTC). These data suggest that a specific IL1B haplotype associated with increased IL1B gene expression increases the risk of NSCLC.

A combination of functional polymorphisms in the CASP8, MMP1, IL10 and SEPS1 genes affects risk of non-small cell lung cancer.

Exposure to tobacco smoke as well as environmental and occupational factors is the major cause of lung cancer. Non-small cell lung cancer (NSCLC) is the major histological type. Genes in pathways affecting inflammation, cellular stress and apoptosis are important, and the extent of inflammation in the lung could be affected by polymorphisms modifying these responses. In the present study we have investigated whether a combination of potential functional polymorphisms in genes related to inflammation may modulate risk of NSCLC. Eleven functional polymorphisms in nine genes were analyzed for association with risk of NSCLC in 882 subjects from the Norwegian population. The results showed that individuals carrying combination of three functional polymorphisms in the caspase-8, matrix metalloproteinase-1, seleno-protein S1, and interleukin-10 genes had two-fold increased risk of NSCLC (OR 2.06 (95% CI, 1.19-3.47) whereas individuals with four risk genotypes had 4.62-fold increased risk (OR 4.62, 95% CI, 1.69-12.63). These results highlight the need to investigate the combinatory effects of multiple SNPs in the carcinogenesis of the lung.

Functional characterization of polymorphisms in the human TFPI gene

Tissue factor pathway inhibitor (TFPI) is the primary physiological inhibitor of tissue factor (TF) induced coagulation. Low plasma TFPI levels have been shown to be associated with increased risk of arterial and venous thrombosis. Several clinical studies have reported that single nucleotide polymorphisms (SNPs) in the regulatory regions of the gene, such as the -287T/C, the -399C/T, and the -33T/C SNPs, may affect plasma TFPI levels. However, molecular studies investigating the functionality of the polymorphisms are lacking. In this study, we found that the -287C and -399T alleles affected the activity of the promoter using a reporter gene system. This was also the case for the -33T/C polymorphism. An association regarding the transcriptional activity of the reporter gene was detected between the -287C allele and the -33T/C polymorphism. Analysis of the polymorphic sites with electrophoretic mobility shift assay (EMSA) showed that all three polymorphisms potentially alter DNA-protein interactions. Based on these findings, we speculate that the -287C and the -33C alleles can be associated with lowered risk of thrombosis.

Signalling pathways involved in 1-nitropyrene (1-NP)-induced and 3-nitrofluoranthene (3-NF)-induced cell death in Hepa1c1c7 cells

We previously reported that 1-nitropyrene (1-NP) and 3-nitrofluoranthene (3-NF) elicited apoptotic cell death as well as non-apoptotic programmed cell deaths (PCDs) with paraptotic and necroptotic characteristics, respectively. In the present study, we have further confirmed and extended these findings. Flow cytometric analyses of 1-NP-exposed/3NF-exposed Hepa1c1c7 cells revealed that caspase-3 was only activated in the subpopulation of cells corresponding to that with classic apoptotic morphology. Immunocytochemical analysis indicated that leucocyte elastase inhibitor-derived DNaseII (LEI/L-DNaseII), apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were more clearly translocated to the nucleus following 3-NF exposure than after 1-NP. These 3-NF-induced changes in AIF and EndoG translocation were reduced by necrostatin-1, an inhibitor of necroptotic cell death. Both compounds lead to accumulation of lipid droplets and induced DNA damage. Activation of checkpoint kinase (CHK) 1 and H2AX, but not ataxia telangiectasia mutated and CHK2, were observed. Furthermore, inhibition of p53 using pifithrin-alpha reduced the cell death induced by both compounds, suggesting a role of DNA damage/CHK1/p53 pathway in the death process. 1-NP-induced cell death was in addition characterized by increased oxidative damage and intracellular accumulation of Ca(2+). These findings further support the notion that 1-NP elicited apoptotic cell death and PCD with paraptotic characteristics, while 3-NF induced apoptosis and a PCD with necroptotic features.

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

Interleukin‐1β 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.

Functional effect of polymorphisms in 15q25 locus on CHRNA5 mRNA, bulky DNA adducts and TP53 mutations

Genome‐wide association studies have demonstrated that genetic polymorphisms influence the risk of developing lung cancer. Nicotinic acetylcholine receptor alpha3, alpha5 and beta4 genes (CHRNA3, CHRNA5 and CHRNB4) cluster at the 15q25.1 lung cancer susceptibility locus. We genotyped 310 patients with non‐small cell lung cancer and a control group of 348 cancer‐free individuals for seven sequence variants located in CHRNA3 and CHRNA5 genes. Two of the polymorphisms (rs3829787 and rs3841324) statistically influenced the risk of developing lung cancer. We found that four of the variants (rs3829787, rs3841324, rs588765 and rs3743073) were associated with differential levels of genetic alterations measured as the levels of hydrophobic DNA adducts in the adjacent histologically normal tissue of the lung cancer patients and as TP53 mutations in their lung tumors. The seven sequence variants formed three haplotypes with a frequency above 5%. The two most frequent haplotypes were associated with the risk of developing lung cancer and with smoking‐related DNA alterations. We also found an association between CHRNA5 mRNA levels and the sequence variants or haplotypes. In conclusion, our results showed that several of the polymorphisms and their haplotypes in CHRNA5/CHRNA3 genes may have functional effects on (i) CHRNA5 mRNA levels, (ii) polycyclic aromatic hydrocarbon–DNA adduct levels, (iii) TP53 mutations and (iv) susceptibility to lung cancer.

Functional analysis of a lung cancer risk haplotype in the IL1B gene regulatory region

Interleukin-1β (IL-1β), encoded by the IL1B gene, is a cytokine important in regulation of the inflammatory response. Elevated levels of IL1B expression have been associated with risk of gastric and lung cancer. We previously reported that a certain haplotype containing four single-nucleotide polymorphisms (SNPs) (−3893G, −1464G, −511C and −31T; GGCT) in the IL1B gene regulatory region was associated with lung cancer risk and increased expression of the IL1B gene in the lung. In the present study, we have cloned the two haplotypes that were either protective (ACTC) or increasing lung cancer risk (GGCT) in a luciferase reporter vector system. We also cloned the IL1B −3893 and −1464 SNPs that were found to be associated with risk of lung cancer. The haplotype associated with lung cancer risk showed higher transcriptional activity in the human lung epithelial A549 cell line in vitro. We also found that the IL1B −1464C allele increased transcriptional activity compared with the −1464G allele in the tumor necrosis factor α-stimulated cells, as well as specific transcription factor binding patterns to the IL1B −1464C allele. Interestingly, in vitro results showed a similar expression pattern as observed in the normal lung tissues of lung cancer patients reported earlier.

Gene variants and lung cancer risk

Active smoking accounts for 90% of lung cancer deaths, but only 15% of smokers develop lung cancer. Screening for single-nucleotide polymorphisms (SNPs) in candidate pathway genes is an approach to defining high-risk subgroup. Polymorphisms have been investigated to determine whether variations in phase I and phase II metabolism, DNA repair genes and inflammatory genes predict risk of lung cancer, and susceptibility loci have been identified in GWA studies. Pulmonary inflammation together with chemical exposure may potentiate the carcinogenic process, underlying the importance of inflammation in cancer development. Interleucin-1beta (IL-1β) encoded by the IL1B gene, has been associated with chronic inflammation and plays an important role in lung inflammatory diseases, including COPD and lung cancer. There are large interindividual differences in IL1B gene expression, which may be explained genetically by presence of SNPs scattered throughout the regulatory region (rSNPs) of the gene. Our studies have shown that several polymorphisms located in the promoter and the enhancer regions of the IL1B gene may affect risk of non-small cell lung cancer. These rSNPs may affect IL1B expression by changing the binding affinity of transcription factors or creating novel transcription binding sites. Notably, rSNPs at -3893 G, -1464 G, -511 C and -31 T position formed a specific haplotype (GGCT) with near complete linkage disequilibrium in lung cancer patients, but not in controls where the risk haplotype (GGCT) was present in 65% of cases compared with 36% of controls. The risk haplotype was correlated with significantly higher IL1B mRNA levels in the lung of lung cancer patients. Particularly, we found that the T to C SNP at the -31 position which is located close to the transcription start site of the gene resulted in binding of different transcription factors to the C and T variants. We have further characterized the specific transcription factors binding to the -31 SNP. In DNA-protein interaction studies using Electrophoretic Mobility Shift Assay (EMSA) and human lung epithelial cells we found evidence that the transcription factor Ying Yang 1 (YY1) bound preferentially to the C SNP. The binding of YY1 to the C SNP was further confirmed using Chromatin Immunoprecipitation (ChIP) assay in human lung epithelial cells. In summary, we have characterized transcription factors that may interact differentially with base changes in the SNPs and thereby may explain the biological effects of the SNPs in modifying the risk to lung cancer.

Criteria for grouping of manufactured nanomaterials to facilitate hazard and risk assessment, a systematic review of expert opinions

ABSTRACT With the emergence of nanotechnology the number of manufactured nanomaterials (MNM) in production and use is constantly increasing. Exposure of workers to MNM is of concern, because still much is unknown about health effects. MNM may have different properties, testing of each material is time consuming and costly. Experts have proposed various approaches to categorize MNM to facilitate risk assessment of human health effects based on shared properties of various materials. A systematic literature survey was undertaken to identify expert opinions on grouping of MNM published between the years 2000 and 2015. We summarized and synthesized the opinions according to a systematic review of text and opinion. We identified 22 articles that fulfilled our inclusion criteria reporting 17 proposals with three proposals for groups and 14 proposals for criteria for grouping. Five proposals suggested one or more of the following groups of concern: fibrous, biopersistent, high solubility with high toxicity, chemically active. Criteria proposed in multiple studies were: viable testing options, mode of action, physicochemical properties predicting toxicity. We conclude that a limited number of groups have been proposed to categorize MNM according to human health concern. Further research should be conducted to underpin the proposed groups with empirical evidence. Graphical abstract Figure. No Caption available. HighlightsHealth effects of manufactured nanomaterials (MNM) is of concern.To facilitate hazard assessment, various approaches have been proposed to group MNM.This systematic literature search, identified twenty‐two articles reporting 17 proposals.Of these three were proposals for groups and fourteen for criteria for grouping.