Socio-demographic, lifestyle and health characteristics among snus users and dual tobacco users in Stockholm County, Sweden

<p>Abstract</p> <p>Background</p> <p>Socio-demographic and lifestyle characteristics of snus users have not been systematically described. Such knowledge is pivotal for tobacco control efforts and for the assessment of health effects of snus use.</p> <p>Methods</p> <p>A cross-sectional study was conducted, based on the Stockholm Public Health Survey, including a population-based sample of 34,707 men and women aged 18-84 years. We examined how socio-demographic, lifestyle and health-related characteristics were associated with the prevalence of current daily snus use, smoking and dual tobacco use. Logistic regression was used to calculate odds ratios of prevalence (ORs) and 95% confidence intervals (CIs).</p> <p>Results</p> <p>Low educational level (OR = 1.60, CI = 1.41-1.81 and OR = 1.49, CI = 1.17-1.89, for men and women respectively), as well as occupational class and low income were associated with snus use. Some unfavourable lifestyle characteristics, including risky alcohol consumption (males: OR = 1.81, CI = 1.63-2.02; females: OR = 1.79, CI = 1.45-2.20), binge drinking and low consumption of fruit and vegetables were also associated with snus use. In contrast, physical inactivity and overweight/obesity were not, nor was perceived health. The prevalence of smoking followed steeper gradients for social as well as lifestyle characteristics. Overweight and obese men were however less often smokers. Perceived poor general health and psychological distress were highly related to smoking. Social disadvantage, as well as unhealthy lifestyle and self-reported poor health were strongly associated with dual use. There were limited differences between men and women.</p> <p>Conclusions</p> <p>The social, lifestyle and health profiles of exclusive snus users in Stockholm County are less favourable than those of non-users of tobacco, but more advantageous than those of exclusive smokers. This knowledge should guide tobacco control measures as well as the interpretation of health risks linked to snus use.</p

The region of XRCC1 which harbours the three most common nonsynonymous polymorphic variants, is essential for the scaffolding function of XRCC1

XRCC1 functions as a non-enzymatic, scaffold protein in single strand break repair (SSBR) and base excision repair (BER). Here, we examine different regions of XRCC1 for their contribution to the scaffolding functions of the protein. We found that the central BRCT1 domain is essential for recruitment of XRCC1 to sites of DNA damage and DNA replication. Also, we found that ectopic expression of the region from residue 166 to 436 partially rescued the methyl methanesulfonate (MMS) hypersensitivity of XRCC1-deficient EM9 cells, suggesting a key role for this region in mediating DNA repair. The three most common amino acid variants of XRCC1, Arg194Trp, Arg280His and Arg399Gln, are located within the region comprising the NLS and BRCT1 domains, and these variants may be associated with increased incidence of specific types of cancer. While we could not detect differences in the intra-nuclear localization or the ability to support recruitment of POLβ or PNKP to micro-irradiated sites for these variants relative to the conservative protein, we did observe lower foci intensity after micro-irradiation and a reduced stability of the foci with the Arg280His and Arg399Gln variants, respectively. Furthermore, when challenged with MMS or hydrogen peroxide, we detected small but consistent differences in the repair profiles of cells expressing these two variants in comparison to the conservative protein

Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA

<div><p>Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA^−/− cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA^−/− cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.</p> </div

The APIM sequence in XPA is sufficient and necessary for interaction with PCNA.

<p>(A) Sequence alignment of the APIM sequence in XPA (aa 161–170 in human XPA) from different species compared with the APIM sequence in hABH2. The colors are given by Clustal X. (B) Dot blot with the human XPA APIM-peptide. The hABH2 APIM-peptide and its mutant are included as positive and negative controls, respectively (also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049199#pone.0049199-Gilljam1" target="_blank">[27]</a>). Grey lines: dots from the same blot. (C) Images of YFP-tagged XPA_161−167 co-expressed with CFP-tagged PCNA in live cycling HeLa cells. Yellow dots in the merged picture illustrate colocalization. Bar: 5 µM. (D and E) N_FRET measurements in HeLa cells. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET) (D) and 700 (YFP), 800 (CFP), 700 (FRET) (E). CFP/YFP (vectors only) and CFP-PCNA/YFP-PCNA were used as negative and positive controls, respectively (mean ± SEM, n = 24–53 in D and n = 10–34 in E). (F) Overexpressed tagged proteins in live cycling XPA^−/− cells. Yellow dots in the merged picture illustrate colocalization. Bar: 5 µM. (G). N_FRET measurements in XPA^−/− cells. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET) (mean ± SEM, n = 25–66). The P-values (D, E and G) are derived by unpaired t-test.</p

Model describing the role of direct XPA-PCNA interaction for efficient NER after UVR.

<p>To clarify the essence of our hypothesis, only the XPA dimer, XAB2, and RPA of the NER proteins are specified, and the NER complex (yellow) represents the other NER proteins in the model. The grey proteins mark proteins containing the PIP-box, the green mark proteins containing APIM, the blue donut marks PCNA and the red hooks mark 6-4 PPs and CPDs. (A) Optimal NER. (B) Reduced NER due to mutated APIM sequence in XPA.</p

Complete reconstitution of XPA^−/− cells requires XPA with intact APIM.

<p>(A) Cell proliferation after UV-B treatment measured by MTT assay. The data is normalized against untreated day 1. One representative out of three experiments is presented. Data presented is the average of 6 wells ± SD. (B) Normalized XPA intensity measured by in-cell western (LI-COR Bioscience) (mean ± SD, n = 6). The XPA intensity is normalized against the DNA content using Draq5. (C) <i>Left panel:</i> Histograms of 6-4 PP positive cells, untreated, and 0, 2 and 4 h after UV-B. The cells with fluorescent intensity above the dashed line are defined as 6-4 PP positive. The numbers in the bottom row indicate % 6-4 PP positive cells 4 h after UVR. <i>Right panel:</i> Graphic presentation of data in left panel showing reduction of 6-4 PP positive cells as a function of time. (D) <i>Left panel:</i> Histograms illustrating cell cycle distribution of CPD positive and negative cells, untreated, 0 and 24 h after UV-B. Lower UVR-dose was applied for the XPA^−/− cells to avoid excessive apoptosis. The dashed lines separate the cell cycle phases. % CPD positive cells are given in bottom row. <i>Right panel</i>: Bars illustrating the relative cell-phase distribution of the CPD positive cells.</p

After UVR, cells complemented with APIM-mutated XPA accumulate γH2AX foci at the site of replication.

<p>(A) Normalized γH2AX intensity measured by in-cell western (LI-COR Bioscience) (mean ± SD, n = 4) 24 h after exposure to UV-B. The γH2AX intensity is normalized against the DNA content using Draq5 and the intensity of untreated cells. (B) Images of immunostained cells. The cells were exposed to UV-B 24 h prior to fixation. Lower UVR-dose was applied for the XPA^−/− cells to avoid excessive apoptosis. Bar: 5 µm. (C) Fractions of replication foci (PCNA) colocalizing with γH2AX. Each dot represents one cell, on average 35 foci were counted in each cell (mean ± SEM, n = 5 and 15). The P-value is derived by unpaired t-test. Only cells resembling S phase cells and expressing comparable levels of the YFP constructs were included.</p

XPA colocalizes and directly interacts with PCNA in replication foci

<p>. (A) Overexpressed tagged proteins in live cycling HeLa cells. (B) Immunostained HeLa cells. The intensity of α-XPA and α-PCNA along the line in the merged picture is illustrated in the graph. The inserts show an enlargement of the area close to foci 3 and 4. (A and B) Bar: 5 µm. (C) iPOND from cells labeled with EdU (pulse) before fixation. One sample was additionally followed by a chase in thymidine-containing medium (pulse-chase). The WB shows proteins captured due to EdU proximity. The upper and lower panels are from individual iPOND experiments. All bands within one panel (black frame) are from the same WB, lanes and rows are separated by grey lines (also in D and E). (D) Co-IP of endogenous XPA from HeLa cells stably expressing YFP-PCNA using α-YFP beads. SF: soluble fraction, CF: chromatin-enriched fraction, Y: YFP (negative control), Y-P: YFP-PCNA. (E) Co-IP of endogenous XPA from untransfected HeLa cells using α-PCNA beads (pulling down endogenous PCNA). IP with α-YFP was used as control for unspecific binding to the beads. (F) Normalized FRET (N_FRET) measurements in HeLa cells. CFP/YFP (vectors only) and CFP-PCNA/YFP-PCNA were used as negative and positive controls, respectively. Detector gain: 800 (YFP), 700 (CFP), 700 (FRET). The P-value is derived by unpaired t-test. Data presented is from three independent experiments (mean ± SEM, n = 55–75).</p

Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment

ATX-101 induces apoptosis in the MM cell line JJN-3.

<p>(A–C) Flow cytometric measurement of the apoptotic cell population by annexin V-Pacific Blue labeling. (A) JJN-3 cells treated with 6 µM ATX-101 and 0.5 µM melphalan alone or combined were incubated for 1, 2, and 3 days. Control cells were left unexposed. (B and C) JJN-3 cells treated with 6 and 10 µM ATX-101 were incubated for 1, 2, and 4 h. In addition to annexin V labeling, cells were stained with DRAQ5 for DNA profile. (C) The histograms show the cell cycle distribution of live (blue) and apoptotic (pink) cells after 1 h of ATX-101 treatments. (A–C) show data from representative experiments out of three. (D) Flow cytometric measurement of caspase 8, 9, and 3/7 activity by Fluorescent Labeled Inhibitor of Caspases (FLICA) assay. JJN-3 cells were left unexposed and exposed to 8 µM ATX-101 for 2 and 4 h before the FLICA probe was added for staining. The FLICA probe binds irreversible only to the activated caspase and labels apoptotic cells. Data is from four independent experiments for caspase 8 activity and three independent experiments for caspase 9 and 3/7 activity (mean ± SD, ** P < 0.01, Student’s t-test).</p