, 1991) data were collected. 3-HC/Cotinine and Nicotine Levels Saliva samples collected during eligibility assessment were examined to determine 3-HC/cotinine using liquid chromatography CP-868596 with tandem mass spectrometry (Dempsey et al., 2004). The 3-HC/cotinine ratio has been shown to be independent from time since last cigarette and stable over repeated measures (Lea et al., 2006; Mooney et al., 2008). While plasma was used previously to ascertain the rate of nicotine metabolism, the reliability and validity of using saliva samples for 3-HC/cotinine has also been demonstrated (Dempsey et al., 2004) and was selected for this study given its ease of collection, storage, and shipping. A 3-HC/cotinine value of �� .

18 was used, a priori, to select participants with 3-HC/cotinine values that were within the top three quartiles of the 3-HC/cotinine saliva distribution (Dempsey et al., 2004). It is worth noting that this value is lower than the value used to identify the top three quartiles of the 3-HC/cotinine distribution when plasma is used (i.e., 0.23�C0.26; Lerman et al., 2006; Schnoll et al., 2009). Nicotine and cotinine levels in saliva at baseline and Week 1 were also determined using liquid chromatography with tandem mass spectrometry (Dempsey et al., 2004) in order to determine nicotine and cotinine percent replacement, versus baseline levels. To ensure that the Week 1 measures of nicotine and cotinine levels were not confounded by smoking, Week 1 saliva samples were collected only among participants who were confirmed abstinent using CO breath samples (�� 10 ppm).

Patch Adherence Patch adherence was measured by self-report. At each assessment from Week 0�C8, participants indicated if they used the patches on each day. Participants were classified as compliant for a week if they used patches on 6 days or more each week (Schnoll et al., 2010). Side Effects Side effects were assessed using a symptom checklist from past trials (Schnoll et al., 2010). The checklist was administered at Weeks ?1, 0, 1, 3, 5, and 8. Each symptom (e.g., nausea, skin reaction) was rated from 1 (none) to 4 (severe). Participants were instructed to contact study personnel if they experienced any serious medical problems between assessments. Adverse events were considered serious if the participant considered them debilitating or if they required hospitalization.

Batimastat Serious adverse events were reported to the University of Pennsylvania IRB and were classified as related or unrelated to treatment arm allocation. At Weeks 1 and 8, an ECG was administered and blood pressure was assessed. Smoking Cessation At Week 1 and Week 8 (end of treatment), participant quit rates were assessed. A time-line follow-back measure was used to assess daily smoking from Week 0 to Week 8. A breath sample was collected at Week 1 and Week 8 to verify self-reported abstinence using a CO monitor. Two primary outcome measures were used for this study.

The apoptotic rates were analyzed by flow cytometry using an annexin V-FITC/PI kit. Staining was performed according to the manufacturer��s customer review instructions, and flow cytometry was conducted with a flow cytometer (Beckman-Coulter, Brea, USA). Cells with annexin V (?) and PI (?) were deemed viable cells. Cells with annexin V (+) and PI (?) were deemed early apoptotic cells. Cells with both annexin V (+) and PI (+) were deemed late apoptotic cells. TUNEL assay To identify apoptosis in the transfected cells, we utilized the dead-end colorimetric TUNEL system kit (Promega, Madison, USA) to measure DNA fragmentation and caspase-3 activation in the GKN1 transfected cells, according to the manufacturer��s instructions. Briefly, cells were fixed in 4% paraformaldehyde solution for 25min at room temperature, rinsed in PBS, and permeabilized by incubating the slides in 0.

2% Triton X-100 solution. Cells were then incubated with a terminal deoxynucleotidyl transferase (TdT) reaction mixture containing biotinylated nucleotides and TdT at 37��C for 60min, and rinsed with 1��SSC (sodium chloride-sodium citrate buffer) and PBS. Next, streptavidin HRP was added to the cells, and the cell slides were stained with 3,3��-diaminobenzidine color solution. Finally, cells were examined under a light microscope and the number of positive cells was counted and summarized from a total of 10 high power fields. Cell cycle analysis To analyze cell cycle distribution, transfected cells were grown and treated with 25M olomoucine (Santa Cruz Biotechnologies, Santa Cruz, USA) for 1h, and then incubated with regular culture medium for an additional 1h [13].

Cells were then collected and subjected to cell cycle analysis by flow cytometry as described in the previous section. Sensitivity to 5-FU treatment To detect the role of GKN1 in mediating sensitivity of gastric cancer cells to 5-FU treatment, we grew and treated GKN1 transfected tumor cells with 5-FU (Sigma) or DMSO for 24h and 48h. Concentrations of 5-FU ranged from 0.25 to 1.0mmol/L. The apoptosis rate from these cells was detected by flow cytometry as previously described. cDNA microarray analysis To perform cDNA microarray analysis, total cellular RNA from GKN1-transfected and vector-control tumor cells were isolated with the Trizol? Reagent (Invitrogen).

RNA was then reversely transcribed into cDNA using the TrueLabeling-AMP Linear RNA amplification kit (Superarray, Frederick, MD, USA), and then converted into biotin-labeled cRNA using biotin-16-UTP and an in vitro transcription kit (Roche, Basel, Switzerland). The newly synthesized cRNA probes were then purified with the ArrayGrade cRNA cleanup kit (Superarray) and then added to the pretreated Batimastat Oligo GEArrays Human Apoptosis Microarray (OHS-012 from Superarray) that contains 112 apoptosis-related genes. The microarray was then hybridized overnight at 42oC.