These data implicate dysregulation

These data implicate dysregulation of the UPR pathway in the pathogenesis of human lung cancers and indicate that phospho-eIF2[alpha] and BiP (and possibly eIF2[alpha]), may have diagnostic and/or therapeutic potential [32, 33]. Furthermore, the activation of the UPR program via eIF2[alpha] phosphorylation indicates a previously unknown pathogenic effect of CS and suggests that chronic induction of one or more protein effectors of the UPR pathway may play an etiological role in lung cancer. Finally, the upregulation of several UPR regulators (in particular) in lung cancers may provide a pro-survival advantage by increasing cellular resistance to various cytotoxic stresses such as hypoxia, chemotherapeutic drugs, and immune attack [34, 35, 36, 37, 38], especially in tumor cells that have one or more pro-apoptotic pathways disabled, which is a common feature of lung neoplasms [39].
Methods
Cell Culture and Smoke Treatment
A549 cells were purchased from American Type Culture Collection (ATCC no. CCL-185, Manassas, VA) and were cultured in Ham's F12K medium with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate (Gibco/Invitrogen, Carlsbad, CA) and supplemented with 10% fetal bovine serum (ATCC). NHBE cells from nonsmoking, nondiabetic donors were purchased from Cambrex Corporation (Walkersville, MD). Cells were cultured in complete Bronchial Epithelial Cell Growth Medium, prepared by supplementing Bronchial Epithelial Basal Medium with retinoic acid, epidermal growth factor, epinephrine, transferrin, T3, insulin, hydrocortisone, antimicrobial agents and bovine pituitary extract by addition of SingleQuots,[TM] (Cambrex Corporation). All cell cultures were treated before their sixth passage. All incubations were at 37[degrees]C in a humidified atmosphere of 5% CO2 in air. CS treatment was performed as follows: cells were seeded into 35 mm Petri dishes (Fisher Scientific, Falcon #35-3001, Pittsburg, PA) at a density of 105 cells/dish and were typically at 70% confluency at the time of exposure to CS. At least three replicate dishes were treated for each condition, and each replicate was analyzed using a separate microarray (i.e., the RNA from the dishes was not pooled). The cell culture medium was replaced with 37[degrees]C Dulbecco's PBS (D-PBS) containing calcium and magnesium (Gibco/Invitrogen) for the smoke exposure. The covers were removed from the Petri dishes and they were placed in a smoke exposure chamber designed to deliver a consistent dose of diluted CS. CS was generated under Federal Trade Commission (FTC)[40] smoking conditions (35 [+ -] 0.3 cc puff, one puff every 60 seconds, 2-second puff duration with none of the ventilation holes blocked) using a KC 5 Port Smoker (KC Automation, Richmond, VA), from 2R4F reference research cigarette (designed to represent the average 'lights' cigarette marketed in the U.S. with FTC values of 9.7 mg 'tar' and 0.85 mg nicotine; University of Kentucky, Louisville, KY) using FTC machine smoking conditions, or two leading commercially available U.S. cigarette brands representative of either the 'lights' (FTC values of 11 mg 'tar' and 0.8 mg nicotine) or 'full-flavor' (FTC values of 15 mg 'tar' and 1.1 mg nicotine) styles of cigarettes. Cigarettes were smoked to within 3 mm of the filter tip. All cigarettes had been equilibrated at 23.9[degrees]C [+ -] 1.1[degrees]C and 60% [+ -] 2% relative humidity for a minimum of 24 hours and a maximum of 14 days. The smoke exposure chamber was designed to deliver smoke uniformly diluted with 5% CO2 in air and passed through the cell exposure chamber at a constant flow rate of 500 cc/min. Briefly, each 35 cc puff was first drawn into a 250 cc round chamber containing 5% CO2 in air and mixed via a stir bar. The standard smoke dilution used in most of our experiments was 35 cc delivered over 1 min in a 250 cc or 500 cc volume, and the intensity of exposure was varied by varying the length of time the cells spent in the exposure chamber (typically 15 min or 20 min). The time and distance that the smoke traveled from the end of the cigarette to the exposure chamber was minimized by using the shortest lengths of tubing possible between the parts of the apparatus. Mock-exposed cells were treated under identical conditions as the exposed cells except for the absence of a cigarette in the smoking port. Following treatment or mock treatment, the D-PBS covering the cells was aspirated and replaced with 1 ml per chamber of fresh culture medium at 37[degrees]C. The cells were placed in the 37[degrees]C, 5% CO2 incubator for the times indicated.
Thapsigargin, tunicamycin, and dithiothreitol treatment
UPR-stimulating reagents thapsigargin, tunicamycin, and dithiothreitol (DTT) were obtained from Sigma, St. Louis, MO. Treatment conditions used in our experiments were 1 uM thapsigargin, 10 [mu]g/mL tunicamycin, or 2 mM DTT added to the cell culture medium in DMSO (thapsigargin and tunicamycin) or water (DTT), and the cells were placed

Results (Indonesian) 2:

Data ini melibatkan disregulasi dari jalur UPR dalam patogenesis kanker paru-paru manusia dan menunjukkan bahwa phospho-eIF2 [alpha] dan BiP (dan mungkin eIF2 [alpha]), mungkin memiliki potensi diagnostik dan / atau terapi [32, 33]. Selanjutnya, aktivasi program UPR melalui eIF2 [alpha] fosforilasi menunjukkan efek patogen yang sebelumnya tidak diketahui dari CS dan menunjukkan bahwa induksi kronis dari satu atau lebih effectors protein dari jalur UPR dapat memainkan peran etiologi dalam kanker paru-paru. Akhirnya, upregulation beberapa regulator UPR (khususnya) dalam kanker paru-paru dapat memberikan keuntungan pro-hidup dengan meningkatkan resistensi seluler terhadap berbagai tekanan sitotoksik seperti hipoksia, obat kemoterapi, dan menyerang kekebalan tubuh [34, 35, 36, 37, 38 ], terutama pada sel tumor yang memiliki satu atau jalur yang lebih pro-apoptosis dinonaktifkan, yang merupakan fitur umum dari neoplasma paru-paru [39].
Metode
Kultur sel dan Smoke Pengobatan
sel A549 yang dibeli dari Amerika Type Culture Collection (ATCC ada. CCL- 185, Manassas, VA) dan dikultur dalam medium F12K Ham dengan 2 mM L-glutamine disesuaikan mengandung 1,5 g / L natrium bikarbonat (Gibco / Invitrogen, Carlsbad, CA) dan ditambah dengan 10% serum janin sapi (ATCC). Sel NHBE dari tempat, donor nondiabetes yang dibeli dari CAMBREX Corporation (Walkersville, MD). Sel dikultur secara lengkap bronkial Epitel Cell Growth Medium, disiapkan dengan menambah bronkial epitel basal Medium dengan asam retinoat, faktor pertumbuhan epidermal, epinefrin, transferin, T3, insulin, hidrokortison, agen antimikroba dan ekstrak hipofisis sapi dengan penambahan SingleQuots, [TM] (CAMBREX Perusahaan). Semua kultur sel diperlakukan sebelum bagian keenam mereka. Semua incubations berada di 37 [derajat] C dalam suasana lembab dari 5% CO2 di udara. Pengobatan CS dilakukan sebagai berikut: sel unggulan ke 35 mm piring Petri (Fisher Scientific, Falcon # 35-3001, Pittsburg, PA) dengan kepadatan 105 sel / piring dan biasanya di 70% confluency pada saat paparan CS. Setidaknya tiga piring meniru dirawat karena setiap kondisi, dan masing-masing ulangan dianalisis menggunakan microarray terpisah (yaitu, RNA dari piring tidak menggenang). Medium kultur sel diganti dengan 37 [derajat] C Dulbecco PBS (D-PBS) yang mengandung kalsium dan magnesium (Gibco / Invitrogen) untuk paparan asap. Selimut telah dihapus dari cawan Petri dan mereka ditempatkan dalam ruang paparan asap dirancang untuk memberikan dosis yang konsisten dari diencerkan CS. CS dihasilkan di bawah Federal Trade Commission (FTC) [40] kondisi merokok (35 [+ -] 0,3 cc puff, satu isapan setiap 60 detik, durasi engah 2 detik dengan tidak ada lubang ventilasi diblokir) menggunakan KC 5 Pelabuhan Perokok (KC Automation, Richmond, VA), dari 2R4F rokok penelitian referensi (yang dirancang untuk mewakili rokok rata-rata 'lampu' dipasarkan di AS dengan nilai FTC dari 9,7 mg 'tar' dan 0,85 mg nikotin; University of Kentucky, Louisville, KY) menggunakan FTC kondisi mesin merokok, atau dua terkemuka tersedia secara komersial merek rokok US perwakilan baik 'lampu' (FTC nilai 11 mg 'tar' dan 0,8 mg nikotin) atau 'penuh rasa' (nilai FTC dari 'tar' 15 mg dan 1,1 mg nikotin) gaya rokok. Rokok yang merokok dalam waktu 3 mm dari ujung filter. Semua rokok telah diseimbangkan di 23,9 [derajat] C [+ -] 1,1 [derajat] C dan 60% [+ -] 2% kelembaban relatif selama minimal 24 jam dan maksimal 14 hari. Ruang paparan asap dirancang untuk memberikan asap seragam diencerkan dengan 5% CO2 di udara dan melewati ruang paparan sel pada laju alir konstan 500 cc / menit. Secara singkat, setiap isapan 35 cc pertama kali ditarik ke dalam ruang putaran 250 cc yang mengandung 5% CO2 di udara dan campuran melalui bar aduk. Pengenceran asap standar yang digunakan di sebagian besar percobaan kami adalah 35 cc disampaikan lebih 1 menit dalam volume 250 cc atau 500 cc, dan intensitas paparan bervariasi dengan memvariasikan lamanya waktu sel-sel yang dihabiskan di ruang paparan (biasanya 15 menit atau 20 menit). Waktu dan jarak yang asap perjalanan dari ujung rokok ke ruang paparan diminimalkan dengan menggunakan panjang terpendek dari tabung mungkin antara bagian-bagian dari peralatan. Sel mock-terkena diperlakukan dalam kondisi yang sama sebagai sel terkena kecuali tidak adanya rokok di pelabuhan merokok. Setelah pengobatan atau perawatan pura-pura, D-PBS meliputi sel itu disedot dan diganti dengan 1 ml per ruang medium kultur segar pada 37 [derajat] C. Sel-sel ditempatkan di 37 [derajat] C, 5% CO2 inkubator untuk waktu yang ditunjukkan.
Thapsigargin, tunicamycin, dan pengobatan dithiothreitol
UPR-Stimulating reagen thapsigargin, tunicamycin, dan dithiothreitol (DTT) diperoleh dari Sigma, St Louis, MO. Kondisi pengobatan yang digunakan dalam percobaan kami adalah 1 uM thapsigargin, 10 [mu] g / mL tunicamycin, atau 2 mM DTT ditambahkan ke dalam media kultur sel dalam DMSO (thapsigargin dan tunicamycin) atau air (DTT), dan sel-sel ditempatkan