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Introduction:

Objectives: 1. Maintain primary lung epithelial cells. 2. Perform dose dependant studies on primary lung epithelial cells by treating with 0-20nM uPA for 28 hr and 48hr. cell apoptosis and cell proliferation assay will be done for all concentration of uPA 3. Determine uPA, uPAR, PAI-1 and p53 expression in Beas2B cells after the treatment with uPA using western blot. Cells will be treated with 0, 0.5, 1, 2, 5, 10, 15 and 20nM concentration of uPA and bleomycin (40ug/ml) and the condition media, lysates and membrane proteins will be analyzed. B-actin will be used as loading control for analysis of gels.

PROPOSED METHODS OF STUDY Cell culture and Maintenance
The human bronchial epithelial (Beas2B) or primary small airway (SAE) cells will be cultured and maintained in LHC-9 medium supplemented with PSN antibiotics at 370C. The cells will be fed for every 48hrs until it grows as monolayers and reach 80% confluency. Then cells will be subcultured in 100 mm plates and 6 well plates by detaching them using trypsin-EDTA(Trypsin 0.5 g/L; EDTA 0.2g/L), collecting them in 4ml of LHC-9 medium, centrifuge for 10 minutes at 1000 rpm at room temperature and re-suspending the pellet in 4ml of LHC-9 medium. Cells will be cryopreserved for future use using cryoprotectant medium consisting of fresh LHC-9 medium and 10% (v/v) DMSO (dimethyl sulfoxide). The cells will be stored in liquid nitrogen vapour phase.
Treatment of cell lines with uPA Once the SAE cells in 100mm plates grow to 70-80% confluent, it will be treated with 0, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0 and 20.0nM of uPA. After 28 hrs of incubation cells will be detatched and apoptotic cells will be analyzed by flow cytometry after staining with annexin V and propidium iodide.
SAE cells cultured in 6 well plates will be treated with 0, 0.5, 1.0, 2.0, 5.0, 10.0, 15.0 and 20.0nM of uPA for 48 hr in the presence of H-thymidine. The effect of uPA on SAE cell proliferation will be determined by measuring the H-thymidine incorporation Total protein extraction and western blotting SAE cells grown to confluence in 100mm plates will be treated with 0-20nM uPA for 24hr. Following these treatments condition media (CM) will be collected, cells will be detached, washed with PBS and suspended in lysis buffer (10mM Tris HCL pH 7.4, containing 15mM NaCl, 1% Triton X-100, 15% glycerol, 1mM Na3VO4, 1mM NaF, 1mM EDTA, 1 mM phenylmethylsulfonyl fluoride and 3-10ug of aprotinin per 100 ml). The cell lysates will be prepared using three cycles of freezing and thawing using dry ice, and a 37° C water bath and centrifuging at 15,000 RPM for 15 minutes at 4.C.
Membrane proteins will be isolated by lysing the cells in uPAR extraction buffer (pH7.4) containing 50mM Tris HCL, 150mM NaCl, 100mM octyl-β-D-pyranoside, 1mM MgCl2, 1mM MnCl2, and 1 mM PMSF plus 100ug/ml each of aprotinin, pepstatin, cystatin, and leupeptin. Following 30 minutes of incubation at 40.C with constant agitation the lysate will be centrifuged at 35,000g for 20 minutes at 4.C. The clear supernatants will collected as cellular protein extract and stored -70° C. The protein will be quantified by conducting a BCA Assay. Proteins from beas2b condition media and cell lysates will be separated by SDS PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 1% BSA in wash buffer for 1 hr at room temperature followed by overnight hybridization with uPA, uPAR, PAI-1 AND p53 monoclonal antibody in the same buffer at 4 °C and washed, and uPA, uPAR, PAI-1 and p53 proteins will be detected by enhanced chemiluminescence (ECL). The membrane will be stripped with β-mercaptoethanol and subjected to western blotting with β-actin monoclonal antibody.
In separate experiments, beas2b cells cultured in 100mm dishes will be treated with or without uPA (20nM), alone or in combination with CSP or CP for 2h as a pretreatment. These cells will be later exposed to bleomycin (40ug/ml) for 24hr. following this treatment condition media, cell lysate and membrane proteins will be analyzed for uPA, uPAR, PAI-1, p53 and β-actin protein expression by western blotting as described above. Model of Bleomycin – induced injury in mice: six week old CD-1 mice (charles river, Boston, MA) weighing 20g is kept on a 12:12 hr light: dark cycle with free excess to food and water. These mice will be anaesthetized with an intraperitoneal injection of ketamin/xylazine mixture. Mice will be given 40ug of bleomycin hydrochloride in 50ul of sterile saline through intranasal insufflations. Twenty four hours after bleomycin exposure, mice will be intraperitoneally injected with control scaffolding peptide (CSP) or the control scrambled peptides (CP). Mice will be sacrificed on day 3, 7, 14 and 21 following bleomycin treatment and the lungs were used for the experiments below. Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling (TUNEL) Assay
The lungs will be fixed overnight at 4°C in 2% para farmaldehyde in PBS, and were embedded in paraffin. Paraffin will be removed by immersing slides in fresh xylene in a Coplin jar for 5 minutes at room temperature and Repeat one time for a total of two xylene washes. Wash the samples by immersing the slides in 100% ethanol for 5 minutes at room temperature in a Coplin jar. Rehydrate the samples by sequentially immersing the slides through graded ethanol washes (100%, 95%, 85%, 70%, 50%) for 3 minutes each at room temperature.
Wash the samples by immersing the slides in 0.85% NaCl for 5 minutes at room temperature. Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Fix the tissue sections by immersing the slides in 4% methanol-free formaldehyde solution in PBS for 15 minutes at room temperature.
Note: Paraformaldehyde can be directly substituted for methanol-free formaldehyde. Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Repeat once for a total of two PBS washes. Remove the liquid from the tissue and place the slides on a flat surface. Prepare a 20μg/ml Proteinase K solution from the reconstituted Proteinase K (10mg/ml; see Section 2) by diluting 1:500 in PBS. Add 100μl of the 20μg/ml Proteinase K to each slide to cover the tissue section. Incubate slides for 8–10 minutes at room temperature.
Note: Proteinase K helps permeabilize tissues and cells to the staining reagents in subsequent steps. For best results, optimize the length of incubation with Proteinase K. Longer incubations may be needed for tissue sections thicker than 4–6μm; however, with prolonged Proteinase K incubations, the risk of releasing the tissue sections from the slides increases in subsequent wash steps.
Wash the samples by immersing the slides in PBS for 5 minutes at room temperature in a Coplin jar.
Fix the tissue sections after washing by immersing the slides in 4% methanol-free formaldehyde solution in PBS for 5 minutes at room temperature. Pretreatment of Paraffin-Embedded Tissues (continued)
Wash the samples by immersing the slides in PBS for 5 minutes at room temperature. Note: An optional positive control slide may be prepared at Step 11 by treating a sample with DNase I to cause DNA fragmentation. A protocol for DNase treatment is given in Section 4.E.
Follow Steps 5–16 of Section 4.A to analyze apoptosis of these pretreated tissue sections.