Introduction Certain populations such as chemotherapy patients and atomic bomb survivors have been exposed to ionizing radiation and experience tissue damage and cancer initiation and progression. One cancer that can be initiated from radiation is esophageal squamous cell carcinoma (ESCC), an epithelial cancer that has a survival rate as low as 20% (Chunping et al., 2009). Researchers have found that when protein tyrosine kinase receptors (RPTK) activate oncogenes, they can create epithelial tumors and cause deadly cancers like ESCC (Tannock et al., 2005). The RPTK family has one group, MET, that has only two receptors, MET and RON, present in the human body. MET’s ligand is the hepatocyte growth factor (HGF) and RON’s ligand is the macrophage-stimulating protein (MSP-1). Both HGF and MSP-1 have been shown to activate their receptors and are implicated in certain processes [What processes are these?] (Wang et al., 2003). Since radiation damages cells throughout the biological system (Camphausen et. al, 2001), researchers are investigating whether or not HGF and MSP-1 protects or kills certain normal and cancerous cells by being part of cell recovery processes. One research group recently reviewed that the HGF-MET pathway has an important role in the embryonic development in the liver, migration of myogenic precursor cells, regulation of epithelial morphogenesis and growth, and regeneration and protection in tissues (Nakamura et. al, 2011). In addition, since the RON receptor is more commonly expressed in cells of epithelial origin, and when activated is part of epithelial cell matrix invasion, dissociation, and migration processes, scientists conclude that RON might be one of the factors causing epithelial cancer initiation in the biological system (Wang et. al, 2003). In order to examine HGF and MSP-1’s effect on cancer initiation and progression we used two immortalized esophageal epithelial cell lines. One is a normal human cell line (EPC2-hTERT), while the other had a p53 mutation at the 175th amino acid position (EPC2-hTERT- p53R175H). For this investigation, we used 0(control), 2, and 4 Gray doses of gamma (Cs137) radiation and selected various concentrations from 0-100 ng/mL of HGF and MSP-1 in our assays. 1 Since the HGF and MSP-1 pathways have proliferative roles in epithelial cells, we conducted the MTT proliferation assay to see if either drug enhances or inhibits cell proliferation over time. Also, a MTT cytotoxicity assay was necessary to observe whether the drugs are protecting the cells from radiation and if a trend is occurring depending upon the amount of dose added. In addition, a wound healing assay was done since both drugs have been to known to promote cell motility. Since cell damage occurs when radiation is added, apoptosis and micronuclei assays are vital to see if HGF and MSP-1 increase or decrease cell death and damage in normal and pre-cancerous cells and by how much based on the radiation dosage. Overall, we used the MTT, wound healing, apoptosis and micronuclei assays to investigate the effects ofHGF and MSP-1 on irradiated esophageal epithelial cells. Materials and Methods  Cell Lines: Normal (EPC2-hTERT) and mutated (EPC2-hTERT-p53R175H) esophageal epithelial cell lines were used this investigation. The cells were cultured and maintained according to the Phelan, 1998 cell culture protocol.  Radiation Dosages: The gamma (Cs137) radiation doses ranged from 0-4 Gys. (0-400 rads).  Drugs: Hepatocyte growth factor (HGF) and macrophage-stimulating protein (MSP-1) concentrations ranged from 10-100 ng/mL depending on the assay.  Incubation Settings: 37° Celsius, 5% CO , 98% humidity 2  Controls: No drug or cells with KBM (keratinocyte basal medium)  4 assays: MTT Proliferation and Cytotoxicity, Wound Healing, Micronuclei assay, and Apoptosis Assay 2 1. MTT Proliferation and Cytotoxicity Materials:  7 (4 for proliferation and 3 for cytotoxicity) 96-well plates  MTT Solvent: 4mM HCl, 0.1% Nandet P-40 (NP40) all in isopropanol  MTT Reagant*: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)  PBS solution  Aluminum/tin foil  TECAN Spectroflorometer *Reagant is prepared with a concentration 5 mg/mL in PBS and is covered with foil Methods: 1. Culture the cells (2000 cells/well) with KSFM (keratinocyte serum free medium) in the plates and incubate for 24 hours. 2. Add HGF and MSP-1 (10, 50, and 100 ng/ml) in each well according to the plate setup of both proliferation and cytotoxicity assays. Incubate again for 24 hours. Proliferation: 1. Take one plate for the 24 hour reading and add 20 µL of the 5 mg/mL MTT Reagent per well. After putting it on the shaker for a few minutes, incubate it for 3.5 hours. 2. Remove the media from the wells and add 150 µL MTT solvent per well quickly since the plate is photosensitive. Put it on the shaker for 15 minutes. 3. Read the plate by using the TECAN spectrofluorometer. 4. At 48, 72, and 96 hours, repeat the same steps taken for the 24 hour reading. Cytotoxicity: 3 1. Irradiate one plate each at 0, 2, and 4 Gray doses. 2. After 72 hours, repeat the same steps taken for the 24 hour proliferation plate for the 3 cytotoxicity plates. Analysis: To test how many live cells were present over the 96-hour or 72-hour period, we measured the intensity of the purple formazan color, which is produced when living mitochondria converts the yellow MTT reagent to purple by using enzymes during the 3.5 hour incubation period. The TECAN spectroflorometer then uses an absorbance at an optical density of 590 nm to measure this intensity and give values. For the proliferation assay, we can observe if HGF and/or MSP-1 promotes cell proliferation without any stress added to the experiment. In the cytotoxicity assay, we are adding the radiation factor to see if at one particular time if either drug protects both types of cells (Wallert and Provost Lab, 2007). 2. Wound Healing Materials: • 3 24-well plates (2 with no radiation and 1 with a dose of 2 Gray) • Camera • PBS solution • 200 µL pipette tip • Marker with a visible color as biomarker • Media that contains KBM with HGF or MSP-1 (both drugs at 50 ng/mL) Methods: 1. Draw a line with a marker on the bottom of each well 4 2. Use the tip to scratch once on each well to create a wound 3. Rinse the whole plate of cells with PBS solution 4. Replace the PBS solution with 1.5 mL of media containing the drug 5. Take a picture of each well at 0, 3, 6, 9, 12, and 24 hrs. . [This is not the case – media was left on the whole time] Analysis: We used Image J (NIH) to measure the length of the wound from each picture taken**. After drawing two lines parallel to each other where the cells are bordered and one line connecting both perpendicularly, the software measures the length of the gap between the two sides of cells. We can then plot these values and find the percentage of the wound healed. For example, if 100% is the percentage at 24 hours, it means that the drug facilitated the cells to migrate and fill the gap completely over 24 hours. The wound assay can help us determine whether HGF and MSP-1 promote cell motility (Wallert and Provost Lab, 2004). **Error: One picture of the p53 plate at 9 hours was missing. 2. Micronuclei Assay Materials: • (6) 4-chamber well slides • 1X PBS [Wash buffer] • Carnoy’s fixative: 3:1 methanol : acetic acid • DAPI in PBS (1:10 dilution working stock; 1:3000 dilution final) • SlowFade Gold with DAPI 5 • Oil immersol with dropper • OLYMPUS AX70 Upright Compound Microscope Methods: Preparing slides: 1. After culturing and seeding the cells, add HGF or MSP-1 (50 ng/mL) to their selected wells after 1 hour. Then wait for 24 hours. 2. Irradiate 2 slides (2 Gy.) and another 2 (4 Gy.), leaving 2 slides as control with no dosage. 3. After 48 hours, fix the cells in Carnoy’s fixative. 4. Wash with PBS three times, with each for 5 minutes. Add DAPI solution (350 µL/well) and incubate the slides for 5 minutes. Wash the cells again with PBS three times (five minutes each). 5. Mount each slide by adding 3-4 drops of SlowFade Gold and using a 22x50 mm cover glass. Seal each slide with nailpolish on the outer boundary. Counting: 1. Turn on the microscope. 2. Add one drop of oil immersol on Coverslip. 3. Touch the 100x objective lenses with the oil and turn on the fluorescence blue light 4. Count the number of micronuclei and binucleated cells. Picture 1: A field of view showing 1 binucleated cell with one micronuclei. Analysis: 6 When the fluorescence light is turned on, we counted the blue cells that are binucleated and also the ones that have micronuclei (Picture 1). We analyzed the percentage of micronuclei of all the binucleated cells to find quantitatively how much damage occurred since micronuclei is composed of DNA fragments that are all clumped together. (Genpharmtox). 3. Apoptosis Assay Materials: • (6) 4-well chamber well slides • 1X PBS [Wash buffer] • 4% paraformaldehydeProlong Gold with DAPI • 0.5% Triton (Permeabilizing buffer) • 3% BSA (Blocking buffer) • Primary antibody solution (Cleaved caspase-3, 1:300 dilution in blocking buffer) • Secondary antibody solution ( Alexa-Fluor 488 goat anti-rabbit IgG antibody, 1:1000 in blocking buffer) Methods: Preparing slides: 1. After culturing and seeding the cells, add HGF or MSP-1 (50 ng/mL) to each selected well (based on plate setup) after 1 hour. 2. After 24 hours, irradiate 2 slides with 2 Gray while and another 2 with 4 Gray, leaving two as control with no dosage. After 48 hrs, wash with PBS and fix the cells with 4% paraformaldehyde for 10 min. at room temperature. *** 3. Wash the cells three times for five minutes each with PBS. 7 4. Add 400 µL/well permeabilizing buffer for 3 minutes at room temperature. Add 400 µL blocking buffer to each well and incubate for 30 minutes to 1 hour. 5. Add primary antibody solution and incubate for 1-2 hours at room temperature. Wash three times for five minutes each with PBS. 6. Next, add secondary antibody solution (150 µL/well) and incubate 30 minutes to 1 hour at room temperature without being exposed to light. 7. Wash three times for five minutes each with PBS. Use 3-4 drops of Prolong Gold with DAPI/slide, and use 22x50mm cover glass to mount and seal each slide with nail polish. Counting: same as in Micronuclei assay except: 1. Touch the 60x objective lenses with the oil and switch between the blue & green lights 2. Count positive (green) cells/1000 blue (normal) cells = apoptotic index. ***If storing, replace paraformaldehyde with PBS and cover with parafilm, and store it at 4° C. Analysis: Picture 2: A field of view showing 2 positive apoptotic cells and many other blue cells. For this assay, we counted the positive apoptotic (green) and negative normal (blue) cells to see if any cleaved caspase-3 enzymatic activity occurred. For every field of view, we counted the number of blue cells first while the blue light is on. Then, we switched to the green light to see if any positive cells were there in the same field of view. If there is at least one bright green cell, we switched back to the blue light to see if a blue cell is overlapping in the same position to clarify if it actually a cell (Picture 2) (Promega, 2011). Picture 2: A field of view showing normal Results (blue) and two apoptotic (green) cells. 1. MTT Proliferation and Cytotoxicity 8 Proliferation: The MTT proliferation assay was conducted with 10, 50, or 100 ng/mL concentrations of HGF and MSP-1 in both normal and mutated cell lines over a 96-hour period. The results present that cell proliferation increased over time in both normal and mutated cells. Interestingly, there was no positive or negative effect in both cell lines as the concentrations of either drugs (10, 50, and 100ng/mL) increased. When considering HGF and MSP-1, our results show that both drugs do not aid in cell proliferation in both normal and mutated epithelial cells (Figure 1.1a & 1.1b). EPC2-hTERT 14 No Drug 90 12 D5 HGF10 O 10 e HGF 50 c n 8 HGF 100 a b or 6 MSP 10 s b MSP 50 A 4 MSP 100 2 0 24 hr 48 hr 72 hr 96 hr EPC2-hTTiEmRe T-p53R175H Figure 1.112a: The Effect of HGF and MSP at concentrations of 10 ng/mL, 50 ng/mL and 100 No Drug ng/mL over a 96 hour period on normal EPC2-hTERT esophageal epithelial cells. D590 10 HGF10 e O 8 HGF 50 nc HGF 100 a 6 b or MSP 10 s b 4 MSP 50 A MSP 100 2 0 24 hr 48 hr 72 hr 96 hr Time 9 Figure 1.1b: The Effect of HGF and MSP at concentrations of 50 ng/mL and 100 ng/mL over a 96 hour period on mutated EPC2-hTERT-p53R175H esophageal epithelial cells. Cytotoxicity: For the MTT Cytotoxicity assay, the cells were given 2 and 4 Gray dosages of ga mma radiation with two HGF and MSP-1 (50 or 100 ng/mL) concentrations. The results presented in Figures 1.2a & 1.2b show that as radiation dosage increased in both cell lines with no drug, the number of EPC2-hTERTdecreased significantly while there was little change in the p53-mutated cells. When considering HGF and MSP-1’s effects, they interestingly protected the normal cells more than the mutated cells from radiation (Figure 1.2a & 1.2b). 1.4 EPC2-hTERT 1.2 D590 1 No Drug O HGF 50 ce 0.8 HGF 100 n ba 0.6 MSP 50 or s MSP 100 b 0.4 A 0.2 0 Figure 1.2a: The Effec0t G oyf HGF and M2S GPy (50 and 100 4n Ggy/mL) with gamma radiation (Cs137) dosages of 2 and 4 GrRaayd aiatt i7o2n hDoosuer s( Cosn1 3c7y)totoxicty of irradiated normal EPC2-hTERT esophageal epithelial cells. 1.4 EPC2-hTERT-p53R175H 1.2 D590 1 No Drug O HGF 50 e 0.8 nc HGF 100 ba 0.6 MSP 50 or bs 0.4 MSP 100 A 0.2 0 0 Gy 2 Gy 4 Gy Radiation Dose (Cs137) Figure 1.2b: The Effect of HGF and MSP (50 and 100 ng/mL) with gamma radiation (Cs137) dosages of 2 and 4 Gray at 72 hours on cytoxicity of p53-mutated esophageal epithelial cells. 10