Presentation on theme: "ADAR1 Modulation of p21WAF1 Modulation Luis Espinoza-Delgado Central Catholic High School Grade 12."— Presentation transcript:
ADAR1 Modulation of p21WAF1 Modulation Luis Espinoza-Delgado Central Catholic High School Grade 12
Introduction ADAR1 (Adenosine Deaminase Acting on RNA 1) is a mRNA editing protein that post-transcriptionally modifies nucleic acid by catalyzing the conversion of adenosine to inosine, allowing for a greater variety of proteins. ADAR1 contains both a catalytic (editing) domain and a RNA/DNA binding domain. It exists as a long (p150) and short (p110) isoform. We are using constructs that express either wild type ADAR1 p150 or a mutant that cannot edit RNA (MUT).
Introduction p21WAF1 is a cyclin-dependent kinase inhibitor has been linked to senescence and to either increased or decreased growth arrest and to increased or decreased cell death. Preliminary studies indicate a negative correlation between levels of p21WAF1 expression and those of ADAR1 (Sharma et. al. in preparation). Loss of ADAR1 has been linked to embryonic lethality and can result in death of liver cells and of leukemia cells.
Introduction Chronic myelogenous leukemia (CML) is a cancer of the white blood cells caused by a genetic translocation (the Philadelphia chromosome). Rearrangement makes a new gene Bcr Abl—a tyrosine kinase that is always on. CML patients respond well to tyrosine kinase inhibitors (TKIs) of the Bcr-Abl oncoprotein. TKIs fail to eradicate immature leukemia-initiating cells that arise from myeloid progenitor cells—sensitive to the deletion of mRNA editing protein ADAR1. Chronic myelogenous leukemia cells die without ADAR1 and leukemic cells depend on ADAR1 more than normal white blood cells (Steinman et. al. submitted). Targets of ADAR1 are largely unknown.
Project Goals 1.Validate inverse relation between ADAR1 and p21WAF1 2.Map ADAR1 effect as a transcriptional, post- transcriptional, or translational mechanism 3.Establish whether ADAR1 suppression of p21WAF1 involves RNA editing domain
Hypothesis Null: ADAR1 will not have a significant effect on p21 transcription.
Strategic Plan Significant Variation Insignificant Variation
Preliminary Procedure WT and -/- ADAR1 mouse embryonic fibroblasts were obtained from Dr. Qingde Wang. A Western Blot using Ponceau Staining was performed to validate intended presence/absence of ADAR1. Wt -/- MEFs p110 p150 Ponceau stain
Preliminary Procedure Plasmid constructs were obtained and digested with restriction enzymes to alter the promoter length of p21 (FL 2.4kB). luc p21 WT p21 ▽ Hinf1 p21 ▽ 1.1 p21 ▽ 2.2 pgl2basic
Materials Western Blot Kit Lipofectamine 2000 Transfection Kit Luciferase Assay Kit ß- galactosidase Assay Kit ADAR-modified Mouse Embryonic Fibroblasts p21 promoter and ‘UTR constructs ADAR plasmid vectors Clontech X Transfection Kit
Procedure Lipofectamine 2000 Transfection 1. Adherent cells: One day before transfection, plate 0.5–2 × 10 5 cells in 500 μL of growth medium without antibiotics so that cells will be 90–95% confluent at the time of transfection. 2. For each transfection sample, prepare complexes as follows: a. Dilute DNA in 50 μL of Opti-MEM®I Reduced Serum Medium without serum. Mix gently. b. Dilute the appropriate amount in 50 μL of Opti-MEM®I Reduced Serum Medium without serum. Incubate for 5 minutes at room temperature. c. After the 5 minute incubation, combine the diluted DNA with diluted Lipofectamine®2000 Transfection Reagent (total volume = 100 μL). Mix gently and incubate for 20 minutes at room temperature. 3. Add the 100 μL of complexes to each well containing cells and medium. 4. Incubate cells at 37°C in a CO2 incubator for 18–48 hours prior to testing for transgene expression. The medium may be changed after 4–6 hours. 5. For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection. Add selective medium (if desired) the following day
Procedure Cell Lysis Preparation Rinse cells with 500mL PBS. Add 200ul 1x reporter lysis buffer to each well and scrape cells into 1.7mL eppendorf tubes. Freeze samples at -80˚C for 30 minutes. Thaw samples 37˚C incubator and spin down cell debris at 14K for 10 minutes at 4˚C. Luciferase and ß-gal Assays Mix 20ul of cell lysate and 100ul of Luciferase Assay reagent in cell culture tubes. Record values as relative light units. Mix 10ul of cell lysate, 90ul distilled water, and 200ul ß-gal reagent in 1.7mL eppendorf tubes. Water bath at 28˚C for 30 minutes. Stop reaction by adding 500ul of 1M Na 2 CO 3. Record absorption at 420nm.
n=3 P<.05 P>.05
P<.05 n=3 n=1
ADAR1 does not affect p21 expression through 3’UTR P>.05
Conclusions Lipofectamine 2000 transfections suggested ADAR1 involvement in p21 transcription. Observation: Lipofectamine 2000 transfected cells looked sick. Toxic effect that possibly increases p21 levels through p53. Less toxic transfection reagent (Clontech X-fect) indicated that ADAR1 did not affect p21 transcription. p53 levels are possibly elevated in response to the toxicity brought about by Lipofectamine ADAR1 somehow opposes this action.
Future Directions To test this opposition against p53, ADAR1 must be added to p21 promoter constructs without both of p53 binding sites. It is possible that ADAR1 suppresses the p21 promoter directly, but future work is needed. ADAR1 did not significantly affect p21 expression at the level of the 3’-UTR. It is possible that ADAR1 suppresses p21 protein translation or protein stability.
Acknowledgements Richard A Steinman MD, PhD Qingde Wang MD, PhD Christine Stehle Mark Krotec
References "Chronic Myelogenous Leukemia." National Cancer Institute. 03 July ent/page1 Richard A Steinman et. al. “Deletion of the RNA-editing enzyme ADAR1 causes regression of established chronic myelogenous leukemia in mice” (Under review, 2011) Rohit Sharma, Qingde Wang "RNA-editing enzyme ADAR1 supports cell division by inhibiting p21" (In preparation, 2011) Weinberg, Robert A. The Biology of Cancer. New York: Garland Science, 2007
Procedure Clontech X Transfection 1. Prepare cells for transfection. Adherent cells: One day prior to the transfection, plate cells in 1 ml of complete growth medium so that the cells will be 50–80% confluent at the time of transfection. Suspension cells: Just prior to preparing complexes (step 2), plate 5 x 105–1.25 x 106 cells in 1 ml of growth medium. 2. Thoroughly vortex Xfect Polymer. 3. For each transfection sample, prepare two microcentrifuge tubes with appropriate dilutions. 4. Vortex each tube well to mix. 5. Add the Polymer solution to the DNA solution and vortex well at a medium speed for 10 sec. 6. Incubate the samples for 10 min at room temperature to allow nanoparticle complexes to form. 7. Add the entire 200 μl of nanoparticle complex solution. 8. Incubate the plate at 37°C for 4 hr to overnight. 9. Remove nanoparticle complexes from cells by aspiration, replace with 2 ml fresh complete growth medium, and return the plate to the 37°C incubator until time of analysis. Peak expression is typically reached 48 hr posttransfection.