1. Effect of Irradiation Dose on Breast Cancer Cell Proliferation Erin Rieke Mentor: Dr. Christine Kelly

2. Breast Cancer Most prevalent cancer in female population, except skin cancer Most prevalent cancer in female population, except skin cancer 1 in 7 (13.4%) chance of developing invasive breast cancer 1 in 7 (13.4%) chance of developing invasive breast cancer Currently, 2 million women living with breast cancer Currently, 2 million women living with breast cancer Chance of dying is 1 in 33 (3%) Chance of dying is 1 in 33 (3%)

3. Background Current treatment of breast cancer Current treatment of breast cancer Removal of tumor followed by external beam radiotherapy to the whole breast Removal of tumor followed by external beam radiotherapy to the whole breast

4. Recent Therapy Brachytherapy Brachytherapy Radiation is delivered through catheters inserted through the target area Radiation is delivered through catheters inserted through the target area Radioactive solution flows through the catheters for a short period of time and irradiates the tumor cavity Radioactive solution flows through the catheters for a short period of time and irradiates the tumor cavity

5. Background cont. Problem with catheter based brachytherapy Problem with catheter based brachytherapy Requires the catheters to be inserted and remain in the breast for the length of treatment Requires the catheters to be inserted and remain in the breast for the length of treatment Requires one to two day hospital stay Requires one to two day hospital stay Problem Solution Problem Solution Radioactive pellet instead of radiation fluid Radioactive pellet instead of radiation fluid However, has only been tested with prostate cancer – needs to be tested with breast cancer However, has only been tested with prostate cancer – needs to be tested with breast cancer

6. Objectives Culture breast cancer cells Culture breast cancer cells Observe and analyze the effect of irradiation on breast cancer cell proliferation Observe and analyze the effect of irradiation on breast cancer cell proliferation Characteristics to be examined Characteristics to be examined Time After Exposure Time After Exposure Exposure Strength Exposure Strength

7. Cell Culturing Cells cultured in T flasks Cells cultured in T flasks Medium changed every 2-3 days Medium changed every 2-3 days At confluency, cells passaged and split into more flasks At confluency, cells passaged and split into more flasks When adequate cell number reached, cells frozen and stored in liquid nitrogen When adequate cell number reached, cells frozen and stored in liquid nitrogen

8. Optimizing Culture Environment Adherent cells – form a discrete net on the bottom of the culture flask Adherent cells – form a discrete net on the bottom of the culture flask However, sometimes they don ’ t like to stick – reduced number of retained cells However, sometimes they don ’ t like to stick – reduced number of retained cells Attachment factors used to increase number of cells that lay down Attachment factors used to increase number of cells that lay down Fibronectin used to coat flasks before seeded with cells Fibronectin used to coat flasks before seeded with cells

9. Fibronectin A multi-domain glycoprotein found in connective tissue, on cell surfaces, and in plasma and other body fluids A multi-domain glycoprotein found in connective tissue, on cell surfaces, and in plasma and other body fluids Interacts with a variety of macromolecules including components of the cytoskeleton and the extracellular matrix Interacts with a variety of macromolecules including components of the cytoskeleton and the extracellular matrix Binds cell surfaces and various compounds including collagen, fibrin, heparin, DNA, and actin Binds cell surfaces and various compounds including collagen, fibrin, heparin, DNA, and actin

10. Fibronectin and Cell Adhesion Fibronectin required for cell adhesion Fibronectin required for cell adhesion Most cells do not produce enough Most cells do not produce enough Surface coated with 1-5 ug/cm 2 fibronectin Surface coated with 1-5 ug/cm 2 fibronectin Test run to examine usefulness of fibronectin coating with breast cancer cells Test run to examine usefulness of fibronectin coating with breast cancer cells Two T-25 flasks seeded with same number of cells – one coated and one not Two T-25 flasks seeded with same number of cells – one coated and one not After 4 days, cell count performed to determine number of adherent cells After 4 days, cell count performed to determine number of adherent cells

11. Fibronectin Test Results

12. General Experimental Methods Culture breast cancer cells in Culture breast cancer cells in laboratory laboratory Cell line ZR-75-1 Cell line ZR-75-1 Plate in 48-well and 96-well plate for testing Plate in 48-well and 96-well plate for testing Expose cells to radioactive source at various strengths Expose cells to radioactive source at various strengths Perform proliferation and cytotoxicity assays at various time points after the irradiation Perform proliferation and cytotoxicity assays at various time points after the irradiation

13. Analysis Efficacy of irradiation to be tested with various assays Efficacy of irradiation to be tested with various assays The multi-well plates will be useful in following the effects over a period of time The multi-well plates will be useful in following the effects over a period of time Assays to be performed: Assays to be performed: LDH Cytotoxicity Assay LDH Cytotoxicity Assay MTT Cell Proliferation Assay MTT Cell Proliferation Assay Live/Dead Staining with Trypan Blue Live/Dead Staining with Trypan Blue Caspase 3/CPP32 Colorimetric Assay Caspase 3/CPP32 Colorimetric Assay

14. LDH Cytotoxicity Assay Tests levels of lactate dehydrogenase, enzyme present in all cells in the medium Tests levels of lactate dehydrogenase, enzyme present in all cells in the medium Facilitates conversion of lactate into pyruvate, creating NADH in the process Facilitates conversion of lactate into pyruvate, creating NADH in the process

15. LDH Cytotoxicity Assay LDH usually impermeable to cell membrane LDH usually impermeable to cell membrane When cell membrane damaged, released into surrounding medium When cell membrane damaged, released into surrounding medium NADH used to convert tetrazolium salt INT into a formazan product (purple color). NADH used to convert tetrazolium salt INT into a formazan product (purple color).

16. LDH Protocol Samples of cell-free medium taken at 0,12,48,72, and 96 hours after exposure and frozen Samples of cell-free medium taken at 0,12,48,72, and 96 hours after exposure and frozen Samples thawed and placed into 96-well plate Samples thawed and placed into 96-well plate Added LDH dye solution (Biovision K311-400) and allowed 30 min for color development Added LDH dye solution (Biovision K311-400) and allowed 30 min for color development Analyzed with a microplate reader at 490 nm Analyzed with a microplate reader at 490 nm

17. Percent Cytotoxicity Percent cytotoxicity calculated: Percent cytotoxicity calculated: Low control = normal cells (normal LDH levels) Low control = normal cells (normal LDH levels) High Control = cells treated with 1% Triton X- 100 (full LDH release) High Control = cells treated with 1% Triton X- 100 (full LDH release)

18. LDH Results

19. MTT Cell Proliferation Assay Tests for metabolic activity of viable cells Tests for metabolic activity of viable cells Yellow tetrazolium salt MTT cleaved into purple formazan by mitochondrial dehyrogenases found in active cells Yellow tetrazolium salt MTT cleaved into purple formazan by mitochondrial dehyrogenases found in active cells Similar idea to LDH Cytotoxicity Assay Similar idea to LDH Cytotoxicity Assay

20. MTT Protocol 10 uL of 5 mg/ml MTT solution (Chemicon CT02) added to test and control wells 10 uL of 5 mg/ml MTT solution (Chemicon CT02) added to test and control wells Allowed to react for 4 hours – black crystals form on bottom of flask Allowed to react for 4 hours – black crystals form on bottom of flask Isopropanol and HCl solution added to dissolve crystals and negate neutralize medium color Isopropanol and HCl solution added to dissolve crystals and negate neutralize medium color Color development analyzed by spectrophotometer at 570 nm Color development analyzed by spectrophotometer at 570 nm

21. MTT Results

22. Apoptotic DNA Ladder Kit When cell undergo apoptosis, DNA is fragmented When cell undergo apoptosis, DNA is fragmented Assay for fragmented DNA by lysing cells and preparing DNA (Quick Apoptotic DNA Ladder Detection Kit, Biovision K120-50) Assay for fragmented DNA by lysing cells and preparing DNA (Quick Apoptotic DNA Ladder Detection Kit, Biovision K120-50) View DNA through agarose gel electrophoresis View DNA through agarose gel electrophoresis Apoptosis was induced with camptothecin (2 mM) for 0 hr (Lane 1), 6 hrs (Lane 2) and 12 hrs (Lane 3); 20 ml of each sample was electrophoresed on a 1.2% agarose/EtBr gel.

23. Live/Dead Staining Trypan Blue used to stain dead cells. Trypan Blue used to stain dead cells. Compromised cell membranes allow uptake of blue dye Compromised cell membranes allow uptake of blue dye Cell counted using a hemocytometer and % viability found Cell counted using a hemocytometer and % viability found

24. Live/Dead Staining Results

25. Caspase 3/CPP32 Colorimetric Assay Caspase 3 know mediator of apoptosis (programmed cell death) Caspase 3 know mediator of apoptosis (programmed cell death) Member of family of asparate- specific cysteinyl proteases Member of family of asparate- specific cysteinyl proteases Can cleave artificial substrates consisting of an appropriate sequence of four amino acids Can cleave artificial substrates consisting of an appropriate sequence of four amino acids Resulting compounds can be analyzed fluorometrically or colorimetrically Resulting compounds can be analyzed fluorometrically or colorimetrically

26. Caspase 3/CPP32 Colorimetric Assay Protocol Cells collected, pelletted, and lysed Cells collected, pelletted, and lysed Cytosolic extract allowed to react with DEVD- pNA (N-acetyl-Asp-Glu-Val-Asp-pNA) Cytosolic extract allowed to react with DEVD- pNA (N-acetyl-Asp-Glu-Val-Asp-pNA) Active caspase 3 cleaves at Asp residue and leaves free pNA (p-nitroanilide) – chromogenic Active caspase 3 cleaves at Asp residue and leaves free pNA (p-nitroanilide) – chromogenic pNA levels analyzed with spectrophotometer at 400 nm pNA levels analyzed with spectrophotometer at 400 nm

27. Caspase 3/CPP32 Colorimetric Assay Results

28. Summary LDH – Cell stress/death increased 48 hours and beyond with 20 Gy LDH – Cell stress/death increased 48 hours and beyond with 20 Gy MTT – No significant change in cell proliferation within 96 hours of exposure to 20 Gy MTT – No significant change in cell proliferation within 96 hours of exposure to 20 Gy Live/Dead Staining – Cell viability declined starting at 48 hours with 20 Gy, compared to the control Live/Dead Staining – Cell viability declined starting at 48 hours with 20 Gy, compared to the control Caspase3/CPP32 Assay – No significant increase in caspase activity seen with in 72 hours of exposure to 30 Gy Caspase3/CPP32 Assay – No significant increase in caspase activity seen with in 72 hours of exposure to 30 Gy

29. Further Research Examine multiple exposure strengths Examine multiple exposure strengths Determine optimum culture conditions for irradiation experiments Determine optimum culture conditions for irradiation experiments Perform experiments with Matrigel basement membrane matrix – resembles the mammalian cellular basement membrane Perform experiments with Matrigel basement membrane matrix – resembles the mammalian cellular basement membrane

30. Thank You Dr. Christine Kelly – OSU Chemical Engineering Department Dr. Christine Kelly – OSU Chemical Engineering Department Dr. Frank Chaplen – OSU Biological Engineering Department Dr. Frank Chaplen – OSU Biological Engineering Department HHMI Program HHMI Program Dr. Chris Mathews – OSU Biochemistry and Biophysics Dr. Chris Mathews – OSU Biochemistry and Biophysics Dr. Kevin Ahern – OSU Biochemistry and Biophysics Dr. Kevin Ahern – OSU Biochemistry and Biophysics Dr. Alena Paulenova – OSU Nuclear Engineering Department Dr. Alena Paulenova – OSU Nuclear Engineering Department

31. References Ingham, Kenneth. Fibronectin – Molecular Interactions. http://home.comcast.net/ ~kennethingham/newsite/intro/. Visited 08/18/05 Ingham, Kenneth. Fibronectin – Molecular Interactions. http://home.comcast.net/ ~kennethingham/newsite/intro/. Visited 08/18/05 “What Are the Key Statistics for Breast Cancer?” American Cancer Society. http://www.cancer.org/docroot/CRI/content/CRI_2_4_1X_ What_are_the_key_statistics_for_breast_cancer_5.asp?rnav=cri. Visited 07/05/05 “What Are the Key Statistics for Breast Cancer?” American Cancer Society. http://www.cancer.org/docroot/CRI/content/CRI_2_4_1X_ What_are_the_key_statistics_for_breast_cancer_5.asp?rnav=cri. Visited 07/05/05 Medicine.Net. http://www.medterms.com/script/ main/art.asp?articlekey=23606. Visited -9/18/05 Medicine.Net. http://www.medterms.com/script/ main/art.asp?articlekey=23606. Visited -9/18/05

32. Non-Sacrificial Assays Bioluminescence Cytotoxicity Assay Bioluminescence Cytotoxicity Assay Tests for adenylate kinase Tests for adenylate kinase Also an enzyme found in all cells Also an enzyme found in all cells Interconverts ATP to ADP Interconverts ATP to ADP Released from cells when plasma membrane is damaged Released from cells when plasma membrane is damaged Uses fluorescent plate reader to observe results Uses fluorescent plate reader to observe results