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

1. Effect of Irradiation Dose on Breast Cancer Cell ProliferationErin RiekeMentor: Dr. Christine Kelly

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

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

4. Recent Therapy • Brachytherapy • 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

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

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

7. Cell Culturing • Cells cultured in T flasks • Medium changed every 2-3 days • At confluency, cells passaged and split into more flasks • 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 • However, sometimes they don’t like to stick – reduced number of retained cells • Attachment factors used to increase number of cells that lay down • 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 • 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

10. Fibronectin and Cell Adhesion • Fibronectin required for cell adhesion • Most cells do not produce enough • Surface coated with 1-5 ug/cm2 fibronectin • 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 • 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 laboratory • Cell line ZR-75-1 • Plate in 48-well and 96-well plate for testing • Expose cells to radioactive source at various strengths • Perform proliferation and cytotoxicity assays at various time points after the irradiation

13. Analysis • 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 • Assays to be performed: • LDH Cytotoxicity Assay • MTT Cell Proliferation Assay • Live/Dead Staining with Trypan Blue • Caspase 3/CPP32 Colorimetric Assay

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

15. LDH Cytotoxicity Assay • LDH usually impermeable to cell membrane • When cell membrane damaged, released into surrounding medium • 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 thawed and placed into 96-well plate • Added LDH dye solution (Biovision K311-400) and allowed 30 min for color development • Analyzed with a microplate reader at 490 nm

17. Percent Cytotoxicity • Percent cytotoxicity calculated: • Low control = normal cells (normal LDH levels) • 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 • Yellow tetrazolium salt MTT cleaved into purple formazan by mitochondrial dehyrogenases found in active cells • 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 • 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 • Color development analyzed by spectrophotometer at 570 nm

21. MTT Results

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

23. Live/Dead Staining Results

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

25. Caspase 3/CPP32 Colorimetric Assay Protocol • Cells collected, pelletted, and lysed • 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 • pNA levels analyzed with spectrophotometer at 400 nm

26. Caspase 3/CPP32 Colorimetric Assay Results

27. Summary • 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 • 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

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

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

30. References • 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 • Medicine.Net. http://www.medterms.com/script/ main/art.asp?articlekey=23606. Visited -9/18/05