effect of irradiation dose on breast cancer cell proliferation erin rieke mentor dr christine kelly l.
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Effect of Irradiation Dose on Breast Cancer Cell Proliferation Erin Rieke Mentor: Dr. Christine Kelly. 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

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effect of irradiation dose on breast cancer cell proliferation erin rieke mentor dr christine kelly

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

breast cancer
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%)
background
Background
  • Current treatment of breast cancer
    • Removal of tumor followed by external beam radiotherapy to the whole breast
recent therapy
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
background cont
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
objectives
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
cell culturing
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
optimizing culture environment
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
fibronectin
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
fibronectin and cell adhesion
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
general experimental methods
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
analysis
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
ldh cytotoxicity assay
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
ldh cytotoxicity assay15
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).
ldh protocol
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
percent cytotoxicity
Percent Cytotoxicity
  • Percent cytotoxicity calculated:
  • Low control = normal cells (normal LDH levels)
  • High Control = cells treated with 1% Triton X-100 (full LDH release)
mtt cell proliferation assay
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
mtt protocol
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
live dead staining
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
caspase 3 cpp32 colorimetric assay
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
caspase 3 cpp32 colorimetric assay protocol
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
summary
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
further research
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
thank you
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
references
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