Effect of Irradiation Dose on Breast Cancer Cell Proliferation Erin Rieke Mentor: Dr. Christine Kelly.
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Effect of Effect of Irradiation Dose Irradiation Dose on Breast Cancer on Breast Cancer Cell ProliferationCell Proliferation
Erin RiekeErin RiekeMentor: Dr. Christine KellyMentor: Dr. Christine Kelly
Breast CancerBreast Cancer
Most prevalent cancer in female Most prevalent cancer in female population, except skin cancerpopulation, except skin cancer
1 in 7 (13.4%) chance of developing 1 in 7 (13.4%) chance of developing invasive breast cancerinvasive breast cancer
Currently, 2 million women living Currently, 2 million women living with breast cancerwith breast cancer
Chance of dying is 1 in 33 (3%)Chance of dying is 1 in 33 (3%)
BackgroundBackground
Current treatment of breast cancerCurrent treatment of breast cancer Removal of tumor followed by external beam Removal of tumor followed by external beam
radiotherapy to the whole breastradiotherapy to the whole breast
Recent TherapyRecent Therapy BrachytherapyBrachytherapy
Radiation is delivered through catheters inserted Radiation is delivered through catheters inserted through the target area through the target area
Radioactive solution flows through the catheters Radioactive solution flows through the catheters for a short period of time and irradiates the tumor for a short period of time and irradiates the tumor cavitycavity
Background cont.Background cont. Problem with catheter Problem with catheter
based brachytherapybased brachytherapy Requires the catheters to Requires the catheters to
be inserted and remain in be inserted and remain in the breast for the length of the breast for the length of treatmenttreatment
Requires one to two day Requires one to two day hospital stayhospital stay
Problem SolutionProblem Solution Radioactive pellet instead Radioactive pellet instead
of radiation fluidof radiation fluid However, has only been However, has only been
tested with prostate cancer tested with prostate cancer – needs to be tested with – needs to be tested with breast cancerbreast cancer
ObjectivesObjectives
Culture breast cancer cellsCulture breast cancer cells Observe and analyze the effect of Observe and analyze the effect of
irradiation on breast cancer cell irradiation on breast cancer cell proliferationproliferation
Characteristics to be examinedCharacteristics to be examined Time After ExposureTime After Exposure Exposure StrengthExposure Strength
Cell CulturingCell Culturing Cells cultured in T Cells cultured in T
flasks flasks Medium changed Medium changed
every 2-3 daysevery 2-3 days At confluency, cells At confluency, cells
passaged and split passaged and split into more flasksinto more flasks
When adequate cell When adequate cell number reached, number reached, cells frozen and cells frozen and stored in liquid stored in liquid nitrogennitrogen
Optimizing Culture Optimizing Culture EnvironmentEnvironment
Adherent cells – form a discrete net Adherent cells – form a discrete net on the bottom of the culture flaskon the bottom of the culture flask
However, sometimes they don’t like to However, sometimes they don’t like to stick – reduced number of retained stick – reduced number of retained cellscells
Attachment factors used to increase Attachment factors used to increase number of cells that lay downnumber of cells that lay down
Fibronectin used to coat flasks before Fibronectin used to coat flasks before seeded with cells seeded with cells
FibronectinFibronectin
A multi-domain glycoprotein found in A multi-domain glycoprotein found in connective tissue, on cell surfaces, connective tissue, on cell surfaces, and in plasma and other body fluidsand in plasma and other body fluids
Interacts with a variety of Interacts with a variety of macromolecules including macromolecules including components of the cytoskeleton and components of the cytoskeleton and the extracellular matrix the extracellular matrix
Binds cell surfaces and various Binds cell surfaces and various compounds including collagen, fibrin, compounds including collagen, fibrin, heparin, DNA, and actin heparin, DNA, and actin
Fibronectin and Cell Fibronectin and Cell AdhesionAdhesion
Fibronectin required for cell adhesionFibronectin required for cell adhesion Most cells do not produce enoughMost cells do not produce enough Surface coated with 1-5 ug/cmSurface coated with 1-5 ug/cm22
fibronectinfibronectin Test run to examine usefulness of Test run to examine usefulness of
fibronectin coating with breast cancer fibronectin coating with breast cancer cellscells
Two T-25 flasks seeded with same number Two T-25 flasks seeded with same number of cells – one coated and one notof cells – one coated and one not
After 4 days, cell count performed to After 4 days, cell count performed to determine number of adherent cellsdetermine number of adherent cells
Fibronectin Test ResultsFibronectin Test Results
0.0E+00
1.0E+05
2.0E+05
3.0E+05
4.0E+05
5.0E+05
6.0E+05
7.0E+05
8.0E+05
9.0E+05
Coated Non-Coated
Number of Adherent Cells
General Experimental General Experimental MethodsMethods
Culture breast cancer cells Culture breast cancer cells in in
laboratorylaboratory Cell line ZR-75-1Cell line ZR-75-1 Plate in 48-well and 96-well Plate in 48-well and 96-well
plate for testingplate for testing Expose cells to radioactive Expose cells to radioactive
source at various strengthssource at various strengths Perform proliferation and Perform proliferation and
cytotoxicity assays at cytotoxicity assays at various time points after the various time points after the irradiationirradiation
AnalysisAnalysis Efficacy of irradiation to be tested with Efficacy of irradiation to be tested with
various assaysvarious assays
The multi-well plates will be useful in The multi-well plates will be useful in following the effects over a period of timefollowing the effects over a period of time
Assays to be performed:Assays to be performed: LDH Cytotoxicity AssayLDH Cytotoxicity Assay MTT Cell Proliferation AssayMTT Cell Proliferation Assay Live/Dead Staining with Trypan BlueLive/Dead Staining with Trypan Blue Caspase 3/CPP32 Colorimetric AssayCaspase 3/CPP32 Colorimetric Assay
LDH Cytotoxicity AssayLDH Cytotoxicity Assay
Tests levels of lactate dehydrogenase, Tests levels of lactate dehydrogenase, enzyme present in all cells in the mediumenzyme present in all cells in the medium
Facilitates conversion of lactate into Facilitates conversion of lactate into pyruvate, creating NADH in the process pyruvate, creating NADH in the process
LDH Cytotoxicity AssayLDH Cytotoxicity Assay LDH usually impermeable to cell membraneLDH usually impermeable to cell membrane When cell membrane damaged, released into When cell membrane damaged, released into
surrounding mediumsurrounding medium NADH used to convert tetrazolium salt INT NADH used to convert tetrazolium salt INT
into a formazan product (purple color).into a formazan product (purple color).
LDH ProtocolLDH Protocol
Samples of cell-free medium taken at Samples of cell-free medium taken at 0,12,48,72, and 96 hours after exposure 0,12,48,72, and 96 hours after exposure and frozenand frozen
Samples thawed and placed into 96-well Samples thawed and placed into 96-well plateplate
Added LDH dye solution (Biovision K311-Added LDH dye solution (Biovision K311-400) and allowed 30 min for color 400) and allowed 30 min for color developmentdevelopment
Analyzed with a microplate reader at 490 Analyzed with a microplate reader at 490 nmnm
Percent CytotoxicityPercent Cytotoxicity
Percent cytotoxicity calculated:Percent cytotoxicity calculated:
Low control = normal cells (normal Low control = normal cells (normal LDH levels)LDH levels)
High Control = cells treated with 1% High Control = cells treated with 1% Triton X-100 (full LDH release)Triton X-100 (full LDH release)
LDH ResultsLDH ResultsLDH Levels After Irradiaiton Dose of 20 Gy
-10
0
10
20
30
40
50
60
70
0 12 24 36 48 60 72 84 96 108
Time After Exposure (hrs)
Cytotoxicity (%)
MTT Cell Proliferation MTT Cell Proliferation AssayAssay
Tests for metabolic Tests for metabolic activity of viable cellsactivity of viable cells
Yellow tetrazolium Yellow tetrazolium salt MTT cleaved into salt MTT cleaved into purple formazan by purple formazan by mitochondrial mitochondrial dehyrogenases found dehyrogenases found in active cellsin active cells
Similar idea to LDH Similar idea to LDH Cytotoxicity AssayCytotoxicity Assay
MTT ProtocolMTT Protocol
10 uL of 5 mg/ml MTT solution 10 uL of 5 mg/ml MTT solution (Chemicon CT02) added to test and (Chemicon CT02) added to test and control wellscontrol wells
Allowed to react for 4 hours – black Allowed to react for 4 hours – black crystals form on bottom of flaskcrystals form on bottom of flask
Isopropanol and HCl solution added to Isopropanol and HCl solution added to dissolve crystals and negate neutralize dissolve crystals and negate neutralize medium colormedium color
Color development analyzed by Color development analyzed by spectrophotometer at 570 nmspectrophotometer at 570 nm
MTT ResultsMTT ResultsCell Proliferation Response to 20 Gy
00.5
11.5
22.5
33.5
44.5
0 24 48 72 96 120Time After Exposure (hrs)
Cell Number (OD570nm)
Control Experimental
Live/Dead StainingLive/Dead Staining
Trypan Blue used to stain dead cells.Trypan Blue used to stain dead cells.
Compromised cell membranes allow Compromised cell membranes allow uptake of blue dyeuptake of blue dye
Cell counted using a hemocytometer Cell counted using a hemocytometer and % viability foundand % viability found
Live/Dead Staining Live/Dead Staining ResultsResultsPerecnt Viability from Live/Dead Staining with
Trypan Blue - 20 Gy
0
20
40
60
80
100
120
0 24 48 72 96 120
Time After Exposure (hrs)
Percent Viability
Control Experimental
Caspase 3/CPP32 Colorimetric Caspase 3/CPP32 Colorimetric AssayAssay
Caspase 3 know Caspase 3 know mediator of apoptosis mediator of apoptosis (programmed cell death)(programmed cell death)
Member of family of Member of family of asparate-specific asparate-specific cysteinyl proteases cysteinyl proteases
Can cleave artificial Can cleave artificial substrates consisting of substrates consisting of an appropriate sequence an appropriate sequence of four amino acidsof four amino acids
Resulting compounds Resulting compounds can be analyzed can be analyzed fluorometrically or fluorometrically or colorimetricallycolorimetrically
Caspase 3/CPP32 Caspase 3/CPP32 Colorimetric Assay ProtocolColorimetric Assay Protocol Cells collected, pelletted, and lysedCells collected, pelletted, and lysed Cytosolic extract allowed to react Cytosolic extract allowed to react
with DEVD-pNA (N-acetyl-Asp-Glu-with DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-pNA)Val-Asp-pNA)
Active caspase 3 cleaves at Asp Active caspase 3 cleaves at Asp residue and leaves free pNA (p-residue and leaves free pNA (p-nitroanilide) – chromogenicnitroanilide) – chromogenic
pNA levels analyzed with pNA levels analyzed with spectrophotometer at 400 nmspectrophotometer at 400 nm
Caspase 3/CPP32 Caspase 3/CPP32 Colorimetric Assay ResultsColorimetric Assay Results
Caspase 3 Response to 30 Gy
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
24 hours 72 hours
Caspase Activity (OD 400 nm)
Experimental Control
SummarySummary LDH – Cell stress/death increased 48 LDH – Cell stress/death increased 48
hours and beyond with 20 Gyhours and beyond with 20 Gy MTT – No significant change in cell MTT – No significant change in cell
proliferation within 96 hours of proliferation within 96 hours of exposure to 20 Gyexposure to 20 Gy
Live/Dead Staining – Cell viability Live/Dead Staining – Cell viability declined starting at 48 hours with 20 declined starting at 48 hours with 20 Gy, compared to the controlGy, compared to the control
Caspase3/CPP32 Assay – No Caspase3/CPP32 Assay – No significant increase in caspase significant increase in caspase activity seen with in 72 hours of activity seen with in 72 hours of exposure to 30 Gy exposure to 30 Gy
Further ResearchFurther Research
Examine multiple exposure Examine multiple exposure strengthsstrengths
Determine optimum culture Determine optimum culture conditions for irradiation conditions for irradiation experimentsexperiments
Perform experiments with Matrigel Perform experiments with Matrigel basement membrane matrix – basement membrane matrix – resembles the mammalian cellular resembles the mammalian cellular basement membrane basement membrane
Thank YouThank You Dr. Christine Kelly – OSU Chemical Dr. Christine Kelly – OSU Chemical
Engineering DepartmentEngineering Department Dr. Frank Chaplen – OSU Biological Dr. Frank Chaplen – OSU Biological
Engineering DepartmentEngineering Department HHMI ProgramHHMI Program Dr. Chris Mathews – OSU Biochemistry and Dr. Chris Mathews – OSU Biochemistry and
BiophysicsBiophysics Dr. Kevin Ahern – OSU Biochemistry and Dr. Kevin Ahern – OSU Biochemistry and
BiophysicsBiophysics Dr. Alena Paulenova – OSU Nuclear Dr. Alena Paulenova – OSU Nuclear
Engineering DepartmentEngineering Department
ReferencesReferences Ingham, Kenneth. Fibronectin – Molecular Ingham, Kenneth. Fibronectin – Molecular
Interactions. http://home.comcast.net/ Interactions. http://home.comcast.net/ ~kennethingham/newsite/intro/. Visited 08/18/05~kennethingham/newsite/intro/. Visited 08/18/05
““What Are the Key Statistics for Breast Cancer?” What Are the Key Statistics for Breast Cancer?” American Cancer Society.American Cancer Society. http://www.cancer.org/docroot/CRI/content/CRI_2http://www.cancer.org/docroot/CRI/content/CRI_2_4_1X_What_are_the_key_statistics_for_breast_ca_4_1X_What_are_the_key_statistics_for_breast_cancer_5.asp?rnav=cri. Visited 07/05/05ncer_5.asp?rnav=cri. Visited 07/05/05
Medicine.Net. http://www.medterms.com/script/ Medicine.Net. http://www.medterms.com/script/ main/art.asp?articlekey=23606. Visited -9/18/05main/art.asp?articlekey=23606. Visited -9/18/05
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