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
Chance of dying is 1 in 33 (3%)
Background

Current treatment of breast cancer

Removal of tumor followed by external beam radiotherapy to
the whole breast
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.

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

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




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




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



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 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
Fibronectin Test Results
Number of Adherent Cells
9.0E+05
8.0E+05
7.0E+05
6.0E+05
5.0E+05
4.0E+05
3.0E+05
2.0E+05
1.0E+05
0.0E+00
Coated
Non-Coated
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

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


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 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




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 calculated:

Low control = normal cells (normal LDH levels)
High Control = cells treated with 1% Triton X100 (full LDH release)

LDH Results
LDH Levels After Irradiaiton Dose of 20 Gy
70
Cytotoxicity (%)
60
50
40
30
20
10
0
-10
0
12
24
36
48
60
72
Time After Exposure (hrs)
84
96
108
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




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
MTT Results
Cell Number (OD570nm)
Cell Proliferation Response to 20 Gy
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Control
0
Experimental
24
48
72
Time After Exposure (hrs)
96
120
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
Live/Dead Staining Results
Perecnt Viability from Live/Dead Staining with
Trypan Blue - 20 Gy
120
Percent Viability
100
80
60
Control
Experimental
40
20
0
0
24
48
72
Time After Exposure (hrs)
96
120
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



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Cells collected, pelletted, and lysed
Cytosolic extract allowed to react with DEVDpNA (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
Caspase 3/CPP32 Colorimetric
Assay Results
Caspase 3 Response to 30 Gy
Caspase Activity (OD 400 nm)
0.45
Experimental
0.4
Control
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
24 hours
72 hours
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



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






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



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_W
hat_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