Download Characterization of a potential new drug in cancer therapy

Characterization of a
potential new drug in
cancer therapy
Lab 2
Salah Farag
Objectives
Evaluate a new potential anti cancer drug (PIA):
• Effect on cell cycle.
• Detection of DNA damage.
Scope of work
• Culture and maintain Jurkat cells.
• Count cells using Burker chamber.
• Stimulating Jurkat cells with different anti cancer drugs.
• Flow cytometry using FACS (simple stain using
Propidium iodide).
• Immunocyto chemistry (Immuno staining).
Causes of cancer
• Environmental stimulants (carcinogens)
• Genetic mutations (somatic-germ line)
Hallmarks of Cancer Cells
• What a “perfect cancer cell” is;
o
o
o
o
o
o
Self-sufficient for growth
Insensitive to anti-growth signals
Limitless replication
Sustained angiogenesis (uncontrolled division)
Resistant to apoptosis
Tissue invasion and metastsias
Wire dancing of the cells
• To avoid these processes of being malignant, cells have
an intrinsic balance mechanism
o Controlled growth and proliferation
o Tumour suppressors; cell –cycle arrest, repair, apoptosis
• Once this balance mechanism falls down transformation
begins…
How to overcome cancer?
• If body itself cannot overcome the problem with
transformed cells,
o
o
o
o
o
o
Radiation therapy
Surgery
Chemotherapy
Phototherapy
Targeted therapy
Transplantation
Common drugs used in
chemotherapy
• Chemotherapy refers to the use of chemical substances in
treatment of disease.
o Chemical treatment can be combined with radiation therapy in treatment of
human cancer.
• Examples of drugs used in the clinic;
o Alkylating agents, e.g. Cisplatin (crosslinking DNA  apoptosis)
o Alkaloids, e.g. Taxol (cytostatic - stabilizes microtubules)
o Antineoplastics, e.g. Doxorubicin (intercalates DNA)
Cell cycle
DNA Damage
Double strand breaks are the most serious kind of DNA damage
Staining of Microtubules and
Visualization of an M block
• Cytostatic drugs acting on the
cytoskeleton usually disrupts
normal spindle formation
• This in turn causes an M-block
in the cell cycle
• Staining of the microtubule
system can visualize abnormal
spindle formation
Multipolar spindles observed in
cells treated with taxol
Characterization of DNA
damage
DNA
damage
• Common method is the use of
H2AX foci assay
• H2AX is histone phosphorylation in
response to double strand DNA
damage
Phosphorylation
P
DNA
H2AX H2AX
P
H2AX H2AX
• Following phosphorylation
additional components are recruited
to the site of damage in order to
start repair
γ-IR Treated lymphoma cells
PIA
A potential new drug for cancer therapy
PIA
• Chemical compound found in HT screen of a chemical
library
• High efficacy and low toxicity in primary trials from
mouse models of lymphoma.
• The molecular mechanism of PIA (p53 independent
Inducer of Apoptosis) is yet unknown
First Assignment
• To run a FACS assay and analyse the
results the effect of PIA on Jurkat cells
Measuring DNA content by Flow Cytometry
Fluorescence-Activated Cell Sorting (FACS)
Effect of DNA damage and
cytostatic drugs in K562 cells
K562 cells
G2/M
block
24h γ-IR
24h Taxol
2N 4N
DNA content
K562; mylogeous leukemia
A mitotic block lowers the
granularity
• During mitosis membrane
fragmentation increases and
• Granularity decreases.
• This enables a separation
between cells stuck in G2 and
M
Side Scatter (SSC)
R3 = G2 phase cells
R2 = M phase cells
Ctrl
24h γ-IR
24h Taxol
DNA content
Different Drug Treatments in
Jurkat Cells
R6 = M phase cells
R7 = G2 phase cells
Ctrl
Ctrl
Hydroxyurea
Taxol
Doxorubicin
Cell Number
Taxol
Doxorubicin
DNA Content
Side Scatter (SSC)
HydroxyUrea
G2 Cells (%) M Cells (%)
21.13
2.014
7.24
0.16
19.14
32.67
59.33
1.43
You will get
• A T-cell leukaemia cell line, Jurkat cells.
• PIA, and other known drugs
• Access to Mol.biol FACS facility
• All required reagents and equipment needed to perform a FACS
analysis.
• How would you set-up an experiment to test PIA
on Jurkat cells by flow cytometer?
Second Assignment
• To find supporting and
complementary evidence of molecular
function of PIA by Fluorescence
Microscopy
Additional materials available
• All required reagents and equipment needed to perform an
immunofluorescence analysis of the cells
• Access to a Fluorescent microscope
• What are the possible functions of PIA anticancer agents?
• How would you plan an immunofluorescence
assay to investigate these possible functions of
the drug?
Suggested Readings
• Hannahan D, Weinberg RA. 2011. Hallmarks of cancer:
the next generation. Cell, 144, 646-674 (Review)
• Aylon Y, Oren M. 2007. Living with p53, dying of p53. Cell,
130. (Review)
• Castedo M. et al., 2004. Cell death by mitotic catastrophe: a
molecular definition. Oncogene 23, 2825-2837