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Characterization of a potential new drug in cancer
therapy
Lab 2
Salah Farag
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Objectives
Evaluate a new potential anti cancer drug (PIA):
• Effect on cell cycle.
• Detection of DNA damage.
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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).
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Causes of cancer
• Environmental stimulants (carcinogens)
• Genetic mutations (somatic-germ line)
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Hallmarks of Cancer Cells
• What a “perfect cancer cell” is;
o Self-sufficient for growtho Insensitive to anti-growth signalso Limitless replicationo Sustained angiogenesis (uncontrolled division)o Resistant to apoptosiso Tissue invasion and metastsias
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• To avoid these processes of being malignant, cells have an intrinsic balance mechanismo Controlled growth and proliferationo Tumour suppressors; cell –cycle arrest, repair, apoptosis
• Once this balance mechanism falls down transformation begins…
Wire dancing of the cells
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How to overcome cancer?
• If body itself cannot overcome the problem with transformed cells,
o Radiation therapyo Surgeryo Chemotherapyo Phototherapyo Targeted therapyo Transplantation
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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)
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Cell cycle
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Double strand breaks are the most serious kind of DNA damage
DNA Damage
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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
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Characterization of DNA damage
• Common method is the use of H2AX foci assay
• H2AX is histone phosphorylation in response to double strand DNA damage
• Following phosphorylation additional components are recruited to the site of damage in order to start repair
DNA H2AX H2AX H2AX H2AX
γ-IR Treated lymphoma cells
DNA damage
Phosphorylation
PP
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PIAA potential new drug for cancer
therapy
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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
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First Assignment
• To run a FACS assay and analyse the results the effect of PIA on Jurkat cells
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Measuring DNA content by Flow Cytometry
Fluorescence-Activated Cell Sorting (FACS)
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Effect of DNA damage and cytostatic drugs in K562
cells K562 cells
24h γ-IR
24h Taxol
2N 4N
G2/M block
DNA content
Cell
cou
nt
K562; mylogeous leukemia
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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
DNA content
Sid
e S
catt
er
(SS
C)
Ctrl
24h γ-IR
24h Taxol
R3 = G2 phase cellsR2 = M phase cells
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Different Drug Treatments in Jurkat Cells
Sid
e S
catt
er
(SS
C)
Cell
Nu
mb
er
DNA Content
Ctrl
Taxol
HydroxyUrea
Doxorubicin
R6 = M phase cellsR7 = G2 phase cells
G2 Cells (%) M Cells (%)Ctrl 21.13 2.014Hydroxyurea 7.24 0.16Taxol 19.14 32.67Doxorubicin 59.33 1.43
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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?
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Second Assignment
• To find supporting and complementary evidence of molecular function of PIA by
Fluorescence Microscopy
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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?
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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