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 Self-sufficient for growtho Insensitive to anti-growth signalso Limitless replicationo Sustained angiogenesis (uncontrolled division)o Resistant to apoptosiso Tissue invasion and metastsias

• 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

How to overcome cancer?

• If body itself cannot overcome the problem with transformed cells,

o Radiation therapyo Surgeryo Chemotherapyo Phototherapyo Targeted therapyo 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

Double strand breaks are the most serious kind of DNA damage

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

• 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

PIAA 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

24h γ-IR

24h Taxol

2N 4N

G2/M block

DNA content

Cell

cou

nt

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

DNA content

Sid

e S

catt

er

(SS

C)

Ctrl

24h γ-IR

24h Taxol

R3 = G2 phase cellsR2 = M phase cells

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

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