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 growth o Insensitive to anti-growth signals o Limitless replication o Sustained angiogenesis (uncontrolled division) o Resistant to apoptosis o Tissue invasion and metastsias

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… Wire dancing of the cells

How to overcome cancer? If body itself cannot overcome the problem with transformed cells, o Radiation therapy o Surgery o Chemotherapy o Phototherapy o Targeted therapy o 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 γ-IR Treated lymphoma cells DNA damage Phosphorylation P P

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 24h γ-IR 24h Taxol 2N4N G2/M block DNA content Cell count 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 Side Scatter (SSC) Ctrl 24h γ-IR 24h Taxol R3 = G2 phase cells R2 = M phase cells

Different Drug Treatments in Jurkat Cells Side Scatter (SSC) Cell Number DNA Content Ctrl Taxol HydroxyUrea Doxorubicin R6 = M phase cells R7 = G2 phase cells

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 Hallmarks of cancer: the next generation. Cell, 144, (Review) Aylon Y, Oren M Living with p53, dying of p53. Cell, 130. (Review) Castedo M. et al., Cell death by mitotic catastrophe: a molecular definition. Oncogene 23,