Ahmed final_bp

An in vitro screen to identify gene mutations causing resistance to cisplatin chemotherapy

AL Hilli [email protected]

Supervisor Pickard B.BM 913. August 2015

© Ahmed Al Hilli , Strathclyde University

Cancer

Solution

Chemotherapy

• Uncontrolled multiplication of cells.

• Major cause of death:

of the deaths in the UK are due to cancer, (Rang, H P.et al., 2014).

Powerful chemotherapy drugs kill cancer cells by arresting their growth at one or more checkpoints in their cell cycle.( Paxton, J., 2012).

Cisplatin [cDDP]

A key therapy :

• Testicular, bladder, ovarian, head and neck, cervical, lung & colorectal cancer. (Koberle, B. et al, 2010).

• Binding to DNA forming adducts causing different types of DNA lesions.

Problem

The efficacy of cDDP limited due to:Acquired or Intrinsic resistance.

Possible mechanisms:

• Intracellular accumulation.• DNA repair.• Calcium signalling and Apoptosis pathway.(Koberle, B. et al. ,2010).

Resistance To Cisplatin

The Project’s Aim

To identify genes causing cisplatin resistance in HEK293 cells, which can be used in the future to investigate the resistance phenotype in patient tumours.

Gene Trapping used

What is gene trapping?

Technique used to randomly disrupt genes throughout the genome.• Insert a DNA element (pGTIV3) reporter gene & selectable

marker.• If it inserts in a gene intron, the gene will be disrupted.• It will also produce a gene trap – gene fusion mRNA which can

be amplified by PCR and sequenced to identify the disrupted gene.

(Gentile, A. et al., 2013).

An objective, economic & high-throughput tool to dissect genetic influences on cell function

My Research ( Story of George )

HEK293 cells mutated with Gene Traps plated on 10 x 6-well plates.

SCREEN: 4.44µM concentration of cDDP added: cytotoxic.

• All wild type cells dead (Control).• Some mutated cells surviving. ‘George’

My Research ( Story of George )

George and Colleagues picked, expanded and archived.

cDNA

PCR(RAEC1&2)

Purification

Vector ligationInto Bacteria, onto agar plate, sequencing of colonies

THEN …..

Results: 4 resistance genes found so far…

(George)

Results: MRPS27 validation in literature

(George)

Mitochondrial translation protein, known to associate with Death Associated Protein 3 (DAP3): Apoptosis role??

Results: NACA validation in literature

(George) Nascent polypeptide-associated complex alpha subunit (NACA): stops incorrect targeting of proteins, also role in apoptosis??

Clinical Implications

Two fundamental clinical implications:

1. Help identify type of resistance in a patient tumour2. Design new chemotherapy drugs that avoid

resistance

Acknowledgment

To people who care , because it is not fair to thanks ,all people those who care and who not . To the un known person , who had these cells in our life . To My supervisor .In the memorial of Dr Elizabeth Ellis.

References

Rang, H.P., Ritter, J.M., Flower, R.J. &Henderson, G.(2014). Rang and dale’s . Pharmacology (8th ed.) (pp.6766-678).