Cell Research – Prof Nicolas Nowitzky
Prof Nicolas Nowitzky
UCT Medical School, Division of Haematology
- Prof N.Novitzky (Principal investigator)
- Dr Karen Shires (Project leader, student supervisor)
- Sammy Njikan (Masters student)
Title of project
The search for a CML-specific antigen. An investigation into the changes that occur at the cell surface as a result of transformation with active BCR-ABL
What is the aim of this project?
Identification of CML-specific cell surface antigens, using a human fibroblast cell line expressing the oncogenic BCR-ABL protein.
- Construct and fully characterise a stable human fibroblast cell line, which is expressing the oncogenic BCR-ABL protein.
- Use a bi-directional approach to define the protein changes that occur on the fibroblast cellular surface as a result of BCR-ABL expression: A) Phage display technology, using peptide and antibody libraries, to identify novel cell-surface binding ligands (receptor identification), B) Two-dimensional protein analysis to directly identify BCR-ABL-associated cell surface proteins.
- Expression of BCR-ABL-associated proteins, identified in the cell line model, will be assessed in CML patients (chronic and blastic crisis phases) to validate their clinical significance as CML-specific antigens.
What has been achieved to date?
After initial plasmid purification issues, we have successfully characterised all the plasmids involved in the study and expanded the necessary cell lines. We have transfected MIGR1 and MIG210 into NIH3T3 as a positive control to test our transfection methodology (Fugene, Roche) and have successfully generated GFP-expressing transfectants, which were sorted using the FACS cell sorter. Unfortunately these GFP-expressing sorted cells could not be expanded (on several occasions). This is due to the generation of unstable transient transfections, as well as cell damage caused during cell sorting. The transfection efficiencies using these plasmids and Fugene in the 3T3 cell line are probably not high enough to generate stable transfectants, using the GFP-expression as the only selection criteria. Several different approaches have been used to try and increase the transfection efficiency, but to no avail (all below 40%). It will therefore be necessary to concentrate on the drug selection plasmids, using them alone or in co-transfections with the GFP-expressing control plasmids, to generate stable transfections. G418 survival curves are now being performed on all the cell lines, to establish the optimal selection concentration prior to transfection. Other methodologies such as Nucleofector and electroporation are being investigated as alternative methods to liposome-mediated transfection, to improve the transfection efficiencies.
While waiting for the transfection results, the student revived 3T3-pGD210 cells that were received from Prof Warren Pear’s research group in the USA and began setting up the assays that are needed for transfectant characterisation. These included: western analysis of BCR/ABL, ABL and p27; RT-PCR analysis of PRAME and BCR/ABL; AV/PI staining for apoptosis. These techniques will be used on our transfected cell-lines once they have been expanded.
The student has also successfully completed a well-written project proposal that was accepted by the University Research Committee as his Masters proposal. He has also finished his literature review, methodology and the plasmid characterisation results section towards his thesis. He has also given 2 journal club presentations on his work.
What do you hope to achieve in the next year?
The following are our goals until June 2010:
- Transfection of all 4 cell lines with pGD210 and/ or co-transfection with MIGp210 (GFP) and pCVT (G418 resistance), using liposome-mediated transfection methodology.
- If necessary, electroporation transfection of human fibroblasts
- Characterisation of the transfectants as follows:
– BCR/ABL integration into genomic DNA
– BCR/ABL mRNA expression
– BCR/ABL protein expression
– Effects of BCR/ABL expression on cellular proliferation: P27 expression
– Effects of BCR/ABL expression on cytoskeletal arrangements: actin filament staining
- If stable transfection of the 3 human cell lines is unsuccessful, Objective 2 (protein analysis of cell membrane proteins) will be performed on the 3T3-pGD210 cell line supplied by Dr Warren Pear (once fully characterised).
- Completion of MSc degree for Sammy Njikan