Conference 2022 Live Talk
Investigating the cellular effects of Fra-1 in the CGL1 radiation induced tumorigenesis cell model
Authors and Affiliations
Wuroud Al-khayyat 1, Jake Pirkkanen 2, Douglas Boreham 1,2,3,4,5,6, T.C. Tai1,2,3,4,6 Christopher Thome1,2,5,6,7, Sujeenthar Tharmalingam 1,2,3,4,6
1. Department of Biology, Biomolecular Sciences, Laurentian University, Sudbury, Ontario, Canada 2. Medical Sciences Division, Northern Ontario School of Medicine, Sudbury, Ontario, Canada 3. Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada 4. Health Sciences North Research Institute, Sudbury, Ontario 5. Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario, Canada 6. Bruce Power, PO Box 1540, 177 Tie Rd, R.R. 2, Tiverton, Ontario, N0G 2T0, Canada. 7. Department of Physics and Astronomy, McMaster University, Hamilton, Ontario, Canada
The CGL1 cell line is a somatic hybrid system for quantitative analysis of neoplastic transformation frequency. The CGL1 cell was derived from fusion of normal human male fibroblast skin cells and cervical cancer HeLa cells. Irradiation of CGL1 cells with 7Gy of gamma radiation produced tumorigenic segregates called gamma induced mutants (GIM), and non-tumorigenic cells called gamma irradiated control (CON). We published a whole transcriptome study between CGL1, GIM, and CON cells, and identified 1067 differentially expressed genes (DEGs) between the GIM and CON cell lines. In general, GIMs are characterised by the loss of chromosome 11; transfer of chromosome 11 into GIM reduced neoplastic transformation frequency. Similarly, removal of chromosome 11 from CON cells increased transformation frequency. Therefore, we hypothesize that chromosome 11 may contain potential tumour suppressor genes that prevent radiation induced tumorigenesis in the CGL1 cell model. The FOSL1 gene, located on chromosome 11, is down-regulated in tumorigenic cells. FOSL1 codes for Fos-related antigen-1(Fra-1) and is part of the activator protein (AP-1) transcription factor family. We hypothesize that deletion of FOSL1 in CGL1 promotes radiation induced transformation frequency, while overexpression will decrease the transformation frequency. In this study, we generated unique CGL1 cell lines with either Fra-1 knockout or Fra-1 overexpression
Using CRISPR/dCas9 and Cas9 gene editing, CGL1 cells were infected with an in-house produced FRA-1 overexpression and FRA-1 knockout lentivirus. FRA-1 expression was validated using RT-qPCR, and Western blotting. After the cells were validated, we investigated the effect of Fra-1 overexpression and deletion on CGL1 growth and proliferation we performed clonogenic survival assay with 0Gy, 0.5Gy, 1Gy, 2Gy, 4Gy, 6Gy, and 9Gy of X-radiation. Also, to determine the contribution of Fra-1 in any cellular pathways within the CGL1 cells we ran RT-qPCR using a gene arrays of families that include DNA damage response, apoptosis, cell cycle regulation, and AP-1 complex. And we investigated HeLa carcinogenesis cellular marker using the alkaline phosphatase assay. Finally, we shall be conducting a neoplastic transformation assay to determine the cellular function of Fra-1 in controlling tumorigenicity frequency.
RT-qPCR and Western blot analysis showed that our in-house made FRA-1 lentivirus particles showed about 2 fold increase in FRA-1 protein within the CGL1FRA-1 overexpression cells and about 80% FRA-1 protein deletion in our CGL1FRA-1 knockout cells. Clonogenic survival assay between CGL1, CGL1dCas9, CGL1Cas9 and FRA-1 overexpression and knockout cells did not show any statistically significant when exposed to 0.5Gy, 1Gy, 2Gy, 4Gy, 6Gy, and 9Gy radiation. Alkaline phosphatase assay showed that alkaline phosphatase is about 1.3 fold increased within CGL1FRA-1 knockout cells compared to the control CGL1Cas9 cells, while it is about 0.7 decreased compared to the control CGL1dCas9 cells, however the results do not show any statistical significant. Gene expression time course experiment and neoplastic transformation assay are still in progress.
This project will increase the scientific knowledge about the cellular effects of Fra-1 protein and its contribution to cellular transformation from a non-malignant cell into a malignant cells, investigate the effect of activator protein complex function in CGL1 and determine any potential cellular pathways for carcinogenesis development.