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Use of green fluorescent protein to mark hypoxic tumour cells Lange, Arney Harry


Objectives: The lack of oxygen in regions of many solid tumours has been shown to promote metastasis and genomic instability and is also associated with poor response to radiotherapy and some chemotherapy agents. The goal of this thesis was to develop and characterize a mouse xenograft tumour model that could be used to rapidly identify viable hypoxic cells. This model could then be applied in subsequent studies to evaluate therapies directed towards eradicating these resistant cells. Methods: A hypoxia reporter plasmid was constructed and transfected into SiHa human cervical carcinoma cells. The plasmid contained four hypoxia response elements (HRE's) upstream from the herpes simplex virus thymidine kinase promoter that was used to drive expression of the gene for green fluorescent protein (GFP). Upregulation of hypoxia inducible factor-1α (HIF-1α) was measured using Western blotting, and fluorescence activated cell sorting was used to verify that GFP expressing cells were resistant to radiation and able to bind the hypoxia marker pimonidazole. Results: SiHa cells showed increased expression of HIF-1α under hypoxic conditions. SiHa tumour cells were transfected and selected for their ability to express GFP under hypoxic conditions. Frozen sections showed that GFP expression was maximum for cells distant from tumour blood vessels. Cells sorted on the basis of high levels of GFP fluorescence were more resistant to killing by ionizing radiation, consistent with the known radioresistance of hypoxic tumour cells. Highly GFP positive tumour cells also bound significantly higher levels of the hypoxia marker pimonidazole. Conclusions: A stable human tumour cell transfectant was identified that preferentially expressed green fluorescent protein under hypoxic conditions. Cell sorting confirmed that highly GFP positive cells were more radioresistant and more likely to bind a hypoxia marker. Future studies could use this model and other hypoxia driven gene constructs for hypoxic cell killing.

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