Irradiation effect on salivary gland characteristics and α6β1 cell culturing

Each year 500,000 new cases of head and neck cancer occur worldwide. Most of these patients have to undergo irradiation leading to irreversible damage to the salivary glands. In animal and human models α6β1, P63 positive cells were suggested as a source for salivary gland progenitor cells. Objective: to examine the effect of 15 Gy head and neck irradiation on body weight, salivary gland weight, saliva secretion and α6β1 cell growth in vitro before and after irradiation treatment. Methods: rats were irradiated and saliva was collected 24 hours before and 7, 21 days post irradiation. Rats were killed; glands were weighted and α6β1 positive cells were isolated. We further examined α6β1 positive cell growth in vitro before and after head and neck irradiation treatment and checked the possibility of labeling these cells with Renilla luciferase-EGFP chimera plasmid. Results: salivary gland function, gland weight, and body weight were reduced by head and neck irradiation. α6β1 positive isolated cells cultured from post irradiated rats resulted in no colony formation compared to control animals which produced confluent cultures in two weeks period. To examine labeling methodology, α6β1 and 293 cells were transfected by the Renilla luciferase-EGFP chimera plasmid and were monitored by a fluorescence microscope for EGFP expression and Renilla luciferase expression by luminescence emission. Two days later both EGFP and Renilla luciferas were detected as seen by microscopic appearance and luminescence measurements. Protein expression decreased about 50 fold 6 days after transfection. Conclusions: this study demonstrated that 15 Gy irradiation treatments damaged both salivary gland function and possibly its regeneration potential. To examine future transplantation solution, Cell labeling of α6β1 positive cells in a live animal should be developed. For that we show that EGFP-Renilla luciferas construct visualization of α6β1 is possible.