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Title: The interaction of mTOR and autophagy in salivary glands
Author: Kawashima, Naomasa
ISNI:       0000 0004 7427 9905
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2018
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Radiation therapy to treat head and neck cancer damages salivary glands, leading to decreased salivary flow which can cause xerostomia (perception of dry mouth), mucositis and dysphagia. These symptoms of salivary gland hypofunction are a major cause of patients stopping their treatment and greatly decrease their quality of life after treatment. Protecting from damage is essential to avoid these problems so this thesis examined some of the damage mechanisms present in salivary glands. Two intracellular processes seem to be particularly relevant. Mammalian Target of Rapamycin (mTOR) is a growth pathway often activated in cancers, repair and regeneration whereas autophagy is a self-digestion of cellular contents often associated with damage that helps to protect cells from apoptosis. Normally the two processes are linked by an enzyme UNC-like kinase (ULK) in a mutually exclusive way so that growth and degradation do not occur at the same time. However, both mTOR and autophagy have been shown to have beneficial effects after irradiation. Therefore, we decided to study the interactions between mTOR and autophagy in order to find an efficient way to uncouple mTOR and autophagy to protect irradiated salivary glands. Since ULK was an important link between mTOR and autophagy, an interesting new ULK inhibitor MRT67307 was used on salivary glands. In the first part of this study, we evaluated the effects of MRT67307 on cell cultures in order to collect enough data before trying the drug in vivo. Initially NIH 3T3 cells, a well-studied cell model, were cultured to verify the effects of MRT67307. As previously reported, the drug blocked starvation- and Torin 1 (an mTOR inhibitor)- induced autophagy. The next step was to test the effects of the drug on salivary acinar cells which were known to be very sensitive to irradiation. SMG-C6 cells were chosen since they were previously derived from rat submandibular acinar cells. In these cells, in contrast to NIH 3T3 cells, Torin 1 failed to upregulate autophagy, suggesting that mTOR and autophagy were not linked by ULK. This finding was interesting and novel and was further tested in primary-cultured cells (ie in vitro) from mouse submandibular glands. Again, administration of Torin 1 inhibited mTOR but did not activate autophagy and MRT67307 had no effect on marker of autophagy (LC3-I/LC3-II ratios). It could be inferred from these experiments that MRT67307 is a useful tool in examining mTOR/autophagy interactions through ULK1 and that in salivary glands autophagy and mTOR could be activated simultaneously. In the second part of this study, we carried out whole body irradiation of mice to study damage, mTOR-autophagy interactions and saliva flow variation in irradiated salivary glands. A dose escalation study appeared to cause minimal damage to salivary glands when the maximum dose of 11 Gy was given. To determine if salivary hypofunction had occurred whole mouth saliva was collected under temporary gaseous anaesthesia by the administration of pilocarpine (I.P.). Surprisingly, despite minimal histological indications of damage an increase in salivary function occurred. Biochemical analyses of the salivary glands indicated autophagy was transiently and weakly activated a few hours after irradiation whereas mTOR activity occurred a few days later. The use of a whole body irradiator limited the dose of irradiation to the salivary glands. Thus as a model system, transient mTOR activation probably had a beneficial effect, since pilocarpine stimulated saliva flow experiment showed a transient increase of saliva flow. However, this model system did not yield the expected salivary gland damage seen in other studies so instead another model of salivary gland damage, ductal ligation was studied. The third part of this study attempted to use autophagy-inhibitors in vivo, using the ligated salivary gland since autophagy activation was weak and did not last in irradiated salivary gland. Whole body injection of autophagy inhibitors chloroquine and MRT67307 (at two different doses and injection intervals) did not appear to have any beneficial effect on submandibular glands, except a slight delay of atrophy in both chloroquine and MRT67307 treated glands. Autophagy appeared to be mainly mTOR independent since MRT67307 failed to inhibit autophagy. This thesis contains novel data to indicate that autophagy and mTOR are independent of each in mouse submandibular glands. To the best of our knowledge, this is the first time that MRT67307 was used in vivo and no paper demonstrates an mTOR independent activation of autophagy in salivary glands. It provides the basis for further studies to protect salivary glands from irradiation damage by upregulating both mTOR and autophagy simultaneously, something that has not, so far, been tested.
Supervisor: Carpenter, Guy Howard ; Proctor, Gordon Burgess Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available