A study of the cytotoxic effects of methionine depletion in human brain tumour cell lines

The aim of this project was to investigate the importance of methionine depletion as a cause of cytotoxicity for paediatric CNS tumour cell lines, and also to investigate the in vitro cellular biochemical responses, as measured by changes in cellular levels of L-methionine, glutathione and O6 - alkylguanine DNA - alkyltransferase (MGMT) activity in glioma (D54) and medulloblastoma (Daoy) cell lines. A characteristic feature of many solid tumours is their requirement for both endogenous and exogenous L-methionine in order to support cellular proliferation. Normal cells are generally methionine-independent and utilise L-homocysteine to produce sufficient levels of L-methionine for cellular proliferation. The work presented in this thesis shows that Daoy and D54 cells are methionine-dependent cell lines in that they stop proliferating in methionine-depleted media supplemented with L-homocysteine. Whereas D54 cells do not exhibit detectable MGMT activity, methionine depletion markedly down-regulates the activity of this DNA repair enzyme in Daoy cells. Methionine depletion gives rise to a demonstrable decrease in methionine levels and an increase in glutathione levels for both tumour cell lines. Moreover, Daoy and D54 cells are found to be significantly more resistant to methotrexate (MTX), temozolomide (TMZ), and cisplatin (CDDP) under conditions of methionine depletion, compared to controls under normal cell culture conditions, a finding that may, at least in part, be related to the increased glutathione levels found. Further studies are required to determine the relative contribution of glutathione levels and modulation of apoptosis to explain the reasons for the reduced chemosensitivity to MTX, CDDP and TMZ for both Daoy and D54 cells. Evidence suggests that TMZ, CDDP and MTX may modulate cellular determinants of chemosensitivity through effects on methionine metabolism and MGMT levels. Therefore, the potential synergies of TMZ, CDDP and/or MTX for Daoy and D54 cells were investigated in methionine-replete conditions, and the combination of MTX and TMZ in particular was found to demonstrate synergistic effects. Further studies are needed to determine the reasons for this effect, and these may give insights into the further clinical development of these drugs in the setting of childhood CNS tumour therapy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo