Cellular and Molecular Studies on the Mechanistic Basis of Clinical Radioresistance in Human Glioma

Human malignant glioma exhibits considerable clinical radioresistance. It has been postulated that the phenomenon of intrinsic cellular radioresistance might underlie treatment failure in these tumours. The phenomena of cellular radioresistance has been investigated in this thesis, using as models both continuous human glioma-derived cell lines, and primary cultures. The cellular radiosensitivity of five continuous glioma lines has been evaluated by clonogenic assay; this has been compared with results obtained in eight other human tumour cell lines, derived from a range of human tumour types of varying clinical radiocurability, including embryonic tumours and carcinomas. Parameters of cellular radiosensitivity were derived by fitting multitarget, and linear-quadratic models of radiation action to survival data; the model-free parameter of cellular radiosensitivity, the surviving fraction of clonogenic cells at the clinically relevant dose of 2Gy (SF2), was also measured. Considerable heterogeneity of intrinsic cellular radiosensitivity was observed in the thirteen human tumour lines studied. All five continuous glioma lines displayed values of cellular radiosensitivity within the ranges previously observed in radioresistant human cell lines. By comparison, cell lines derived from three embryonic tumours exhibited marked cellular radiosensitivity. Considerable overlap in cellular radiosensitivity was noted between five carcinoma-derived lines, and the human glioma lines.Cellular radiosensitivity of 9 primary human glioma cultures was also assessed by soft agar cloning. The intrinsic cellular radiosensitivity of both primary cultures, and continuous cell lines derived from human malignant glioma were observed to fall within the range of values previously reported for radioresistant human tumours; it is therefore suggested that intrinsic cellular radioresistance might contribute to the profound clinical radioresistance observed in human glioma. Cellular recovery from ionising radiation damage has been proposed as a determinant of intrinsic radiosensitivity. The ability of human tumour cell lines of differing intrinsic radiosensitivity to recover from sublethal radiation damage (SLD) was investigated by clonogenic cell survival following split dose irradiation. Five glioma lines exhibited an ability to recover from sublethal radiation damage, which increased with radiation dose, as predicted by the linear-quadratic model of radiation action. SLD recovery was assessed in three radiosensitive human cell lines, derived from a teratoma, SUSA, a neuroblastoma, IMR32, and an ovarian carcinoma A2780. All three lines exhibited cellular recovery, but the extent of recovery at iso-effective radiation dose levels producing 90% cell kill were lower than observed in the glioma lines.Values for recovery half-times obtained in the glioma cell lines did not differ significantly from those obtained in the three radiosensitive cell lines. Proficient recovery from sublethal damage may therefore contribute to the cellular radioresistance observed in human glioma cell lines. The molecular basis of the differences in radiosensitivity observed in human tumours remains unclear A body of evidence supports the notion that DNA double strand breaks (dsb) are a class of radiation-induced lesions which can lead to both chromosomal aberrations, and cell death. An in vitro assay has been established to assess the ability of nuclear protein extracts derived from human cells to rejoin dsb. The model dsb substrate was pIC2OH, a 2.7Kb recombinant plasmid DNA molecule bearing two bacterial genes encoding ampicillin resistance and ?-galactosidase activity; dsb of defined end-structure were produced by restriction endonuclease digestion to yield both cohesive and noncohesive termini for rejoining reactions. Rejoining activity was assessed both qualitatively, and quantitatively. Conformation of the plasmid substrate molecule was evaluated following rejoining reactions by electron microscopy, and Southern blotting. Accurate dsb rejoining enabled the plasmid molecule to confer both ampicillin resistance and beta-galactosidase activity on JM83 E.coli transformants. A protein concentration-dependent increase in rejoining of cohesive dsb (EcoR I) was detected in nuclear extracts from 4 human tumour lines studied; no significant rejoining activity for a noncohesive terminus (Nru I) was observed in any extracts. No significant difference has been detected in maximal rejoining or rejoin fidelity of cohesive dsb mediated by extracts derived from 2 radiosensitive and 2 radioresistant human tumour cell lines. Such an in vitro system provides a powerful tool for the further analysis of the molecular mechanisms involved in the recognition, and repair of ionising radiation-induced DNA lesions.