title: Mechanisms of radiation resistance in cultured cells
Creators: name: Russell, James
Publisher: name: University of Glasgow
Year: 1995
Subjects: subject: Biophysics.

description: To investigate cellular radioresistance, three approaches were adopted. These were: to examine claims in the literature that growing cells as spheroids enhances their resistance to ionising radiation; to follow reports that cells transfected with ras oncogenes became more radioresistant; and to isolate a radioresistant subline from a radiosensitive human neuroblastoma cell line. It is known that for some cell lineages, culturing the cells as three-dimensional aggregates (spheroids), leads to an increase in their radioresistance, relative to monolayers, and it has been proposed that this "contact effect" is related to the presence of inter-cellular gap junctional communication in the spheroid. This claim was investigated by testing two cell lines which were shown to form gap junctions. No contact effect could be demonstrated in either case. The existence of gap junctional communication is not sufficient to confer a contact effect, though the possibility remains that it is a necessary component. The prevalence of the contact effect in human tumour cell lines was examined by means of a literature survey. Of 27 lines for which published data was available, approximately one third showed a contact effect, and these lines were, on average, relatively radiosensitive, when grown as monolayers. However, an attempt to identify more instances of the contact effect by examining human neuroblastoma cell lines failed. (Neuroblastoma lines are known to be, in general, more than usually radiosensitive.) The effect of transfection with the oncogenes H-ras and c-myc was examined using a mink epithelial line, Mv1Lu. No alteration in radiosensitivity was observed in the transfected lines. These transfected lines were monoclonal in origin. There seemed to be little clonal variation in the parent line in terms of radiosensitivity. Five independent clones were established. Analysis of variance showed that in terms of radiosensitivity, they all belonged to the same population. This suggests that background variation in the radiosensitivity of the Mv1Lu cells is unlikely to have obscured an oncogene-mediated effect. However, analysis of several oncogene-transfected clones would be desirable to confirm this conclusion. By subjecting a radiosensitive human neuroblastoma derived cell line, NB1-G, to a regime of fractionated X-irradiation, a radioresistant variant, XRNB1-G was obtained. Beginning with an original population of approximately 107 cells, selection was carried out by once or twice weekly irradiation with doses of 2 Gy. After 15 such treatments, an increase in the radioresistance of the population was noted. A monoclonal line was derived from the resistant population, (clone F) which remained resistant to ionising radiation over 40 passages, after the end of the fractionation protocol. The D0 of the parent line was 0.7 Gy +/-0.1 (95% confidence Hmits); the resistant line had a of 1.1 Gy +/- 0.3. Both the parent and resistant lines had almost exponential survival curves with no shoulder region and little overall curvature. Increased radioresistance was confirmed independently of colony formation by means of the spheroid growth delay assay. Using flow cylometry, the cell cycle distributions of the lines were found to be almost identical. Studies with neutral filter elution showed no difference between the lines in the level of initial DNA double strand breaks. Double strand break repair did not differ between the cell lines. The resistant and parent lines sustained equal amounts of chromosome damage, as judged by the micronucleus assay. However there seemed to be substantially more radiation-induced apoptosis in the parent cells. A reduced tendency to undergo apoptosis in response to radiation presumably underlies the resistant phenotype of the XRNB1-G/clone F cells. Because of the established link between p53 and radiation-induced apoptosis, the p53 status of the NB1-G and clone F lines was investigated. Most p53 mutations occur in exons 5 to 8. cDNA sequencing of that region did not identify any mutation in either line. Western analysis showed p53 protein was induced equally in both lines following radiation exposure. The difference between the resistant and parental lines seems to lie in the chain of events which leads from radiation damage to apoptotic death; however which step is altered in the resistant cells remains unknown.