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The human thyroid epithelial cell HTori-3 has been transformed with doses of either chronic and acute x-rays or strontium beta particles. Models of the past have relied upon animal cell systems to mimic in vitro carcinogenesis. The HTori-3 system hoped to overcome the limitations associated with these types of models by using a human thyroid cell line immortalised with the SV40 virus. HTori-3 human thyroid epithelial cells were irradiated in vitro, passaged and then transplanted into nude mice. Tumours that grew over a 2-6 month period were excised and re-established in culture. Samples were stored and all tumours were taken for histological examination. Chromosome spreads confirmed the human nature of all tumours. Following exposure to acute x-rays in the range of 0.25-2.0 Gy 13 tumours were observed in 25 recipients. Following 0.25-2.0 Gy of chronic x-rays 10 tumours from 25 recipients were observed. From a single 2 Gy exposure of strontium beta particles 3 primary tumours from 5 recipients were observed. The largest of these was re-transplanted in nude mice resulting in 100% incidence. All tumours were classified as undifferentiated anaplastic carcinomas. A small number of tumours were observed in the control cell lines, these may be the result of a general instability found with the partial transformed parental cell line. All 2Gy tumours and those previously established from this laboratory after alpha or gamma radiation were used to test for the presence of the delayed lethal death phenotype. A number of cell and molecular endpoints were used. These included plating efficiency, cell adherence, micronucleus formation and p53 status. In all incidences, the reproductive viability of irradiated cells was below that of non- irradiated cells at up to 4 weeks post-irradiation. The HTori-3 cell line and the techniques used to study the delayed effects of radiation may be applicable to other cell systems and may be a useful model to study the long-term effects of radiation induced genomic instability.

The objective of this project was to investigate the chromatin structure of the mutant Chinese hamster ovary cell line, xrs, the project consisted of two parts by; a radiobiological study, and a protein chemistry study and to specifically ask, whether or not the increased susceptibility of the x family of mutants compared to wt could be attributed to modifications to structure of chromatin or are the modifications a pleiotropic effect of the mutated gene. The radiobiological study used the change in DNA radiosensitivity with progressive removal of DNA bound proteins as a probe to investigate the chromatin conformation of CHO KI and xrs. The DNA alkaline unwinding assay, showed that there was a difference in sensitivity to ionising radiation (IR)-induced damage between xrs-5 and CHO KI after treatment with various concentrations of salt (0.25-0.75 M NaCl) and y-rays (0.25-0.75 Gy), 0.75M NaCl and 0.75 Gy gave maximal effects. The sensitivity difference between mutant and wt was less obvious with NaCl treatment at 0.75 Gy y-irradiation but nonetheless apparent. This radiosensitivity could be attributed to structural modifications in the mutant cell lines. These structural modifications are represented graphically in two algebraic forms i.e. a quadratic (y=mx2) and a quartic sine (y= ax4 + bx3 + cx4 + dx+ C) in CHO KI and xrs-5 respectively. The decrease in unwinding in hypertonic solutions was thought to be attributable to either ongoing fast ssb repair, a faster unwinding population, or, loss of DNA supercoiling due to high salt and irradiation, or negative interaction between one unwound DNA population and a adjacent break points leading to saturation. Partial reversion of .xrs-5 did not alter the unwinding profile markedly. As deduced from the observation that despite the partial reversion the mutant was consistently more susceptible to IR-induced DNA damage compared to wt. The difference in DNA radiosensitivity in this assay of mutant compared to wt may not necessarily reflect the radiosensitivity of the xrs cells. The protein chemistry study showed that there was a difference observed between protein extracts of mutant xrs-6 and wt, with respect to histone-related proteins. Specifically, a species of 31 kDa was detectable in extracts of wt but not xrs-6 mutant cells. The possible identity of this polypeptide and the role of candidate proteins in chromatin structure and radiosensitivity is discussed.

Transformation is a complex multistage process in vitro by which benign cells gradually acquire characteristics of tumour cells. Transformed C3H10T1/2 cells appear in vitro as multilayers of cells termed foci. Two main aspects of transformation of C3H10T1/2 cells in vitro have been investigated. Firstly, the quantitative assessment of the dose and dose-rate effects after irradiation with 250 kVp X-rays were examined and secondly the relationship between various properties of transformed cells in vitro and their tumourigenic potential in vivo. The induction of transformation was found to be linear with dose for 0.25 to 5 Gy X-rays. Lowering the rate at which the X-ray dose is delivered to the C3H10T1/2 cells lowers the observed transformation frequency by at least a factor of two. A variety of transformed phenotypes are observed in vitro and samples of these phenotypes were developed as cell lines and assessed for a number of properties. These properties were the ability to induce tumours in C3H mice and the ability to reconstruct foci in vitro. Other properties examined were growth in vitro parameters (lag time, doubling time and saturation density) as well as chromosome number and distribution. Tumour cell lines were also developed and assessed for the above properties. Transformation phenotypes induced by X-rays and alpha-particles were compared. Differences were found between some of the properties of the X-ray and alpha- particle induced transformants. In particular, higher proportions of X-ray induced transformants were tumourigenic while most of the alpha-particle induced transformants were non-tumourigenic and also tumours induced by the X-ray induced transformants appeared earlier and grew faster than the alpha-particle induced equivalent. The ability of the transformation phenotypes to reconstruct foci in vitro was greater for alpha-particle induced transformants (not including tumour cell lines) than for the X-ray induced transformants. The reverse was true for tumour cells where X-rays produced higher frequencies of reconstructed foci than alpha-particles. No differences were noted in in vitro growth parameters irrespective of transformation phenotype or radiation type apart from differences in saturation density where the transformation phenotypes (not including tumour cells) generally produced higher densities than the tumour cells for both X-rays and alpha-particles. Chromosome numbers in cells of the different transformation phenotypes (including tumour cells) induced by both X-rays and alpha-particles showed a greater spread and a general shift of the mean and modal chromosome number to lower values than that of untransformed C3H10T1/2 cells. The presence of metacentric chromosomes (Robertsonian chromosomes) was not unique to the radiation induced transformation phenotypes as most of the cell lines examined showed fewer of these chromosomes than the untransformed cells. The X-ray induced transformants (including tumour cells) generally produced more Robertsonian chromosomes than the alpha-particle equivalent. Correlation tests of the above properties with tumourigenicity of the transformed cells revealed a positive correlation of tumourigenicity with the ability to reconstruct foci. A negative correlation was noted between the ability to reconstruct foci and the mean and modal chromosome numbers.

Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the basic epithelial morphology of the parent HToriS cells. Investigation of radiosensitivity showed that none of the 6 tumour cell lines examined had a higher radiosensitivity compared to the parent HToriS cells. This excludes the possibility that the observed transformation was the result of the selection of a pre-existing transformed subpopulation of the parent cells but that radiation-induced transformants were being induced de novo. The tumour cell lines were screened for mutations in H- and K-ras oncogenes using restriction enzyme analysis of PCR amplified DNA. No mutations were detected in 26 tumour cell lines suggesting that mutations in these two genes do not appear to be involved in radiation- induced neoplastic transformation in human thyroid epithelial cells. Screening for mutations in p53 protein using immunoprecipitation method detected no mutations in 6 tumour cell lines. This human thyroid epithelial cell line may thus be useful for the in vitro study of cellular and molecular mechanisms that are involved in human epithelial cell carcinogenesis.

The damage caused to cells by ionising radiation is believed to centre on damage to the DNA. In particular, the induction of DNA double strand breaks (DSB) have been implicated in biological end-points such as cell killing and the formation of chromosomal aberrations. The xrs-5 cell line is a mutant Chinese hamster ovary fibroblast (CHO-K1) mutant which exhibits sensitivity to ionising radiation and a number of other DNA damaging agents. This mutation, postulated to involve the hamster homologue of the human XRCC5 gene, is believed to be involved in the repair of the DSB. In addition, there are constitutive differences between the wild type and xrs cells involving the structure and function of the nucleus and higher order chromatin structures. The aims of this thesis were to study further the xrs-5 cell line and its response to DNA damage and to investigate the possible link between chromatin structure and DSB repair. By the examination of the response of xrs-5 cells to a number of DNA damaging agents and potential modulators of this response using the cytokinesis block micronucleus assay [Fenech and Morley, 1985] a possible cell cycle defect was identified in addition to elevated levels of chromosomal damage. Xrs-5 cells appeared to be partially defective in the cell cycle checkpoints involving the passage from G2 phase to mitosis. By the use of a modified neutral filter elution procedure variations in the repair of DSB were observed between xrs-5 and CHO. Conventional neutral filter elution requires harsh lysis conditions to remove higher order chromatin structures which interfere with the elution of DNA containing DSB. By lysing cells with non-ionic detergent in the presence of 2 M NaC1, histone depleted structures which retain the higher order nuclear matrix organisation, including chromatin loops, can be produced. Elution from these structures will only occur if two or more DSB lie within a single looped domain delineated by points of attachment to the nuclear matrix. Repair experiments indicate that in CHO cells repair of DSB in loops containing multiple DSB are repaired with "slow" kinetics (t1/2 = 5 hrs) whilst DSB occurring in loops containing single DSB are repaired with "fast" kinetics (t1/2 " 10 min). Xrs- 5 cells are incapable of repairing these multiply damaged loops. This work indicates that the spatial orientation of DSB in higher order structures of chromatin are a possible factor in the repair of these lesions. By construction of a mathematical model of the process of elution from chromatin loops it was possible to postulate the size of the loops to approximate to 2.5-3 Mbp. Further evidence of a potential structural defect in the chromatin of xrs-5 cells was provided by examination of the polypeptide composition and DNA binding activity of nuclear extracts. The affinity of extracted proteins for double-stranded calf-thymus DNA was measured in nuclear extracts of xrs-5 and CHO cells. There was an alteration in the DNA binding activity of salt extractable proteins from xrs-5 as measured by a filter binding assay. By the use of SDS-PAGE and the technique of South-Western blotting, it was possible to identify the approximate molecular weights of these DNA binding proteins. Differences were found in DNA binding between proteins from CHO and xrs-5 extracts of both non-irradiated and irradiated cells. Two proteins with apparent molecular weights of 32.2 and 31.8 kDa exhibited a lower DNA binding activity in xrs-5 than proteins of similar extracts from CHO. The amount of the 32.2 kDa protein was less in the xrs-5 extracts than in CHO extracts, as measured by Coomassie blue staining. The two proteins have not yet been identified but comprise a major DNA binding activity in CHO extracts obtained by detergent-free extraction procedures. This work provides circumstantial evidence that suggests these two polypeptides may form part of the histone H1 family.

The effects of gamma radiation and restriction endonuclease (RE) induced DNA double strand breaks (dsb) upon the mutation frequency and the surviving fraction of three Chinese hamster cell lines V79-4, CHO-K1 and an X-ray sensitive dsb repair deficient cell line xrs-5 were studied. The X-ray sensitive xrs-5 cell line was shown to be more sensitive to both the lethal and the mutagenic effects of gamma radiation having a substantially lower surviving fraction and a higher thymidine kinase (tk) mutation frequency per unit dose than the parental CHO-K1 cells. The frequency of induced hprt- mutations in the V79-4 cell line was comparable to the induced frequency of tk mutations in the CHO-K1 cells. The effect of blunt- and cohesive- ended dsb upon the surviving fraction and the induced mutation frequency was studied by porating different Chinese hamster cell lines (CHO-K1, V79-4 and xrs-5) with RE using Streptolysin O (SLO). The surviving fraction of the different cell lines was reduced with increasing concentrations of Pvu II. Increases in the concentration of Pvu II produced increases in the frequency of hypoxyanthine guanine phosphoribosyl transferase (hprt) mutations in the V79-4 cells and tk mutations in the CHO-K1 and xrs-5 cells. However, the xrs-5 cells were shown to be hypomutable to Pvu II compared with the parental CHO-K1 cells. EcoR1 was ineffective at inducing tk mutations in the CHO-Kl cells but was as effective as Pvu II at inducing hprt mutations in the V79-4 cells. None of the spontaneously induced V79-4 hprt- mutant cells were shown to have observable molecular deletions when analysed by PCR deletion screening. One third of the radiation induced hprt - mutants were shown to be deletions. However, too few mutant cells were analysed for any non-random distribution of deletions to be observed. Half of the hprt- mutants induced by SLO poration alone were shown to be due to deletions of oi\e or more exons. The distribution of the DNA deletions in SLO hprt- mutations appeared to be nonrandom. The PCR amplification products of exons 7&8 were more frequently lost than any of the other exons in the hprt gene. It has been suggested that the SLO provided by the manufacturer used to porate the cells was contaminated with small amounts of endonucleases and exonucleases. The Pvu II induced deletions also appeared to be non-randomly distributed. The PCR amplification products of exon 2 were more frequently absent than any of the other exon products. Sequence data from the EMBL library indicated that Pvu II had a restriction site adjacent to the exon 2 nucleotide sequence of the Chinese hamster hprt gene but not in or bordering the other exons. This provides evidence that the blunt-ended dsb plays a role in the production of mutations. Mutation studies indicated that there is only one active copy of the autosomally located thymidine kinase gene in the CHO-K1 cells and their daughter cell lines (Singh and Bryant, 1991). However, whether the other homologous copy has been inactivated or deleted is not known. Experiments attempting to locate the thymidine kinase gene(s) were performed using FISH and a mouse tk cDNA probe. However, these attempts were unsuccessful.

Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or mutant conformation in all the above cells lines and two other control lines HOS (a human osteosarcoma cell line) and H Tori-3 (SV40 immortalised thyroid epithelial cells).

Cells of ataxia telangiectasia (AT) individuals are hypersensitive to a variety of DNA damaging agents such as ionizing radiation and bleomycin, presumed to be due to an intrinsic defect in repair of DNA damage. The nature of the DNA lesion(s) to which AT cells are abnormally sensitive, and the defect in DNA repair are presently unclear. The major part of this project aimed at investigating the sensitivity of AT cells to DNA double-strand breaks (dsb) generated by restriction endonucleases (RE), thereby verifying the hypothesis that AT cells are deficient in the processing of dsb. AT lymphoblastoid cell lines (AT-PA and AT-KM) used in this study were initially characterized and found to be approximately 3 times more sensitive to ionizing radiation in the induction of micronuclei (Mn) and chromosomal aberrations (CA) compared with a normal lymphoblastoid cell line (N-SW). Other cellular characteristics were observed in AT-PA cells following-irradiation such as normal induction and rejoining of dsb and reduced inhibition of DNA synthesis. By using SLO poration, RE were introduced into the AT and normal cell lines and the yield of CA resulting from RE-induced dsb were subsequently investigated. The frequencies of CA induced by Pvu II were 2 - 4 fold higher in AT-PA than in N-SW cells at both 5 h and 24 h sampling times. The enhanced frequency of CA in AT cells treated with Pvu II was principally a result of an increase of chromatid aberrations, rather than chromosome aberrations at 24 h. higher frequencies of chromatid exchanges appeared in AT-PA than in N-SW cells. The results suggest that AT cells are characterized by a defect in dsb processing that converts a higher number of dsb into CA than in the normal cell line. With respect to the different end-structures of RE-induced dsb, cohesive-ended dsb generated by BamH I and Pst I were found to induce lower frequencies of CA than blunt-ended dsb generated by Pvu II and EcoR V in both the AT cell lines and the normal cell line. The results support the previous observations that cohesive-ended dsb are less clastogenic than blunt-ended dsb (Bryant 1984). Although inducing lower frequencies of CA than Pvu II and EcoR V, BamH I and Pst I induced higher number of CA in both AT-PA and AT-KM cells when compared with N-SW cells, again indicating a defect in processing cohesive-ended dsb exists in AT cells. A potent DNA repair inhibitor, Ara A, was found to potentiate the production of CA by RE in AT and normal cells. The enhancement ratios (by ara A) for CA induced by Pvu II and Pst 1 were higher in N-SW cells than in AT-PA and AT-KM cells. Ara A appeared to have no effect on the frequencies of CA induced by BamH I in any of the cell lines tested. Based on these findings, a mechanism for the rejoining of RE-induced dsb in which DNA repair synthesis may be involved is proposed, and it is postulated that dsb in AT cells are subjected to greater end degradation. Inhibition of DNA synthesis was observed in normal cells after treatment with Pvu II and EcoR V, while EcoR I and BamH I had only minor effect. AT-PA cells were found to be resistant to RE-induced inhibition of DNA synthesis, as in the case of ionizing radiation. This result suggests that RE-induced blunt-ended dsb mimic radiation-induced lesions in supressing DNA synthesis in normal cells and that AT cells respond to RE-induced dsb in a similar way to damage induced by ionizing radiation. Finally, when a nuclear extract from N-SW cells was introduced into Pvu Il-treated AT-PA cells, it was able to confer a normal frequency of CA. In contrast, neither whole cell nor nuclear extracts from normal cells influenced the production of CA induced by y-rays. These findings provide evidence for the presence of factor(s) in normal nuclear extract which complements the defect in processing of RE-induced dsb in AT cells.

DNA double-strand breaks (dsb) are thought to be major radiation-induced lesions in biological end-points such as cell lethality and chromosome aberrations. Based on this notion, this project aimed to extend further the investigation of the role of dsb in radiation-induced mutagenesis. The initial part of the project involved optimising conditions for the mutation assay so as to select for 'true' tk-mutants in Chinese hamster cells, following treatment with X-rays. This was important, due to the insufficient previous mutation data involving this locus in Chinese hamster cells. Furthermore, the choice of the tk locus over the more commonly used hprt locus was based on existing evidence of its higher sensitivity, as found in mutation experiments with the L5178Y mouse lymphoma cell line (Evans et al, 1986). An Initial comparative study was carried out to measure the induced mutation frequency following X-ray irradiation in both the parent Chinese hamster Ovary (CHO KI) cell line and its X-ray- sensitive mutant (xrs 5) cell line. This mutant line was chosen because of its characteristic marked deficiency in dsb repair, yet normal ability to rejoin single-strand breaks (Kemp et al, 1984., Costa and Bryant, 1988). This allowed the study of the role of dsb in mutation induction. The enhanced mutation induction observed in xrs 5 over that in CHO KI cells suggested the Importance of dsb in radiation-induction mutagenesis. The next experimental strategy adopted involved the use of the DNA synthesis inhibitor, 9-D-arabinofuranosyladenine (ara A). The choice of this drug was based on previous work by Bryant and Blocher (1982) and Iliakis and Bryant (1983) who, using DNA unwinding and neutral velocity sedimentation, showed ara A to strongly inhibit dsb repair, Plateau-phase CHO KI cells were exposed to X-rays alone or in combination width ara A , the latter treatment showing an increased induction of mutations. This suggested the possible existence of dsb which are fixed as mutations in the absence of DNA polymerization, suggesting a sub-class of dsb which may be critical in the steps leading to the induction of a mutation. XV The third approach was to use restriction endonucleases (RE) which were introduced into cells by electroporation. This method unlike ionising radiation, induced 'pure' dsb. The use of this method was based on the work of Bryant (1984), who used RE to mimic radiation-induced damage in the induction of chromosomal aberrations. Two different types of RE were used: those which produce blunt- and those which produce cohesive-ended dsb. In all mutation experiments with these enzymes, blunt-ended dsb were found to be more effective in generating mutations compared to cohesive-ended dsb. This suggests a possible further resolution of type(s) of dsb that would be induced by radiation in the ability to induce mutations i.e dependent on the end-structure of the induced dsb. Blunt-ended dsb may thus represent the major type of critical pre-mutational lesions which may be fixed as a mutation, as a result of misrepair. Cohesive- ended dsb may be of lesser importance. Finally, a RE (Pvu II) which generates blunt-ended dsb was used to induce mutations at the hprt locus in Chinese hamster (V79) cells. DNA from mutant cells was analysed using Southern blot and PCR analysis of 3 exons in the hprt gene. Some of the mutants (5/15) showed large deletions (representing complete loss of the gene), a change similar to that observed in mutants Induced following treatment with ionising radiation (e.g. Thacker, 1986). However, the percentage of large deletion mutants (70%) observed in radiation- induced mutants was higher than that (~34%) obtained with RE- induced mutation data. This preliminary data on the analysis of RE- induced mutations suggests that blunt-ended dsb mimics radiation- induced pre-mutational lesions, resulting in some large genomic changes (e.g. large deletions). However, a larger number of RE- induced mutants would have to be analysed before a more accurate comparison between RE and ionising mutation data can be made. In summary, this study provides evidence for dsb as a major pre-mutational lesion in cells exposed to ionising radiation, and suggests the existence of a sub-class of dsb in relation to mutation induction. In addition, RE offer the possibility of gaining further understanding of the role of dsb in the origin of mutations such as those caused by deletions.

When cells are trypsinized before irradiation, potentiation of cell killing is seen; this is known as the 'trypsin effect'. The trypsin effect is re-examined here in the light of experiments in which enzymatic modifications of DNA in permeabilized cells has become a powerful experimental tool (Bryant et al, 1978, Ahnstrom and Bryant,1982; Natarajan et al, 1980; Bryant, 1984, 1985; Natarajan and Obe, 1984) and where in some cases it is suspected that trypsinization as part of the technique could significantly alter cell membrane permeability and chromatin structure (Obe et al, 1985; Obe and Winkel, 1985; Bryant and Christie, 1989). The trypsin effect was investigated at various cellular levels, assaying for cell survival (to verify the potentiation), anaphase chromosomal aberrations, DNA damage and repair and lastly using a nucleoid assay to investigate the effect of trypsin on DNA-nuclear matrix interactions. Each of these are considered in separate chapters as individual studies, then all compared in the final discussion. A small potentiation effect of X-ray damage on cell killing was seen when using Chinese Hamster Ovary (CHO) cells but no potentiating effect was found in the murine Ehrlich ascites tumour (EAT) cell line. Trypsinization was found to increase the number of X-ray induced chromosomal anaphase abnormalities in EAT cells. To investigate the possibility that the basis of the trypsin effect lies in its action at the DNA level, further experiments were performed to monitor DNA damage and repair using the DNA unwinding and neutral elution techniques. No difference was seen in the unwinding kinetics or in the DNA unwinding dose-effect curves for induction of DNA single strand breakage (ssb); when using neutral elution however, treatment of cells with trypsin or buffer alone increased the incidence of X-ray induced double strand breaks (dsb) at higher doses. Trypsinized EAT cells were found to repair ssb after 12 Gy less rapidly than those treated with buffer (EDTA) indicating an inhibitory effect of trypsin on repair. A progressive decrease in repair capacity with increase in time of trypsin treatment was seen. The dsb repair kinetics as measured by the DNA unwinding technique after 50 Gy showed that either trypsin of buffer (EDTA) alone reduced the dsb repair rate, no difference between their repair kinetics being evident (this was also seen with neutral elution repair after 40 Gy). This indicates that the EDTA/buffer solution in which the trypsin is dissolved may also be contributing to the trypsin effect. A new nucleoid assay was developed and used to investigate the effect of prolonged trypsinization and electroporation on nucleoid morphology. The results confirm that routine trypsinization of cells enhances X-ray induced cell killing in some cell lines. It is postulated that this may occur by reducing the repair capacity of the cells rather than by increasing the amount of damage initially caused.

The non-denaturing filter elution technique of Bradley and Kohn (1979) is widely used as an assay for DNA double-strand breaks (dsb) in mammalian cells. Results characteristically obtained with this assay following exposure of cells to ionising radiation, namely that of non-linear induction of dsb and rapid biphasic repair, are not in agreement with those of the neutral velocity sedimentation technique where the biophysical basis of measurement is understood. The discrepancy in the results obtained with these two techniques with regard to both Induction and repair of dsb has led to a controversy. In particular concerning the manner in which the data obtained with the neutral elution technique should be interpreted (AhnstrSm's Comment on Radford 1985; Hutchinson 1989). The aim of this project was therefore to attempt to test the assumed specificity of the non-denaturing filter elution technique as an assay for dsb. Optimization of the lysis and eluting conditions was followed by detailed X-ray dose-response and repair experiments with the CHO K1 cell line. A comparative study was performed using the xrs 5 cell line, a radiosensitive mutant of the CHO K1 line chosen for its characteristic marked deficiency in dsb repair, yet normal ability to rejoin single-strand breaks (Kemp et al 1984; Costa and Bryant 1988). Previous reports by Bryant and Blocher (1982), and Iliakis and Bryant (1983) revealed that the DNA synthesis Inhibitors ara A and ara C strongly inhibit dsb repair as assayed by neutral velocity sedimentation. I thus adopted an experimental strategy In which the effect of ara A and ara C on putative dsb repair was examined using the non- denaturing filter elution assay. Only limited inhibition of dsb repair by these nucleoside analogues was observed with the non-denaturing filter elution technique tn contrast to the complete inhibition of dsb repair as measured by neutral velocity sedimentation for the same concentrations of DNA synthesis inhibitor (Bryant and Blocher 1982; Iliakis and Bryant 1983). These results suggest that the two above mentioned techniques are detecting disparate types of dsb, as manifested by the differential requirement of the repair mechanism of these breaks for DNA polymerization. A further approach was the introduction of restriction endonucleases (RE) into mammalian cells by electroporation, to induce dsb in the absence of other types of lesions. The observed increase in the rate of elution of the DNA of RE-treated cells substantiates the ability of the non-denaturing filter elution assay to detect cellular dsb. Surprisingly Pvu II was found to remain active inside the cell for up to 24 h, and the continual incision of the DNA by the enzyme thwarted the possibility of monitoring the repair of these dsb. A noteworthy result was the relative inability of RE which generate cohesive-ended dsb (e.g. Bam HI and Eco R1) to Induced measurable numbers of dsb as compared with the blunt-end cutter Pvu II. A hypothesis of a competition between the induction of dsb by RE and the subsequent repair is offered as explanation, where the repair of cohesive-ended dsb is assumed to take place at a higher rate than that of blunt-ended dsb. A comparative study using the xrs 5 mutant cell line, known to be deficient in dsb repair, revealed enhanced levels of RE-induced dsb which would support the notion that the levels of dsb reflect a competition between RE-incision and dsb repair. In summary, this study validates the measurement of dsb by the non-denaturing filter elution method and provides new evidence for the mode of induction of dsb by RE which has not been hitherto possible. Finally, the work indicates the way in which cells handle different types of dsb which may be similar to the manner In which the variety of dsb induced by ionising radiation are dealt with.