Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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Typescript. / Thesis (Ph. D.)--University of Hawaii, 1964. / Bibliography: leaves [80]-86. / vi, 86 l mounted illus

Thesis (Ph. D.)--Georgia State University, 2006. / Title from title screen. William H. Nelson, committee chair; Thomas L. Netzel , A.G. Unil Perera, Brian D. Thoms, Gary Hastings, committee members. Electronic text (243 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Aug. 16, 2007. Includes bibliographical references.

Nuclear Factor I is a cellular transcription factor involved in the initiation of adenovirus type 2 replication. It exists as a family of proteins and comprises two distinct domains. The N-terminal domain is highly conserved between species and is involved in DNA binding, dimerisation, and stimulates adenovirus replication. The C terminal domain is responsible for the transcriptional activation. In this work, the N terminal, DNA binding domain of Nuclear Factor I, was cloned in two different expression systems. Using recombinant baculovirus, the unmodified protein and a GST fusion product were expressed in insect cells. Although both proteins were expressed at high levels, it was impossible to purify the fusion protein, while after purification, the unmodified protein remained heterogeneous. Expression in E. coli using the expression vector pET22b and pGEX2T produced inclusion bodies. The insoluble material was extracted, solubilised using guanidine HCl, then folded in vitro using new additives known as non-detergent sulphobetaines. In vitro folding was optimised and yields of up to 8% could be obtained. The affinity of the refolded material for a specific DNA oligonucleotide was determined in a gel electrophoresis DNA binding assay and was identical to the native protein purified from baculovirus infected insect cells. The structure of the Nuclear Factor I DNA Binding Domain was investigated using limited proteolysis followed by separation on SDS PAGE, N terminal sequencing and mass spectrometry. Residues 1 to 165 formed a compact domain. Residues 166 - 181 were extremely sensitive to degradation in the absence of DNA but fully protected in the presence of specific DNA. The C terminal region from residue 182 could be degraded in both conditions. Along with two other regions previously determined which bind DNA, the region between residues 166 and 181 is required for DNA binding. Differential labelling using iodoacetate showed that cysteine residues 95 and 111 were modified in the free protein which resulted in an inactive protein but were protected in the presence of DNA. This demonstrates their direct involvement in DNA binding.

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.

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.

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).