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The effects of a range of complexing agents on the radiation damage routes in frozen aqueous DNA have been analyzed by ESR techniques in a semi-quantitative way. Establishment of additive-free DNA standards for this study revealed an ESR pattern different from the four elementary shapes which contributed ∼ 13X of the total spin seen after annealing to 210 K. It is proposed that this represents a population of sugar radicals; hitherto unseen intermediates in additive-free systems between secondary base radicals and strand breaks. The intercalating aminoanthraquinones, mitoxantrone and ametantrone, were found to compete very efficiently with thymine for electron capture, and behaved as protecting agents. Concomitant strand break studies did not support this, and more extensive ESR studies suggested that the intercalator radical centre could also lead to strand breaks. It is therefore proposed that the mode of action of these drugs does not involve free radical processes in DNA. Use of these agents provided evidence that electron migra tion in DNA is much more extensive than previously thought. Copper ions were found to reduce the amounts of T/TH detected, with no effect on G . During this evaluation, evidence appeared for the existence of a site on DNA that could bind two Cu(II) ions so close together that they became ESR silent. Of special interest was the finding that this binding was preferred over that of single Cu(II) ions, and it is proposed that this might have a physiological significance. The dominance of thymine over cytosine as the site of electron capture is reinforced by the above studies, but it is suggested that it is the nature of the system under study that determines the ratio of T to C.

The phenomenon of induced peroxisome proliferation in rodents has invariably been associated with upregulation of member(s) of the CYP4A subfamily of hemo- proteins. However, regulation of the members of the rat CYP4A family has not been fully examined at the molecular level. Despite the publication of the rat CYP4A1 and CYP4A2 genomic sequences (Kimura et al., 1989a), no functional assays have been performed to determine the mechanism of peroxisome proliferator modulated transcriptional activation of these genes. The objective of this study was to isolate members of the rat CYP4A subfamily of genes and investigate their inducibility by peroxisome proliferators at the molecular level. In this thesis I present results to demonstrate: 1) the isolation and partial characterisation of recombinant DNA clones corresponding to 3 different rat genomic sequences, showing a degree of similarity to the rat CYP4A1 cDNA, 2) identification, by sequencing, of one of the groups of clones (group C) to correspond to CYP4A2 and another (group B) to be, possibly, the genomic sequence of CYP4A3. The identification of the remaining group (group D) remains unknown, but most likely represents a novel member of the rat CYP4A subfamily, 3) the apparent absence of a peroxisome proliferator response element (PPRE) in the 5' flanking DNA sequence of the clones, as determined by hybridisation to the rat acyl-CoA oxidase PPRE oligonucleotide (GTOX285), 4) a lack of a response of a -5.8kb upstream sequence of the newly-isolated human CYP4A11 gene in a co-transfection assay with the mouse PPAR and in the presence of the peroxisome proliferator Wy 14643. The results further extend knowledge on the multiplicity of CYP4A genes in the rat and provide data on the transcriptional regulation of CYP4A genes in rodents and man.

Various potential metabolites of steroids with alkylating properties were synthesised, with important cellular in order that their reaction constituents and their mutagenicity could be investigated by Dr. Grover of the Chester Beatty Research Institute. Promising compounds were also investigated by Dr. Traynor, of Loughborough University, as part of an ongoing project to find selectively toxic compounds to estrogen dependant tumours.

The search for the cure for cancer is currently a multi-billion dollar industry and the search for the elusive “magic bullet”, i.e. the perfect cancer drug that would interact therapeutically with cancerous tissues while having a minimal effect on healthy cells, is the topic of many research studies in the world today. A large number of novel drugs or drug complexes and conjugates are being synthesized and subjected to rigorous evaluation in the race to find the perfect cure. ECDG (Ethylene diCysteine DeoxyGlucose) seems to have promising cancer targeting ability. Even though this compound has been described in a few publications, we could not find any reference to the current use of ECDG in oncology clinics, either as a therapeutic agent, or as a diagnostic tool for imaging purposes. It was also not possible to purchase pure ECDG anywhere in the world. This prompted us to further investigate ECDG as a possible candidate for cancer targeting research, either as an imaging agent for cancer diagnosis or complexed with an anti-cancer agent for therapeutic purposes. Detailed investigations done in our laboratory can be divided into the following categories: - Development of a synthetic method for ECDG on a multigram scale ; - Purification of prepared ECDG not using the described dialysis method that only allows the purification of small quantities of ECDG (mg scale) ; Detailed investigation of the chemistry involved in the preparation of pure ECDG and its metal complexes ; - Investigation of the stability of ECDG and its metal complexes that is essential data required for any pharmaceutical agent ; - Preparation of ECDG complexes for use as a diagnostic tool, i.e. complexation with 99mTc ; Investigation of the bio distribution of ECDG-ReO complexes ; - Preparation of an ECDG kit as a diagnostic tool for use in oncology clinics. The development of novel aromatic ligands having similar characteristics compared to ECDG, containing an N2S2 chromophore as donor atoms, to further investigate their targeting capabilities, have also been investigated. All intermediates and final compounds were characterized mainly by ESI MS, in some cases IR and NMR whenever available. Successful preparation and purification of ECDG ands its metal complexes was achieved and extensively characterized and evaluated. Efforts directed towards the development of ECDG at NECSA, South Africa, were also rewarded with significant success. Furthermore, significant development regarding the synthesis of two novel compounds with ECDG-like characteristics was also completed.

Type I collagen is the predominant collagen within the stroma and plays an important role in the processes of tumour cell invasion and metastasis during which the collagens within the stroma is degraded. Total RNA was extracted from different stages of breast cancer and adjacent normal tissue for analysis of collagen gene expression by Northern blot hybridisation. Stage I breast tumours had increased α1(I) and α2(I) collagen mRNA, whereas stages II and III tumours had decreased mRNA levels when compared to the adjacent normal tissue. This stage-specific change in collagen gene expression was confirmed by non-radioactive in situ hybridisation and the results indicated that α1(I) and α2(I) collagen mRNA was produced by the stromal fibroblasts and not the tumour cells. To determine whether this altered collagen gene expression was manifested in other cancers, α1(I) and α2(I) collagen mRNA levels were analysed in colorectal carcinoma samples by in situ hybridisation. Colon cancer as in the case of breast cancer, also showed stage specific changes in collagen gene expression. Dukes C and D colon cancer samples had decreased collagen mRNA levels compared to Dukes A and B. Mutated Ras has been shown to affect collagen mRNA levels in vitro (Slack et al, 1992), therefore the colon samples were analysed for Ras mutations in an attempt to correlate Ras mutations with the decreased levels of α1(I) and α2(I) collagen mRNA. Colorectal DNA samples were screened for Ras mutations by SSCP and direct sequence analysis. No possible association was found between the presence of Ras mutations and the decreased collagen gene expression. To gain greater insight into exactly how tumour cells modulate the collagen produced by normal fibroblasts, primary breast fibroblasts (prepared from breast tissue) were cocultured with various breast tumour cell lines. The fibroblasts were also incubated with conditioned media prepared from the tumour cells. Collagen production was analysed using the collagenase assay and the results showed that co-cultured tumour cells, as well as growth in the presence of tumour cell conditioned media, resulted in decreased type I collagen production by the fibroblasts. Type III collagen is often produced in conjunction with type I collagen and we have found that the breast tumour cells modulated type III collagen in the same way as type I collagen. These results demonstrated that a factor(s) was secreted by the tumour cells which affected collagen production. This factor was further shown to stimulate the fibroblasts to produce type I collagenase as analysis of the medium from co-cultured fibroblasts and tumour cells indicated the presence of collagenases. The tumour cell conditioned media was subsequently shown by Western blot analysis to contain a protein of similar molecular weight to the tumour cell derived collagenase stimulatory factor (known as EMMPRIN or extracellular matrix metalloproteinase inducer) which stimulates fibroblasts to secrete collagenases and has been shown to play a crucial role in tumour invasion (Biswas 1982, 1984 and Biswas et al, 1995). In order to determine whether fibroblasts of different origins reacted similarity when cocultured with breast tumour cell lines, WI-38 lung fibroblasts and FGo skin fibroblasts were co-cultured with breast tumour cells. WI-38 fibroblasts responded in the same way as breast fibroblasts (having decreased collagen production), FGo fibroblasts had no effect or slightly elevated collagen production, depending on the tumour cell line. These results suggested that the response to tumour cells is tissue specific. The decrease in type I collagen produced by the fibroblasts when incubated with the tumour cell conditioned media was not due to a decrease in α1(I) and α2(I) collagen mRNA as shown by Northern hybridisation. Type III collagen mRNA was affected differently, the levels were either decreased or increased depending on the tumour cell line being used. We postulate that the fibroblasts and tumour cells required contact for type I collagen mRNA to be decreased. Northern hybridisation showed that types I and III collagen mRNA levels were decreased when tumour cells were co-cultured with the fibroblasts. To demonstrate that specific contact was in fact required, the tumour cells were separated from the fibroblasts by a diffusible membrane and the levels of collagen mRNA were not adversely affected. Tumour cells, therefore can modulate collagen production by normal fibroblasts in two ways I); cause the fibroblasts to secrete collagenases which will degrade the collagen and 2); decrease collagen mRNA. Both of these mechanisms would aid the tumour in invasion and metastasis.

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No description available.

No description available.