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In vitro data from our laboratory demonstrate that vitamin B6 (B6) supplementation of estrogen receptor - positive and - negative breast cancer cells is growth inhibitory. Others have reported that dietary B6 supplementation resulted in increased fibrosarcoma pyridoxal phosphate (PLP) concentrations and a significant inverse relationship between tumor PLP concentration and tumor volume in mice. This suggests that, in contrast to data reported for normal cells, tumor cells are capable of accumulating supplemental B6. In the current study, we investigated the effects of dietary B6 supplementation on 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in rats. Specifically, we aimed to identify the effect of pyridoxine (PN) supplementation on tumor growth and vitamin uptake by tumor cells. To accomplish this, 50 d old female Sprague Dawley rats were gavaged with 15 mg DMBA and fed a diet containing either 7, 350, or 1050 mg PN-HCl/kg diet, which is the equivalent of 1, 50, or 150x the National Research Council's B6 requirement for rats, respectively. These levels of PN have previously been shown to produce no overt signs of toxicity in rats. Throughout the experiment, the percent of rats with tumors and the average number of tumors per rat remained similar between groups. Mammary tumor growth rates were significantly increased in response to dietary B6 supplementation (P < 0.05). Liver PLP and pyridoxal (PL) concentrations did not differ between dietary treatment groups. Plasma PL and PLP concentrations were significantly higher in the group fed the 150x diet compared with the 1x diet (P < 0.001, P < 0.05). Mammary tissue PL concentrations of the 150x group were significantly higher (P < 0.05) than the 1x group, but no differences were observed in mammary PLP concentrations. Similarly to mammary tissue, no differences between groups were observed in tumor PLP concentration. However, tumor PL concentrations in both the 50x and 150x dietary treatment groups were significantly higher than those from the rats fed the 1x diet (P < 0.002). These data demonstrate that previously reported inhibitory effects of supplemental B6 on breast cancer growth in vitro do not occur in response to dietary supplementation at 50 or 150 times the B6 requirement in vivo. In fact, dietary B6 at 150x the requirement may actually promote mammary tumor growth. In light of these results, investigation of the effects of supplemental B6 on cancer growth in humans is warranted. Supported by American Cancer Society Grant # IRG-99-225-01. / Master of Science

This dissertation describes the analysis of the carbohydrate portion of glycosphingolipids from colorectal carcinoma cells, SW1116, by metabolically labelling the cells with radioactive monosaccharide precursors. SW1116 cells (1 x 10⁶) metabolically labelled with 222 μCi/ml of either 6-[³H]-D-galactose (25 Ci/mMol) or 6-[³H]-D-glucosamine (38 Ci/mMol) for 30 hours, incorporated 1%-3% of the radioactivity into their glycoconjugates. Approximately 63% of the radioactivity recovered in the glycoconjugates corresponded to glycolipids when cells were labelled with 6-[³H]-D-galactose, and about 12% when cells were radiolabelled with 6- [³H]-D-glucosamine. Metabolically radiolabelled glycolipids were separated into neutral (88-91% of the radioactivity recovered in glycolipids) and acidic (9-12% of the radioactivity in glycolipids) fractions by ion exchange chromatography. Glycolipids in these fractions were subjected to ozonolysis and alkali fragmentation to release the oligosaccharide chains from the ceramide portion. Oligosaccharides obtained from the neutral glycolipids were separated into single components by a combination of high performance liquid chromatography (HPLC) and Ricinus communis agglutinin I (RCA-I)-agarose affinity chromatography. Oligosaccharides were identified based on the monosaccharide composition, methylation analysis, and exoglycosidase digestions. Major glycolipid components present in the neutral fraction were, glucosylceramide (Glc-Cer), galactosylceramide(Gal-Cer), galabiosylceramide (Galαl-4Gal-Cer), lacto-N-tetraosylceramide (Galβ1-3GIcNAcβ1-3Galβ1-4Glc-Cer), Le^a- pentaglycosylceramide (Galβ1-3[Fucal-4]GlcNAcβ1-3Galβ1-4Glc-Cer), HIpentaglycosylceramide (Fucal-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer), a difucosylated lacto-N-tetraosylceramide, and a fucosylated lacto-Nnorhexaglycosylceramide. Minor components detected in this fraction corresponded to lactosylceramide (Galfp1-4Glc-Cer), lacto-Nneotetraosylceramide (Galβ1-4GlcNAcβ1-3Galβ1-4Glc-Cer), and fucosylated and difucosylated lacto-N-neotetraosylceramides. The acidic fraction was separated into monosialylgangliosides and _ disialylgangliosides by ion exchange chromatography. Monosialyloligosaccharides were further purified on HPLC, and biochemically characterized by methylation analysis, exoglycosidase digestions, and monosaccharide composition. The major component of this fraction corresponded to the sialyl-Le^a glycolipid (NeuAcα2-3Galβ1-3[Fucαl-4]GlcNAcβ1-3Galβ1-4Glc-Cer) as previously reported by Magnani et al. [183]. GM3 (NeuAcα2-3Galβ1-4Glc-Cer) (0.42% of radioactivity recovered in glycolipids), sialyltetraosylceramide a (NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer) (0.46% of radioactivity in glycolipids), sialyltetraosylceramide b (Galβ1-3[NeuAcα2-6]GIcNAcβ1-3Galβ1- 4Glc-Cer) (0.21% of radioactivity in glycolipids), and sialyllated fucosylhexaglycosylceramide, were present in minor quantities. Results from this study demonstrate that metabolic radiolabelling provides a method for the structural analysis of glycolipids, as sensitive as the immunostaining procedures, as unmistakable as physical techniques (Mass Spectrometry, and Nuclear Magnetic Resonance), and that permits the identification of the majority of glycolipids expressed by a cell line, using relatively small number of cells in culture (6 x 10⁶). Application of this method could be extended to the study of changes in glycolipid accompanying oncogenic transformation and differentiation, glycolipid biosynthesis, intracellular sorting of glycolipids, recycling and turnover. / Ph. D.

Thesis (Ph. D.)--University of Wisconsin--Madison, 2001. / Includes bibliographical references (p. 217-245). Also available on Internet.

Thesis (Ph. D.)--University of Wisconsin--Madison, 2001. / Includes bibliographical references (p. 217-245).

Thesis (M.A)--University of Tennessee. / "Date Issued: Jul. 19, 1960." Bibliography: p. 66.

Recent morphologic and molecular genetic studies have led to a paradigm shift in our conceptualization of the carcinogenesis and histogenesis of pelvic (non-uterine) serous carcinomas. It appears that both low-grade and high-grade pelvic serous carcinomas that have traditionally been classified as ovarian in origin, actually originate, at least in a significant subset, from the distal fallopian tube. Clonal expansions of the tubal secretory cell probably give rise to serous carcinomas, and the degree of ciliated conversion is a function of the degree to which the genetic hits deregulate normal differentiation. In this article, the authors review the evidentiary basis for aforementioned paradigm shift, as well as its potential clinical implications.

Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Prostate cancer (CaP) has the highest incidence of any malignancy affecting South African males. The aetiology of prostate carcinoma indicate that ethnicity is one of the most important risk factors. The causes of these ethnic differences are unknown but presumably involve both environmental and genetic factors. Carcinoma of the prostate is androgen dependent, and it has been suggested that variations in androgen metabolism and synthesis may affect an individuals' risk. Therefore, genes involved in these pathways are candidates for determining CaP susceptibility. In this study two candidate genes in the androgen biosynthetic and metabolic pathway were analysed, viz., the androgen receptor gene (AR), involved in androgen transport and transcriptional activation, and the cytochrome p450c17a gene (CYP17), important for testosterone biosynthesis. Comprehensive mutation detection assays were designed (appropriate for analysis of archival paraffin-embedded material) for almost the entire coding region (excluding polymorphic repeat sequences), and including all splice site junctions of the AR gene, as well as the entire coding region of CYP17. The aim of this study was thus to determine the type and frequencies of genetic variants of these androgen metabolism genes within the diverse South African population, and to determine if the observed ethnic variation in the incidence and progression of CaP can be explained by ethnic-based genetic differences. For high sensitivity mutation detection, the most powerful of the pre-screening methods was used, namely denaturing gradient gel electrophoresis (DGGE). 20 CaP and 25 control benign prostatic hyperplasia (BPH) tissue samples were screened in order to identify possible mutations. Blood samples from the same patients were analysed in order to determine whether mutations are germline and therefore present in all cells of the body. Additional blood samples from the Western Province Blood Transfusion Service (WPBTS) (Refer to section 2.1.2, Table) were also analysed in order to determine the frequency of identified polymorphisms within the general population. Certain polymorphisms were further analysed in paraffin-embedded wax material (exclusively from Blacks) to determine the distribution of these polymorphisms in the Black population. Direct sequencing of mutant-containing DNA fragments was performed to determine the exact location and nature of mutation. Using the AR- DGGE assay 4 novel mutations were identified as well as a previously reported codon 211 (E211) polymorphism. With the CYP17- DGGE assay, 3 novel single nucleotide polymorphisms (SNPs) were detected. Three base variants occured, in codons 36 (L36), 46 (H46) and 65 (S65), as well as intronic substitutions in intron 4 (IVS+58G4C) and intron 6 (IVS-25C7A). Frequencies of SNPs were measured in the CaP and BPH samples. In conclusion, the identified polymorphisms could be used as markers in determining CaP susceptibility and may thus facilitate the identification of individuals with a high- or low-risk of developing carcinoma of the prostate. / AFRIKAANSE OPSOMMING: Prostaatkanker vertoon die hoogste voorkoms van enige kwaardaardigheid wat Suid-Afrikaanse mans aantas. Die etiologie van prostaatkarsinoom dui aan dat etnisiteit een van die mees belangrike risikofaktore is. Oorsake van hierdie etniese verskille is onbekend, maar vermoedelik is omgewing en genetiese faktore albei betrokke. Karsinoom van die prostaat is androgeenafhanklik en daar is voorgestel dat variasies in androgeenmetabolisme en androgeensintese 'n persoon se risiko mag affekteer. Gevolglik, is gene betrokke in hierdie paaie kandidate vir die bepaling van prostaatkanker vatbaarheid. In hierdie studie het ons twee kandidaat gene in die androgeen biosintetiese en metaboliese pad geanaliseer, naamlik, die androgeen reseptor geen (AR), betrokke in androgeen vervoer en aktivering van transkripsie, en die sitokroom p450c17a geen (CYP17), belangrik vir testosteroon biosintese. Ons het omvattende mutasie-bespeurings-essai-sisteme ontwikkel (ook uitvoerbaar op argivale paraffien-bewaarde materiaal), wat amper vir die hele koderende streek van die AR geen gebruik kan word (uitsluitend herhalende polimorfiese reekse) en wat alle splytpunt-aansluitings van die AR geen insluit, asook vir die hele koderende streek van CYP17. Die doel van hierdie studie was dus om die tipe en frekwensies van genetiese variante van androgeen metabolisme gene in ons diverse Suid-Afrikaanse bevolking te bepaal, en om vas te stel of die waarneembare etniese wisseling in die insidensie en vordering van prostaatkanker verstaan kan word deur etnies gebaseerde genetiese verskille. Die mees sensitiewe tegniek wat tans beskikbaar is vir vooraf-sifting vir onbekende mutasies is gekies, naamlik denaturerende gradiënt gel elektroforese (DGGE). Om moontlike mutasies op te spoor, het ons 20 prostaatkanker en 25 benijne prostaathiperplasie (BPH) monsters geanaliseer. Analise was gedoen op bloedmonsters van dieselfde pasiënte om vas te stel of kiemlyn mutasies (in alle liggaamselle) teenwoordig is. Bykomstige bloedmonsters (van die Westelike Provinsie Bloedoortappingsdiens) is ook geanaliseer om die frekwensie van bespeurde polimorfismes in die algemene bevolking te bepaal. Argivale paraffien-bewaarde materiaal (eksklusief van Swartes) is ook geanaliseer om die verspreiding van sekere polimorfismes in die Swart bevolking te bepaal. Direkte DNA volgorde bepaling van mutante DNA fragmente is uitgevoer om die ligging en tipe van mutasies te bepaal. Met die toepassing van ons AR-DGGE mutasiesisteem het ons 4 nuwe mutasies ontdek asook 'n kodon 211 (E211) polimorfisme wat voorheen gevind is. Vyf enkel nukleotied polimorfismes is met die CYP17-DGGE mutasiesisteem opgespoor. Die polimorfismes sluit in: drie basis veranderinge wat voorkom in kodons 36 (L36), 46 (H46) en 65 (S65), asook introniese substitutisies in intron 4 (IVS+58G4C) en intron 6 (IVS-25C7 A). Frekwensies van die polimorfismes was bereken in die prostaatkanker en BPH monsters. Die resultate aangebied in hierdie tesis dui aan dat die gevonde polimorfismes as merkers gebruik kan word om prostaatkanker vatbaarheid te bepaal en daardeur individue te identifiseer met 'n hoë of lae risiko vir prostaatkarsinoom ontwikkeling.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy