A dissertation submitted in fulfilment of the requirements for the degree of Masters in Science in the School of Molecular and Cell Biology, University of the Witwatersrand Johannesburg, 2013 / Cervical cancer is a gynaecological malignant disorder and is a common cause of death in women of the sub-Saharan Africa, striking nearly half a million of females each year worldwide. Cervical cancer is due to the persistence infection of human papillomavirus (HPV), a formidable virus that targets the cervix and is present in most cancers of the cervix. In South Africa, plants used to treat cancer are rare and there is a need for screening further plant extracts in order to identify potentially new anti-cancer drug discovery leads. The purpose of this study was to screen Tulbaghia violacea (TV) and Agave palmeri (AG) for anti-cancer therapy in the cervical cancer cell lines HeLa and ME-180 and in the fibroblast cell line KMST-6. Staurosporine (ST) was used as a positive control. AG and TV crude plant extracts were screened for apoptosis induction, followed by elucidation of the role of Bax, Bcl-2, p53, Rb, RBBP and Mdm2 genes in cervical cancer. Plant extracts of TV and AG were time (24 hours) and dose (50, 100, 150 μg/ml) dependently screened against cervical cancer cell lines HeLa, ME-180 and in KMST-6 for anti-cancer activity using the thiazolyl blue test (MTT) assay. With an IC50 ~ 150 μg/ml, T. violacea extract exhibited significant cytotoxicity on both HeLa and ME-180 cancer cell lines, whilst A. palmeri was cytotoxic to ME-180 cells and 25nM ST as a positive control had a cytoxicity effect on all cell lines including the KMST-6, yet TV and AG had no cytotoxic effect on KMST-6. The annexin-V/FITC detection assay was performed to evaluate the occurrence of apoptosis. Crude extracts of TV and AG together with ST induced significant apoptosis of HeLa, ME-180 and KMST-6 cells. The crude extracts were further analysed for DNA fragmentation, protein expression and gene expression by Western Blot and RT-PCR respectively, to investigate whether these extracts have an effect on the on the expression of Bax, Bcl-2, p53, Rb, RBBP6, Mdm2 and the relationship between p53 and RBBP6. Morphological and biochemical changes were seen in this study. A further mixed response by several genes was observed following treatment with the two plant extracts, where RBBP6 was seen to be spliced in cancer cells while Bax was induced and Bcl-2 was inhibited, but the levels of p53 remained the same. Preliminary, the extracts of TV and AG induce cell death by down-regulating Bcl-2 and Mdm2. Quantitative RT-PCR showed that when p53 was silenced RBBP6 was up-regulated and vice versa. From these results it was deduced that RBBP6 gene interacts with p53 during cervical cancer development. The anti-proliferative activity together with the characterization of p53, RBBP6 and Mdm2 and concentrations of these plant extract could be manipulated as diagnostic markers and potential therapeutic targets for cancer treatment; however, further studies on these plant extracts need to be performed to validate results obtained in this study.

Thesis (B.S.) Magna Cum Laude --Butler University, 2009. / Includes bibliographical references (leaf 26).

published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Since high-risk Human Papillomavirus (HPV) plays a critical role in cervical carcinogenesis, it is essential to improve our understandings on the role of HPV-related molecular markers in cervical neoplasia. The aim of this study was to investigate the expression and prognostic significance of selected HPV-related markers, including HPV16/18 E2 binding sites (E2BS) methylation, Pak 1, c-FLIP, Notch 1 and Brd4 expressions, as well as the potential functions of Pak 1 in cervical neoplasia. First, the differential expressions of these markers among clinical samples of normal cervical epithelium, low-grade and high-grade cervical intraepithelial neoplasia (CINs) and cervical cancer were studied. Methylation status of E2BS 1, 2 and 4 was determined by pyrosequencing. Expressions of the target proteins were assessed by immunohistochemistry. Both HPV16/18 E2BS 1&2 and E2BS4 methylation progressively increased from normal cervix through CINs to cancer. More importantly, HPV16 E2BS1&2 (for transcriptional repression of E6/E7 oncoproteins) became more heavily methylated than E2BS4 (for transcriptional activation of E6/E7) in cervical cancer, favouring the differential binding of E2 protein to E2BS4. Pak 1, c-FLIP, Notch 1 and Brd4 were all overexpressed in cervical cancer. Their expressions increased progressively from normal cervix to low-grade +/- high-grade CINs. Pak 1 and c-FLIP expression was positively correlated with HPV18 E6 and HPV16 E7 expression respectively. Notch 1 expression was inversely correlated with HPV16 E7 and HPV18 E6 expressions. Brd4 expression was positively correlated with HPV16 E2 and inversely correlated with HPV16 E7 expressions. The prognostic significance of the molecular markers was investigated by correlation with clinical parameters. Heavier methylation at E2BS1&2 relative to E2BS4 was associated with better overall and disease-free survival in cervical cancer patients. HPV16 E2BS1&2 hypermethylation, weak cytoplasmic Brd4 expression, strong c-FLIP or Notch 1 expression were associated with higher risk of recurrent abnormal smears after treatment of CINs. The role of Pak 1 in cervical cancer was further explored by comparing its functions between cervical cancer cell lines with and without transient knock-down of Pak 1 by siRNAs. It was demonstrated that the significant functions of Pak 1 in cervical cancer were on promoting cell proliferation and inhibiting apoptosis. Transient HPV16 E6 inhibition showed no effect on total / phosphorylated Pak 1 expressions. Lastly, the function of Pak 1 on the regulation of cervical cancer cell radiosensitivity was also investigated. Pak 1 inhibition increased cell sensitivity in response to irradiation by enhancing apoptosis and inhibiting cell proliferation. Conclusively, differential methylation status at HPV16/18 E2BS, as well as Pak 1, c-FLIP, Notch 1 and Brd4 proteins contribute to cervical carcinogenesis, and are potential prognostic markers in cervical cancer and CIN patients. Pak 1 inhibitor may be a potential adjunctive agent to improve radiotherapy. / published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

Not available / text

Thesis (Ph. D.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 182-218).

Thesis (Ph.D.)--University of Hong Kong, 1999. / Includes bibliographical references (leaves 182-218) Also available in print.

Thesis (doctoral)--Rijksuniversiteit te Groningen.

Thesis (MTech (Biomedical Technology))--Cape Technikon, Cape Town, 2002 / Infection \\'ith specific high risk human papilloma"iruses (HPV) has been shown to play a causal role in the development of ceJVical intraepithelial neoplasia (CIN) and cenical cancer in women. The development of a prophylactic vaccine to immull.ize women against HPV infection would play a \'ita! role in protecting women against HPV infection and ultimately ceMcal cancer. Despite cancer of the cer\'ix being the second most common cancer in South African women, a literature search reveals that few studies have been performed in South Africa on the types of HPV prevalent in women with CIN or cancer ofthe ceMx. HPVs that infect the anogenital tract have also been shown to infect the oral ca\'ity. However, the HPV prevalence rates vary greatly between studies and the significance of the presence ofHPV in the oral ca\'ity is still not understood. The primary objectives of this study were to establish the HPV prevalence rate infecting women with CIN lesions using a sensitive nested polymerase chain reaction (PCR) and to develop a novel restriction fragment length polymorphism (RFLP) method to type the high risk mucosal HPVs detected in these women. The secondary objective of this study was to establish the prevalence rate and HPV types infecting the oral mucosa of women with CIN lesions and to compare these HPV types with those detected in the ceMx. Cemcal punch biopsies were taken from 163 women with CIN lesions and buccal cells were collected from 33 of these participants. DNAwas extracted from the biopsies and buccal samples and PCR using CCRS primers performed to ensure sample adequacy. Nested PCR usmg consensus degenerate primers for HPV was performed on all samples sho\\'wg sufficient amplifiable DNA A novel restriction fragment length pol)morphism (RFLP) method was developed to identify the 10 high risk mucosal HPVs considered human carcinogens of group 1 by the International Agency for Research on Cancer (lARC) as well as HPV 11 which is commonly found in the oral cavity.