KNA interference (RNAi) is a ubiquitous gene silencing mechanism, in which small interfering RNAs (siRNAs) guide sequence-specific cleavage of mRNA transcripts. Since naked siRNAs are inefficiently internalised by mammalian cells, successful delivery of siRNA to target cells is the biggest challenge for therapeutic application.

High-risk human papillomavirus type 16 (HPV16) is the primary causative agent of cervical cancer and is therefore associated with high mortality worldwide. Cellular transformation of host cells is directly mediated by the expression of three viral oncoproteins, of which HPV16 E5 (16E5) is the least characterised. The hydrophobic protein consists of 83 amino acids, which constitute three, alpha helical transmembrane domains. The protein is able to enhance proliferative signalling and subvert recognition processes in host cells. A number of 16E5-host interactions and SDS-resistant 16E5 oligomers have been identified, however the molecular basis of 16E5 function remains unclear. Often disregarded as an artefact of over-expression, we show that the 16E5-16E5 interaction, in mammalian cell Iysates, is lipid and not concentration dependent. Biochemical analysis of 16E5 oligomers has been hindered by the lack of a robust purification system. Here, we describe several, novel E5 expression and purification systems, used to purify significant quantities of detergent-free recombinant protein, for study in membrane mimetic systems. Purified 16E5 was able to associate with and integrate into liposomal membranes and formed ring-like structures visible by transmission electron microscopy (TEM). Complexes owned a defined lumenal diameter, allowing fluorescent dye release from carboxyfluorescein (CF) loaded liposomes and a hexameric stoichiometry was inferred by native PAGE. Furthermore, 16E5 channel activity was activated upon lowered hydrogen ion concentration, suggesting a potential pH dependent gating mechanism. These data identified a novel function for 16E5, as a member of the viroporin family of virally encoded channels. Known viroporin inhibitors, rimantadine and NN-DNJ, prevented 16E5 channel function in a dose-dependent manner and in the absence of high- resolution structural data, established de novo molecular modelling and rational drug design were used to develop the first specific 16E5 inhibitors. Compound MV006 did not prevent 16E5 association with liposomes or oligomerisation, validating docking studies. Functional determinants of channel activity and 16E5 oligomerisation were predicted using an in silico model. Identification of 16E5 viroporin function will enhance the understanding of the physiological role of 16E5 in HPV infections, persistence and carcinogenesis. Furthermore, 16E5 channel activity may represent an important target for antiviral intervention.

The class 1 PDZ domain Tip-1 protein was firstly identified as a binding partner for the Human T-Cell Leukaemia Virus type 1 (HTLVl) Tax oncoprotein. It was later also shown to interact with: the RhoA signalling effector rhotekin, the wnt signalling effector β-catenin and the E6 oncoprotein from high-risk HPV16 but not low risk HPV6. These observations suggested that Tip-1 may be an important "hub" protein that is involved in pathways with a proven link to carcinogenesis. Based on these finding, it was decided to further characterise the cellular role of Tip-1 by carrying out a yeast two-hybrid screen to identify new binding partners in order to uncover potentially novel functions of this protein. This identified an intracellular fragment of the transmembrane receptor Plexin Dl and a C-terminal fragment of the AT Hook DNA binding containing l(AHDCl) protein which also had a carboxyl terminal PDZ domain binding site consensus sequence. Both of these interactions were confirmed by yeast mating assay which was also used to show that mutant constructs of AHDCl lacking the carboxyl PDZ binding site did not bind Tip-L The Tip-1 protein was subsequently expressed in bacteria and purified as a glutathione transferase (GST) fusion product whilst full length AHDCl was cloned and expressed by in-vitro transcription / translation (IVTT).

Somatic mutations in the PTEN gene (Phosphatase and Tensin Homologue Deleted on Chromosome Ten) have been found in many types of cancer, but most frequently in cancer of the endometrium. The PTEN gene encodes a D3 lipid phosphatase of the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3), which activates protein kinase B/Akt. Constitutive activation of Akt has been found in cells that lack functional PTEN, thus by dephosphorylating PIP3, this enzyme modulates several cellular functions e.g. proliferation, differentiation and migration. The PTEN protein comprises a 403 amino acid, 55KDa protein which is present in the cytoplasm but has also been detected in the nucleus of some cells. The function of nuclear versus cytoplasmic PTEN has not yet been determined. Little is known about modulation of PTEN expression by molecules such as hormones and cytokines. It has been reported, however, that NGF, BDNF and vitamin D3 analogues can up-regulate PTEN. The steroid hormones oestrogen and progesterone have been proposed as mediators of PTEN transcription, since their expression in endometrium reflects the menstrual cycle. The role of TGF-beta1 in PTEN expression of PTEN in human cells has been also investigated, but the results are contradictory. Compounds which stimulate up-regulation of PTEN represent potential anti-tumour therapies and therefore merit investigation. To further investigate the role of PTEN in endometrial cancer the following approaches were taken. The effect of TGF-beta1 on two endometrial carcinoma cell lines, HEC-1B and Ishikawa were investigated. The cell lines were stimulated with TGF-beta1 in the presence or absence of serum, and changes in mRNA and protein levels of PTEN and other genes analysed by RT-PCR and Western blotting. The morphology, cell number and cell viability were also assessed. Modest up-regulation of PTEN mRNA was detected in both cell lines, but little change in protein levels was observed. In accordance with published data, TGF-beta1 suppressed the growth of, and changed the morphology of both cell lines. To study PTEN sub-cellular localisation, full-length human PTEN cDNA was used in RT-PCR to generate a 1.2Kb fragment which was cloned into a green fluorescent protein expression vector pEGFP-N1 to create pRC-2. Sequencing of pRC-2 confirmed the in-frame cloning of wild-type PTEN. Lipid-basedtransfection was used to transiently transfect HEC-1B, Ishikawa and Cos-7 cells. Strong perinuclear and cytoplasmic localisation was detected in these cell lines, and localisation to the endoplasmic reticulum was observed. Stimulation with TGF-beta and 17-beta-estradiol had no discernable effect on sub-cellular localisation of PTEN in either HEC-1B or Ishikawa cell lines. A mutational study was performed using a large repository of archival endometrial carcinomas and normal cervical controls. PCR was used to amplify PTEN exons 5 and 8 from extracted DNA and the fragments separated by single-strand conformation polymorphism (SSCP) analysis. A number of samples exhibiting bandshifts were detected in both exons.

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Disruption of gap junctional intercellular communication and/or Connexins (the channel proteins of gap junctions) is frequently reported in malignant cell lines and tumours. Many tumor cells exhibit aberrant gap junctional intercellular communication (GJIC), which can be restored by transfection with Connexin genes. Of the Connexin family, Connexin 43 has attracted the most attention as it is widely expressed in many tissues and Connexin 43 gap junctions correlate with various physiological functions. Connexin 43 is a short-lived protein (half-life of 1-3 h in cultured cells), both lysosomes and proteasomes having been reported to be involved in its degradation. Certain human papillomaviruses (HPV) associated with the development of cancers, especially of the cervix, have been reported to downregulate GJIC in vitro. There is also evidence for reduced gap junctions in cervical dysplasia. The association between HPV and GJIC, and the mechanism and consequence of deregulated GJIC in cervical tumour progression, remains unclear. In HPV-16 associated cervical cancer, the viral oncogene E6 inactivates the tumour suppressor p53, but also has p53- independent functions in tumour progression. One of these may involve interaction with hDlg (the human homologue of Drosophila Discs Large), a tumour suppressor present in epithelial cells at sites of cell-cell contact and which regulates cell polarity and attachment. hDlg contains three PDZ protein-protein interaction domains, the second PDZ domain of which binds E6. Connexin 43 also has a PDZ binding domain in its C-terminus. Previously, it was demonstrated that Connexin 43 relocates from the membrane to the cytoplasm in a novel HPV-16 E6-containing cervical epithelial cell line (named WI2GPXY) that is fully transformed and invasive and deficient in gap-junctional intercellular communication (Aasen T et aI., 2003 Oncogene 22, 7969- 7980). The overall aim of this project was to investigate the relationship of loss of gap junctions to malignant progression by comparing the properties of W12GPXY with those of the non-malignant parental cell line, WI2G, from which W12GPXY was derived. First, microarray was used to identify global differences in RNA expression between the two cell lines, large differences were seen in expression of angiogenesis-related genes and they were confirmed by Real-Time RT-PCR for three genes, IL-8, VEGF and FGF-2. No significant differences were noted for connexin genes but there were differences in MAGUK family members including hDlg. However, protein expression studies by western blot and immunofluorescence staining showed a significant increase (2.9 fold) of HPV-16 E6 in W12GPXY cells, which co-localises with hDlg in the cytoplasm. Connexin 43 also binds hDlg. Treatment ofW12GPXY cells with Leptomycin B to trap E6 in the nucleus or siRNA knockdown of E6 abrogate the relocation and co-location of hDlg and endogenous wild type Connexin 43 and restore Connexin 43 gap junction at points of cell-cell contact. Further, when C33a cells (HPV -negative cervical tumour cells which normally retain large Connexin 43 gap junctions) are transfected with HPV -18 wild type E6, changes in localisation of Connexin 43 and hDlg are consistent with those in W12GPXY cells. However, C33a cells transfected with a mutant E6 lacking the hDlg binding site retain Connexin 43 gap junction plaques. Finally, Connexin 43 associates with hDlg through its PDZ-binding domain and this is required for its relocalisation to the cytoplasm in W12GPXY cells. The results suggest that increased cytoplasmic E6 associated with malignant progression of W12GPXY cells redistributes Connexin 43 from membrane junctions into the cytoplasm by a mechanism involving binding of hDlg to the Connexin 43 C-tenninal tail. The findings have uncovered a new role for hDlg in trafficking of Connexin 43. It also provides a novel mechanism for the loss of gap junctional intercellular communication during malignant progression of squamous epithelial cells. The specific roles played by lysosomes and proteasomes in the degradation of Connexin 43 in W12GPXY cells were also studied. The results suggest the involvement of both proteolytic pathways, although the lysosome seems to be the major compartment for Connexin 43 degradation. Association with HPV-16 E6 and hDlg together with proteasome activity seems to be required for Connexin 43 redirection from the cell membrane and transport into the lysosomal degradation pathway. Taken together, these results suggest that Connexin 43 gap junction intercellular communication was lost from the cell membrane requiring maintenance of E6 and hDlg complexes for proteasomal internalization and consequently transport into lysosomal compartment for degradation in W12GPXY cells.

Human papillomavirus type 16 (HPVI6) is a causative agent of cervical cancer. The HPV16 viral transcription/replication factor E2 is essential for the viral life cycle. The function of E2 is dependenton the interaction with cellular partner proteins. The amino- terminal domain of E2 is an acidic protein-protein interaction domain essential for all of the functions of E2. A yeast-2-hybrid screen using the amino-terminal domain of HPV16 E2 as bait identified multiple possible partner proteins for E2 function (Boner & Morgan 2002) including the DNA replication/repair protein TopBPl. E2 molecules from HPVI6 and HPV18 interact with multiple proteins involved in the cellular response to DNA damage (e.g. p53, BRCAl, PARP and TopBPl). The role of TopBPl in E2 function and the functional response of E2 to DNA damage stimuli were investigated. Overexpression of TopBPl enhances the transcription and replication activation functions of E2. Overexpression of an amino-terminal truncation mutant of TopBPl has no effect on the transcription/replication functions of E2. During this study a novel method for the detection of E1/E2 DNA replication function by real-time PCR was developed. The UVB irradiation of cells resulted in the significant reduction of both E2 transactivation function and E2 protein amount. These results demonstrated that E2 function is altered by cellular DNA damage response proteins and signaling pathways. In many HPV induced cancers the HPV genome is either present integrated into the cellular chromosomes or is maintained episomally with large DNA deletions/rearrangements. Therefore HPV genome stability is a significant risk factor for the development of HPV induced cancer. Thus the fidelity of DNA replication activated by the HPV 16 E1/E2 replication factors was investigated on undamaged and UVC damaged templates in a variety of genetic backgrounds. On undamaged DNA templates there were a significant amount of mutations due to DNA deletions/rearrangements and the frequency of mutation increased when the template was damaged. This increase on damaged templates was marked in cells with defects in key DNA replication or repair proteins. These results highlight the instability of HPV16 genome replication. The interaction of E2 with DNA damage response proteins and the reduction of E2 function in response to DNA damage may be an evolutionary response by the virus to ensure genetic integrity and host cell viability.

Cervical cancer is the most common malignancy after breast cancer in women worldwide. It accounts for 83% of all new cases and 85% cancer death in developing countries. In South Africa, cervical cancer is the common cancer in women with an annual crude incidence rate of 30.2 per 100,000 women and the highest rate were found to be in women between the ages of 66-69 years. Approximately 6800 women in S.A face new case of cervical cancer while accounting for 3700 cancer death annually. Because of unequal access to the health facilities, socio-economic differences, HPV and HIV infection, the rate of cervical cancer in black women is higher (42.1%) compared to the low rate in white women. Because of the name “cancer bush’ given to Sutherlandia Frutescence(S.F) plant by the traditional healers as well as Xhosas, Zulu, Sotho and cape Dutch for its anti-cancer activity, the plant was in this study to confirm its cytotoxic effect on the cervical cancer cell lines. Aim of the study: to evaluate the methanolic extracts of Sutherlandia Frutescens on cervical cancer cell lines. Materials and Methods: The MTT assay was performed to evaluate SiHa cell lines treated with methanolic extract of S.F (50μg/ml, 100μg/ml, 150μg/ml and 200μg/ml). The three compounds (Canavanine, GABA and Pinitol) were also evaluated for its anti-tumour activity. The cell growth was then showed in real time using Xcilligence. Flow cytometry was employed to determine the mode of action. Caspase 3/7 assay was performed to confirm if cell death was via caspase-dependent or independent and ATP was done to assess the ATP level in S.F treated cells. Results: MTT shows a significant decline in cells treated with 50μg/ml, 100μg/ml and 200μg/ml of the extract and 50μg/ml was concluded to be the concentration at which 50% of the cells die. The ATP results are inconsistent with MTT result; the ATP level increased in S.F treated cells. Cell index which represents the quantitative measure of cell growth in real time decline upon treatment with 50μg/ml. Flow cytometry showed cells are dying by apoptosis and the cell cycle arrest is mostly in the S phase. The cell death was caspase-dependent as it shows an increased luminescence which is proportional to the number of caspase. The concentrations of the compounds used, Canavanine (1000μM, 1500μM and 2500μM), GABA (100μM, 300μM and 500μM) and Pinitol (30μM, 90μM and 120μM) induce cell death and cell death shows to decrease after the maximum concentration. Conclusion: Sutherlandia Frutescence has proven with number of research that it induces cell death via apoptosis. After evaluating its cytotoxicity, the plant shows to be a promising anti-tumor agent that needs to be clinically proven. / Life Sciences / M. Sc. (Life Science)