Browsing by Author "Prince, Sharon"
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- ItemRestrictedThe anti-cancer activity of a novel palladacycle, BTC2, in oestrogen receptor positive and triple negative breast cancers(2017) Irene, Ikponmwosa; Prince, Sharon; Kimani, SerahBreast cancer remains the leading cause of cancer death among women worldwide. This is in part due to late diagnosis, high recurrence rate and the development of drug resistance. Indeed, even though oestrogen receptor-positive breast cancers are known to respond to hormonal therapy, drug resistance is a common occurrence. Furthermore, triple negative breast cancers lack a specific therapeutic target, which has led to poor treatment outcomes. Hence, there is a critical need for new therapeutic approaches. Our laboratory previously identified a novel palladium-based compound, AJ-5, that exhibit potent anti-cancer activity in triple negative and oestrogen receptor-positive breast cancer cells. However, AJ-5 is poorly soluble, therefore, a series of water soluble AJ-5-based compounds were synthesized. The aim of this study was to test and characterise the anti-cancer activity of one of these AJ-5 analogues, BTC2, in triple negative (MDA-MB-231) and oestrogen receptor-positive (MCF7) breast cancer cell lines. Cytotoxicity assays were performed and BTC2 was shown to inhibit the proliferative rates of breast cancer cells with calculated IC50 values of 0.49μM in MCF7 cells and 0.58μM in MDA-MB-231 cells. BTC2 did not display considerable selectivity to breast cancer cells as the calculated IC50 value for the normal fibroblast cell line (FG0) was found to be 0.85μM and thus the selectivity index was less than 2 in both cell lines. Clonogenic assays were performed and BTC2 was shown to inhibit the long term (10 to 21 days) survival of MCF7 and MDA-MB-231 cells as it reduced their colony forming ability. Western blot analyses and immunofluorescence with an antibody to ƴH2AX, a robust marker of DNA double strand breaks, indicated that BTC2 acts by inducing DNA damage as the levels of this protein increased in drug treated cells. Light microscopy revealed that BTC2 induced morphological features of apoptosis (membrane blebbing and cell shrinkage) and autophagy (vacuoles reminiscent of autophagosomes). To further characterise the molecular mechanism underpinning the cytotoxic effects of BTC2, western blotting was performed with antibodies against key protein markers of stress signalling, cell cycle, apoptosis and autophagy. The results indicated that BTC2 activated the p38 MAP kinase signalling pathway and the p53 response in MCF7 cells. It is worth noting that MDA-MB-231 cells have a mutant p53 but that the p53 target protein, p21, was upregulated in both MCF7 and MDA-MB-231 cells. This suggests that p21 is regulated by a p53-independent mechanism in the MDA-MB-231 cells. BTC2 was shown to induce apoptosis and autophagy in both breast cancer cell lines as demonstrated by increased levels of cleaved PARP and LC3-II respectively. Apoptosis was confirmed by Annexin V-FITC/ propidium iodide double staining using flow cytometry. Taken together, data from this study suggest that BTC2 represents a promising anti-cancer drug for the treatment of triple negative and oestrogen receptor-positive breast cancer cells.
- ItemOpen AccessCancer cell behaviour following parasite exposure(2018) Jacobs, Brittany-Amber; Smith, Katherine; Prince, SharonInfectious diseases, including helminthiases, are estimated to cause 16.1% of global cancer cases. While certain helminths are conclusive causes of cancer, others have been shown to reduce the disease. It is currently unknown why differing helminth infections promote or prevent cancer development and progression, or which cellular mechanisms are altered following exposure. Using several in vitro and in vivo techniques, this study aimed to determine the effect that certain helminths have on the progression of cervical and colorectal cancer. The results revealed that antigen from the hookworm Nippostrongylus brasiliensis significantly reduced cervical cancer cell migration and the expression of two markers of metastasis: vimentin and N-cadherin. Importantly, N. brasiliensis antigen significantly lowered the expression of cell-surface vimentin, while decreasing Human Papillomavirus type16 pseudovirion internalization. In vivo infection with N. brasiliensis significantly decreased vimentin expression within the female genital tract, confirming the relevance of these in vitro findings. Furthermore, exposure to antigen from the gastrointestinal nematode Heligmosomoides polygyrus decreased the in vitro proliferation of human and mouse colorectal cancer cells and simultaneously increased the expression of cell cycle regulator proteins, p53 and p21. Surprisingly, while antigen from H. polygyrus inhibited human colorectal cancer cell migration, it had the opposite effect on mouse colorectal cancer cells, suggesting that its impact on colorectal cancer migration may be, at the very least, species dependent. Using a syngeneic tumour model, the excretory-secretory product from H. polygyrus was shown to significantly increase tumour growth and the expansion of regulatory T cells and neutrophils in the tumour. Similarly, in a model of colitis-associated colorectal cancer this antigen significantly worsened pathology in a TGF-β dependent manner. Undoubtedly, the knowledge gained from this study will contribute to the limited understanding about helminths and the effect that these parasites have on cancer progression.
- ItemOpen AccessCharacterisation of the 3'-UTR of the COL5A1 gene: implication for musculoskeletal soft tissue injuries(2015) Laguette, Mary-Jessica Nancy; Collins, Malcolm; Prince, SharonCOL5A1 encodes the α1 chain of type V collagen, a minor fibrillar collagen that is an important regulator of collagen fibril assembly. A polymorphism (rs12722, C/T) within the 3'-untranslated region (UTR) of COL5A1 is associated with chronic Achilles tendinopathy (TEN) and other soft tissue injuries as well as exercise-related phenotypes. These phenotypes are directly or indirectly associated with the mechanical properties of musculoskeletal soft tissue. It has therefore been hypothesised that variants in the COL5A1 gene, specifically the 3'-UTR, regulate synthesis of the α1(V) chain and type V collagen production. Type V collagen levels in turn regulate fibril architecture and structure and, thereby, mechanical properties of musculoskeletal soft tissues. Although the 3'-UTR of many eukaryotic genes have been shown to play an important regulatory role, the function of the COL5A1 3'-UTR is currently unknown. Aim. The primary aim of this thesis was therefore to determine whether the COL5A1 3'-UTR was functional and to identify functional differences between the COL5A1 3'-UTR cloned from participants with TEN and healthy asymptomatic control individuals. The secondary aim was to start mapping the functional regions within the 3'-UTR, focusing on regions which are potentially responsible for contributing to the tendinopathic phenotype.
- ItemOpen AccessThe effect of seminal fluid on TBx2 and TBX3 expression and activity in cervical cancer cells(2017) Cooper, George William; Katz, Arieh; Prince, SharonCervical cancer is one of the most common female cancers in Africa, both in terms of incidence and mortality, and is disproportionately prevalent in developing nations due to a lack of adequate access to healthcare. While new vaccine technologies are rapidly reducing the incidence of Human Papilloma Virus (HPV) infection, the primary causative agent of cervical cancer, new cases continue to accumulate in the developing world. Beyond the role of HPV in the early stages of cancer development, the molecular aetiology of this disease is poorly understood. Frequent exposure to seminal fluid (SF), the liquid component of semen, has been proposed as a potential driver of oncogenesis in cervical cancers and has been shown to exacerbate some aspects of cervical cancers. While some of the cellular signaling pathways responsible for these phenomena have been identified, much remains to be elucidated. We hypothesized that TBX2 and TBX3, two highly homologous transcription factors frequently implicated in other cancers, may be responsible for mediating some of the effects of SF on cervical cancer cells. We established that TBX3 protein is significantly overexpressed in both primary cervical adenocarcinomas and squamous cell carcinomas compared to normal tissue. SF was shown to increase expression of both TBX2 and TBX3 mRNA in HeLa and CaSki, but not C-33 A, cervical cancer cell lines. Furthermore, SF upregulated TBX3 protein expression in both of these cell lines. In contrast, TBX2 protein was undetectable in these cell lines. In addition, our results showed that SF treatment of HeLa cells increases the expression of the known TBX3 target gene, p21CIP1/WAF1 (p21), while having no effect on PTEN expression. Transient knockdown of TBX3 resulted in decreased p21 expression in SF-treated cells suggesting that SF upregulation of p21 is dependent on TBX3. This is the first study to investigate TBX3 protein expression in primary cervical tissues and SF regulation of TBX3. However, further research is required in order to elucidate the role of SF-induced TBX3 in cervical cancer development. The identification of the role of TBX3 in cervical cancer development could aid in the development of more effective treatments for cervical cancers and could potentially impact sexual health policy recommendations for women with cervical cancer.
- ItemOpen AccessIdentification and characterization of a novel Palladacycle, AJ-5, to treat advanced Melanoma and breast Cancer(2014) Aliwaini, Saeb; Prince, Sharon; Mapolie, Selwyn
- ItemOpen AccessIdentification of signalling pathways regulating TBX2 gene expression and its target genes(2008) Teng, Huajian; Prince, Sharon; Parker, M IMembers of the T-box family of transcription factors provide an important link between development and cancer. T-box factors play critical roles in embryonic development and results from recent studies suggest that they function in controlling cell cycle progression and also in the genesis of cancer. Importantly, the T-box factors Tbx2 and Tbx3 are overexpressed in several cancers including melanoma, small cell lung carcinoma, breast, pancreatic, liver and bladder cancers and can suppress senescence, a cellular process which serves as a barrier to cancer development. However, the precise role of most T-box factors is poorly defined, in part, because their target genes are still poorly characterised and very little is known of the signalling pathways that regulate their expression and activity. The broad aim of this study was therefore to contribute towards the identification of Tbx2 target genes as well as to identify signalling pathways that regulate TBX2 expression. The specific aims were thus to (1) investigate the regulation of type1 collagen gene expression by Tbx2; (2) clone the human TBX2 regulatory region and to identify cis-acting elements involved in the basal transcription of the TBX2 gene and (3) investigate the regulation of TBX2 gene expression by signalling pathways.
- ItemOpen AccessIdentifying novel drugs for the treatment of rhabdomyosarcoma(2019) Bleloch,Jenna; Prince, SharonRhabdomyosarcoma (RMS) forms in skeletal muscle and is the most common soft tissue sarcoma in children and adolescents. Current treatment is associated with debilitating side effects and treatment outcomes for patients with metastatic disease are dismal. Other than a need for alternative and more effective therapies there is also a growing appreciation for the need to understand the molecular underpinnings of RMS with the aim of identifying, in part, novel targets to develop highly specific and effective treatments with negligible adverse effects. The aim of this study was to identify novel drugs for the treatment of the two major RMS subtypes viz alveolar (ARMS) and embryonal (ERMS) RMS and to do so it adopted a two-pronged approach. Firstly, a novel binuclear palladacycle, AJ-5, was investigated for its anti-cancer activity and its mechanism(s) of action in RMS cells. The second approach involved a target-based drug repurposing strategy where a library of FDA-approved drugs was screened to identify leads that were able to negatively regulate the oncogenic TBX2 and TBX3 transcription factors that are known drivers of RMS. The binuclear palladacycle, AJ-5, was recently shown to exert potent cytotoxicity in melanoma and breast cancer and to present with negligible adverse effects in mice. To investigate the anti-cancer activity of AJ-5 in RMS cells, MTT assays were firstly performed in ERMS and ARMS as well as 'normal' cells. IC50 values determined from these experiments showed values of ≤ 0.2μM for the RMS cells and a favourable selectivity index of > 2. Clonogenic and migration assays showed that AJ-5 inhibited the ability of RMS cells to survive and migrate respectively. Western blotting revealed that AJ-5 induced levels of key DNA damage response proteins (γH2AX, p-ATM and p-Chk2) and the p38/MAPK stress pathway. This correlated with an upregulation of p21 and a G1 cell cycle arrest. Annexin V-FITC/propidium iodide staining revealed that AJ-5 induced apoptotic and necrotic cell death. Apoptosis was confirmed by the detection of cleaved PARP and increased levels and activity of cleaved caspases-3, -7, -8 and -9. Increased levels of necroptotic markers p-RIP3 and p-MLKL and inhibition of necroptosis with necrostatin-1 with a corresponding significant increase in cell viability suggests that AJ-5 is also capable of triggering a form of programmed necrosis. Furthermore, AJ-5 reduced autophagic flux as shown by reduced LC3II accumulation in the presence of bafilomycin A1, and a significant reduction in autophagosome flux J. Pharmacokinetic studies in mice show that AJ-5 has a promising half-life and that its volume of distribution is high, its clearance low and its intraperitoneal absorption is good. With the intention of improving the drug-like properties of AJ-5, specifically its water solubility, a derivative of AJ-5, BTC2, was synthesised and identified to display comparable anti-cancer activity against ERMS and ARMS cells. Together these findings suggest that AJ-5 and BTC2 may be effective chemotherapeutics with a desirable and novel mechanism of action for treating drug resistant and advanced RMS. The highly homologous T-box transcription factors TBX2 and TBX3 have both been implicated as key drivers of RMS and they have been identified as novel therapeutic targets for the treatment of this sarcoma subtype. Indeed, TBX2 or TBX3 overexpression in normal myoblasts inhibits muscle differentiation and overexpression and knock-down cell culture and mouse models show that RMS cells are addicted to them for their cancer phenotype. However, targeting transcription factors is notoriously challenging because unlike enzymes they do not have catalytic activity and deep binding pockets to which small molecule inhibitors can be designed which is further exacerbated by the length of time and costs associated with de novo drug development. Therefore, this study adopted a novel strategy to circumvent these challenges by combining a drug repurposing with a targeted approach to TBX2/3. Briefly, a high throughput cell-based immunofluorescence screen was designed and conducted to identify FDA-approved drugs that could negatively regulate TBX2 and/or TBX3 protein levels or nuclear localisation. Cells were engineered to express induced exogenous FLAG-tagged TBX2 and TBX3 using a Tet-On system and they were screened with the Pharmakon 1600 drug library at a concentration of 10μM. 'Hits' were identified by z-scores and amongst these, niclosamide, piroctone olamine and pyrvinium pamoate were validated to be potent inhibitors of TBX2/3 and were shown to display anti-cancer activity in RMS. These drugs have the potential to be repurposed for the treatment of RMS and other TBX2/3 driven cancers either as single agents or in combination with currently used chemotherapeutics.
- ItemOpen AccessIdentifying the molecular basis for treatment resistance in a subset of myasthenia gravis patients of African ancestory(2013) Auret, Jennifer Mary; Heckmann, Jeannine; Prince, Sharon; Abrahams, AmaalMyasthenia gravis (MG) is an autoimmune disease in which pathogenic antibodies block, target or destroy the acetylcholine receptors of the muscle endplate resulting in failure of neuromuscular transmission and fatigable weakness. We have previously shown that a subpopulation of South African MG patients of African genetic ancestry develop a severe extraocular muscle (EOM) phenotype whilst receiving standard immunosuppressive drug therapies. This phenotype associates with a functional c.-198C>G SNP (C>G SNP) in the regulatory region of decay accelerating factor (DAF), a complement regulatory protein, which is essential for protection against complementmediated damage. MG patients are treated with prednisone as the first-line immunosuppressant and frequently, an additional steroid-sparing agent, such as azathioprine, cyclosporin A or methotrexate. We hypothesised that MG patients with the C>G SNP when treated with immunosuppressant drugs, may have lower DAF protein levels contributing to increased susceptibility to autologous complement-mediated damage at their EOMs. This study tests this by comparing the effect of prednisone, azathioprine, cyclosporine and methotrexate individually and prednisone in combination with each of these steroid-sparing agents on wild-type and C>G DAF protein (western blotting) and mRNA (quantitative real time PCR) levels and promoter activity (luciferase reporter assays). These experiments were performed in EBV transformed lymphoblastoid cell lines from control individuals with wild-type DAF and MG patients carrying the C>G SNP. As a more representative model for this study the experiments were repeated in mouse skeletal muscle cells because there was no available EOM cell line. In support of the hypothesis of this study, prednisone, cyclosporin A and azathioprine individually was shown to repress C>G DAF protein levels while having either no effect on wild-type DAF or slightly activating it. Importantly, methotrexate was the only drug that was able to increase C>G DAF lymphoblast protein expression and therefore holds promise as a potentially effective treatment for MG patients with the C>G SNP. Moreover, using a calcein assay, clinically relevant doses of prednisone in combination with MG patient sera was shown to significantly increase the susceptibility of C>G DAF lymphoblasts to cell lysis (82% C>G DAF lymphoblasts vs. 9% wild-type DAF lymphoblasts). These results suggest that MG patient sera contain factor(s) that together with prednisone may also be responsible for the susceptibility of the EOMs in these patients to injury. The results show that the levels of DAF protein and mRNA did not always match which suggests that the drugs tested may regulate the DAF protein at a posttranscriptional level. In a mouse skeletal muscle model, Daf (equivalent to human wild-type DAF) protein expression was consistently repressed by prednisone treatment but activated by cyclosporine A, azathioprine and methotrexate. Furthermore, co-treatment of prednisone with either of the steroid-sparing drugs showed that azathioprine, and low doses of cyclosporin A and methotrexate were able to overcome the repressive effect of prednisone-only treatment on Daf expression. These observations indicate that the regulation of Daf may be species and/or cell-type specific. Together the results from this thesis suggest that in EOMs, where DAF is already downregulated, compared to other muscles, our MG patients with the C>G SNP may have an increased susceptibility to complement-mediated damage when treated with prednisone, which further represses C>G DAF expression.
- ItemOpen AccessInvestigating the functionality of African-specific variants in the TGFB1 regulatory region and their potential role in HIV-associated kidney complications(2016) Buys, Joy-Mari; Heckmann, Jeannine M; Prince, Sharon; Nel, MelissaTransforming growth factor beta-1 (TGF-β1) is a cytokine involved in immune modulation and tissue regeneration and has a polymorphic TGFB1 promoter. African-specific single nucleotide polymorphisms (SNPs) have been identified in the TGFB1 promoter and they occur at higher frequencies in South Africans with African-genetic ancestry (≈17%) compared with West and East African genomes (<5%). However, the functional significance of these SNPs has only been partially explored. In this study it was hypothesized that higher frequencies of immunemediated disease complications in South Africans, such as HIV-associated nephropathy (HIVAN), may be influenced by functional genetic variations in TGFB1. To address this, the African-specific TGFB1 haplotypes containing singular, or combinations of, -1287 G>A (rs11466314), -1154 C>T (rs35318502), -387 C>T (rs11466316) and -14 G>A (rs9282871) were investigated for their effect on TGFB1 promoter activity. Briefly, an extended TGFB1 regulatory region driving a luciferase reporter was used as a template to generate six TGFB1 promoter haplotypes (referred to as H-1 through H-6 in this thesis) by site-directed mutagenesis and luciferase activity was used to measure promoter activity. The functional TGFB1 -1347 C>T variant was also investigated (H-5 and H-6 containing -1347 C and T alleles, respectively) because all four of the African variants more frequently co-occurred with the previously reported "lower expressing" TGFB1 -1347 C variant (H-1 through H- 4). Transient transfection of the TGFB1 promoter reporter constructs in two renal cell lines (RCC4+VHL and Caki- 2) showed no difference between -1347 C (H-5) and T (H-6) basal promoter activity. Having at least one African variant resulted in ~5-fold loss of basal TGFB1 promoter activity in renal cells when compared to the most common haplotype (H-5) (p<0.05). The repressive effect is mainly attributed to the -387 T variant (H-1) as the addition of other African variants on this haplotype showed no additional TGFB1 promoter repression. The repressive effect of the TGFB1 -387 T variant was maintained even after in-vitro treatment of transfected renal cells with recombinant human TGF-β1 (rhTGF-β1). To determine whether the African-specific TGFB1 promoter haplotypes (H-AFR), containing TGFB1 -387 T and tested in luciferase assays, also impact on endogenous TGF-β1 protein levels, western blot analysis was performed on human dermal fibroblasts from patients who had been genotyped. Similar to the promoter studies, basal TGF- β1 protein levels in cells with the H-AFR were ~47% lower compared to cells without (p=0.04) and no difference was seen in response to hrTGF-β1. Interestingly, when western blots were screened for phosphorylated Smad3 (pSmad3) protein levels, as an indicator of the activated TGF-β1 canonical pathway, similar pSmad3 levels were observed under basal and hrTGF-β1 stimulated conditions for all haplotypes (p>0.05). The possible interaction of HIV tat on the African TGFB1 promoter variants was also assessed. Luciferase activity was measured after co-transfecting renal RCC4+VHL and HT1080 fibroblast cells with a HIV Tat expression vector and the H-6, H-5 and H-AFR promoter luciferase constructs. Results showed that the promoter activity for all TGFB1 haplotypes was upregulated in the renal cells (≥1.6-fold; p<0.001). The same result was, however, only observed for TGFB1 haplotypes H-5 and H-AFR in the fibroblast cells (≥1.4-fold; p<0.01). This is interesting because no difference was seen between TGFB1 H-5 and H-6 basal promoter activity. To investigate whether histopathological severity of HIVAN correlated with TGF-β1 staining patterns, immunohistochemistry was performed on 20 renal biopsies from patients with HIVAN. A semi-quantitative "histo" score (H-score) was calculated by multiplying the percentage of positive cells with the intensity of the stain before comparing the scores with control biopsies (HIV-negative, n=3 and HIV-positive without HIVAN, n=3). Compared to the HIV-positive controls the kidney tubules of HIVAN biopsies had higher H-scores. Strikingly, the interstitium of HIVAN samples stained much more prominently (17/18) than both control groups suggesting that TGF-β1 staining in the renal interstitium appear to be specific for HIVAN. In conclusion this study shows that the African-specific haplotypes effect basal TGFB1 promoter activity and TGF-β1 protein levels. However, they do not seem to affect the cytoplasmic TGF-β1/pSmad3 protein levels in response to rhTGF-β1 autocrine stimulation. Immunohistochemistry results suggest that TGF-β1 pathway may be prominently dysregulated in the renal interstitium of HIVAN cases.
- ItemOpen AccessInvestigating the functionality of candidate susceptibility genes in ophthalmoplegic myasthenia gravis using patient-derived material(2019) Moyakhe, Lihle Bayavuya; Heckmann, J M; Nel, M; Prince, SharonOphthalmoplegic myasthenia gravis (OP-MG) is a subphenotype of an autoimmune disease, myasthenia gravis (MG). This subphenotype described by our group is characterised by extraocular muscle (EOM) weakness which does not respond to standard immunosuppressive therapy whilst the non-ocular muscles do respond to treatment. OP-MG predominantly affects patients of African genetic ancestry and most commonly those with juvenile-onset. The cause of OP-MG is unknown. Previously, in efforts to understand the pathophysiology of this subphenotype, whole-exome sequencing was undertaken which identified amongst others a putatively functional multi-allelic deletion in the 5’ untranslated regulatory region of the ST8SIA1 gene. The most frequent deletion was c.-477_475 delCCC. The first aim of this thesis was to assess the functionality of this variant. To this end, in vitro promoter-reporter assays were performed after the delCCC deletion (MT) was created by site-directed mutagenesis of the wild-type (WT) promoter. Transient transfection assays showed that basal promoter activity of the MT promoter was lower than the WT promoter in the human fibroblast cell line (HT1080, p=0.031) but not in a mouse myoblast cell line (C2C12, p=0.33). Next, gene expression analysis was performed in opportunistically sampled orbital fibroblasts (n=5 controls, n= 2 OP-MG) and the endogenous expression levels of ST8SIA1 was determined under basal conditions and following treatment with homologous MG sera to mimic MG conditions. Although there was no significant difference in the basal ST8SIA1 expression levels in OP-MG ocular fibroblasts when compared to the control ocular fibroblasts (p=0.091), there were significantly lower ST8SIA1 expression levels in OPMG ocular fibroblasts in response to MG sera (p=0.024). Previous work by others also identified two other African specific gene variants in IL6R and TGFB1 which associated with OP-MG and in which luciferase reporter work showed functional implications. This thesis conducted the first examination of the endogenous expression levels of these genes in patientderived ocular fibroblasts. There was no difference in TGFB1 expression levels between OP- MG and control ocular fibroblasts under basal conditions or following treatment with MG sera (p>0.3). However, IL6R expression showed a similar trend to the luciferase reporter assays results in HT1080 cells. Both the OP-MG ocular fibroblasts tested harboured the 3’UTR IL6R c.*3043T>C variant, which was predicted to alter a putative miRNA binding site and showed a trend towards repression of IL6R expression in response to MG sera (p=0.083). In conclusion, although faced with the scarcity of sample tissue, we were able to use patient-derived ocular fibroblasts to validate previous genetic association studies and in vitro assays. Although the ocular fibroblasts are not EOM, they are both specialised tissue existing in the same microenvironment. Since MG sera modulate the expression of ST8SIA1 and IL6R differently between OP-MG and controls, our findings suggest that these genes may play a role in the OP-MG pathogenesis. Larger sample sizes are required for a more accurate representation of endogenous gene expression within these cell lines as the sample size is a factor when determining the significance of data. This is, however, a challenge due to the scarcity and unavailability of EOM for cell culture.
- ItemOpen AccessAn investigation into the role of TBX3 in breast carcinogenesis and its regulation by the TGF-?1 signalling pathway(2014) Li, Jarod Ang; Prince, SharonCancer is the second leading cause of death among both men and women and accounts for 13% of total deaths worldwide. Enormous efforts have therefore been made to cope with this problem, but unfortunately limited success has been achieved with most of the current therapeutic strategies. The T-box family of developmentally important transcription factors plays a role in the genesis of cancer and shows much promise as a focus for targeted therapeutic approaches to treat cancer. For example, the T-box factor TBX3 is overexpressed in a number of cancers including breast cancer but the mechanism(s) responsible for this upregulation as well as the precise role of TBX3 in the progression of this disease still need to be elucidated. This study provides novel data that show that TBX3 is specifically involved in breast cancer cell proliferation and migration and that its upregulation by the TGF-β1 signalling pathway mediates the TGF-β1-regulated anti-proliferative and pro-migratory effects. Furthermore, this study demonstrates that TBX3 mediates the anti-proliferative function of TGF-β1 through repressing transcription of its homologue, TBX2, which allows for the de-repression of p21 and a G1 cell cycle arrest. The findings of the current study are of great significance as it identifies TBX3 as a potential target for the development of novel breast cancer therapeutics. In order to ascertain the function of upregulated expression of TBX3, cell culture models were established in which TBX3 was either (1) stably silenced in an invasive breast ductal carcinoma cell line (MCF-7) which was previously shown to overexpress TBX3 or (2) overexpressed in a normal human breast epithelial cell line (MCF-12A). The resultant cells were then compared to control cells and tested for key characteristics of cancer. The data generated provide evidence that increased TBX3 levels inhibit breast epithelial cell proliferation in growth curve, BrdU incorporation and MTT assays. However, in vitro motility assays show that TBX3 may contribute to breast cancer progression by enhancing the migratory ability of these cells.
- ItemOpen AccessMolecular analysis of decay accelerating factor as a potential susceptibility factor to developing treatment resistant extraocular muscle involvement in Myasthenia Gravis(2009) Uwimpuhwe, Henriette; Heckmann, Jeannine; Ballo, Robea; Prince, SharonMyasthenia gravis (MG) is an autoimmune disorder in which auto-antibodies directed at the acetylcholine receptors (AChR) of the neuromuscular junction (NMJ) block, alter or destroy their targets. The anti-AChR antibodies cause activation of the classical complement pathway leading to inflammatory injury at the NMJ. Decay Accelerating Factor (DAF), a member of complement regulatory proteins, prevents activation of autologous components of complement pathways. The absence of DAF, in knock-out mouse models, has been shown to significantly increase the susceptibility to experimental autoimmune MG. A previous study showed that a high proportion of South African MG patients of African genetic ancestry develop immunosuppressive therapyresistant extraocular muscle (EOM) dysfunction. We hypothesized that these patients have deficient DAF expression in their EOMs resulting in less protection from complement injury.
- ItemOpen Accessp53 requires the stress sensor USF1 to direct appropriate cell fate decision(Public Library of Science, 2014) Bouafia, Amine; Corre, Sébastien; Gilot, David; Mouchet, Nicolas; Prince, Sharon; Galibert, Marie-DominiqueGenomic instability is a major hallmark of cancer. To maintain genomic integrity, cells are equipped with dedicated sensors to monitor DNA repair or to force damaged cells into death programs. The tumor suppressor p53 is central in this process. Here, we report that the ubiquitous transcription factor Upstream Stimulatory factor 1 (USF1) coordinates p53 function in making proper cell fate decisions. USF1 stabilizes the p53 protein and promotes a transient cell cycle arrest, in the presence of DNA damage. Thus, cell proliferation is maintained inappropriately in Usf1 KO mice and in USF1-deficient melanoma cells challenged by genotoxic stress. We further demonstrate that the loss of USF1 compromises p53 stability by enhancing p53-MDM2 complex formation and MDM2-mediated degradation of p53. In USF1-deficient cells, the level of p53 can be restored by the re-expression of full-length USF1 protein similarly to what is observed using Nutlin-3, a specific inhibitor that prevents p53-MDM2 interaction. Consistent with a new function for USF1, a USF1 truncated protein lacking its DNA-binding and transactivation domains can also restore the induction and activity of p53. These findings establish that p53 function requires the ubiquitous stress sensor USF1 for appropriate cell fate decisions in response to DNA-damage. They underscore the new role of USF1 and give new clues of how p53 loss of function can occur in any cell type. Finally, these findings are of clinical relevance because they provide new therapeutic prospects in stabilizing and reactivating the p53 pathway.
- ItemOpen AccessProfiling of patient-specific myocytes identifies altered gene expression in the ophthalmoplegic subphenotype of myasthenia gravis(BioMed Central, 2019-01-29) Nel, Melissa; Prince, Sharon; Heckmann, Jeannine MBackground: While extraocular muscles are affected early in myasthenia gravis (MG), but respond to treatment, we observe a high incidence of treatment-resistant ophthalmoplegia (OP-MG) among MG subjects with African genetic ancestry. Previously, using whole exome sequencing, we reported potentially functional variants which associated with OP-MG. The aim of this study was to profile the expression of genes harbouring the OP-MG associated variants using patient-derived subphenotype-specific ‘myocyte’ cultures. Methods From well-characterised MG patients we developed the ‘myocyte’ culture models by transdifferentiating dermal fibroblasts using an adenovirus expressing MyoD. These myocyte cultures were treated with homologous acetylcholine receptor antibody-positive myasthenic sera to induce muscle transcripts in response to an MG stimulus. Gene expression in myocytes derived from OP-MG (n = 10) and control MG subjects (MG without ophthalmoplegia; n = 6) was quantified using a custom qPCR array profiling 93 potentially relevant genes which included the putative OP-MG susceptibility genes and other previously reported genes of interest in MG and experimental autoimmune myasthenia gravis (EAMG). Results OP-MG myocytes compared to control MG myocytes showed altered expression of four OP-MG susceptibility genes (PPP6R2, CANX, FAM136A and FAM69A) as well as several MG and EAMG genes (p < 0.05). A correlation matrix of gene pair expression levels revealed that 15% of gene pairs were strongly correlated in OP-MG samples (r > 0.78, p < 0.01), but not in control MG samples. OP-MG susceptibility genes and MG-associated genes accounted for the top three significantly correlated gene pairs (r ≥ 0.98, p < 1 × 10− 6) reflecting crosstalk between OP-MG and myasthenia pathways, which was not evident in control MG cells. The genes with altered expression dynamics between the two subphenotypes included those with a known role in gangliosphingolipid biosynthesis, mitochondrial metabolism and the IGF1-signalling pathway. Conclusion Using a surrogate cell culture model our findings suggest that muscle gene expression and co-expression differ between OP-MG and control MG individuals. These findings implicate pathways not previously considered in extraocular muscle involvement in myasthenia gravis and will inform future studies.
- ItemOpen AccessRegulation of melanogenesis in conditionally immortalised mouse melanocytes expressing a temperature-sensitive SV40 large T antigen(1999) Prince, Sharon; Kidson, SueThe transformation of a normal melanocyte to a malignant melanoma involves a series of poorly understood genotypic and phenotypic alterations. In vitro models of melanoma formation generated by transforming mouse melanocytes with exogenous oncogenes have revealed that this process is frequently accompanied by a loss of pigmentation. The aim of this study was to establish, and to make use of unique cell lines to gain further insight into the mechanism(s) by which oncoproteins alter melanocyte differentiation. Primary cultures of mouse epidermal and dermal melanocytes were infected with a retrovirus carrying a temperature-sensitive mutant SV40 large T antigen. Six immortalised cell lines thus generated were analysed by northern and western blots and by enzymatic assays at the permissive temperature of the oncoprotein. Three epidermal and two dermal melanocyte clones remained pigmented and expressed tyrosinase, TRP-1 and -2 genes and the proteins encoded by them. In addition they expressed the mi gene and the c-kit receptor. In contrast, one dermal melanocyte clone (DMEL-3) gradually depigmented: this was accompanied by enhanced growth and down-regulation of melanocyte-specific gene expression. At the non-permissive temperature of the oncoprotein, proliferation ceased and DMEL-3 cells repigmented with a time-dependent increase in melanocyte-specific gene expression. Moreover, mi gene expression was down-regulated in the DMEL-3 cell line at the permissive temperature and was re-expressed at the non-permissive temperature. These results provided direct evidence for the role of the SV40 large T antigen in melanocyte dedifferentiation and emphasized the pivotal role of Mi in this process. Northern blot analysis of DMEL-3 cells cultured at the permissive and non-permissive temperatures revealed that there were no detectable levels of Pax3 transcripts at either temperature. In addition, Pax3 expression was absent in the highly pigmented DMEL-2 and melan-a cell lines. These results suggest that Pax3 is not required for mi expression and that it is unlikely to be a target of the T antigen-mediated repression of mi. To explore the possibility that other melanocyte markers are also altered as a consequence of alterations in mi expression, the DMEL-3 cells were examined for changes in the α-MSH and c-kit receptors. Melanin synthesis and tyrosinase activity assays showed that alterations in mi expression did not correlate to responsiveness to α-MSH, suggesting that the MSH receptor gene is not regulated by Mi. Furthermore, northern blot analysis showed that DMEL-3 cells did not express c-kit at either the permissive or nonpermissive temperature, suggesting that Mi does not regulate c-kit expression. To address the possible role of RB family members in melanocyte differentiation, it was investigated whether melanocyte differentiation is accompanied by an increase in their mRNAs and protein levels. Northern blot analysis strongly suggested that expression of the RB1, p130 and p107 is not altered when DMEL-3 cells were induced to differentiate at the non-permissive temperature. The results from western blot anaysis were inconclusive and require further investigations. Finally, the pigmented cell lines established in the present study provided a unique opportunity to investigate the stimulatory effect of TPA on melanogenesis because growth curves showed that the cells become TPA-independent. The results showed that stimulation of melanogenesis by TPA in a pigmented melanocyte line, DMEL-2, resulted in an increase in tyrosinase, TRP-1 and TRP-2 proteins and mRNAs. Additionally, TPA increased mi gene expression which suggests that Mi is necessary for the TPA-triggered signalling cascade that induces expression of the tyrosinase gene family. These results disclose, for the first time, a mechanistic link between TPA and the transcriptional induction of pigmentation.
- ItemOpen AccessThe regulation of the COL5A1 gene via the 3' - UTR and its impact on Achilles Tendinopathy and other exercise-related phenotypes Yoonus Abrahams.(2013) Abrahams, Yoonus; Collins, Malcolm; Prince, SharonIncludes abstract. Includes bibliographical references.
- ItemOpen AccessRegulation of the cyclin-dependent kinase inhibitor p21 by TBX3(2013) Hare, Shannagh; Prince, SharonTBX3, a member of the T-box family of transcription factors, is essential in development and has emerged as an important player in the oncogenic process. It is vital for the development of the heart, breasts, limbs, teeth and genitalia and TBX3 mutations lead to Ulnar-Mammary Syndrome, which is characterised by malformations of these organs and body structures. TBX3 is overexpressed in several cancers and has been implicated in a number of oncogenic processes ranging from the bypass of senescence to the inhibition of apoptosis and more recently TBX3 has been shown to contribute directly to tumour formation, migration and invasion of melanoma and breast cancer cells. However, little is known about the molecular basis for its role in development and oncogenesis because there is a paucity of information regarding its target genes. Of interest to the current study, preliminary evidence suggest that TBX3 may repress the cyclin-dependent kinase inhibitor p21WAF1, which has a role in a myriad of processes including cell cycle arrest, senescence, apoptosis, differentiation and DNA replication and repair. The current study therefore aimed to explore this possibility and to reveal a mechanism by which TBX3 regulates p21WAF1. Results show, using luciferase reporter gene assays, that TBX3 is a potent transcriptional repressor of the p21WAF1 promoter and that this repression is due to a direct interaction between TBX3 and the p21WAF1 promoter via the TBX3 DNA-binding domain. This study also provides evidence that the ability of TBX3 to repress p21WAF1 may require the TBX3 N-terminal repression domain. In addition, the site through which TBX3 acts to repress p21WAF1 was identified close to the initiator of the p21WAF1 promoter and TBX3 is shown to bind this site in vitro and in vivo. Furthermore, this study provides evidence that a serine proline motif (S190) located within the DNA-binding domain of TBX3, may play an important role in regulating the ability of TBX3 to transcriptionally repress p21WAF1 by inhibiting binding. Lastly, the repression of p21WAF1 by TBX3 is also shown to be biologically significant in TBX3 overexpression and knock down cell culture models. Results from this study provide a detailed mechanism of how TBX3 directly transcriptionally represses p21WAF1 which adds to our understanding of how TBX3 may contribute to oncogenesis.
- ItemOpen AccessThe role of T-box transcription factor TBX3 in rhabdomyosarcoma(2016) Sims, Danica Anne; Prince, Sharon; Peres, JadeCancer remains one of the leading causes of death worldwide due to late diagnosis and ineffective treatment options. To address this problem requires the elucidation of the molecular mechanisms, including the signaling pathways and transcription factors that drive cancer initiation and progression. In this regard, our laboratory has been particularly interested in the embryonically important T - box family of transcription factors which has been heavily implicated in promoting initiation and progression of a long list of cancers. For example, the overexpression of the T - box factor TBX3, has been reported to function in promoting immortalization, migration, invasion and tumour formation in a number of epithelial - derived malignancies. Furthermore, our laboratory recently reported that TB X3 is also overexpressed in a wide range of sarcoma subtypes including rhabdomyosarcomas. This suggests that TBX3 may also contribute to the development and/or progression of sarcomas and potentially may serve as a biomarker for their diagnosis and targete d therapy. This is exciting because sarcomas are diverse and heterogeneous cancers with varying clinical behaviours, high rates of metastasis and recurrence and are notoriously resistant to current chemotherapies. However, whether TBX3 is a molecular drive r of these mesenchymal - derived cancers remains to be determined. This project therefore aimed to elucidate the role of TBX3 overexpression in embryonal rhabdomyosarcomas (ERMS) which is the most common soft tissue sarcoma in children and adolescents. To this end, ERMS cell culture models were established in which TBX3 was either stably knocked down or stably overexpressed and the resulting cells were tested for several features of the cancer phenotype using in vitro and in vivo experiments. The results show that TBX3 promotes cell proliferation, anchorage independent growth and cell migration in vitro and tumour formation and invasion in vivo. This study also provides evidence that nucleolin binds to, and co - operates with, TBX3 to promote proliferation and migration of ERMS cells. Furthermore, data from initial experiments reveal that Hsc70 interacts with TBX3, to possibly increase its protein stability, and that oncogenic c - Myc and AKT 1 positively regulat e TBX3 levels in ERMS. This, albeit preliminary data, suggest that Hsc70, c - Myc and AKT1 are responsible, in part, for the overexpression of TBX3 in ERMS. Together findings from this study implicate TBX3 as an oncogene in ERMS and suggest that TBX3, nucleolin, Hsc70, c - Myc and AKT may be used in combination as biomarkers for the diagnosis and targeted therapy of ERMS.
- ItemOpen AccessThe role of the developmentally important transcription factor, TBX2, in the cell cycle and cancer(2009) Davis, Emily; Prince, SharonT-box factors play crucial roles in embryogenesis and mutations in T-box factor genes have been implicated in multiple human disorders. In addition, an increasing body of evidence implicates the T-box family in cell cycle regulation and in cancer...The aim of this study was therefore to address this question by establishing TBX2 over-expression and knockdown cell culture models. The results show that TBX2 does indeed contribute directly to the oncogenic process and further reveals a novel mechanism by which it contributes to tumourigenesis.
- ItemRestrictedThe role of the transcription factor TBX2 in breast cancer and melanoma and its regulation by the UV-induced DNA damage pathway.(2013) Wansleben, Sabina Maria; Prince, SharonT-box genes constitute an ancient family of developmentally important transcription factors that have gained prominence in cancer biology. For example, endogenous TBX2 is overexpressed in a growing list of cancers and when ectopically overexpressed it can bypass senescence and the tetraploidy checkpoints that protect against cancer. When the current study was initiated it was unclear as to whether the endogenous overexpression of TBX2 was a cause or consequence of the oncogenic process and whether it may be a suitable anti-cancer drug target. The objectives of this study were therefore (1) to explore the role of increased TBX2 levels in melanomagenesis by assessing the effect of knocking it down in melanoma cell lines, (2) to determine the mechanism by which TBX2 is upregulated by the UV-induced DNA damage pathway in melanoma cells and (3) to verify whether TBX2 is a suitable drug target by assessing the cellular sensitivity of control and TBX2-depleted cells in response to the chemotherapeutic drug cisplatin.