Browsing by Author "Parker, Iqbal"

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    Open Access
    Characterisation of CIS- and trans-acting factors that regulate the human alpha 2(1) procollagen gene
    (1999) Masemola, Agatha Maripanyane; Parker, Iqbal
    The differential expression of the a2(I) procollagen gene in normal and transformed human fibroblasts has been correlated with differential in vitro DNA-protein interactions on the basal promoter region between -100 and -67. A 23 bp region of the a.2(1) procollagen promoter encompassing the G/CBE (CCTCCATTGG) and the Ctv'IE (GGAGGCCCTTTT) has previously been shown to engage in specific DNA protein interactions that determined the transcriptional activity of the promoter. The CME forms two distinct DNA-protein complexes that might be crucial in the regulation of the a2(I) procollagen gene in a cell specific manner. The hypothesis was, therefore, that depending on the protein that participates in complex formation with the CME, the gene would be activated or repressed. The objective of this study was to investigate the role of this 23 bp region in the regulation of expression. of the a2(I) procollagen gene in transformed fibroblasts. In addition, the study sought to establish the role of the proto-oncogene c-fos-in the regulation of expression of the a2(I) procollagen gene. In contrast to previous observations, this study demonstrated that only one DNA protein complex is formed on the CME and the second complex is a specific proteolytic cleavage of the product of the larger complex. Preparation of nuclear extracts in the absence of protease inhibitors, specifically leupeptin, resulted in the formation of a smaller complex, previously shown to bind the CME. The importance of this proteolytic fragment that still retains DNA binding activity is yet to be determined. In addition, the CME binding proteins were fairly ubiquitously expressed in both a.2(1) collagen producing and non-producing cells. CT-1 fibroblasts (transformed by y-irradiation) synthesise over 80% of total a2(I) collagen produced by its untransformed counterpart (WI-38 fibroblasts), whereas the gene, is down regulated in the human embryonic lung fibroblasts transformed with SV40 (SVWI-38 fibroblasts). These cell lines are therefore ideal for studying regulation of a.2(I) procollagen gene. To analyse the importance of the G/CBE and CME regions of the a.2(1) procollagen gene promoter, point mutations were introduced by site-directed mutagenesis. Mutated promoter DNA was cloned into a p8CAT reporter vector, and the activity of the promoter determined in transient transfection experiments. Mutations introduced in the G/CBE region of the a.2(1) procollagen promoter resulted in a 3-12-fold decrease in the activity of the promoter. The decrease was observed with both proximal (-343 bp) and basal (-107 bp) promoter constructs~ a significant reduction in promoter activity was observed in both CT-1 and SVWI-38 fibroblasts. These results imply that the G/CBE region of the promoter is required for the activation of transcription of the a.2(1) procollagen gene and therefore the factor that interacts with the G/CBE functions as a transcriptional activator. Previously, this factor was shown to complex with antibodies raised against the mouse CCAAT binding factor (CBF), suggesting that the protein belongs to the CBF family of transcription factors. Furthermore, these results demonstrate that the adjacent, upstream inverted GGAGG sequence is crucial for activation of the gene through the CCAAT binding element. The inhibition of promoter activity in constructs with a mutated G/CBE element was correlated with lack of protein binding to the mutated sequence as confirmed by electrophoretic mobility shift assays. Transfection of a.2(1) procollagen promoter constructs containing mutations in the CME region, however, resulted in a significant increase in promoter activity in both CT-1 and SVWI-38 fibroblasts. A much higher increase, 3-fold, was observed for the SVWI-38 cell line compared to a 1.5-fold increase observed for CT-1 fibroblasts. These results suggested that the factor that interacts with the CME functions as a repressor of the a.2(1) procollagen gene. Interestingly, the promoter activity in SVWI38 fibroblasts transfected with mutated CME constructs was similar to that observed in CT-1 fibroblasts transfected with the wild type promoter construct. An interesting observation was that repression of the a.2(1) procollagen gene via the CME required upstream elements since transfection of the basal mutated promoter did not result in increased promoter activity. From these results, it can be concluded that the CME binding protein is involved in cell-specific repression of the a2(I) procollagen gene and that the mechanism of repression appears to be dependent on the presence of upstream elements. Mutations in the G/CBE and CME pointed out the significance of these elements in the expression of the a2(I) procollagen gene and since a number of studies have characterised the mouse CCAAT binding protein, this study focused on purification and identification of the CME binding protein(s). Purification was performed by conventional biochemical techniques using heparin-agarose and sequence-specific DNA affinity chromatography, as well as separation on SDS-polyacrylamide gels. Two cycles of DNA affinity chromatography yielded two polypeptides with apparent molecular weights of 50 and 67 kDa. Automated N-terminal sequencing of the polypeptides indicated that they were blocked and therefore no sequence could be obtained. In addition, these polypeptides failed to raise an immune response in mice and rabbits. Subsequently, polypeptides were digested with trypsin in situ in polyacrylamide gels and the eluted peptides were analysed by MAWITOF-mass spectrometry. The mass:charge ratios (mlz ratios) obtained were used to search the database using a mass tolerance of 1.5 Da and only one hit was obtained. The match obtained was that of a mouse zinc finger protein of which not much is known, except that it might be a transcription factor. This result supports previous observations of Collins et al (J Cell Biochem 1998, 70: 455-467) that complex formation requires the presence of zinc. The primary structure of the CME binding protein remains to be determined. Transformation of fibroblasts is normally accompanied by changes in the expression of extracellular proteins, including type I procoUagen. Although CT-I fibroblasts, show very little change in a2(I) procollagen gene expression, the c-f os gene is drastically down-regulated. This study sought to establish if there is any relationship between the unusually high levels of the a2(I) procollagen gene in this transformed cell line and failure of the cells to stimulate c-fos expression in response to serum. CT-1 :fibroblasts that overexpressed wild type Fos were established and changes in the expression of the a,2(1) procollagen gene were measured. Overexpression of Fos down-regulated the a,2(1) procollagen gene, which was not due to increased turnover of the a1(I) procollagen mRNA. Analysis of promoter activity showed that the promoter and first intron, which has been reported to contain negative regulatory elements, did not harbour any Fas-responsive elements. The -343 bp and the -2300 bp promoter constructs were transactivated in cells overexpressing Fos. Thus, although overexpression of Fos resulted in a significant decrease in the levels of the a2(1) procollagen WA, it does not involve the region between -2300 bp and +1800 bp of the a2(1) procollagen gene. Furthermore, there was no change in the stability of the message, indicating that constitutive expression of Fos did not activate a factor that could play a role in altered turnover of the a2(I) procollagen mRNA. It is therefore possible that constitutively high levels of Fos may trigger the expression of a number of other genes, which have a negative impact on the expression of the a2(I) procollagen gene.
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    The development of a radiolabelled macromolecule as a therapeutic agent for the treatment of cancer
    (2015) Driver, Cathryn Helena Stanford; Parker, Iqbal; Hunter, Roger; Zeevaart, Jan Rijn
    One of the major focus areas of anticancer therapy is the design of new radiotherapeutic agents that are able to specifically target and destroy cancer cells with minimal side effects and damage to healthy, normal cells. This thesis describes studies towards the synthesis of a macromolecular bioconjugate that was designed to: i) co-ordinate a radioisotope through a tetra-amine macrocycle (cyclam), ii) lead to passive tumour targeting via the EPR effect and a suitably large carrier such as human serum albumin and iii) induce active targeting through a glucose moiety recognised by the over-expressed glucose transporters on the surface of highly metabolically active cancer cells. The various cyclam functionalisation strategies explored were relatively unsuccessful, but eventually a bis-aminal cyclam was successfully converted, through nucleophilic substitution, into a precursor pro-conjugate: a di-functionalised cyclam containing a β-glycoside tether and a long chain primary alkylamine. The glycoside tether was synthesised via glycosylation of a glycosyl iodide with decandiol followed by oxidation of the terminal hydroxyl group to an acid chloride for cyclam acylation. The second linker attached to cyclam was synthesised by conversion of decanediol to a brominated alkyl amine. This amine would then be converted into a maleimide functionality suitable for Michael addition with a free thiol group contained within the proposed bio-carrier to form the desired bioconjugate. Further studies described towards the synthetic construction of the bioconjugate include: 1) The construction of a maleimide group 2) The attachment of an imaging radioisotope, ⠹⠹m Tc, or therapeutic isotope, ¹⠰³ Pd, to the pro- conjugate and other glucose-cyclam precursors 3) The determination of the potential uptake of the bioconjugate through glucose transporters by using a fluorescent dansyl-glucose compound as a model and monitoring its uptake into WHCO1 oesophageal cancer cells. 4) The HPLC analysis of the coupling of a glucose-maleimide model compound to bovine serum albumin to investigate the Michael addition of the free thiol in HSA to a maleimide 5) The development of a potentially alternative nanoparticle carrier by synthesis of palladium and magnetic nanoparticles with commercially available thioglucose or glucuronic acid moieties as the surface targeting and stabilising agent. In summary, this thesis outlines a number of synthetic, radiological and biological aspects towards the development of a fully functioning radiolabelled macromolecular bioconjugate that could be tested for improved targeted cancer radiotherapy.
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    Differentially expressed genes in oesophageal cancer
    (2002) Wamunyokoli, Fred Alexander Wafula; Parker, Iqbal; Hendricks, Denver T
    Oesophageal cancer (OC) is a leading cause of cancer death in the black population in South Africa. The lifetime risk of the disease amongst black males is 1 in 59. High incidence areas are the Transkei, Ciskei and KwaZulu-Natal where it is responsible for over 45 of all malignancies. Cancer develops through a multistep process of genomic instability of clonal evolution. Several oncogenes, tumour suppressor genes and transcription factors have been shown to be altered in oesophageal cancer. Their role however, in the development and/or susceptibility to the development of oesophageal cancer is poorly understood. The aim if this project is to identify candidate genes that are differentially expressed in oesophageal cancer patients with a view to understanding the development of oesophageal cancer. The ultimate objective of the project is to use this data to develop possible biomarkers for the disease.
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    Fluorescent ajoenes as a mechanistic probe for cancer
    (2010) Cotton, Jonathan; Hunter, Roger; Caira, Mino R; Parker, Iqbal
    This thesis describes the development and synthesis of several novel ajoene mimics. The first of which, (E/Z)-1,8-(Bis-p-methoxyphenyl)-2,3,7-trithia-octa-4-ene 7-oxide 17, was developed as a continuation of SAR studies performed by our group, involved the placing p-methoxyphenly groups on the termini of the ajoene pharmacophore.
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    Genetic polymorphisms in the drug metabolizing genes and their roles in the development of oesophageal cancer
    (2008) Li, Dong-Ping; Parker, Iqbal; Dandara, Collet
    Although the incidence and mortality due to the oesophageal squamous cell carcinoma (OSCC) in Black South Africans is extremely high, very little is known about the aetiology and molecular biology of the disease. In order to make a contribution to the understanding to the causes of this disease we investigated the role of the polymorphisms in the genes coding for the cytochrome P450 (CYP1A1, CYP1A2, CYP1B1, CYP2E1), sulphotransferase 1A1 (SULT1A1), glutathione S-transferases (GSTT1. GSTM1 and GSTP1) alcohol dehydrogenase (ADH2 and ADH3) and aldehyde dehydrogenase (ALDH2) because the products of these genes are involved in the metabolism or biotransformation of harmful compounds.
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    Human Papillomaviruses in oesophageal cancer
    (2003) Matsha, Tandi Edith; Parker, Iqbal
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    Investigating the Role of Cigarette Smoke and Alcohol in Oesophageal Squamous Cell Carcinoma
    (2022) Gamieldien, Raabie'ah; Hendricks, Denver; Parker, Iqbal
    The World Health Organisation (WHO) has estimated cancer to be one of the leading causes of death to people under the age of 70. Every year there are about 18.1 million new cancer cases worldwide; oesophageal cancer (OC) ranks seventh out of all incidence cases and sixth in mortality. OC has a poor 5-year overall survival rate of about a 15% (Arnal et al., 2015). This occurs as OC is largely asymptomatic and patients often seek medical assistance at a late stage of their cancer. This late diagnosis and a lack of efficient treatment has rendered OC a serious world health problem. Oesophageal Squamous Cell Carcinoma (OSCC) is the more common subtype of and accounts for about 90% of incident oesophageal cancers every year (Abnet et al., 2018) with the majority of OSCC cases occur in developing countries. The objective of this study was to investigate cigarette smoking and alcohol consumption as they have widely been reported as risk factors for OSCC. The project explored the impact of cigarette smoke condensate (CSC) treatment and EtOH on the expression of genes in cultured oesophageal cancer cells. Prior to treating the cells for expression analysis, cytotoxicity experiments were conducted to determine treatment conditions of CSC and EtOH which were sub-lethal, for the cell types and timeframes investigated. It was found that concentrations of 40 µg/ml CSC and 50 mM EtOH did not cause cell death for the time period of three days. Furthermore, we also showed that cell viability was maintained up to 10 days of treatment. RNA-sequencing then revealed a wide variety of genes that were differentially expressed in the OSCC cells treated with these selected concentrations of CSC and EtOH. One gene found to be differentially expressed in two RNA-Seq analyses was confirmed to be upregulated by RT-qPCR. Seven genes, AHRR, ALDH3A1, CYP1A1, CYP1B1, GSTM3, GSTM4 and UGT1 A6, involved in xenobiotic metabolism and a number of other metabolic pathways were also altered in response to CSC and EtOH treatment. However, these genes' involvement require further confirmation by RT-qPCR. This result of this study confirms that we have designed a reliable experimental system to investigate the role of EtOH and CSC in the development of OSCC. These results gave us a deeper insight into the genes and pathways affected by CSC and EtOH which may contribute to OSCC. Additionally, the cytotoxicity data can be use for future experimental work and the RNA-seq data can be used for further investigation into the development of OSCC.
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    An investigation into the anti-cancer mechanism of garlic-related organosulfur compounds
    (2014) Smith, Muneerah; Parker, Iqbal; Kaschula, Catherine Hart
    Crushed garlic contains organosulfur compounds (OSC), which are reported to have cancer chemotherapeutic properties both in vitro and in vivo. A library of 15 organosulfur analogues were obtained as mechanistic probes in WHCO1 oesophageal cancer cells. Structure-activity studies showed a positive correlation between the anti-proliferative-IC50 of disulfides and the relative stability of their anion leaving groups, as assessed through resonance and quantified by predictive pKa-values.
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    Matrix-mediated regulation of type 1 collagen synthesis and degradation in cultured fibroblasts
    (2009) Dzobo, Kevin; Parker, Iqbal
    Stromal cells and the extracellular matrix (ECM) components provide the microenvironment that is pivotal for cell growth, motility, attachment and differentiation. Fibroblasts are some of the cells responsible for the synthesis of most of the extracellular matrix proteins. Type I collagen is the most abundant extracellular matrix protein in the human body and is found in tissues requiring high tensile strength. In this study we investigated the effect of a pre-formed fibroblast-derived extracellular matrix on the expression of type I collagen and associated matrix metalloproteinases in fibroblasts.
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    Molecular mechanisms involved in the anticancer activity of BISPMB in oesophageal cancer cells
    (2016) Siyo, Vuyolwethu Penelope; Parker, Iqbal; Kaschula, Catherine Hart
    BisPMB (E, Z)-1,8-(Bis-p-methoxyphenyl)-2,3,7-trithiaocta-4-ene 7-oxide) is a synthetic analogue of the garlic compound ajoene. It is 12 times more active at inhibiting the growth of oesophageal squamous cell carcinoma WHCO1 cells and displays selectivity for cancer cells over normal cells. BisPMB is therefore attractive as a potential cancer therapeutic. In this study, bisPMB was found to inhibit WHCO1 cancer cell proliferation in a time and concentration dependent manner with 24 hour IC50's between 6.7 - 8.1 μM against a range of oesophageal cancer cell lines including WHCO1, KYSE30 and WHCO6. The normal oesophageal epithelial cell line, HET1A was found to be five times less responsive to bisPMB. Furthermore, bisPMB was found to induce apoptosis and G2/M cell cycle arrest in WHCO1 cells. Gene expression data obtained from the microarray analysis showed that bisPMB primarily targets the unfolded protein response (UPR) in WHCO1 cells. We also found that bisPMB deregulated the ER stress genes involved in protein processing in the endoplasmic reticulum and also deregulated MAPK pathways in WHCO1 cells. At a protein level, bisPMB was found to induce an increase in protein ubiquitination and in the expression of ER stress and UPR genes ATF4, Grp78 and CHOP in WHCO1 cells. We also observed a decrease in ATF6 90 kDa protein and transient XBP-1 mRNA splicing. The activation of p38, JNK and ERK MAPK pathways in bisPMB treated WHCO1 cells was also observed. Furthermore siRNA mediated knock-down of CHOP abolished the anti-proliferative effect of bisPMB in WHCO1 cells. However, inhibition of JNK and p38 MAPK by chemical inhibitors, SP600125 and SB 203580 respectively, had no effect on bisPMB antiproliferative activity against WHCO1 cells. On the other hand, inhibition of ERK1/2 MAPK by U0126 enhanced the anti-proliferative effect of bisPMB in WHCO1 cells. These results support the hypothesis that ER stress and MAPK signalling pathways are essential for bisPMB induced cytotoxicity in oesophageal cancer cells.
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    The regulation of type I collagen gene expression in stromal fibroblast by breast tumour cells
    (2011) Rose, Beverley Ann; Parker, Iqbal; Leaner, Virna
    Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix (ECM) components found in the stroma, including type I collagen. Previous in vivo studies in our laboratory have shown that type I collagen mRNA levels are decreased in stage II and III breast tumour tissue compared to adjacent normal tissue.
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    The role of viral sequences in genetic aberrations and malignant transformation
    (2014) Mwapagha, Lamech Malagho; Parker, Iqbal
    Cancer is a leading cause of death worldwide and viral infections such as HBV/HCV and HPV have been known to be responsible for up to 20% of cancers in low- and middle-income countries. Approximately 500,000 of these deaths are due to oesophageal squamous cell carcinoma (OSSC) alone, one of the major cancers in Eastern and Southern Africa, Latin America and Asia. Previous studies have shown HPV DNA to be integrated in nearly 40% of oesophageal tumours whereas it was present in only 3% of normal healthy asymptomatic individuals, implicating it as a possible risk factor. The aim of this study was to compare the roles and effects of the E6 gene from the low risk HPV11 and high risk HPV18 on the cellular gene expression profile in order to identify genes required for the initiation of cellular transformation and also to identify genomic alterations associated with oesophageal squamous cell carcinoma. Cancer is a leading cause of death worldwide and viral infections such as HBV/HCV and HPV have been known to be responsible for up to 20% of cancers in low- and middle-income countries. Approximately 500,000 of these deaths are due to oesophageal squamous cell carcinoma (OSSC) alone, one of the major cancers in Eastern and Southern Africa, Latin America and Asia. Previous studies have shown HPV DNA to be integrated in nearly 40% of oesophageal tumours whereas it was present in only 3% of normal healthy asymptomatic individuals, implicating it as a possible risk factor. The aim of this study was to compare the roles and effects of the E6 gene from the low risk HPV11 and high risk HPV18 on the cellular gene expression profile in order to identify genes required for the initiation of cellular transformation and also to identify genomic alterations associated with oesophageal squamous cell carcinoma.
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    Somatic expansion of premutation alleles and the role of the mismatch repair and base excision repair proteins on repeat expansion in a mouse model of the fragile X-related disorders
    (2016) Lokanga, Rachel Adihe; Parker, Iqbal; ; Usdin, Karen
    The Fragile X-related disorders arise from an unusual mutation in the X-linked FMR1 gene. The mutation involves expansion, or an increase in the number of repeats, in a CGG•CCG repeat tract located in its 5' untranslated region. FMR1 alleles carrying 55-200 repeats are called Premutation (PM) alleles, and cause Fragile X associated tremor/ataxia syndrome (FXTAS) and Fragile X-associated primary ovarian insufficiency (FXPOI). FMR1 alleles having more than 200 repeats are referred to as full mutation (FM) alleles and cause Fragile X syndrome (FXS). These different alleles arise by intergenerational expansion of the repeat tract from smaller unstable alleles by a mechanism that is unknown. We have shown that in addition to germ line expansion, somatic expansion also occurs in a human cell line in vivo and in a FX PM mouse model. In the mouse model, we found that the extent of somatic instability is dependent on age, gender and tissue. Specifically, organs such as brain, liver and gonads are susceptible to expand more than heart and kidney and expansion is much more frequent in males than in females. No differences were found between male and female mice in the levels of the DNA repair proteins that had already been implicated in repeat expansion in model systems of other disorders thought to arise via a similar mechanism. Neither were there any differences between males and females in the amounts of proteins produced from X-linked DNA repair genes. We also showed that estrogen did not protect against expansion. However, we found that PM alleles expanded exclusively when they were located on the active X chromosome. Thus some of the differences between males and xii females in the level of somatic expansion might be due to the fact that females undergo X inactivation and thus have the PM allele on the inactive X chromosome in half (~50%) of their cells. It also indicates that transcription and/or an open chromatin configuration is required for expansion in the FX PM mouse.