No description available.

A number of amino acid deprivations have been tested in our laboratory, for the selective eradication of tumour cells in vitro. Withdrawal of the essential amino acid, L-arginine, produced the greatest differential effect on cell proliferation. Normal cells ceased to proliferate and remained viable in G, of the cell cycle, while tumour cells attempted to proliferate in conditions unfavourable for growth, resulting in rapid cell death. Of the six tumour cell lines studied here, four displayed the latter response, while the other two responded in an apparently similar manner to normal cells. Most tumour cells cannot arrest in G1 and are therefore defective in G1 cell cycle control (particularly at the R-point). Analysis of a normal cell line, and the two tumour cell lines that survived arginine deprivation, revealed that cdk4 was downregulated, and the cells were found to possess functional p53. The other four tumour cell lines had dysfunctional p53 and did not downregulate cdk4 upon arginine withdrawal, or relied upon cdk6 for pRb phosphorylation. Arginine is required for histone synthesis during S phase. Histone synthesis in the absence of arginine was compared in a normal and a tumour cell line. Normal fibroblasts synthesise histones to support previously initiated DNA synthesis for the first 24 h of arginine deprivation until the cells reach the R-point. However, HeLa cells cannot synthesise adequate amounts of histone proteins, despite continued DNA synthesis and this is to their detriment. A novel cancer therapy has been developed which exploits the differential response of normal and tumour cells to arginine deprivation. Extracorporeal dialysis was used to reduce blood arginine levels in normal and tumour-bearing dogs. Arginine was successfully reduced to <10 M within the first 12 h of dialysis and this low level was maintained for up to 5 days, but arginine was not reduced for a long enough period of time to see significant tumour regression.

Unnatural amino acids (UAAs) have been used in bacteria and yeast to pinpoint protein binding sites, identify binding partners, PEGylate proteins site-specifically (vs. randomly), and attach small molecule fluorophores to proteins. The process of UAA incorporation involves the manipulation of the genetic code, which is established by the proper function of aminoacyl tRNA synthetases (RSs) and their cognate transfer RNAs (tRNAs). It has been discovered that certain regions of RS proteins can either block or enable cross-species reactivity of RSs. In essence, a bacterial RS can function with a human tRNA by transferring the human CP1 region to the bacterial RS, and vice versa. This knowledge has been used to engineer a tRNA capable of recognizing a stop codon (tRNA*), rather than an amino acid codon, and a cognate RS capable of recognizing only tRNA* and no endogenous tRNAs. We have previously described the use of this methodology to engineer a UAA incorporation system capable of amber stop codon suppression in HEK293T cells. Since UAAs are so useful, and their use has now been enabled in mammalian systems, we applied UAA incorporation to pluripotent cells. Stem and pluripotent cells have been the focus of cutting edge research for years, but much of the work done on these cell lines is done in the ignorance of basic biological processes underlying differentiation, dedifferentiation, and tumorigenesis. In order to facilitate the study of these basic biological processes and enable more adept manipulation of differentiation, dedifferentiation, and tumorigenesis, the development and use of two separate UAA incorporation systems is described herein. The overarching goal of this project is to facilitate the study of protein-protein interactions in stem and pluripotent cells. Since we have also previously described the development of a mammalian two-hybrid system, the use of that system in pluripotent cells is also described. / text

Since the introduction of Auranofin in 1985 there has been no new clinically approved gold containing drugs introduced. Although promising results were achieved with a gold(I) phosphine complex [Au(dppe) 2]Cl (Hoke et al., 1991; Mckeage et al., 2002), this compound was never entered into clinical trials due to its toxicity to normal tissue such as the liver and heart (Smith et al.,1989). Six novel derivatives of [Au(dppe) 2]Cl were developed and synthesized to identify possible new candidates with improved tumour specificity compared to [Au(dppe) 2]Cl and cisplatin. Human cervical carcinoma cells (HeLa) were used for an initial toxicity screening. IC50’s obtained for [Au(dppe) 2]Cl and cisplatin were 0.661 and 0.710 µM respectively. Three mixed novel derivatives (MM4, MM5 and MM6) displayed IC50’s ranging between 0.026 and 0.103 µM. These compounds were then selected to be tested further for selectivity and cytotoxicity on various malignant and normal cell lines. MM4 showed selectivity for ovarian, prostate, cervical and breast cancer cells, while MM5 was the most effective against ovarian, colon, prostate, cervical and breast cancer cells. MM6 was most active against ovarian, colon, prostate, cervical and breast cancer cells. The experimental compounds had much higher IC50’s when tested on the normal cells, which indicates selectivity for cancer cells. The octanol/water partition coefficient (lipophilicity) of all the experimental compounds was measured to determine the lipophilicity of the compounds. [Au(dppe) 2]Cl was found to be strongly lipophilic; while the novel compounds had varying degrees of lipo- and hydrophilicity. The octanol/water partition coefficient (lipophilicity) was also used to establish whether there is a correlation between the lipophilicity, IC50 and tumour specificity. In this study no correlation was found between these parameters. [Au(dppe) 2]Cl is known to have an effect on the mitochondrial membrane potential of cells. MM4, MM5, MM6 and cisplatin were compared to [Au(dppe) 2]Cl for effects on mitochondrial membrane potential. PHA stimulated human lymphocytes and a human undifferentiated leukemia T-cell line (Jurkat cells) were used in these experiments. [Au(dppe) 2]Cl, MM4, MM5 and MM6 depolarized the mitochondrial membranes of PHA stimulated lymphocytes significantly, while only [Au(dppe) 2]Cl depolarized the mitochondrial membranes of the Jurkat cells significantly, indicating that a different mechanism of action might be operational. MM4, MM5, MM6 and cisplatin were compared to [Au(dppe) 2]Cl for effects on plasma membrane potential. PHA stimulated human lymphocytes and Jurkat cells were used in these experiments. [Au(dppe) 2]Cl and MM6 depolarized the plasma membranes of both PHA stimulated lymphocytes and the Jurkat cells significantly. In order to determine whether the depolarization of mitochondrial and plasma membranes was a precursor for apoptosis, experiments were done to determine whether MM4, MM5 and MM6 induce apoptosis in Jurkat cells. [Au(dppe) 2]Cl and cisplatin were added for comparison. [Au(dppe) 2]Cl, cisplatin, MM4 and MM6 did induce apoptosis in Jurkat cells, but MM5 did not. The effect of [Au(dppe) 2]Cl, cisplatin, MM4, MM5 and MM6 on the cell cycle of Jurkat cells was determined to establish whether the experimental compounds altered this process. [Au(dppe) 2]Cl, MM4, MM5 and MM6 arrested the cell cycle in the G1 phase and cisplatin did so in the S phase. In order to determine whether the inhibition of cell growth and partition coefficient of the experimental compounds is related to the uptake of the drug, radio labeled drug uptake experiments were carried out with 198Au labeled [Au(dppe) 2]Cl, MM5 and MM6. Two different types of ovarian cancer cells were used for these studies. One cell line was sensitive to cisplatin (A2780) and the other was resistant to cisplatin (A2780 cis). Results obtained from these experiments showed that the uptake of these experimental compounds was dependent on their octanol/water partition coefficient. However, the inhibition of cell growth did not correlate with the uptake of these compounds by the cells that were tested. To confirm the octanol/water partition coefficient and drug uptake results, 198Au labelled [Au(dppe) 2]Cl, MM5 and MM6 were testedin vivo for bio distribution in rats. [Au(dppe) 2]Cl (lipophilic) had higher bio distribution compared to MM5 and MM6 (hydrophilic). Conclusion The experimental compounds show low IC50’s combined with increased tumour specificity. This indicates that these compounds have great potential to target tumour cells selectively and should be investigated further as anti-cancer agents. / Dissertation (MSc)--University of Pretoria, 2008. / Pharmacology / unrestricted

Breast cancer is the most common non-skin cancer in women and the second most common cause of cancer-related death in U.S. women. Despite numerous advances in treatment strategies against breast cancer, the presence of undetected distant metastasis of the primary tumor remains the main cause of mortality. Current screening and detection methods such as mammograms are simply not sensitive enough to detect formation of metastasis. Further, currently available therapies against metastatic breast cancer do not provide a complete cure for the disease. Thus, understanding the biology and molecular factors involved in cancer metastasis will help aid in preventing the onset of metastasis and discovering an effective treatment for this deadly disease. My research focused on understanding the mechanism of action of one such factor, breast cancer metastasis suppressor 1 (BRMS1), a suppressor gene found deleted in late stage breast cancers. The goal of my dissertation was to investigate the role of membrane signaling lipids phosphoinositides, specifically phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) in BRMS1-mediated metastasis suppression in MDA-MB-435 and MDA-MB-231 human breast carcinoma cells. My studies revealed BRMS1 selectively reduced receptor tyrosine kinases (RTK) and Gprotein coupled receptors (GPCR) expression and downstream signaling in human breast carcinoma cells. My observations are critical as many of these receptors are upregulated in metastatic breast cancer and PI(4,5)P2 is a critical constituent for mediating their downstream signaling events. Further, using immunoblotting studies, I uncovered a possible compensatory mechanism in tumor cells to overcome downregulation of PI(4,5)P2 by BRMS1 and maintain its downstream signaling. When studied for BRMS1 regulation of enzymes involved in PI(4,5)P2 synthesis, I showed BRMS1 completely inhibits phosphatidylinositol 4-phosphate 5-kinase β (PIP5Kβ) expression. Using overexpression studies, I showed PIP5Kβ to be the major contributor to the cellular PI(4,5)P2 pool required for agonist-induced intracellular calcium rise. Taken together, my dissertation research has identified some critical breast cancer markers and revealed signaling pathways altered by BRMS1 in human breast carcinoma cells that can be studied as potential therapeutic targets against breast cancer metastasis.

Thesis (D.Sc.(Odontology))--University of Pretoria, 2003. / "Published work submitted to the University of Pretoria for the degree of Doctor of Science in Odontology (Oral pathology)". Includes bibliographical references. Also available online.

Philosophiae Doctor - PhD / Globally, breast cancer is the most common cancer affecting women and it is predicted that in 2030 about 12 million deaths will occur with approximately 21.7 million new cases [2]. Genetic risk factors as well as race and ethnicity, account for about 5-10% of all breast cancer occurrences. Triple negative breast cancer (TNBC), tumors that tested negative for oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), contribute to 10-20% of all breast carcinomas [3,4] and is known to be a more aggressive type of cancer with varying degree of response to chemotherapeutic and radiation therapy [5,6] / 2022-02-24

Os microRNAs (miRs) desempenham um papel importante na biologia das células-tronco por meio da interação com seus mRNAs alvos, induzindo inibição da tradução e/ou degradação destes transcritos. Durante a diferenciação de células pluripotentes, os miRs podem ser induzidos ou reprimidos, no entanto, suas funções específicas são amplamente inexploradas. Nós investigamos os papéis funcionais de um conjunto selecionado de miRs na pluripotência e diferenciação celular, usando microscopia de fluorescência quantitativa (High Content Analysis). Para isso, foram empregadas a NTera-2 (células de carcinoma embrionário humano, CCE) e a H1 (células-tronco embrionárias humanas, CTEh) como modelos. Essas células foram transfectadas reversamente com trinta moléculas de miRs distintas (individualmente) ou moléculas controles. Após 3-4 dias de cultura, as células foram fixadas, permeabilizadas e coradas com Hoechst / CellMask Blue (núcleo/citoplasma), anti-OCT4, anti-Ciclina B1 e imageadas com um sistema ImageXpress Micro HCA. O CellProfiler foi utilizado para quantificar vários parâmetros morfométricos e medidas de intensidade de OCT4 e Ciclina B1 em compartimentos nucleares e citoplasmáticos. Esses dados foram usados para gerar perfis fenotípicos multiparamétricos específicos de cada miR (usando KNIME) e o agrupamento desses dados levou à identificação de vias e processos envolvidos na indução de características de pluripotência ou diferenciação celular causadas por miRs com efeitos fenotípicos similares. Como exemplo, as vias de PI3K-AKT, WNT, TGF? e DICER foram encontradas como moduladas por alguns clusters fenotípicos e os transcritos de alguns alvos foram avaliados por qPCR para validar os achados. Parte do trabalho foi focada na regulação da via Notch por miRNAs em células pluripotentes, o que levou à observação de que o miR- 363-3p inibe a sinalização de Notch e promove pluripotência nessas células. A transfecção de miR-363-3p não apenas elevou as características de pluripotência em NTera-2 e H1, mas também protegeu as CCE da diferenciação induzida por cocultivo com OP9 expressando DLL1 e causou a diminuição no nível de transcritos de PSEN1. Em conclusão, o ensaio desenvolvido aqui provou ser uma ferramenta robusta na detecção de mecanismos moleculares, baseando-se na combinação de análises fenotípicas funcionais e bioinformáticas. / microRNAs (miRs) play an important role in stem cell\'s biology by binding to target mRNAs transcripts, inducing translation blockage and/or transcripts degradation. Upon differentiation of pluripotent cells, miRNAs can be induced or repressed, however, their specific roles are largely unexplored. We investigated the functional roles of a selected set of miRs in pluripotency and differentiation, using quantitative automated fluorescence microscopy (High Content Analysis). For this, we used NTera-2 (human embryonal carcinoma cells, ECC) and H1 (embryonic stem cells; ESC) as models. These cells were reverse-transfected with thirty distinct miRs mimics (individually) or control molecules. Following 3-4 days of culture, cells were fixed, permeabilized and stained with Hoechst/CellMask Blue (nucleus/cytoplasm), antiOCT4, anti-Cyclin B1 and imaged using an ImageXpress Micro HCA System. CellProfiler was used to quantify several morphometric parameters and intensity measurements of OCT4 and CYCB1 in nuclear and cytoplasmic compartments. Quantified parameters were used to generate miR-specific multiparametric phenotypic profiles (using KNIME) and clustering these data led to identification of pathways and processes involved in the induction of pluripotency or cell diferention features caused by miRs with similar phenotypic effects. As an example, PI3K-AKT, WNT, TGF? and DICER pathways were found to be regulated by some phenotypic clusters and transcripts level of some of miR targets were evaluated by qPCR to validate de findings. Part of the work was focused in the regulation of Notch pathway by miRNAs in pluripotent cells, which led the observation that miR-363-3p inhibits Notch signaling and promotes pluripotency feature, as the transfection with miR-363-3p mimic not only enhanced pluripotent phenotype in NTera-2 and H1, but also protected de ECCs from differentiation induced by coculture with OP9 expressing DLL1 and decreased PSEN1 transcripts level.In conclusion, The assay developed here proved to be a robust tool in the detection of molecular mechanisms based on combined functional phenotypic and bioinformatic analyzes.

No description available.

Os microRNAs (miRs) desempenham um papel importante na biologia das células-tronco por meio da interação com seus mRNAs alvos, induzindo inibição da tradução e/ou degradação destes transcritos. Durante a diferenciação de células pluripotentes, os miRs podem ser induzidos ou reprimidos, no entanto, suas funções específicas são amplamente inexploradas. Nós investigamos os papéis funcionais de um conjunto selecionado de miRs na pluripotência e diferenciação celular, usando microscopia de fluorescência quantitativa (High Content Analysis). Para isso, foram empregadas a NTera-2 (células de carcinoma embrionário humano, CCE) e a H1 (células-tronco embrionárias humanas, CTEh) como modelos. Essas células foram transfectadas reversamente com trinta moléculas de miRs distintas (individualmente) ou moléculas controles. Após 3-4 dias de cultura, as células foram fixadas, permeabilizadas e coradas com Hoechst / CellMask Blue (núcleo/citoplasma), anti-OCT4, anti-Ciclina B1 e imageadas com um sistema ImageXpress Micro HCA. O CellProfiler foi utilizado para quantificar vários parâmetros morfométricos e medidas de intensidade de OCT4 e Ciclina B1 em compartimentos nucleares e citoplasmáticos. Esses dados foram usados para gerar perfis fenotípicos multiparamétricos específicos de cada miR (usando KNIME) e o agrupamento desses dados levou à identificação de vias e processos envolvidos na indução de características de pluripotência ou diferenciação celular causadas por miRs com efeitos fenotípicos similares. Como exemplo, as vias de PI3K-AKT, WNT, TGF? e DICER foram encontradas como moduladas por alguns clusters fenotípicos e os transcritos de alguns alvos foram avaliados por qPCR para validar os achados. Parte do trabalho foi focada na regulação da via Notch por miRNAs em células pluripotentes, o que levou à observação de que o miR- 363-3p inibe a sinalização de Notch e promove pluripotência nessas células. A transfecção de miR-363-3p não apenas elevou as características de pluripotência em NTera-2 e H1, mas também protegeu as CCE da diferenciação induzida por cocultivo com OP9 expressando DLL1 e causou a diminuição no nível de transcritos de PSEN1. Em conclusão, o ensaio desenvolvido aqui provou ser uma ferramenta robusta na detecção de mecanismos moleculares, baseando-se na combinação de análises fenotípicas funcionais e bioinformáticas. / microRNAs (miRs) play an important role in stem cell\'s biology by binding to target mRNAs transcripts, inducing translation blockage and/or transcripts degradation. Upon differentiation of pluripotent cells, miRNAs can be induced or repressed, however, their specific roles are largely unexplored. We investigated the functional roles of a selected set of miRs in pluripotency and differentiation, using quantitative automated fluorescence microscopy (High Content Analysis). For this, we used NTera-2 (human embryonal carcinoma cells, ECC) and H1 (embryonic stem cells; ESC) as models. These cells were reverse-transfected with thirty distinct miRs mimics (individually) or control molecules. Following 3-4 days of culture, cells were fixed, permeabilized and stained with Hoechst/CellMask Blue (nucleus/cytoplasm), antiOCT4, anti-Cyclin B1 and imaged using an ImageXpress Micro HCA System. CellProfiler was used to quantify several morphometric parameters and intensity measurements of OCT4 and CYCB1 in nuclear and cytoplasmic compartments. Quantified parameters were used to generate miR-specific multiparametric phenotypic profiles (using KNIME) and clustering these data led to identification of pathways and processes involved in the induction of pluripotency or cell diferention features caused by miRs with similar phenotypic effects. As an example, PI3K-AKT, WNT, TGF? and DICER pathways were found to be regulated by some phenotypic clusters and transcripts level of some of miR targets were evaluated by qPCR to validate de findings. Part of the work was focused in the regulation of Notch pathway by miRNAs in pluripotent cells, which led the observation that miR-363-3p inhibits Notch signaling and promotes pluripotency feature, as the transfection with miR-363-3p mimic not only enhanced pluripotent phenotype in NTera-2 and H1, but also protected de ECCs from differentiation induced by coculture with OP9 expressing DLL1 and decreased PSEN1 transcripts level.In conclusion, The assay developed here proved to be a robust tool in the detection of molecular mechanisms based on combined functional phenotypic and bioinformatic analyzes.