Graduation date: 1996

Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumour which secretes ACTH and other related peptides. Contrary to normal production by the pituitary, ACTH production is not inhibited by glucocorticoids (Gcs) in SCLC. This insensitivity to Gc action can be attributed to impaired Gc receptor (GR) expression in these cells. Over-expression of the GR induces apoptosis both in vitro and in vivo. Evasion of GR signalling thus confers a significant survival advantage to SCLC cells. Re-expression of endogenous GR in SCLC cells may provoke the same effect. Many tumours silence the expression of tumour suppresser genes by epigenetic mechanisms. Recent evidence suggests that the GR in SCLC cells is epigenetically silenced by hypermethylation of its promoter. The overall aim of this study was to determine whether endogenous GR re-expression induces apoptosis of SCLC cells. The DMS 79 SCLC cell line, and the control HEK and non-SCLC A549 cell lines were treated with the DNA methyltransferase inhibitor (DNMTi), 5-aza-2′-deoxycytidine (5-aza), to determine whether treatment with 5-aza results in re-expression of endogenous GR. Conflicting results were thought to result from the use of possibly degraded 5-aza. However, a quantitative real-time PCR analysis using newly purchased, freshly prepared 5-aza indicated that 5-aza treatment up-regulated GR mRNA expression in the DMS 79 cells (p<0.0005). No significant changes in GR expression were seen in the HEK and/or A549 cells, suggesting that the GR in these cell lines is not methylated. Contrary to expectations and possibly due to the use of degraded stock, Western blot analysis revealed that 5-aza had no effect on GR protein expression in DMS 79 cells, yet affected GR protein expression in HEK and A549 cells (p=0.003 and p=0.042, respectively). Cell viability assays indicated that treatment with varying concentrations of 5-aza had no effect on the viability of DMS 79 and A549 cells, but had a minimal effect on HEK cell (p<0.0005) viability. These data reinforce the hypothesis that stock 5-aza had degraded as 5-aza is known to exert cytotoxic effects at higher concentrations. Using newly purchased, freshly prepared 5-aza, flow cytometry and/or microscopy were performed to establish whether endogenous GR re-expression was sufficient to kill the SCLC cells by apoptosis. FITC Annexin V staining and nuclear morphology showed that significant proportions of the 1 μM (p=0.010 and p=0.027) and 5 μM (p=0.002 and p=0.018) 5-aza treated DMS 79 cells were apoptosing, with little apoptosis seen in HEK cells. 5-Aza induced negligible HEK cell death, as determined by microscopic analyses. The effect of dexamethasone (Dex; a synthetic Gc) on HEK and DMS 79 cells was examined to determine whether Gc treatment could enhance apoptosis. Treatment with Dex alone, and in combination with 5-aza, resulted in significant HEK cell death (p=0.046 and p=0.005 respectively), but not apoptosis. This was unexpected as HEK cells express very little unmethylated GR, and may be due to excessive drug exposure or combined drug toxicity. The same effect was observed with DMS 79 cells (p=0.003 and p<0.0005 respectively), with 5-aza appearing to enhance cell death induced by Dex. No effects on apoptosis were seen confirming earlier reports that GR-mediated apoptosis is ligand-independent. As 5-aza does not selectively demethylate the GR, cells were exposed to the GR antagonist, RU486, to establish whether apoptosis associated with 5-aza treatment is specifically due to demethylation and subsequent expression of the GR. Treatment with RU486 in conjunction with 5-aza induced cell death (p=0.014), but not apoptosis, of HEK cells. Again, this may have been due to excessive drug exposure or combined drug toxicity. Flow cytometric data showed that DMS 79 cell death was induced by both RU486 (p=0.004), and RU486 in combination with 5-aza (p=0.003). Furthermore, although not significant, RU486 treatment appeared to inhibit apoptosis induced by 5-aza in the DMS 79 cells. The data suggest that re-expression of the GR may be responsible for apoptotic induction. Our findings, although not significant, hint that endogenous re-expression of the GR leads to apoptosis. Unlike mutations, epigenetic marks are reversible and clinical trials with DNMTis have shown promising results. The identification of a novel endogenous mechanism that specifically induces apoptosis of SCLC cells offers great promise for the development of targeted therapeutics for the treatment of this deadly disease. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

The effect of feeding colostrum or milk to newborn calves on serum cortisol, thyroxine and immunoglobulin G was investigated. Twenty-four calves (12 males and 12 females) were obtained immediately postpartum and randomly assigned to one of two rations after being blocked by breed and sex. Both rations were force-fed at birth, 12, 24 and 36 h postpartum. Blood sampling was performed at 0 time, 1, 2, 3, 4, 6 and 12 h postfeeding with this regime followed for a 48 h period (4 feedings). The average serum cortisol concentration was highest at birth, 221.9 and 245.6 ng/ml for colostrum and milk-fed calves, respectively. Cortisol levels between treatments were different (P<.05) at 2, 3, 12, 14, 18, 24, 37 and 48 h postpartum. The sex of the calf did not affect the mean cortisol concentrations. No treatment difference was observed for serum thyroxine. A sex difference was observed with the female calves exhibiting higher average thyroxine concentrations over the entire trial. A reduction in thyroxine concentration occurred with time (P<.001) as mean concentrations peaked at 4 h postpartum (22.1 μg/dl) and declined to 10.6 μg/dl by 48 h postpartum. Both treatment groups were born with similar serum immunoglobulin G levels (~0.7 mg/ml). However, at approximately 4 h postpartum, the colostrum-fed calves acquired an increase (P<.001) in serum immunoglobulin G, peaking at 24 h postpartum (26.83 mg/ml) and remaining much higher throughout the entire trial. There was a treatment difference (P<.001) between the two groups following the 4 h sample. Muscle samples (20-30g) were surgically removed from the right semitendinosus at 36 h postpartum from 14 neonatal beef calves (male and female), homogenized, and centrifuged at 105,000 x g at 4 C for 60 min. The supernatant (cytosol) was harvested and receptor quantitation, binding kinetics and ligand specificity assays were performed via [1,2,4,³H] dexamethasone. There were no binding differences between the colostrum and milk-fed calves' muscle samples. The average protein content of the muscle cytosol fraction was 50.82 mg/ml. The binding component displayed a high apparent equilibrium dissociation constant for the binding of [³H] dexamethasone (K_d = 2.34x10 ⁻⁸ ). The apparent maximum number of binding sites determined from Scatchard plots was approximately 37.61 fmol/mg of protein in the case of the dexamethasone receptor. Maximum binding appeared to reached between 16 and 24 h (48.5 and 48.2 %, respectively). Competition assays indicated all of the ligands tested had an affinity for the glucocorticoid receptor. The percent of specific binding for each was: dexamethasone (66+/-14), corticosterone (52+/-10), cortisol (58+/-13), estradiol-17, beta (37+/-7), progesterone (29+/-9), testosterone (10+/-3), and triamcinolone (41+/-11). / Master of Science

A study was conducted to determine the effects of trenbolone acetate (TBA) and estradiol-17B (E2) implantation on the characteristics of the glucocorticoid and E2 receptor, skeletal muscle protein turnover and tissue growth. Twenty-four lambs were utilized. Trenbolone acetate did not ,affect (P>.10) degradation rates in the semitendinosus (ST) and triceps brachii (TB) muscles, the production of cortisol, adrenal weights and cytosolic glucocorticoid binding capacity (Bmax). Trenbolone acetate decreased synthesis rate of muscle protein (P<.Ol), the percent of [3H] dexamethasone binding in the nuclear fraction, Bmax and the disociation constant (Kd) of the cytosolic E2 receptor, only in the TB muscle. Deoxyribonucleic acid (DNA) of the TB was increased (P<.05) with TBA. Pituitary weights were decreased (P<.005) with TBA and increased (P<.Ol) with E2. Estradiol decreased (P<.05) Bmax of the cytosolic E2 receptor in the ST and decreased (P<.05) Bmax of the nuclear E2 receptor in the TB muscle. The TB muscle had greater (P<.05) synthesis rates than the ST and the protein:RNA ratio was decreased (P<.05) in the TB. The TB muscle had greater (P<.005) Bmax for the cytosolic glucocorticoid receptor. / Master of Science

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Background: Although glucocorticoids are the most effective treatment for asthma, some patients show a poor response. In such patients with steroid resistant asthma, this has been ascribed to altered glucocorticoid receptor (GR) ligand-binding affinity induced by IL-2 combined with IL-4 or IL-13 alone- all of which can also modulate glucocorticoid function in vitro. Objective: We sought to assess the ligand-binding affinity in a distinct group of oral steroid-dependent asthmatic subjects and examine the mechanisms by which IL-2 and IL-4 (or IL-13) modify the ligand-binding affinity of the GR. Methods: Using dexamethasone-binding assays, we examined PBMCs ex vivo from healthy subjects, subjects with controlled asthma, and oral steroiddependent subjects with severe asthma. In addition, IL-2 and IL-4 were used to alter GR affinity in vitro. We used mediators or inhibitors of signal transduction to investigate the mechanisms of resistance. We also determined cytokine production of PBMC's by means of ELISA. Results: GR ligand-binding affinity was significantly reduced in the nucleus but not in the cytoplasm of oral steroid-dependent asthmatic subjects compared with that seen in steroid-sensitive and healthy subjects (dissociation constant, 41.37 ± 17.83 vs. 25.36 ± 2.63 nmol/L vs. 9.40 ± 4.01 nmol/L, respectively [p<.05 for both in comparison to normals] ). This difference in ligand-binding affinity could be mimicked by IL-2 and IL-4 co-treatment and was blocked by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. PBMC's rendered resistant in vitro demonstrated lower IL-10 and increased GM-CSF production following LPS or PMA & PHA stimulation compared to cells with normal GR affinity. Resistant cells also showed reduced dexamethasone repression of LPSstimulated IL-10 release. These effects were also reversed by SB203580. Inhibition of the ERK MAPK pathway by PD098059 (10 mol/L), phosphoinositol 3 kinase by wortmannin (5 nmol/L) or treatment with IL-10 (10 ng/mL) failed to modulate the effect of IL-2 and IL-4 on receptor affinity. Ro318220 (10 nmol/L), a specific protein kinase C inhibitor and theophylline, similarly, had no effect on affinity. Conclusion: GR ligand binding affinity is tiered; compared to normal subjects; steroid responsive asthmatics have a mild reduction in ligand binding whereas oral steroid dependent asthmatics have greater reductions. When mononuclear cells are rendered resistant in vitro, cytokine production (low IL-10 and high GM-CSF) favours a pro-inflammatory state. Our data do not support the ERK MAPK, phosphoinositol 3 kinase, protein kinase C pathways in steroid resistance. Treatment with IL-10 and theophylline also failed to modulate the effect of IL-2 and IL-4 on receptor affinity. However, P38 MAPK inhibitors may have potential in reversing glucocorticoid insensitivity and re-establishing the beneficial effects of glucocorticoids in patients with severe asthma. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2007.

Lung cancer is the most common cancer worldwide and is the fourth leading cause of death in South Africa. Lung cancer is categorised into two types; non-small cell lung cancer and small cell lung cancer (SCLC). SCLC constitutes 20% of all lung cancers and is considered to be an aggressive tumour as it gains chemo-resistance and exhibits early metastasis in diagnosed patients. SCLC cells originate from the neuroendocrine cells of the bronchoepithelium and are known to secrete the neuropeptide, proopiomelanocortin (POMC). POMC undergoes proteolytic cleavage to produce the adrenocorticotropin hormone (ACTH). ACTH stimulates the production of the steroid hormone, glucocorticoid hormone (GC), through the hypothalamus-pituitary-adrenal (HPA) axis. The produced GCs mediate a negative feedback system of the HPA axis to sequester ACTH production. SCLC cells are insensitive to this negative feedback stimulus. GCs elicit their actions through the glucocorticoid receptor (GR). Studies have shown that SCLC cells have a reduced expression of GR which perpetuates the GC-insensitivity. Importantly, over-expression of exogenous GR in SCLC cells leads to cell death by apoptosis. It was postulated that SCLC cells select against GR expression for longevity. Cancer cells are known to alter/silence the expression of tumour suppressor genes by a mechanism known as methylation. Methylation occurs when the enzyme, DNA methyltransferase 1, adds a methyl group to a cytosine present in a guanine-cytosine rich region of the gene (CpG island). The GR gene has a 5’-untranslated exon 1 region that consists of eight promoter regions (1A-1J), in these promoter regions are many CpG islands that have the potential to be methylated. The first aim of this study was to determine the promoter/s utilised by SCLC cells to express the GR protein. Conventional PCR revealed that all three cell lines predominantly utilise promoters 1B and 1C for GR expression. Bioinformatic analysis revealed that these promoters contain putative CpG islands and new data suggests that the GR is silenced by methylation and that treatment with a de-methylating agent results in GR re-expression. To determine which promoter is responsible for GR re-expression after de-methylation, the SCLC cell line, DMS79, as well as two control cell lines, A549 and HEK cells, were treated with the de-methylating agent, 5-aza-2’-deoxycytidine, for 72 hours. qPCR analyses revealed that all three cell lines expressed promoters 1B and 1C with A549 cells showing no evidence of methylation. The HEK cells showed methylation in promoter 1C and not promoter 1B. The SCLC cells showed methylation in both promoter 1B and 1C, however, only promoter 1B showed a significantincrease in transcript levels. SCLC cells are induced to undergo GC-mediated apoptosis when GR expression is restored however the mechanism utilised by the GR to induce the apoptotic cascade is unknown. The GR structure is divided into three domains; ligand binding domain (LBD), DNA binding domain (DBD) and amino terminal domain (NTD). The second aim of this study was to determine the component of the GR that induces apoptosis of SCLC cells. HEK and SCLC cells were infected with empty virus and various GR construct viruses; containing either a wild-type GR, ligand binding mutant, DNA binding mutant or a transactivation mutant (NTD); for 72 hours. Both cell lines were quantified for apoptosis and cell death using microscopic analyses. In HEK cells, it was shown that apoptosis occurred in cells expressing the wild-type GR, the DNA binding mutant and transactivation mutant constructs but apoptosis was reduced in cells expressing the ligand binding viruses. This indicates that the LBD may be necessary for inducing apoptosis in HEK cells. In DMS79 cells, apoptosis occurred in cells expressing the wild-type GR, ligand binding mutant and the DNA binding mutant constructs. There was less apoptotic activity exhibited in the transactivation constructs which indicates the NTD may be necessary for apoptosis induction in these cells. The NTD of the GR is responsible for interaction with other transcription factors to mediate GR transcriptional activity and this study has shown that the transactivation domain plays a necessary role in apoptosis induction. An analysis of the various pathways the GR interacts with through the NTD domain could lead to the identification of the pathway which triggers apoptosis in SCLC cells. This discovery, together with knowledge of promoter methylation and expression may contribute to the development of new, more effective therapies for SCLC. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Thesis (PhD (Biomedical Sciences. Medical Physiology))--University of Stellenbosch, 2006. / Early life trauma leads to behavioural abnormalities later in life. These include mood and anxiety disorders such as depression and posttraumatic stress disorder (PTSD). This association may be due in part to the effects of trauma on brain development. Data from basic and clinical experiments suggest that alterations in the hippocampus may be fundamental to the development of these disorders. Here we used an animal model of early life trauma to investigate its effects on the behaviour and neurochemistry of the adult rat. Adolescent rats were subjected to time-dependent sensitization stress consisting of a triple stressor (2 hours restraint, 20 min swim stress and exposure to ether vapour) on post-natal day (PND) 28, a single re-stress on PND 35 (20 min swim stress), and a second re-stress in adulthood (PND 60, 20 min swim stress). The rationale was that the frequency of exposure to situational reminders contributes to the maintenance over time of fear-related behavioural disturbances. The effects of trauma on the hypothalamus-pituitary-adrenal-axis, hippocampal and plasma neurotrophin levels, behaviour and phosphoinositide-3 kinase (PI-3 kinase) signaling proteins were initially investigated. In addition, proteomic technologies such as protein arrays and 2D-SDS PAGE combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) were employed to study trauma-induced effects on the hippocampus. Traumatized animals showed a decrease in glucocorticoid receptors in the dentate gyrus of the hippocampus and an increase in basal corticosterone levels 24 hours after adulthood re-stress. These effects were reversed by pretreatment with the serotonin selective reuptake inhibitor, escitalopram. A decrease in the neurotrophins, BDNF and NT-3 were evident 8 days, but not 24 hours after adulthood re-stress. This decrease was not accompanied by decreases in plasma neurotrophin or PI-3 kinase, protein kinase B (PKB), phosphatase and tensin homologue (PTEN), phospho-forkhead and phospho-AFX protein levels. In addition, traumatized animals showed increased rearing in both the elevated plus maze and open field. Proteomic analysis of trauma-induced changes in the hippocampus show increases in Ca2+ homeostasis / signaling proteins such as S-100B, phospho-JNK and calcineurin. Apoptotic initiator proteins, including caspase 9, -10 and -12 were increased and there was evidence of cytoskeletal protein dysregulation. Furthermore, cell cycle regulators and energy metabolism proteins were decreased. These effects indicate to a cellular state of cell cycle arrest after increased calcium influx to avoid apoptosis. Our data suggest that adolescent trauma with adulthood re-stress may affect numerous systems at different levels. These include neuroendocrine-, protein systems and behaviour, and confirmed that a systems biology approach is needed for a better understanding of the neurobiology of mental disorders.