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.

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.

We explore both robust biologically guided intensity-modulated radiation therapy (BG-IMRT) and pattern recognition to identify responders to cancer treatment for lung cancer. Heterogeneous dose prescriptions that are derived from biological images are subject to uncertainty, due to potential noise in the image. We develop a robust optimization model to design BG-IMRT plans that are de-sensitized to uncertainty. Computational results show improvements in tumor control probability and deviation from prescription dose compared to a non-robust model, while maintaining tissue dose below toxicity levels. We applied machine learning algorithms to 4D gated positron emission tomography/computed tomography (PET/CT) scans. We identified classifiers which could outperform a naive classifier. Our work shows the potential of using machine learning algorithms to predict patient response. This could hopefully lead to more adaptive treatment plans, where the clinician would adapt the treatment based on the prediction provided at certain time intervals in the treatment.

We explore both robust biologically guided intensity-modulated radiation therapy (BG-IMRT) and pattern recognition to identify responders to cancer treatment for lung cancer. Heterogeneous dose prescriptions that are derived from biological images are subject to uncertainty, due to potential noise in the image. We develop a robust optimization model to design BG-IMRT plans that are de-sensitized to uncertainty. Computational results show improvements in tumor control probability and deviation from prescription dose compared to a non-robust model, while maintaining tissue dose below toxicity levels. We applied machine learning algorithms to 4D gated positron emission tomography/computed tomography (PET/CT) scans. We identified classifiers which could outperform a naive classifier. Our work shows the potential of using machine learning algorithms to predict patient response. This could hopefully lead to more adaptive treatment plans, where the clinician would adapt the treatment based on the prediction provided at certain time intervals in the treatment.

Small cell lung cancer (SCLC) is an aggressive form of lung cancer that frequently develops multidrug resistance (MDR) during chemotherapy. Vault RNA1 (vRNA1), a non-structural component of the MDR-associated vault organelle, is believed to act as a microRNA (miRNA) and may contribute to MDR by regulating the expression of genes involved in apoptosis, inflammation, and/or drug metabolism. Since vaults function to aid cells in survival, we hypothesized that vRNA1 might be free in the cytoplasm and able to inhibit expression of pro-survival mRNAs when vaults are open in drug-sensitive GLC4/S cells but not in the MDR GLC4/ADR cells where vaults might be closed with the miRNA sequestered within. In order to establish the role of vRNA1 as a regulator of survival in SCLC cells, siRNA-mediated down-regulation of vRNA1 was employed in GLC4/S and GLC4/ADR SCLC cells. Fluorescence microscopy using a green fluorescent 3’ AlexaFluor-488 negative siRNA control was used to estimate transfection efficiency, yielding 56% for GLC4/S and 89% for GLC4/ADR. However, these values and the level of apoptosis before and after transfection, as judged by trypan blue hemacytometer cell counts, were not entirely reliable due to cell clumping. The latter counts indicated a 2-fold decrease in viability in GLC4/S cell following transfection but no decrease in GLC4/ADR cells (p< 0.05). RT-PCR revealed that transfection significantly (p<0.05) decreased vRNA1 expression in GLC4/S cells but not in GLC4/ADR cells, confirming our hypothesis concerning the availability of vRNA1 in the two cell types. Caspase activity measurements showed vRNA1 down-regulation in the GLC4/ADR cells significantly (p≤0.05) increased survival via a 6.1-fold reduction in caspase 3/7 activity, further supporting our hypothesis. However, GLC4/S cells showed a similar loss of apoptosis when transfected with either sivRNA1 or the negative control siRNA. vRNA1 down-regulation did not significantly (p≤0.05) affect the expression of major pro-survival (Bcl-2, Bcl-xL), pro-apoptotic (Bad), or pro-inflammatory (IL-6, NFĸB p65) factors in either GLC4/S or GLC4/ADR cells. However, the drug metabolism protein CYP3A (previously shown by Persson et al., 2009 to be regulated by vRNA1) was significantly (p≤0.05) lowered (~16%) following vRNA1 down-regulation in the GLC4/S cells. In conclusion, we were successful in down-regulating vRNA1 which enhanced cell survival as hypothesized, but we were not able to identify new proteins regulated by vRNA1. / Department of Biology

Only discovered about 20 years ago, the structure of the eukaryotic vault particle has been studied extensively, but the function has yet to be determined. Vault numbers are up regulated in many types of cancer cells that are treated with chemotherapy agents and it is thought that they may act to transport chemotherapy drugs out of such cells, leading to multi-drug resistance (MDR). To determine a possible role of the vault particle in MDR, the goal of this research was to examine one of the functional vault proteins, vault poly(ADP)ribose (VPARP) for interactions with other proteins. Two forms of small cell lung cancer cells were used; GLC4/S which do not exhibit MDR and the MDR cells GLC4/ADR, which are cultured with the chemotherapy drug doxorubicin. Both cell cultures were subjected to a subcellular fractionation followed by gentle immunoprecipitation with an antibody to VPARP. Immunoprecipitated proteins interacting with VPARP were only observed in GLC4/ADR cells, as seen on a PAGE gel. This sample was taken to Monarch Life Sciences and analyzed by mass spectrometry. One interacting protein was found to be NALP1 pyrin domain (PYD), a member of the death domain family of proteins which is involved in inflammation and apoptosis. The interaction of VPARP with NALP1, which only occurred in MDR cells, suggests an exciting, previously unreported possibility – that VPARP binding may inhibit NALP 1-stimulated apoptosis when MDR is occurring. Future studies are needed to examine if levels of NALP1 vary in GLC4 cells with and without treatment with doxorubicin and in normal lung cells. The cellular location (nucleus or cytoplasm) of the interactions should also be identified. Furthermore, immunoprecipitation of proteins interacting with NALP1 should include VPARP and perhaps identify other proteins interacting in the signaling pathways under MDR and normal culture conditions. This information may contribute insight into the function of VPARP and vaults within the cell. / Department of Biology

This thesis investigated the cooperation of the Kras2 oncogene with the tumor suppressor gene Dlc1 in lung tumor development. Dlc1 is a negative regulator of RhoGTPase proteins, which are mainly involved in the regulation of the actin cytoskeleton and cell migration. We hypothesized that loss of Dlc1 expression leads to more aggressive tumors, which should also result in increased incidence of metastasis. All experiments were performed in mice containing a heterozygous oncogenic Kras allele and a heterozygous gene trapped Dlc1 allele (KD) and in mice only carrying the oncogenic Kras allele (K+). Throughout all experiments we have consistently found no significant differences between the two groups in terms of tumor burden (tumor numbers, sizes and areas), metastases or methylation patterns. These results suggest that heterozygous downregulation of Dlc1 is not enough to increase tumor formation and metastasis development in the Kras lung tumors.

Lung cancer is a disease which patients experience stigma in society and in care. This is because lung cancer is often seen as a self-inflicted disease. The stigma surrounding lung cancer is due to the strong relationship with smoking and have been shown to have a negative impact on the perceived health. The purpose of this study was to illuminate experiences of stigma for patients with lung cancer. The literature review was based on 11 scientific articles. The analysis resulted in three themes: experiences of being excluded out of context, experienced feelings surrounding stigma and the meeting within healthcare. Ignorance from healthcare and the community lead to patients with lung cancer feeling excluded, therefore it is important that nurses carry with them an awareness and good knowledge of stigma. The experience of stigma in patients with lung cancer was found to be reduced by good treatment and care from medical staff. Further qualitative research should be conducted that focuses on patients' experiences of stigma surrounding lung cancer both in the community and in healthcare. / Lungcancer är en sjukdom där patienter upplever sig stigmatiserade både i samhället och i vården eftersom lungcancer ofta ses som en självförvållad sjukdom. Stigmatiseringen kring lungcancer beror på det starka sambandet till rökning och har visat sig ha en negativ inverkan på den upplevda hälsan. Syftet med denna studie var att belysa upplevelser av stigmatisering av patienter med lungcancer. Litteraturstudien baserades på 11 vetenskapliga artiklar. Resultatet utmynnade i tre teman: upplevelser av att bli utesluten ur sammanhang, upplevda känslor kring stigmatisering och mötet i vården. Okunskap från vården samt från samhället leder till att patienter med lungcancer upplever ett utanförskap, det är därför av stor betydelse att sjuksköterskor bär med sig en medvetenhet och god kunskap kring stigmatisering. Upplevelsen av stigmatisering hos patienter med lungcancer visade sig kunna minska genom ett gott bemötande från vårdpersonalen. Mer kvalitativ forskning bör bedrivas som fokuserar på patienters upplevelser kring stigmatisering vid lungcancer såväl i samhället som i vården.

The objective of this thesis was to further develop a methodology for surface tension measurement called Axisymmetric Drop Shape Analysisn(ADSA) and to adapt it to studies of lung surfactants, i.e. the material that coats and facilitates the functioning of the lungs of all mammals. The key property of a functioning lung surfactant is its surface tension, which can reach extremely low values. Such values are difficult to measure; but a certain configuration of ADSA, using a constrained sessile drop (ADSA--CSD), is capable of performing such measurements. Clinically, lung surfactant films can be altered from both sides, i.e. from the airspace as well as from the bulk liquid phase that carries the film. Therefore, being able to access the interface from both sides is important. Here, ADSA--CSD was redesigned to be used as a micro film balance allowing access to the interface from both gas- and liquid-side. This allows deposition from the gas side as well as complete exchange of the bulk liquid phase. The new design was used to study lung surfactant inhibition and inhibition reversal. A dynamic compression-relaxation model (CRM) was developed to describe the mechanical properties of lung surfactant films by investigating the response of surface tension to changes in surface area. The model evaluates the quality of lung surfactant preparations -- beyond the minimum surface tension value -- and calculates the film properties, i.e. elasticity, adsorption and relaxation, independent of the compression protocol. The accuracy of the surface tension measurement can depend on drop size. A detailed analysis of drop shapes and accuracy of measured surface tension values was performed using a shape parameter concept. Based on this analysis, the design of ADSA--CSD was optimized to facilitate more accurate measurements. The validity analysis was further extended to the more conventional pendant drop setup (ADSA--PD). An overall upgrade of both hardware and software of ADSA--CSD, together with extensive numerical work, is described and applied to facilitate a more efficient operation. Finally, it is noted that the ADSA--CSD setup developed here can be used for a wide range of colloid and surface chemical applications.

The objective of this thesis was to further develop a methodology for surface tension measurement called Axisymmetric Drop Shape Analysisn(ADSA) and to adapt it to studies of lung surfactants, i.e. the material that coats and facilitates the functioning of the lungs of all mammals. The key property of a functioning lung surfactant is its surface tension, which can reach extremely low values. Such values are difficult to measure; but a certain configuration of ADSA, using a constrained sessile drop (ADSA--CSD), is capable of performing such measurements. Clinically, lung surfactant films can be altered from both sides, i.e. from the airspace as well as from the bulk liquid phase that carries the film. Therefore, being able to access the interface from both sides is important. Here, ADSA--CSD was redesigned to be used as a micro film balance allowing access to the interface from both gas- and liquid-side. This allows deposition from the gas side as well as complete exchange of the bulk liquid phase. The new design was used to study lung surfactant inhibition and inhibition reversal. A dynamic compression-relaxation model (CRM) was developed to describe the mechanical properties of lung surfactant films by investigating the response of surface tension to changes in surface area. The model evaluates the quality of lung surfactant preparations -- beyond the minimum surface tension value -- and calculates the film properties, i.e. elasticity, adsorption and relaxation, independent of the compression protocol. The accuracy of the surface tension measurement can depend on drop size. A detailed analysis of drop shapes and accuracy of measured surface tension values was performed using a shape parameter concept. Based on this analysis, the design of ADSA--CSD was optimized to facilitate more accurate measurements. The validity analysis was further extended to the more conventional pendant drop setup (ADSA--PD). An overall upgrade of both hardware and software of ADSA--CSD, together with extensive numerical work, is described and applied to facilitate a more efficient operation. Finally, it is noted that the ADSA--CSD setup developed here can be used for a wide range of colloid and surface chemical applications.