Apoptotic cell death of cardiac myocytes plays an important pathological role after a myocardial infarction and during heart failure. Apoptotic myocytes are not regenerated because of the restricted ability of terminally differentiated cardiac myocytes to undergo cell division. Because ventricular function is directly related to the number of active muscle cells, the inappropriate loss or premature death of cardiac myocytes results in reduced cardiac performance. Bnip3 was previously identified by Dr. Lorrie Kirshenbaum’s laboratory as a critical mediator of hypoxia-induced apoptosis in the heart. Importantly, his lab established that the cytoprotective actions of NF-κB during hypoxia included the transcriptional repression of Bnip3. However, the mechanism by which NF-κB acted as a transcriptional repressor was undefined. The present work strongly supports the hypothesis that NF-κB-mediated inhibition of Bnip3 transcription is dependent on the recruitment of the corepressor protein HDAC1. Immunoprecipitation experiments revealed that HDAC1 and p65 NF-κB formed protein-protein interactions. ChIP assays demonstrated that HDAC1 and p65 NF-κB associated with the Bnip3 promoter. HDAC1-mediated repression of Bnip3 was lost in cells deficient for p65 NF-κB, and restored upon repletion of p65. A second avenue of investigation described in this work demonstrated that the cell cycle factor E2F-1 directly activated Bnip3 transcription. Earlier work by Dr. Kirshenbaum found that adenovirus-mediated overexpression of E2F-1 in ventricular myocytes induced apoptosis. Herein, it is shown that E2F-1-mediated cell death is largely Bnip3-dependent because functional loss of Bnip3 inhibited E2F-1-induced cell death. Concerning hypoxia, Bnip3 expression is dependent upon the loss of p65/HDAC1-mediated repression, and on the presence of transcriptionally active E2F-1. During hypoxia, overexpression of p65, HDAC1, or Rb, an endogenous inhibitor of E2F-1-dependent transcription, attenuated hypoxia-induced Bnip3 transcription. Based on these findings, future therapies may be designed to repress Bnip3 gene expression after a myocardial infarction, thereby averting cardiac cell death and preserving cardiac function post-infarction.

Background: Airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated. Results: Primary EBF were cultured on non-collagen coated flasks without serum or in the presence of feta bovine serum (FBS) or horse serum (HS) or in serum depleted medium. EBF cultured in serum-free basal media and those serum deprived were not able to proliferate and even exhibited considerable cell death. In media containing FBS or HS, proliferation of the cells was reproducible between different primary cultures and cells demonstrated expression of vimentin. Large variations were found in the ability of FBS and HS to support growth and differentiation of EBF in monolayer culture. Indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in FBS than in HS. EBF culturing in HS needed longer doubling and confluence time. The protein content of the cell pellets was higher in EBF cultured in medium containing HS than FBS. Alpha-smooth muscle actin seemed to be less expressed in EBF cultured in medium containing FBS than those in HS. Conclusions: In sum, serum addition to basal EBF medium enhanced EBF differentiation into myofibroblasts, and these findings are useful to develop in vitro fibroblast culture models that mimic in vivo physiological processes and to study airway disease mechanisms and remodeling.

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from human prostate cancer cell lines to identify secreted proteins that could serve as novel prostate cancer biomarkers. An initial proof of principle study of the PC3 prostate cancer cell line was conducted. From this study over 200 proteins were identified in the conditioned media. Through gene ontology analysis and literature searches Mac-2 binding protein was selected as a candidate biomarker for validation in the serum of prostate cancer patients. A preliminarily validation showed that Mac-2 binding protein has discriminatory ability in prostate cancer diagnosis. However, an extended validation did not confirm this. Based on our proof of principle study we optimized our workflow and extended our analysis by culturing three different prostate cell lines [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)]. We conducted a bottom-up analysis of each cell line by 2-dimensional liquid chromatography and tandem mass spectrometry. Of the 2124 proteins identified, 12% (329) were classified as extracellular and 18% (504) as membrane-bound. Among the identified proteins were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. Based on these results, several candidates were selected for validation in serum of patients with and without prostate cancer. Of these four novel candidates: follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2 showed discriminatory ability. Of the four candidates, follistatin was further studied in an extended validation in serum of patients with biopsy confirmed prostate cancer and tissues of prostate cancer patients of low and high grade tumours by immunohistochemistry. In addition, follistatin was also investigated in the tissue of colon and lung cancer where intense staining was observed in one specimen of lung squamous carcinoma.

The limbal stem cell population is located in the limbal junctional zone between the cornea and the conjunctiva, and is responsible for maintaining the corneal epithelium. Damage to the limbal stem cell population results in a condition known as limbal stem cell deficiency (LSCD), which is characterised by conjunctivalisation of the cornea, visual impairment and persistent irritation. To treat LSCD, an alternative source of human limbal epithelial (HLE) cells must be transplanted back onto the diseased cornea. Limbal tissue grafts have had a moderate degree of success. However, autologous grafts risk damage to the healthy eye, whilst allogeneic grafts are susceptible to immunological rejection. Cultured HLE grafts offer a promising alternative to whole tissue grafts. The production of cultured HLE grafts involves the removal of a small (1-2 mm2) biopsy from the patient’s healthy limbus, followed by ex vivo expansion to produce an epithelial sheet, which is subsequently transplanted onto the damaged corneal surface. However, the production of cultured HLE grafts usually requires the addition of animal-derived products during cell culture. Animal-derived components, such as foetal bovine serum (FBS) and murine 3T3 feeder cells, introduce the patient to potential crossspecies infection and immune responses to xenogeneic antigens. Consequently, the overall aim of this project has been to develop a culture technique free of xenogeneic products for the establishment and propagation of HLE cells. To achieve this aim, alternatives to FBS in the culture medium and 3T3 feeder cells were pursued. A defined serum-free medium (SFM) containing vitronectin (VN), insulin-like growth factor binding protein 3 (IGFBP3), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) was investigated as an alternative to serumsupplemented medium (SSM) for HLE cell culture. Initial studies focused on the effects of these growth factors on HLE cell metabolic activity and migration. Metabolic activity was primarily stimulated by IGF-I and EGF, with the combination of IGF-I and EGF in solution stimulating metabolic activity to a significantly greater extent than the SSM positive control (p = 0.006). HLE cell migration was also effected by combinations of VN, IGFBP3, IGF-I and EGF. Migration was stimulated above the SFM negative control by the combination of IGFBP3 and IGF-I either with or without the addition of EGF. However, the presence of VN was required for optimal migratory responses (p < 0.003). Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) were also investigated as additional components to the SFM formulation. HGF significantly stimulated HLE cell metabolic activity and migration (p < 0.02). In contrast, KGF did not significantly stimulate either HLE cell metabolic activity or migration. The addition of either HGF or KGF to the SFM supplemented with VN, IGFBP3, IGF-I and EGF did not significantly enhance the metabolic activity of HLE cells. Therefore, HGF and KGF were no longer pursued as additional components to the SFM formulation. Additional studies were conducted to examine the efficacy of replacing murine 3T3 feeder cells with human ocular stromal cells during HLE cell culture. Initially, stromal cells were isolated from the cornea, limbus and sclera to determine whether there were differences between these stromal cell populations. The results indicated that scleral stromal cells had a significantly larger area and perimeter than either corneal or limbal stromal cells (p < 0.001). Scleral stromal cells were also significantly more rounded than either corneal or limbal stromal cells, as determined by the elliptical factor equation (p < 0.001). Immunocytochemistry also revealed that scleral stromal cells expressed significantly more of the myofibroblast marker ..- smooth muscle actin than either corneal or limbal stromal cells (p < 0.001), and significantly less of the fibroblast/myofibroblast marker Thy-1 than corneal or limbal stromal cells (p < 0.001). Therefore, scleral stromal cells were identified as different in comparison to corneal and limbal stromal cells. Primary HLE cells were cultured with irradiated corneal, limbal and scleral stromal cells. HLE cultures established with either corneal or limbal stromal feeder cells contained more cellular protein (as measured by rhodamine B dye absorbance) than cultures established without feeder cells (p < 0.001). The colony forming efficiency (CFE) of HLE cells established with corneal or limbal stromal feeder cells was also significantly greater than HLE cells established without feeder cells (p < 0.001). In contrast, HLE cultures established with scleral stromal feeder cells contained low levels of cellular protein and had a low CFE, which was not significantly different to the HLE cultures established without feeder cells. Immunocytochemistry indicated that HLE cultures established with scleral feeder cells also showed lower expression of the stem cell markers ABCG2 and C/EBP ... These results suggest that freshly isolated HLE cells can be cultured with irradiated corneal or limbal stromal cells as a replacement for murine 3T3 feeder cells. Finally, the SFM supplemented with VN+IGFBP3+IGF-I+EGF was combined with limbal stromal feeder cells, and examined as a culture technique free of animalderived products. Freshly isolated HLE cells established in SFM supplemented with VN+IGFBP3+IGF-I+EGF and limbal feeder cells contained a similar amount of cellular protein (as measured by crystal violet dye absorbance) when compared to the SSM+3T3 positive control. In addition, the CFE of freshly isolated HLE cells established in VN+IGFBP3+IGF-I+EGF and limbal feeder cells was significantly higher than the SSM+3T3 positive control (p = 0.004). However, a live/dead assay revealed a reduced HLE cell viability in SFM supplemented with VN+IGFBP3+IGFI+ EGF and limbal feeder cells after seven days in culture. In addition, immunocytochemistry demonstrated a lower expression of the stem cell markers ABCG2 and C/EBP .. in the SFM treatment with limbal feeder cells. Therefore, freshly isolated HLE cells can be cultured in SFM supplemented with VN+IGFBP3 +IGF-I+EGF and limbal feeder cells. However, this culture technique is less likely to support the growth of immature limbal stem cells when compared to the SSM+3T3 positive control. Overall, this research has attempted to create a culture system free of animal-derived products for the production of cultured HLE grafts to treat limbal stem cell deficiency. The results show that HLE cells respond to a serum-free medium formulation containing VN+IGFBP3+IGF-I+EGF. In addition, this culture medium can be combined with irradiated stromal cells isolated from the limbus to support HLE culture production. However, the combination of VN+IGFBP3+IGF-I+EGF and limbal feeder cells demonstrated a reduced viability, which indicates that further refinement of the formulation is required. This thesis has also demonstrated differences between stromal cells isolated from the cornea, limbus, and sclera, and has generated knowledge which may impact on the understanding of stromalepithelial regulation.

A proteomics approach was used to identify proteins potentially implicated in the cellular response concurrent with elevated production levels of human follicle stimulating hormone in a recombinant Chinese hamster ovary cell line (Darren cells), using zinc and sodium butyrate in the production media to increase expression. To this end, 2-dimensional gel electrophoresis (2-DGE) was utilized. Firstly, several aspects of 2-DGE were developed for this investigation. Gel drying conditions were optimized, and a glycine-free blotting method is described which achieved greater efficiency in rapid transfer of proteins than those previously described. Next, hFSH expression was characterized in Darren cells. An ELISA developed for this investigation examined intracellular (expression) and extracellular (secretion) of hFSH during increased expression. These results show a disproportionate increase in intracellular hFSH (188%) expression above extracellular hFSH (41%).

Male fertility is largely controlled by the hypothalamic-pituitary axis, a careful balance between stimulating and suppressing gene expression and the secretion of hormones. The critical factors for male fertility have in the past been thought to be limited to testosterone and the gonadotropins. Estrogen has only recently been demonstrated to be both a crucial requirement for fertility and a cause of infertility. Reports in the early 1990s demonstrated a decrease in mean sperm counts over the last 50 years. A hypothesis for this observation is the increase of xenoestrogens in the environment that are able to mimic and potential disrupt the natural estrogens involvement in fertility. Although the mechanisms of estrogens involvement are not yet defined, the Sertoli cells are a potential sites of action as they possess receptors for the hormone and are able to locally produce it. Sertoli cells both act to protect and provide for the male germ cells and the developing spermatozoa. Pyrethroids are common synthetic insecticides of which some have previously shown estrogenic activity. Therefore this investigation examined the effects of pyrethoids, whose estrogenicity was confirmed via the yeast assay, on the estrogen receptor expression in mouse Sertoli cells as a model for general effects of estrogenic chemicals on male fertility. The results first confirmed the estrogenicity of some pyrethroids and these pyrethroids when exposed to mouse Sertoli cells effected estrogen receptor mRNA expression however in a different way to the natural ligand 17β-estradiol.

Doctor of Philosophy / Benign tumours grow by expanding and displacing the surrounding tissues, while malignant tumours replace and destroy the surrounding tissues by invasion. Although there is extensive literature on mechanisms of tumour invasion and metastasis, with an emphasis on angiogenesis, adhesion, degradation of the extracellular matrix and migration, an important question not clearly addressed by the literature, but nonetheless approached in this thesis, is that of the fate of normal cells during tissue replacement by migrating invasive malignant cells. Earlier work in the laboratory where this PhD candidature was carried out, investigated the effect of osteosarcoma cells on endothelium. In contrast to the expected angiogenic effect of malignant cells for endothelium, it was found that the human osteosarcoma cell line (SAOS-2) induced apoptosis in human umbilical vein endothelial cells (HUVEC) in contact dependent manner (McEwen et al., 2003). It was suggested that apoptosis of endothelium by malignant tumour cells may facilitate tumour invasion and metastasis (McEwen et al., 2003), and one of the aims of the current study was to extend these findings to include human gingival fibroblasts (HGF) and human umbilical artery smooth muscle cells (HUASMC). The major finding of this thesis was that SAOS-2 induced a reduction in the apparent cell culture density of HGF and HUASMC in a contact-dependent manner. The SW480 colorectal carcinoma cell line did not have any clear effect upon the apparent stromal cell culture density of either HGF or HUASMC, suggesting that the effect under investigation was tumour cell line specific. Surprisingly and in contrast to the similar effect reported for endothelium (Chen et al., 2005; McEwen et al., 2003), the effect of SAOS-2 upon HGF and HUASMC was not due to stromal cell apoptosis. Apoptosis was ruled out as a possible mechanism for the reduced apparent culture density under study, by using widely accepted methods which are dependent upon intermucleosomal fragmentation of DNA, the permeability of plasma membranes to dyes in advanced apoptosis and necrosis, phosphatidylserine translocation as well as inhibitor studies blocking both caspase dependent and independent pathways. While apoptosis was not demonstrated, the possibility emerged that reduced apparent stromal cell culture density reflected fusion events rather than the simple removal of cells as had been earlier reported for HUVEC (McEwen et al., 2003). This idea was supported by reduced SAOS-2 circularity in co-culture. Confocal microscopy of cells pre-labelled with fluorescent dyes further supported this idea, with dual-labelling as evidence of cell fusion. Although occasional homotypic fusion of stromal cells was seen, heterotypic fusion of stromal cells with SAOS-2 was much more prevalent. Time lapse microscopy was performed to further characteristic cell fusion in co-cultures, and revealed multiple transient fusions between SAOS-2 and HGF. To work towards determining the biological relevance of the key observation, two stable SAOS-2 GFP clones were generated for future planned studies using human gingival explants and nude mice. Importantly, the clones were similar to native SAOS-2 with regard to alkaline phosphatise expression and reducing apparent stromal cell culture density. Transient fusions between HGF and SAOS-2, may be a mechanism for cooption of stromal cells into the malignant process, facilitating tumour invasion. Additionally, heterocellular fusion of SAOS-2 with stromal cells may facilitate immune evasion, while it seems likely that despite the absence of an identical activity in SW480 cells, other malignant tumour cells may also express similar activity.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Michigan, 1968.