<p>Sun-damaged skin is a relevant target tissue for studying the development of skin cancer. The aim of the present study was to investigate the epidermal response to ultraviolet radiation (UVR) in human skin<i> in vivo</i> and <i>in vitro</i> and to explore the mutagenic effect of UVA. The prevalence and the genetic background of epidermal p53 clones were furthermore analysed.</p><p>Large inter- and intraindividual differences were observed in the epidermal response to UVR. Repair of UV-induced DNA damage appeared more efficient in chronically sun-exposed skin than in non-sun-exposed skin. Irradiation with UVA1 induced p53 mutations in keratinocytes. The pattern of mutations was indicative of oxidative damage, consistent with UVA acting as a mutagen.</p><p>The prevalence of p53 clones in skin adjacent to basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and benign melanocytic nevus in patients of different age groups was analysed. An age-dependent increase in number and size of p53 clones was observed. Epidermal p53 clones were significantly larger and more frequent in skin adjancent to SCC than adjacent to BCC or melanocytic nevus. Mutation analysis of the entire coding region of the p53 gene showed that 57% of p53 clones in normal skin surrounding BCC and SCC have a mutated p53 gene. </p><p>In conclusion, this study has increased our knowledge of the effects of UVR in chronically sun-exposed skin. The mutation spectra observed in epidermal p53 clones resembled that of non-melanoma skin cancer. The increased prevalence of epidermal p53 clones adjacent to SCC indicates that epidermal p53 clones may represent a step prior to actinic keratosis in the development of SCC.</p>

Sun-damaged skin is a relevant target tissue for studying the development of skin cancer. The aim of the present study was to investigate the epidermal response to ultraviolet radiation (UVR) in human skin in vivo and in vitro and to explore the mutagenic effect of UVA. The prevalence and the genetic background of epidermal p53 clones were furthermore analysed. Large inter- and intraindividual differences were observed in the epidermal response to UVR. Repair of UV-induced DNA damage appeared more efficient in chronically sun-exposed skin than in non-sun-exposed skin. Irradiation with UVA1 induced p53 mutations in keratinocytes. The pattern of mutations was indicative of oxidative damage, consistent with UVA acting as a mutagen. The prevalence of p53 clones in skin adjacent to basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and benign melanocytic nevus in patients of different age groups was analysed. An age-dependent increase in number and size of p53 clones was observed. Epidermal p53 clones were significantly larger and more frequent in skin adjancent to SCC than adjacent to BCC or melanocytic nevus. Mutation analysis of the entire coding region of the p53 gene showed that 57% of p53 clones in normal skin surrounding BCC and SCC have a mutated p53 gene. In conclusion, this study has increased our knowledge of the effects of UVR in chronically sun-exposed skin. The mutation spectra observed in epidermal p53 clones resembled that of non-melanoma skin cancer. The increased prevalence of epidermal p53 clones adjacent to SCC indicates that epidermal p53 clones may represent a step prior to actinic keratosis in the development of SCC.

Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa and also one of the more common mucosal conditions mostly affecting middle aged individuals. Even though OLP is well investigated the etiology of this disease is still unknown, even if autoimmunity as a possible etiologic factor has been suggested. WHO classifies OLP as a pre malignant condition but malignant transformation of OLP is a matter of great controversy. The p53 protein is a tumour suppressor with the potential to induce apoptosis or cell cycle arrest of DNA damaged cells. Another member of the p53 family, p63, comprises six different isoforms, and plays a crucial role in the formation of oral mucosa, salivary glands, teeth and skin. p63 has also been suggested to be involved in development of squamous cell carcinoma of the head and neck (SCCHN). β-catenin, E-cadherin and epidermal growth factor receptor (EGFR) are p63 related proteins and abnormalities in their expression are suggested to be involved in development of SCCHN. Methods. Using immunohistochemistry and antibodies directed against p53 and those distinguishing between the p63 isoforms we analysed biopsies of OLP, SCCHN and normal oral tissue. We also mapped levels of p63 and p53 isoforms using RT-PCR technique. Furthermore expression of the p63 related proteins β-catenin, E-cadherin and EGFR was studied using immunoblot analysis. In an attempt to investigate autoimmunity as a causative factor of OLP we analysed sera from patients diagnosed with OLP and matched control individuals in order to see if there were autoantibodies directed against the p53 family. Results. When mapping p53 and p63 protein status decreased expression of p63 and increased expression of p53 was seen in OLP compared to normal tissue. In accordance with these results, levels of p63 RNA were also lower in OLP lesions compared with normal tissue. Concerning p53 isoforms, the “original” p53 isoform was expressed in all OLP lesions and normal control tissue. Of the other isoforms, p53β and Δ133p53 were expressed in the majority of samples. Our results regarding p63 related proteins showed a generally lower expression of these proteins in OLP lesions compared to normal control tissue. When studying sera from patients with OLP we found circulating autoantibodies against all six p63 and four p73 isoforms in two patients. Conclusions. The potential for malignant transformation of OLP is still a subject of discussion and rather controversial. While some of our results regarding status of p53 and p63 both at protein and RNA levels support this theory, other results concerning for example p63 related proteins point in the opposite direction. Based on our studies it is thus not possible to either support nor contradict the statement that OLP is a clear-cut premalignant condition. In our effort to understand the etiology of OLP we were the first to demonstrate autoantibodies against p63 and p73 in what could be a subgroup of OLP patients. OLP could thus be suggested to be not one distinct disease, but based on our data a disease comprising different subgroups.

<p>Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in humans. Its aggressive and infiltrative growth into the brain, and, at best, only partial sensitivity to radiotherapy and chemotherapy, renders it extremely difficult to treat and survival remains dismal. </p><p>Growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), and their corresponding receptors are seen in glioma tissue, suggesting the presence of autocrine stimulatory loops. <i>PDGFB</i> and a mutated EGF receptor were also identified as cellular homologues of two oncoviruses, thereby indicating a role in tumorigenesis. This thesis presents a brain tumor model in mice, developed using a <i>PDGFB</i> coding retrovirus to induce overexpression of PDGF-B in neonatal mouse brain. Immature tumors, with histological characteristics of primary brain tumors developed at relatively high frequencies. Mice injected with a non-coding retrovirus did not develop tumors, indicating the crucial role of PDGF stimulation in this system. Tumor cells were also shown to continue to depend on PDGF stimulation when cultured <i>in vitro</i>. </p><p>In human glioblastomas, growth factor receptor signaling is present in conjunction with defects in cell cycle arrest pathways. When the <i>PDGFB</i>-virus model was used with <i>p53</i> or <i>Ink4a-Arf</i> deficient mice, tumors arose with shorter latency and higher frequency. Loss of <i>p53</i> or <i>Ink4a-Arf</i> seemed to facilitate signaling through the PI3K/Akt pathway. Thus, a functional role for the co-existence of p53 loss of function and PDGF signaling in a subset of gliomas is presented. </p><p>Human GBM samples were collected and analyzed with respect to expression and activation of the EGFR and PDGFRα. Most tumors expressed the both receptors at moderate to high levels, but high activation of either receptor seemed mutually exclusive.</p>

<p>Mast cells (MCs) are forceful multifunctional effector cells of the immune system. MCs are normally distributed throughout connective and mucosal tissues, but in several pathological conditions accumulation of MCs occur. This accumulation is probable due to directed migration of MCs and they are subjects for migration at least two different occations: 1) when they are recruited as progenitor cells from the blood into the tissue; and 2) when they as mature MCs are recruited to sites of inflammation. The aim of this study was to investigate MC migration to chemoattractants released <i>in vivo</i> or <i>in vitro</i> (body fluids collected from patients with asthma or rheumatoid arthritis and T_H1- and T_H2-cytokines) and to recombinant cytokines (transforming growth factor -β (TGF-β) and CCL5/RANTES).</p><p>This thesis shows that bronchoalveolar lavage (BAL) fluid from asthmatic patients and synovial fluid from patients with rheumatiod arthritis contain MC chemoattractants, and that part of the chemotactic activity can be related to the presence of stem cell factor (SCF) and TGF-β. We also show that MC chemotactic activity during pollen season is significantly increased compared to before pollen season. Furthermore, we demonstrate that TGF-β isoforms, CCL5, TNF-α and IL-4 act as MC chemoattractants in a bellshaped dose- dependent manner. TGF-β proved to be an extremely potent attractant giving an optimal migratory response at 40fM and TGF-β3 being the most effective isoform. The chemokine CCL5 induced migration through interaction with the receptors CCR1 and CCR4 expressed on MC. Furthermore, we also found that TNF-α produced by T_H1-lymphocytes and IL-4 produced by T_H2-lymphocytes are MC chemoattractants.</p><p>In conclusion, with this thesis we have identified six new human mast cell chemoattractants and provide evidence that BAL fluid and synovial fluid from patients with asthma and rheumatoid arthritis, respectivly, contain MC chemoattractants. This information provides important clues in understanding the mechanisms behind MC recruitment to sites of inflammation.</p>

Glioblastoma multiforme (GBM) is the most common form of primary brain tumor in humans. Its aggressive and infiltrative growth into the brain, and, at best, only partial sensitivity to radiotherapy and chemotherapy, renders it extremely difficult to treat and survival remains dismal. Growth factors, such as platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), and their corresponding receptors are seen in glioma tissue, suggesting the presence of autocrine stimulatory loops. PDGFB and a mutated EGF receptor were also identified as cellular homologues of two oncoviruses, thereby indicating a role in tumorigenesis. This thesis presents a brain tumor model in mice, developed using a PDGFB coding retrovirus to induce overexpression of PDGF-B in neonatal mouse brain. Immature tumors, with histological characteristics of primary brain tumors developed at relatively high frequencies. Mice injected with a non-coding retrovirus did not develop tumors, indicating the crucial role of PDGF stimulation in this system. Tumor cells were also shown to continue to depend on PDGF stimulation when cultured in vitro. In human glioblastomas, growth factor receptor signaling is present in conjunction with defects in cell cycle arrest pathways. When the PDGFB-virus model was used with p53 or Ink4a-Arf deficient mice, tumors arose with shorter latency and higher frequency. Loss of p53 or Ink4a-Arf seemed to facilitate signaling through the PI3K/Akt pathway. Thus, a functional role for the co-existence of p53 loss of function and PDGF signaling in a subset of gliomas is presented. Human GBM samples were collected and analyzed with respect to expression and activation of the EGFR and PDGFRα. Most tumors expressed the both receptors at moderate to high levels, but high activation of either receptor seemed mutually exclusive.

Mast cells (MCs) are forceful multifunctional effector cells of the immune system. MCs are normally distributed throughout connective and mucosal tissues, but in several pathological conditions accumulation of MCs occur. This accumulation is probable due to directed migration of MCs and they are subjects for migration at least two different occations: 1) when they are recruited as progenitor cells from the blood into the tissue; and 2) when they as mature MCs are recruited to sites of inflammation. The aim of this study was to investigate MC migration to chemoattractants released in vivo or in vitro (body fluids collected from patients with asthma or rheumatoid arthritis and TH1- and TH2-cytokines) and to recombinant cytokines (transforming growth factor -β (TGF-β) and CCL5/RANTES). This thesis shows that bronchoalveolar lavage (BAL) fluid from asthmatic patients and synovial fluid from patients with rheumatiod arthritis contain MC chemoattractants, and that part of the chemotactic activity can be related to the presence of stem cell factor (SCF) and TGF-β. We also show that MC chemotactic activity during pollen season is significantly increased compared to before pollen season. Furthermore, we demonstrate that TGF-β isoforms, CCL5, TNF-α and IL-4 act as MC chemoattractants in a bellshaped dose- dependent manner. TGF-β proved to be an extremely potent attractant giving an optimal migratory response at 40fM and TGF-β3 being the most effective isoform. The chemokine CCL5 induced migration through interaction with the receptors CCR1 and CCR4 expressed on MC. Furthermore, we also found that TNF-α produced by TH1-lymphocytes and IL-4 produced by TH2-lymphocytes are MC chemoattractants. In conclusion, with this thesis we have identified six new human mast cell chemoattractants and provide evidence that BAL fluid and synovial fluid from patients with asthma and rheumatoid arthritis, respectivly, contain MC chemoattractants. This information provides important clues in understanding the mechanisms behind MC recruitment to sites of inflammation.

<p>This study was carried out to determine the phenotype of special dog mammary gland tumours that were grown in nude mice. 26 tumours were examined by the immunohistochemical ABC-Elite protocol. The tumour tissues were labelled with following anti-human antibodies:</p><p>- AE1/AE3 (pankeratin antibody) labelled epithelial and myoepithelial cells</p><p>- CD 31 labelled endothelial cells</p><p>- desmin labelled cross-striated and smooth muscle cells</p><p>- myosin labelled cross striated muscle cells</p><p>- neurofilament (NF) labelled nerve cells</p><p>- osteopontin labelled preosteoblasts, osteoblasts and osteocytes</p><p>- p63 labelled nuclei of the myoepithelial cells</p><p>- smooth muscle actin (SMA) labelled the cytoplasm of myoepithelial cells</p><p>- type I collagen labelled the extracellular matrix in connective tissue and bone</p><p>- type II collagen labelled the extracellular matrix in cartilage</p><p>- vimentin labelled fibroblasts, fibrocytes, lipocytes, smooth muscle cells, endothelial cells, nerve cells, macrophages and myoepithelial cells</p><p>The tumours were also submitted to a double immunolabelling study using p63 and SMA.</p><p>The study could not give a final conclusion about the origin the tumours. There was still need for more research to answer that question. However, the immunohistochemical technique was analysed in detail, in order to obtain perfect labelings.</p><p>Initially, all the antibodies were tested on normal dog tissue, to acquire the best working dilutions with the lowest background problems. In the tumours, good results were obtained with these dilutions for the antibodies p63, SMA, vimentin, desmin, NF, AE1/AE3 and CD 31. Except for type I collagen, type II collagen and osteopontin that gave too much unspecific labelling of the mouse connective tissue. Even, when using the Vector® M.O.M. blocking kit, the results were still very difficult to interpretate.</p><p>The antigen retrieval methods were evaluated for all the antibodies. The antibodies p63, SMA, vimentin, desmin, AE1/AE3, myosin, neurofilament and CD 31 needed the antigen retrieval treatment. The antibodies type I collagen and type II collagen needed the treatment with the enzyme pepsin, while osteopontin did not need any pretreatment at all.</p><p>The double immunolabelling with p63 and SMA gave excellent results. Different combinations were tried out with different substrates, namely Vector® Nova RED, Vector® DAB and Vector® SG. Vector® methyl green was used as counterstaining, but it interfered with the other substrates, and better results were obtained without this counterstaining.</p>

The human p63 gene discovered in 1997 encodes a series of protein isoforms that differ in their N- and/or C-terminal sequences. These isoforms have widely differing properties in promoting or repressing p53-related functions such as growth arrest and apoptosis. In addition, p63 appears to play important roles in the maintenance and differentiation of epithelial cell populations. Many studies have shown that p63, particularly Np63, is expressed in normal epithelium and also highly expressed in squamous cell carcinomas of surface epithelia. Methods. We have refined the analysis of the expression patterns of p63 isoforms by the use of a quantitative RT-PCR technique applied to micro-dissected normal oral mucosal samples. We have also studied p63 expression in squamous cell carcinoma of the head and neck (SCCHN) compared to normal oral mucosa taken from the same patient. Furthermore, tobacco-exposed buccal mucosa was compared to age and gender matched non exposed controls. RT-PCR for telomerase and immunohistochemical analysis for detection of p53 and Ki-67 proteins was further performed. We also explored the function of p63 in SCCHN cells by using small interfering RNA (siRNA) to silence the expression of different p63 isoforms in cell lines originating from SCCHN. The effect of p63 knockdown was studied using a Fluorescein Diacetate based cytotoxicity assay and immunohistochemistry looking at expression of differentiation markers. The response of the siRNA treated cells to radiation and cytostatic drug was also investigated. We have further studied normal oral wound healing using immunohistochemistry and quantitative PCR. By the use of a macro array comparing siRNA treated cells with non-treated cells a possible connection between the BRCA1 gene and p63 expression was shown and further studied with the use of cHIP and luciferase reporter assays. Results. The p63 isoforms are expressed in normal epithelium, with the highest levels consistently found in basal and parabasal layers. Extensive use of tobacco had no effect on p63. The quantitative PCR showed statistically increased levels of the ΔNp63 and p63isoforms. No correlation was found between p63-isoform expression patterns and proliferation. The exploration of the function of p63 in SCCHN cells by the use of small interfering RNA (siRNA) showed a statistically significant decreased survival for tumour cells when all p63 isoforms, the N-terminal truncated or the  isoforms were inhibited. No effect on cell proliferation or expression of epithelial differentiation markers was observed. We also demonstrated that inhibition of p63 expression sensitizes cells to the effects of ionizing radiation and cisplatin. The study of normal oral wound healing using immunohistochemistry and quantitative PCR showed significant changes in p63 isoform expression. The Np63 isoform was mainly expressed in the basal layer in the non proliferating and migrating cells while TAp63 was almost absent. The BRCA1 study showed p63 to bind to the BRCA1 promoter and activate the expression of BRCA1 protein. Summary. The p63 proteins have different functions and the balance between the isoforms and their localisation within the epithelium seems to be important for normal wound healing as well as cancer cell survival.

Background: The p63 gene is a member of the p53 transcription factor family and can produce six different proteins using two promoters and differential splicing. Expression of p63 is required for proper formation of epithelial tissues. Studies on the transcriptional control of specific genes involved in cell survival, proliferation, differentiation and adhesion have revealed the contributions of p63 to the continuously renewing stratified epithelium. In this thesis, the aim was to improve our understanding of the roles of p63 in epithelial homeostasis by investigating expression of p63 in normal and benign hyper-proliferative epithelia and exploring the influence of p63 deregulation on cancer progression. Materials and methods: Using quantitative real time RT-PCR and immunohistochemistry, we first examined the expression of different p63 isoforms in patients diagnosed with psoriasis - a benign hyper-proliferative and inflammatory skin disease. Afterwards, we investigated responses of p63 in psoriatic epidermis upon Narrowband-UVB (NB-UVB) phototherapy. At the same time, we studied the potential impact of p63 in carcinogenesis by searching for p63 transcriptional targets in a cell line derived from squamous cell carcinoma of the head and neck (SCCHN) - the sixth most common cancer worldwide with over-expression of the ∆Np63α protein as a common feature. p63 gene silencing and microarray were used to identify p63 regulated genes. Real time RT-PCR, western blot, immunohistochemistry, chromatin immunoprecipitation, transient transfection and reporter assays were performed to confirm specific genes as direct p63 targets. Results: Significant down-regulation of p63 mRNA levels was found in psoriatic lesions compared to patients’ own clinically normal skin. Moreover, a trend of decreased TAp63 mRNA levels was seen in patients’ normal skin compared to age- and sex-matched healthy controls. Following NB-UVB phototherapy, an effective first line therapy for psoriasis, expression of p63 was not significantly affected. However, significant changes in p53, FABP5, miR-21 and miR-125b were found. Surprisingly, location and expression levels of p63 proteins detected by immunohistochemistry were similar under all skin conditions. A direct transcriptional regulation of TRAF4 by p63 was seen in the SCCHN cell line and we further found that the localization of the TRAF4 protein was associated with histological differentiation of SCCHN cells. However, unlike its over-expression in SCCHN, similar TRAF4 mRNA expression levels were seen in psoriatic lesions as compared to healthy controls. Besides TRAF4, a total of 127 genes were identified as potentially p63 regulated in the SCCHN cell line and strikingly, about 20% of these genes are involved in cell adhesion or migration. Conclusions: Dysregulation of p63 isoforms in psoriatic epidermis, especially decreased TAp63 expression, and their resistance to NB-UVB phototherapy implicated a contribution of p63 to the psoriasis phenotype. Transcriptional regulation of genes involved in multiple biological pathways indicated that over-expression of p63 in SCCHN might account for altered cell differentiation, adhesion and migration, thus contributing to SCCHN. In conclusion, our studies have found additional mechanisms through which p63 guarded homeostasis of the established epithelium. Deregulation of p63 might play a role in distinct pathological conditions by participating in diverse cellular pathways under different microenvironments.