Le mélanome cutané est l'un des cancers les plus agressifs et mortel capables de dissémination métastatique à distance. L’intravasation cellules tumorales dans le vaisseau sanguin dans les tumeurs primaires et l'extravasation sont des étapes importantes dans la formation de métastases. Ces étapes impliquent la perturbation de la barrière endothéliale par des cellules tumorales afin de faciliter leur migration transendothéliale et la colonisation métastatique. Cependant, le processus par lequel les cellules tumorales modulent l'intégrité des jonctions vasculaires est encore mal compris. Afin de déterminer les facteurs de perméabilité sécrétés par les cellules métastatiques, nous avons identifié la protéine matricielle SPARC comme un facteur critique contribuant à la perméabilité vasculaire et l'extravasation des cellules tumorales. Nous montrons que SPARC sécrété par les cellules de mélanome induit une perméabilité vasculaire via l'ouverture des jonctions intercellulaires des monocouches endothéliales et entraîne la migration transendothéliale de cellules de mélanome. In vivo, l’extinction de SPARC mène à une diminution drastique dans la colonisation à court et long terme des poumons et de la perméabilité des capillaires pulmonaires. A l’inverse, sa surexpression augmente les capacités d’extravasation et les métastases. / Cutaneous melanoma is one of the most aggressive cancers capable of distant and lethal metastatic spread. Tumor cell intravasation into blood vessel at primary tumor sites and subsequent extravasation are critical steps in the formation of metastases. These steps entail disruption of the endothelial barrier by tumor cells to facilitate their transendothelial passage and metastatic seeding. However, the way by which tumor cells modulate vascular junction integrity is still poorly understood. In an attempt to determine permeability factors secreted by metastatic cells, we identified the matricellular protein SPARC as a critical signaling factor that contributes to elevated vascular permeability and tumor cell extravasation. We show that SPARC released by melanoma cells enhances vascular leakiness by inducing opening of intercellular junctions of endothelial monolayers and drives melanoma cell transendothelial migration. In vivo vascular permeability and metastatic assays demonstrate that SPARC deficiency abrogates tumor-induced permeability of lung capillaries and prevents extravasation from blood vessels and metastasis, whereas overexpression of SPARC increases the lung metastatic potential of melanoma cells. Mechanistically, SPARC-induced endothelial gap formation and transmigration is dependent on vascular cell adhesion molecule (VCAM1) and p38 MAPK signaling pathway in endothelial cells. Importantly, blocking VCAM1 impedes melanoma cell extravasation. The clinical relevance of our findings is highlighted by the high levels of SPARC detected in tumor cells from human pulmonary melanoma lesions.

Non-melanoma skin cancer (NMSC) is the most common cancer in the world with a lifetime risk for development as high as 2 in 3 in Queensland, Australia. Mortality is quite low, representing an approximate 360 deaths in Australia annually but cost of treatment is extremely high, estimated at $232 million each year. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) are the two most common forms of NMSC. Although BCC generally do not have the propensity to metastasise, they are highly invasive and can be locally destructive. SCC on the other hand is invasive and has metastatic potential. SCC is generally derived from a precursor lesion, solar keratosis (SK), which is also considered to be a biomarker of BCC, SCC and malignant melanoma. According to one theory, SKs actually represent the first recognisable stage of SCC development and therefore may be indicative of the earliest stage of NMSC development. In addition to these common forms of NMSC, rarer forms such as keratoacanthoma (KA), which spontaneously regress, and SCC in situ, which rarely become invasive, may provide clues into protective mechanisms associated with prevention of development. Like all other cancers, NMSC arises from an accumulation of genetic abnormalities that result in severe cellular dysfunction. A number of genes have been proposed in the development of NMSC, including p53, CDKN2a, Bcl-2 and the Ras family of genes, which are typically associated with proliferative and differentiation processes. Also, a number of genetic disorders that predispose individuals to NMSC have also been identified. Genetic abnormalities in these genes may be a result of somatic mutations that may be promoted by environmental carcinogens. For NMSC, ultraviolet (UV) radiation is the primary environmental stimulus that acts upon skin to generate mutations. UV effects are 2-fold; the first being direct damage produced by UVB radiation and the second being indirect damage as a result of UVA-induced oxidative stress. In addition to mutations of genes that directly result in carcinogenesis, polymorphic variants of genes may also play a role in susceptibility to NMSC. These susceptibility genes may have immunogenic, detoxifying or transcriptional roles that could be involved in increased mutagenesis or activation of cancer causing genes. The purpose of this study was ultimately to identify further molecular based mechanisms associated with the development of non-melanoma skin cancer. Initially, this study aimed to examine the effects of aberrant chromosomal regions on NMSC development and also to identify candidate genes within these regions that may be implicated in the development and progression of NMSC. Also, based on chromosomal and functional implications, a number of candidate genes were assessed using association analysis to determine their involvement in susceptibility to the earliest stages of NMSC development. Implicated susceptibility genes were then further investigated to determine their response to UV radiation. Therefore the methodological approach of these studies was based on three broad technical applications of cytogenetic, association and expression analyses. Previous comparative genomic hybridisation (CGH) studies implicated the 18q chromosomal region in progression of SK to SCC and this region was therefore suspected of harbouring one or more tumour suppressor genes that were associated with a more malignant phenotype. Following on from this analysis, loss of heterozygosity (LOH) analysis was used for further delineation of this region and possibly to implicate candidate genes involved in progression. Additionally, CGH was used to investigate keratoacanthoma to determine aberrant regions that might be involved in progression and also regression of this NMSC. Genes that had potential functional roles in NMSC development and that were located in or near regions implicated by these cytogenetic analyses were further investigated using association analysis. Association analysis was performed using polymerase chain reaction and subsequent restriction enzyme digestion or GeneScan analysis to determine genotype and allele frequencies in an SK affected versus control population for polymorphisms within a number of candidate genes. This population was carefully phenotyped so that not only genotypic factors could be analysed but also their interaction with a number of phenotypic and environmental risk factors. Genes with polymorphisms that did show association with solar keratosis development were then examined functionally. Specifically, gene expression analysis was undertaken to investigate their response to UV radiation. Both UVA only and combined UVA/UVB treatments were used for short term irradiation and also for long term irradiation with recovery to determine differential effects of UV range and dose in human skin. Relative mRNA expression analysis of these genes was performed using quantitative real time reverse transcription polymerase chain reaction to determine if UV radiation imposed gene expression changes in the skin. A combination of these methodologies provided a wide basis for investigation of NMSC. Cytogenetic, association and expression analyses all allow for the identification of molecular risk factors that cause or are associated with NMSC development and progression. These analyses provided diverse results that implicated various molecular mechanisms in the development of NMSC. Cytogenetic analysis is a powerful technique, especially for the identification of a broad range of aberrations throughout the genome. This study employed LOH analysis to investigate an implicated region involved in progression to SCC and to attempt identification of candidate genes that may be involved in this process. LOH analysis was successfully performed on 9 SCCs, 5 SCCs in situ and 2 SKs using 8 microsatellite markers within the 18q region. Polymerase chain reaction (PCR) was used to amplify polymorphic regions of these markers and genotypic composition was determined for normal and cancerous tissue within the specimen. In heterozygote individuals, determined by analysis of normal tissue, the cancerous tissue was examined to determine if alleles within the implicated region had been lost. However, after analysis of multiple different samples, there was no LOH detected in any of the samples examined for this analysis. This does not necessarily reject a role for 18q, or genes within this region, as the localisation of candidate tumour suppressor genes within a small region may indicate a tighter region of involvement than was expected. As such, a more targeted study may further delineate this region and implicate candidate genes in progression of SK to the more malignant phenotype of SCC. Further CGH analysis of keratoacanthoma was also undertaken to identify aberrations associated with development and also regression of this skin cancer. CGH was performed using universal amplification and nick translation to incorporate a fluorescent dye. Differentially labelled normal and tumour DNA were then competitively hybridised to a normal metaphase spread and fluorescence emission indicated either amplification or deletion of specific chromosomal regions. In total, 6 KA samples were analysed, with 2 samples each from evolving, matured and regressing stages of KA development. In general, regressing KAs appeared to be more highly associated with deleted regions than evolving and matured KAs. Specifically, the 15q chromosomal region that was deleted in regressing KAs but amplified in evolving or matured KAs, may be significantly involved in the process of KA regression. Also various candidate genes that were being considered for analysis were located in or near some of these implicated regions, including GSTM1, GSTP1 and SSTR2. As such, these candidate genes were targeted for further investigation. A number of susceptibility genes that were located in or near aberrant regions implicated in NMSC development were investigated using association analysis. These genes included members of the somatostatin receptor family (SSTR1 and SSTR2), members of the glutathione-S-transferase (GST) family (GSTM1, GSTT1, GSTP1 and GSTZ1) and the vitamin D receptor (VDR). Studies detected a number of interesting interactions between genetic, environmental and phenotypic factors in the development of the early stages of non-melanoma skin cancer. Additionally, genes implicated in NMSC development were further investigated using expression analysis to determine response to UV radiation. Association analysis was initially performed on members of the somatostatin receptor family. Somatostatin is a growth inhibiting factor, amongst other things, that mediates its actions through the somatostatin receptors (SSTRs). The presence of these receptors (SSTR1-5) in tumour cells indicates a potential for somatostatin to bind and suppress growth, as well as allowing for therapeutic treatment with somatostatin analogues. Additionally, expression of these receptors in normal tissue, including skin, should allow for potential protection against tumour growth. The genes for SSTR1 and SSTR2 have been shown to contain dinucleotide repeat polymorphisms, and although these polymorphisms may not directly result in altered expression or binding potential, they may be linked to another functional polymorphism that does. Using association analysis the SSTR1 and SSTR2 genes were investigated to determine whether they play a role in the development of solar keratosis. Results showed that there were no significant differences between SSTR1 and SSTR2 polymorphism frequencies in the tested solar keratosis population (P = 0.10 and P = 0.883, respectively) as compared to an unaffected population. Hence, these studies do not support a role for the SSTR1 or SSTR2 genes in solar keratosis development. Further association analysis and subsequent expression analysis was also performed on members of the glutathione-S-transferase family. The GST enzymes play a role in the detoxification of a number of carcinogens and mutagens, including those produced by UV-induced oxidative stress. This study examined the role of GSTM1, GSTT1, GSTP1 and GSTZ1 gene polymorphisms in susceptibility to SK development. Association analysis was performed to detect allele and genotype frequency differences in SK affected and control populations using PCR and restriction enzyme digestion. No significant differences were detected in GSTP1 and GSTZ1 allele or genotype frequencies, however polymorphisms within both genes were found to be in linkage disequilibrium, as previously reported, and a new allelic variant of the GSTZ1 gene was identified. Significant associations between GSTM1 (P = 0.003) and GSTT1 (P = 0.039) genotypes and SK development were detected, with the null variants of both genes conferring an approximate 2-fold increase in risk for solar keratosis development (OR: 2.1; CI: 1.3-3.5 and OR: 2.3; CI: 1.0-5.0 for GSTM1 and GSTT1, respectively). For the GSTM1 gene, this risk was significantly higher in conjunction with high outdoor exposure (OR: 3.4; CI: 1.9-6.3) and although the GSTT1 gene showed a similar trend (OR: 2.9; CI: 1.1-7.7), this did not reach significance. The increased risk of SK development associated with these genes is likely due to a decreased ability of the skin to detoxify mutagenic compounds produced by UV-induced oxidative stress, and hence a decreased ability to protect against carcinogenesis. Implication of the GSTM1 and GSTT1 null variants in solar keratosis development prompted interest in analysis of gene expression changes in response to UV radiation. Due to the high homology of the GSTM1 gene with other GSTM genes, and therefore potential issues with primer specificity, the GSTT1 gene was focussed on for the expression studies. Real time reverse transcription PCR, incorporating SYBR green fluorescence and 18S as a comparative gene, was used to study GSTT1 gene expression changes in response to both UVA and combined UVA/UVB radiation. It was found that only short term UV radiation had an effect on GSTT1 expression changes, whereas no alteration of gene expression was seen after 4 and 12 hours of recovery from long term irradiation between irradiated and matched non-irradiated skin samples. This indicated that changes in gene expression for the GSTT1 gene apparently occur relatively quickly after exposure to UV radiation. Analysis of both UVA only and combined UVA/UVB short term irradiation indicated that an initial decrease in expression, followed by an increase was likely to represent translation into protein and subsequent transcription of mRNA, and in some cases a second decrease indicated further translation. Hence, it appears as though UV radiation does have a significant effect on the expression of at least one GST gene, and that UV radiation in combination with genetic variation of these genes may play a role in the development of NMSC. Finally, association and subsequent expression analysis was also performed on the vitamin D receptor. The hormonal form of vitamin D, 1a25 dihydroxyvitamin D3, has been shown to have numerous cancer-related effects, including antiproliferative, differentiation, proapoptotic and antiangiogenic effects. These effects are mediated through the binding of 1a25 dihydroxyvitamin D3 to the vitamin D receptor and subsequent transcriptional pathways. Polymorphisms within the VDR are known to regulate its transcription and therefore expression, which is linked to the ability of 1a25 dihydroxyvitamin D3 to bind. Association analysis of a 5’ initiation codon variant (Fok I) and two 3’ variants (Apa I and Taq I) was performed in SK affected and control populations. Although the Fok I variant showed no association with SK development, both the Apa I and Taq I variants were found to be associated with SK development (P = 0.043 and P = 0.012, respectively). In particular, the Aa and Tt genotypes were associated with increased risk of SK. These results were however more complicated, as shown by further analysis. This showed that genotypes containing at least one allele that conferred decreased VDR transcription (ie. AA/Aa and Tt/tt) increased risk of SK development by 2-fold in fair skinned individuals (OR: 2.1; CI: 1.2-3.7 and OR: 1.7; CI: 1.1-2.7 for Apa I and Taq I variants, respectively) but also found to decrease the risk of SK development by 2-fold in medium skinned individuals (OR: 0.5; CI: 0.3-1.0 for Apa I variants). Additionally, genotypes containing 2 alleles conferring decreased transcription of the VDR gene were found to further increase the risk for SK development in fair skinned individuals (OR: 2.5; CI: 1.4-4.5 and OR: 2.4; CI: 1.2-5.0 for Apa I and Taq I variants, respectively), indicating a possible additive effect for the alleles. The highly differential association of the VDR gene polymorphisms amongst phenotypes may reflect a combination between the ability of an individual to synthesise 1a25 dihydroxyvitamin D3 with the binding availability of the VDR. To further investigate the role of VDR in NMSC, expression analysis of the VDR gene was undertaken using real time reverse transcription PCR, with SYBR green fluorescence and 18S as a comparative gene, to examine expression pattern changes associated with UV radiation. It was found that short term irradiation, as well as long term irradiation and recovery were associated with gene expression changes. Short term irradiation resulted in patterns indicative of translation and subsequent transcription, whereas long term irradiated samples resulted in reduction of VDR expression that was recovered after an extended period of time. Thus, VDR expression is clearly influenced by UV exposure. It would be very interesting to see more specifically if particular VDR genotypes, which appear to play a role in NMSC risk, also are affected differentially by UV exposure. It is possible that VDR expression is reduced to limit excessive binding of 1a25 dihydroxyvitamin D3, although since both UVA and UVB radiation affect VDR expression, this may not be mediated the effect of 1a25 dihydroxyvitamin D3 but rather a different pathway resulting from a general UV response. In summary, the detection of a number of susceptibility genes involved in SK development and their subsequent expression analysis in response to UV radiation has given further insight into the molecular changes associated with NMSC. In fact, both detoxification genes (GSTM1 and GSTT1) and a transcription related gene (VDR), were found to confer susceptibility to solar keratosis, an early stage skin lesion with tumourigenic potential. This suggests that even the earliest stages of skin cancer are mediated through a wide range of effects. Additionally, expression changes related to these genes indicate that they are associated with the well known environmental carcinogen of UV radiation and that their effects may be mediated through a wide range of pathways. Although implication of the 18q region in SCC progression was not confirmed in this study, it is still likely to play a role in malignant transformation. The implication of this region, as well as the implication of susceptibility genes has vastly increased knowledge into processes associated with NMSC. Although additional analysis can confirm and further implicate these molecular alterations, this study has resulted in a more comprehensive understanding of NMSC that may ultimately be of benefit in terms of prognosis and treatment.

In order to study the incidence of Estrogen Receptors (ER) in breast carcinoma, lung carcinoma and melanoma, an in situ hybridisation technique for ER mRNA (ER mRNA-ISH) was developed. Various technical aspects of the procedure including tissue fixation, hybridisation conditions, and demonstration technique were investigated in order to obtain an optimum technique for routine use. ISH results were compared with ER immunohistochemistry using the monoclonal antibodies ER1D5 and D5. Commercially available biotin labelled antisense oligonucleotides to ER, Poly A (total mRNA), and sense chromogranin (negative control) were applied to frozen and formalin-fixed paraffin sections of breast carcinomas. For frozen sections, various fixatives including formalin, alcohol, Schoobridge, Zamboni's and acetic- alcohol were compared. A direct streptavidin- eroxidase and an indirect demonstration method using anti-biotin were also compared. The effect of differing formamide concentrations and post hybridisation stringency washings were analysed. An optimised ISH technique was then applied to frozen sections of 21 cases of breast carcinoma and 11 cases of lung carcinoma. Results were compared to H222 staining on adjacent sections. The ISH technique was also optimised for use on formalin-fixed, paraffin-embedded sections of 28 breast carcinomas and 17 melanomas. The results were compared with ER1D5 and D5 immunohistochemistry done on adjacent sections. The occurrence of endogenous biotin was also studied on a range of normal tissues. Consistent ISH results were obtained when formamide was omitted from the hybridisation cocktail, high stringency post hybridisation washes were discarded, room temperature hybridisations and an indirect demonstration method were used. Fixation of frozen sections in acetic/ethanol gave more consistent results with good morphology and resulted in positive nucleolar staining in 90% of breast and 45% of lung carcinomas. Positive nucleolar staining was also present in frozen sections of one metastatic melanoma. In formalin fixed paraffin sections, acid hydrolysis and pronase treatment were required prior to ISH. Cytoplasmic and/or nucleolar ER mRNA-ISH staining was seen in 87% of breast carcinoma and 97% of melanoma studied. ER1D5 was present in 54% of breast carcinomas but was absent in all melanomas. D5, on the other hand, was found in 88% of the melanomas. In conclusion, ER mRNA-ISH can be successfully done on acetic/alcohol fixed frozen sections and formalin fixed paraffin sections. Formamide, high stringency washes and elevated hybridisation temperatures are detrimental to a successful ISH reaction and an indirect demonstration method (using anti-biotin) is preferred. Unfortunately, endogenous biotin can cause false positive ISH reactions and needs to be considered during interpretation. Results show that the localisation of ER mRNA in the nucleolus is specific. Both ER mRNA-ISH and ER immunohistochemistry indicate that melanomas and some lung carcinomas contain a receptor possibly similar to that in breast carcinomas. / Thesis (M.Sc.)--Department of Anatomical Sciences, 2004.

Increased expression of autotaxin has been shown to promote metastasis formation and cancer proliferation. These actions could be related to the catalytic activity of autotaxin which converts lysophosphatidylcholine into lysophosphatidate extracellularly or non-catalytic functions of autotaxin may be responsible. Also both LPC and LPA have been reported to stimulate migration through their respective receptors. This work investigates the role of autotaxin in controlling the motility of two cancer cell lines. With the use of autotaxin inhibitors we were able to block LPC-induced migration. Knocking-down autotaxin secretion also blocked stimulation of migration by LPC. Autotaxin inhibitors abolished any migratory effects from media collected from autotaxin secreting cells. We determined that LPC alone is unable to stimulate migration. Also we did not observe non-catalytic effects of autotaxin on migration. This thesis provides strong evidence that the inhibition of autotaxin production or activity would provide a beneficial therapy in the prevention of tumour growth or metastasis in patients with autotaxin expressing tumours.

Angiogenesis, the formation of new blood vessels from pre-existing ones, is a crucial step in many pathologies, including tumor growth and metastasis. We developed an adenovirus vector allowing the 16K hPRL expression, an antiangiogenic factor, in situ. We show 16K hPRL inhibits oxygen-induced retinopathy in mice. Then, using the 16K-Ad vector, we investigated the ability of 16K hPRL to prevent metastatic spread through inhibition of angiogenesis. We show that 16K hPRL administered via adenovirus-mediated gene transfer inhibits tumor growth in a subcutaneous B16-F10 mouse melanoma model by reducing the size and width of tumor vessels. We also show, for the first time, that 16K hPRL considerably reduces the establishment of B16-F10 metastases in an experimental lung metastasis model. These results highlight a potential role for 16K hPRL in anticancer therapy for both primary tumors and metastases. In parallel, we have sought to identify in human 16K PRL (16K hPRL) a peptide that might be responsible for its antiangiogenic activity. Although the 16K fragments of the other three human PRL/GH-family members are also potently antiangiogenic, the sequence similarity of these fragments is low (around 35% similarity between all mammalian PRL/GH sequences). This led us to seek a peculiar common structural feature rather than a similar sequence. We demonstrate that all these fragments possess a 14-amino-acid sequence having the characteristics of a tilted peptide. We show for the first time that tilted peptides exert antiangiogenic activity. The tilted peptides of hPRL and hGH induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo. These antiangiogenic effects are abolished when the peptides hydrophobicity gradient is altered by mutation. We further demonstrate for the first time that the well-known tilted peptides of SIV gp32 and Alzheimers beta-amyloid peptide are also angiogenesis inhibitors. Taken together these results point to a potential new role for tilted peptides in regulating angiogenesis. Langiogenèse, la formation de nouveaux vaisseaux à partir de vaisseaux préexistants, joue un rôle important dans de nombreuses pathologies incluant la croissance tumorale et la dissémination des métastases. Nous avons développé un vecteur adénoviral (16K-Ad) nous permettant de produire le facteur antiangiogène hPRL 16K directement in situ. Une première analyse permettant détudier les effets de la hPRL 16K produite par lapproche adénovirale a été réalisée in vivo. Nous montrons tout dabord que la hPRL 16K produite est capable de prévenir la néovascularisation rétinienne dans un modèle murin de rétinopathie. Par la suite, en utilisant le vecteur 16K-Ad, nous montrons que la hPRL 16K peut inhiber le développement tumoral dans un modèle murin développant des tumeurs formées par les cellules de mélanome B16-F10 dans le tissu sous-cutané. Cette inhibition de la croissance tumorale est corrélée avec une diminution de la taille des vaisseaux. Nous montrons aussi, pour la première fois, que la hPRL 16K peut considérablement réduire létablissement des métastases B16-F10 dans un modèle expérimental de métastases se développant dans le poumon. Ces résultats soulignent le rôle potentiel de la hPRL 16K dans la thérapie anticancéreuse dirigée contre les tumeurs primaires et les métastases. Parallèlement à ces travaux, nous nous sommes attachés à identifier une région responsable de lactivité de la hPRL 16K. Partant du fait que les fragments 16K des trois autres membres de la famille humaine PRL/GH sont aussi de puissants facteurs antiangiogènes, malgré que leur similarité de séquence soit faible (environ 35% de similarité entre les séquences de la PRL et de la GH), nous avons recherché une caractéristique structurale commune partagée par ces différents fragments. Nous avons identifié un domaine susceptible d'adopter une structure en peptide oblique dans les séquences protéiques des fragments de 16 kDa de la famille PRL/GH. Leffet antiangiogène des peptides obliques de la hPRL 16K et de la hGH 16K est montré dans des expériences menées in vitro et in vivo. Nous montrons également leffet antiangiogène des peptides obliques du peptide de fusion gp 32 du virus SIV et du peptide b-amyloïde.

Every year approximately 2800 Swedes are diagnosed with malignant melanoma, the form of cancer that is most rapidly increasing in incidence in the Western world. The earlier we can identify and diagnose a malignant melanoma, the better is the prognosis. In Sweden, 155 000 benign naevi, harmless skin tumours or moles, are surgically excised each year, many of them because melanoma cannot be dismissed by non-invasive methods. The excisions result in substantial medical costs and cause unrest and suffering of the individual patient. For untrained physicians, it is often difficult to make an accurate diagnosis of melanoma, thus a tool that could help to strengthen the diagnosis of suspected melanomas would be highly valuable. This thesis describes the development and assessment of a non-invasive method for early skin cancer detection. Using near infrared (NIR) and skin impedance spectroscopy, healthy and diseased skin of various subjects was examined to develop a new instrument for detecting malignant melanoma. Due to the complex nature of skin and the numerous variables involved, the spectroscopic data were analysed multivariately using Principal Component Analysis (PCA) and partial leas square discriminant analysis (PLS-DA). The reproducibility of the measurements was determined by calculating Scatter Values (SVs), and the significance of separations between overlapping groups in score plots was determined by calculating intra-model distances. The studies indicate that combining skin impedance and NIR spectroscopy measurements adds value, therefore a new probe-head for simultaneous NIR and skin impedance measurements was introduced. Using both spectroscopic techniques it was possible to separate healthy skin at one body location from healthy skin at another location due to the differences in skin characteristics at various body locations. In addition, statistically significant differences between overlapping groups of both age and gender in score plots were detected. However, the differences in skin characteristics at different body locations had stronger effects on the measurements than both age and gender. Intake of coffee and alcohol prior to measurement did not significantly influence the outcome data. Measurements on dysplastic naevi were significantly separated in a score plot and the influence of diseased skin was stronger than that of body location. This was confirmed in a study where measurements were performed on 12 malignant melanomas, 19 dysplastic naevi and 19 benign naevi. The malignant melanomas were significantly separated from both dysplastic naevi and benign naevi. Overall, the presented findings show that the instrument we have developed provides fast, reproducible measurements, capable of distinguishing malignant melanoma from dysplastic naevi and benign naevi non-invasively with 83% sensitivity and 95% specificity. Thus, the results are highly promising and the instrument appears to have high potential diagnostic utility.

The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin. Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis. After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC’s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique. <b>Keywords:</b>single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras

Skin pigmentation is an important issue in most cultures. Until recently we have not understood the most important elements of pigmentation regarding detailed chemical structure. The synthesis of melanin is very complex, and although core enzymes, other important proteins, and parts of the melanin structure have been identified much information in this context awaits disclosure. The function of the melanocyte and the deposition of melanin pigments into the keratinocytes are very important in the protection against UV light. Melanin pigments consist of high-molecular structures often described as brown to black eumelanin and yellow to red pheomelanin. Eumelanin is photoprotective, whereas pheomelanin is believed to be carcinogenic after UV radiation. There is strong evidence that people of fair complexion with freckles who tan poorly are at higher risk of developing melanoma. These people have a higher pheomelanin to eumelanin ratio in their skin. Melanoma, one of the most widely spread cancers, is derived from melanocytes. There is accumulating evidence that pigment constitution is highly involved in the development of melanoma. We found that patients with advanced melanoma secrete substantial amounts of pigment structures into the urine, in particular those with diffuse melanosis. In subsequently performed experiments we purified these pigments and subjected the product to chemical degradation by either hydrogen peroxide oxidation or hydriodic hydrolysis. Several new chromatographic methods were developed for the structural analysis of these products. Structural analysis of new chromatographic peaks was performed. In conclusion, complex pheomelanin structures as well as low molecular weight pigments and free benzothiazoles have been identified in the urine of patients with melanoma and diffuse melanosis. The present thesis provides new insight into melanogenesis and melanoma progression. This opens the doorway to further approaches to the investigation of melanins and can help to understand fundamental problems about the structure and biosynthesis of natural melanins.

Malignant melanoma is the most lethal skin cancer with no effective therapeutic treatment in its metastatic stages. RAS and PI3K pathways have been shown to play a critical role in melanoma development and progression. In this study, we assessed the in vitro and in vivo inhibition potential of a BRAF inhibitor (Sorafenib, Bayer) and a PI3K/mTOR inhibitor (PI-103, PIramed-Genentech) in primary melanoma cell lines. We used primary cell lines isolated from spontaneous melanomas obtained in the UV induced HGF transgenic melanoma mouse model.Although PI-103 and sorafenib inhibited melanoma in vitro cell proliferation and viability, the inhibition of RAS pathway was more effective. The combination of the two drugs showed a synergistic effect inhibiting RAS and PI3K pathways and in vitro melanoma cell proliferation in a cell line dependent manner. However, the combined treatment of orthotopic xenographs in immunocompetent FVB mice did not cooperate blocking tumor growth. Surprisingly, the in vivo treatment with PI-103 enhanced tumor growth. Our results also revealed that PI-103 caused immunosuppression inducing thymus atrophy and upregulating the intratumoral transcriptional levels of inmunosuppressors. In addition, PI-103 induced the antiapoptotic BH3 family proteins Mcl-1, Bcl-2 and BclXL, which correlated with the lower apoptotic rate observed within the PI-103 treated tumors. These data indicates that due to melanoma heterogeneity, some precautions should be taken when using these inhibitors for treatment. Moreover, these results certainly make an argument for investigating unexpected effects of rational drug combinations on immunocompetent animal models before conducting clinical studies.

<p>The investigation of genetic alterations in cancer-relatedgenes is useful for research, prognostic and therapeuticpurposes. However, the genetic heterogeneity that often occursduring tumour progression can make correct analysischallenging. The objective of this work has been to develop,evaluate and apply techniques that are sufficiently sensitiveand specific to detect and analyse genetic alterations in skintumours as well as in normal skin.</p><p>Initially, a method based on laser-assisted microdissectionin combination with conventional dideoxy sequencing wasdeveloped and evaluated for the analysis of the p53 tumoursuppressor gene in small tissue samples. This method was shownto facilitate the analysis of single somatic cells fromhistologic tissue sections. In two subsequent studies themethod was used to analyse single cells to investigate theeffects of ultraviolet (UV) light on normal skin. Single p53immunoreactive and nonimmunoreactive cells from differentlayers of sunexposed skin, as well as skin protected fromexposure, were analysed for mutations in the p53 gene. Theresults revealed the structure of a clandestine p53 clone andprovided new insight into the possible events involved innormal differentiation by suggesting a role for allele dropout.The mutational effect of physiological doses of ultravioletlight A (UVA) on normal skin was then investigated by analysingthe p53 gene status in single immunoreactive cells at differenttime-points. Strong indications were found that UVA (even atlow doses) is indeed a mutagen and that its role should not bedisregarded in skin carcinogenesis.</p><p>After slight modifications, the p53 mutation analysisstrategy was thenused to complement an x-chromosomeinactivation assay for investigation of basal cell cancer (BCC)clonality. The conclusion was that although the majority ofBCC’s are of monoclonal origin, an occasional tumour withapparently polyclonal origin exists. Finally, apyrosequencing-based mutation detection method was developedand evaluated for detection of hot-spot mutations in the N-rasgene of malignant melanoma. More than 80 melanoma metastasissamples were analysed by the standard approach of single strandconformation polymorphism analysis (SSCP)/DNA sequencing and bythis pyrosequencing strategy. Pyrosequencing was found to be agood alternative to SSCP/DNA sequencing and showed equivalentreproducibility and sensitivity in addition to being a simpleand rapid technique.</p><p><b>Keywords:</b>single cell, DNA sequencing, p53, mutation,UV, BCC, pyrosequencing, malignant melanoma, N-ras</p>