Das maligne Melanom ist für 90 % der Sterbefälle bei Hautkrebs verantwortlich (Garbe et al., 2022). Verschiedene Mutationen wirken sich auf die Signalwege aus und fördern die Karzinogenese, was zu einem ständigen Wandel der Melanomtherapie führt. Zusätzlich zu der, in frühen Stadien kurativen, chirurgischen Therapie, erzielt die zielgerichtete Therapie mit BRAF- und MEK-Inhibitoren in den letzten Jahren durchschlagende Erfolge für das Gesamtüberleben der Patienten, bei nicht-resezierbaren Metastasen wird weiterhin die Chemotherapie empfohlen. Zunehmende Therapieresistenzen schränken die Melanomtherapie ein. Müller et al. (2020) weisen auf Wildtyp p53 exprimierende Tumorzellen hin, die mithilfe der Expression von nukleärer Caspase-8 den G2/M-Zellzyklusarrest umgehen und so die Proliferation und Expansion entarteter Tumorzellen fördern. Bei der Untersuchung von 16 Melanomzellproben, die sich in ihrem BRAF-/NRAS- und p53 Mutationsstatus, sowie in ihren klinischen Vortherapien unterscheiden, konnte die potenziell klinische Relevanz der nukleären Caspase-8 herausgearbeitet werden. Wir konnten bestätigen, dass nukleär lokalisierte Caspase-8 bei Wildtyp p53 exprimierenden Tumorzellen zu Chemoresistenz und auch in geringem Maß zur Resistenz gegenüber zielgerichteten Therapien führt. Zunächst wurde die subzelluläre Lokalisation von Caspase-8 in den vorhandenen 16 Melanomzellproben mithilfe immunzytochemischer Färbung bestimmt, wobei sich herausstellte, dass die Melanomzellproben mit nukleärer Caspase-8, alle von Metastasen stammen. Das hohe Metastasierungspotenzial dieser Zellproben wurde weiterhin dadurch betont, dass die Melanomzellproben mit nukleär lokalisierter Caspase-8 die stärkste Migration aufwiesen. Dem gegenüberstehend präsentierten sie die geringste Proliferation, was den direkten Zusammenhang zwischen migrativen und proliferativen Eigenschaften bei Tumorzellen unterstreicht. Ebenso zeigten BRAF-mutierte Zellproben die stärkste Migration und bekräftigen, dass Melanompatienten mit BRAF-Mutation früher Metastasen entwickeln. Das stärkste Wachstum zeigten hingegen die Zellproben mit diffus lokalisierter Caspase-8, was die protektive Relevanz der diffusen Caspase-8 für Tumorzellen verdeutlicht. Ein Einfluss des p53-Mutationsstatus auf die Proliferation und Migration konnte ebenfalls nachgewiesen werden. Die Zellproben mit Wildtyp p53 migrierten stärker, wohingegen die Zellprobe mit mutp53(E285K) stark proliferierte. Bei Korrelation der subzellulären Caspase-8-Lokalisation mit der Caspase-8- und p53-Expression der Melanomzellproben mithilfe Westernblotanalysen zeigte sich, dass metastatische Zellproben verstärkt Caspase-8 exprimieren. Dies betraf besonders die metastatischen Zellproben mit nukleär lokalisierter Caspase-8, die kein p53 oder wenig Wildtyp p53 exprimieren. Diese inverse Korrelation der Caspase-8- und p53 Expression konnte ebenfalls bei den unstimulierten Zellproben mit zytoplasmatischer Caspase-8 festgestellt werden. Zellprobe M31 mit mutp53(E285K) exprimiert konstant stark p53 und wies Chemoresistenz auf. Bei Untersuchung des Zelltods 24 h und 48 h nach Cisplatinstimulation, sowie 48 h nach Stimulation mit Dabrafenib, Trametinib und deren Kombination mithilfe des IncuCyte® Readers, wurde deutlich, dass die Zellproben mit nukleär lokalisierter Caspase-8 chemoresistenter sind als die restlichen Zellproben, wobei die Zellproben mit diffus lokalisierter Caspase-8 am resistentesten gegenüber den zielgerichteten Therapeutika sind. Die protektive Rolle der diffusen Caspase-8 für die Tumorzellen konnte unterstützend durch die Zunahme der Apoptose bei einigen Zellproben nach Herunterregulierung von Caspase-8 nachgewiesen werden. Hervorzuheben ist, dass die Zellproben mit zytoplasmatischer Caspase-8 am sensitivsten, sowohl auf zielgerichtete Therapeutika als auch auf Chemotherapie reagierten. Insgesamt induzierte Cisplatin deutlich mehr Zelltod als die zielgerichteten Therapien, weshalb die potenzielle Überlegenheit der Chemotherapie, besonders bei rezidivierten Melanomen bedacht werden muss. Es bestätigte sich ebenfalls die Relevanz des BRAF-/NRAS Mutationsstatus für die Therapiesensitivität, wobei BRAF-mutierte Zellproben die größte Chemosensitivität präsentierten und NRAS-mutierte Zellproben vor allem nach der Stimulation mit Trametinib und der Kombination Dabrafenib + Trametinib Zelltod aufwiesen. Überraschenderweise zeigten sich die Zellproben vortherapierter Melanompatienten sensitiver gegenüber Dabrafenib und Cisplatin als die Zellproben therapienaiver Melanompatienten, was die Bedeutung von Chemotherapie und Dabrafenib für vortherapierte, rezidivierte Tumore bzw. Metastasen hervorhebt. Weiterhin konnte der Impact des p53-Mutationsstatus herausgearbeitet werden. Die Zellproben mit mutiertem oder ohne p53 wiesen die größte Cisplatinresistenz auf, wohingegen die Zellproben mit Wildtyp p53 am meisten Zelltod zeigten, gefolgt von den Zellproben mit dem Einzelnukleotidpolymorphismus (wt(P72R)). Nach Herunterregulierung des mutierten p53 nahm die Apoptose nach Cisplatinstimulation hoch signifikant zu. Dahingegen zeigten besonders die Zellproben mit zytoplasmatischer Caspase-8 und Wildtyp p53 eine Hochregulierung von p53 nach Cisplatinstimulation im Westernblot. Wir konnten nachweisen, dass die Wildtyp p53 exprimierende Zellprobe M40 nukleär lokalisierte Caspase-8 aufweist und die stärkste Chemoresistenz zeigte, wohingegen die Zellproben mit diffus oder zytoplasmatisch lokalisierter Caspase-8, die Wildtyp p53 oder den Einzelnukleotidpolymorphismus (wt(P72R)) exprimieren, am chemosensitivsten waren. Die Therapieresponsivität von Melanomen wird durch eine Vielzahl an Faktoren beeinflusst, die für die optimale Behandlung der Patienten bei Beginn einer Therapie möglichst genau bestimmt werden sollten. Wir empfehlen für die Risikostratefizierung und Prognose des Krankheitsverlaufes, sowie für die Entscheidung des Therapiewegs, die Bestimmung der subzellulären Caspase-8-Lokalisation in Kombination mit dem p53-Mutationsstatus. Weiterhin weisen wir auf die Bedeutung des BRAF-/NRAS-Mutationsstatus sowie mögliche Vortherapien in Bezug auf Therapieresistenz hin und stellen die Bedeutung der Chemotherapie, besonders für rezidivierte, BRAF-mutierte Melanompatienten bei Resistenzentwicklung gegen BRAF- und/oder MEK-Inhibitoren heraus. / Malignant melanoma is responsible for 90 % of skin cancer deaths (Garbe et al., 2022). Various mutations affect the signaling pathways and promote carcinogenesis, which leads to a constant change in melanoma therapy. In addition to surgical therapy, which is curative in the early stages, targeted therapy with BRAF and MEK inhibitors has achieved resounding successes for the overall survival of patients in recent years while chemotherapy is still recommended for non-resectable metastases. Increasing therapy resistance limits melanoma therapy, with Müller et al. (2020) pointing to wild-type p53-expressing tumor cells that use the expression of nuclear caspase-8 to bypass the G2/M cell cycle arrest and thus promote the proliferation and expansion of degenerated tumor cells. By investigating 16 melanoma cell samples differing in their BRAF/NRAS and p53 mutation status, as well as in their prior clinical therapies, the potential clinical relevance of nuclear caspase-8 could be elaborated. We were able to confirm that nuclear localized caspase-8 in wild-type p53-expressing tumor cells leads to chemoresistance and, to a lesser extent, to resistance to targeted therapies. First, the subcellular localization of caspase-8 in the existing 16 melanoma cell samples was determined using immunocytochemical staining, which revealed that the melanoma cell samples with nuclear caspase-8 all originated from metastases. The high metastatic potential of these cell samples was further emphasized by the fact that the melanoma cell samples with nuclear localized caspase-8 showed the strongest migration. In contrast, they presented the lowest proliferation, underlining the direct correlation between migratory and proliferative properties in tumor cells. Likewise, BRAF-mutated cell samples showed the strongest migration and verified that melanoma patients with BRAF mutations develop metastases earlier. In contrast, the cell samples with diffusely localized caspase-8 showed the strongest growth, which illustrates the relevance of diffusely localized caspase-8 in its protective function in tumor cells. The impact of the p53 mutation status on proliferation and migration could also be demonstrated. The cell samples with wild-type p53 migrated stronger, whereas the cell samples with mutp53(E285K) showed stronger proliferation. Correlation of subcellular caspase-8 localization with caspase-8 and p53 expression of melanoma cell samples using western blot analysis showed that metastatic cell samples express more caspase-8. This was particularly true for the metastatic cell samples with nuclearly localized caspase-8, which express little wild-type p53 or no p53. This inverse correlation of caspase-8 and p53 expression was also observed in the unstimulated cell samples with cytoplasmically localized caspase-8, respectively. Cell sample M31 with mutp53(E285K) consistently expressed high levels of p53 and exhibited chemoresistance. When examining cell death 24 h and 48 h after cisplatin stimulation, as well as 48 h after stimulation with dabrafenib, trametinib and their combination using the IncuCyte® Reader, it became clear that the cell samples with nuclearly localized caspase-8 are the most chemoresistant, whereby the cell samples with diffusely localized caspase-8 are the most resistant to the targeted therapeutics. The protective role of diffusely localized caspase-8 for the tumor cells was supportively demonstrated by the increase in apoptosis in some cell samples after downregulation of caspase-8. It should be emphasized that the cell samples with cytoplasmically localized caspase-8 responded most sensitively to both targeted therapies and chemotherapy. Overall, cisplatin induced significantly more cell death than the targeted therapies, which is why the potential superiority of chemotherapy, especially in recurrent melanoma, must be considered. The relevance of BRAF/NRAS mutation status for therapy sensitivity was also confirmed, with BRAF-mutated cell samples presenting the greatest chemosensitivity and NRAS-mutated cell samples showing cell death particularly after stimulation with trametinib and the combination dabrafenib + trametinib. Surprisingly, the cell samples of pre-treated melanoma patients were more sensitive to dabrafenib and cisplatin than the cell samples of treatment-naive melanoma patients, which highlights the importance of chemotherapy and dabrafenib for pre-treated, recurrent tumors and metastases. Furthermore, the impact of the p53 mutation status could be worked out. The cell samples with mutated or no p53 showed the highest cisplatin resistance, whereas the cell samples with wild-type p53 showed the highest cell death, followed by the cell samples with the single nucleotide polymorphism (wt(P72R)). After downregulation of mutant p53, apoptosis increased highly significantly after cisplatin stimulation. In contrast, especially the cell samples with cytoplasmically localized caspase-8 and wild-type p53 showed an upregulation of p53 in the Western blot after cisplatin stimulation. We demonstrated that wild-type p53 and nuclear caspase-8 expressing cell sample M40 exhibited the strongest chemoresistance, whereas the cell samples with diffusely or cytoplasmically localized caspase-8 expressing wild-type p53 or the single nucleotide polymorphism (wt(P72R)) were the most chemosensitive. In summary, it is clear that the treatment response of melanoma is influenced by a variety of factors that should be determined as precisely as possible for the optimal treatment of patients at the start of therapy. We recommend the determination of subcellular caspase-8 localization in combination with the p53 mutation status for risk stratification and prognosis of disease progression, as well as for the optimal therapy. Furthermore, we point out the importance of BRAF/NRAS mutation status and possible prior therapies with regard to therapy resistance and emphasize the importance of chemotherapy, especially for relapsed, BRAF-mutated melanoma patients who developed resistance to BRAF and/or MEK inhibitors.

Le mélanome malin est l’un des cancers les plus mortels dont l’incidence continue à augmenter chaque année avec peu de traitement efficace à long terme. Il est causé et initié principalement par l’exposition excessive aux rayons ultraviolets engendrant des photoproduits hautement génotoxiques. Il est bien connu que la cascade de signalisation PI3K/Akt joue un rôle crucial dans la régulation des processus qui sont généralement dérégulés durant le développement tumoral comme la prolifération, le contrôle du cycle cellulaire et l’apoptose. Néanmoins, l’implication de cette voie moléculaire dans la réponse aux dommages à l’ADN est peu caractérisée. Chez les mammifères, trois isoformes de la protéine kinase Akt ont été identifiées: Akt1, Akt2 et Akt3. Bien qu’elles soient très homologues en termes de séquence, plusieurs études ont montré que ces isoformes ont des fonctions biologiques distinctes, et nous suggérons qu’elles puissent contribuer différemment à la régulation de la réponse génotoxique. Les objectifs de ce projet étaient de: (i) évaluer l’activation d’Akt dans les cellules de mélanomes (ii) déterminer l’impact de l’inhibition de cette activité sur la régulation de la réponse cellulaire aux UV (iii) vérifier si la perte d’expression de l’un ou de l’autre des isoformes d’Akt peut réguler la réponse aux UV. Nous avons démontré qu’Akt est transitoirement hyperactivée par phosphorylation suite aux irradiations UV dans les lignées cellulaires de mélanomes. Afin de déterminer l'importance de cette activation dans la réponse cellulaire aux UV, notre approche était de diminuer (i) la phosphorylation d’Akt par l’usage d’inhibiteurs pharmacologiques ou (ii) l’expression de chaque isoforme d’Akt par l’approche des ARN interférents. Nous avons montré que l’inhibition de la phosphorylation d’Akt amène à l’augmentation du taux de l’apoptose induit par les UV d’une manière isoforme spécifique, alors qu’elle n’a aucun effet sur la régulation de la voie de réparation par excision de nucléotides (NER), qui est la seule voie humaine pour éliminer les dommages à l’ADN induits par les UV. En somme, notre étude constitue un nouvel aspect qui permet de mieux comprendre les mécanismes moléculaires du développement de mélanomes malins suites aux irradiations ultraviolettes. / Malignant melanoma is one of the deadliest cancers whose incidence continues to rise each year with a few effective long-term treatments. It is caused and initiated mainly by excessive exposure to ultraviolet radiation generating highly genotoxic DNA photoproducts. It is well known that the PI3K/Akt signaling cascade plays a crucial role in the regulation of processes commonly deregulated in tumor development such as proliferation, cell cycle control and apoptosis. Nevertheless, the nuclear involvement of this molecular pathway in the genotoxic response is poorly characterized. In mammals, three Akt kinase isoforms have been identified: Akt1, Akt2 and Akt3. Although these exhibit a high degree of homology, several studies have shown that they have distinct biological functions; therefore, we suggest that these isoforms may contribute differently to the regulation of genotoxic response. The objectives of this project were to: (i) evaluate Akt activation in UV-irradiated melanoma cells, (ii) determine the effect of the Akt phosphorylation inhibition on the regulation of the cellular response to UV, (iii) evaluate whether the loss of the expression of one or more of Akt isoforms can regulate the cellular response to UV. We demonstrated that Akt undergoes transient hyperactivation after UV treatment in melanoma cell lines. To determine the importance of this activation, our approach was to reduce (i) the phosphorylation of Akt by the use of pharmacological inhibitors or (ii) the expression of each individual Akt isoform using RNA interference. We have shown that inhibition of Akt phosphorylation leads to increased rates of UV-induced apoptosis in an isoform specific manner, while exerting no effect on regulation of nucleotide excision repair (NER), the only human pathway for eliminating UV-induced DNA damage. In summary, our study provides a better understanding of the molecular mechanisms of malignant melanoma development in response to UV.

The motivation of this research is to propose innovative nonlinear and optimal control design algorithms, which can be used in real life. The algorithms need to be computationally efficient, should deal with control constraints and should operate under state feedback. To show the efficacy of algorithms, automatic therapy for different cancer problems is chosen to be the field of application. In this thesis, first an advanced control design technique called ’optimal dynamic in-version’ has been successfully experimented with control constraints. The proposed approach has subsequently been shown to be quite effective in proposing automatic drug delivery schemes with simultaneous application of chemo and immunotherapy drugs for complete elimination of cancer cells in melanoma (a skin cancer) as well as glioma (a brain cancer). As per the current practice, the amount of drug dosages are generally given based on some apriori statistical study with a very small sample size, which in reality may either also lead to drug toxicity (due to excessive drug) or may become ineffective (due to insufficient drug) for a particular patient. Subject to the fidelity of the mathematical model (which has been taken from published literature), it has been shown in this thesis that nonlinear control theory can be used for computation of drug dosages, which can then be used in a feedback strategy, thereby customizing the drug for the patient’s condition, to cure the disease successfully. Next, attention has been shifted to impulsive control of systems. Such impulsive con-trol systems appear in many other applications such as control of swings, control of spacecrafts and rockets using reaction control system, radiotherapy in cancer treatment and so on. Two impulsive control design philosophies are proposed in this thesis. In one approach, recently proposed model predictive static programming (MPSP) has been extended for impulsive control systems and has been named as impulsive-MPSP (I-MPSP). In other approach, another recent development, namely the Pseudospectral method has been utilized to consider both the magnitude of the control impulses as well as the time instants at which they are applied as the decision variables. It can be noted, that to the best of the knowledge of the author, the time instants of control application, being considered as decision variables is being proposed for the first time in the nonlinear and optimal control framework. Both I-MPSP and Pseudospectral methods are computationally quite efficient and hence can be used for feedback control (I-MPSP happens to be computationally more efficient than the Pseudospectral method). Applicability of the proposed extensions have been shown by solving various benchmark problems such as (i) a scalar linear problem, (ii) Van der Pol’s oscillator problem and (iii) an inverted pendulum problem. Finally the applicability of the proposed I-MPSP strategy has been shown by solving challenging problems such as radiotherapy treatment of head and neck and adenocarcimona cancers. Radio-therapy model is considered with oxygen effect, in which radiosensitivity parameters are considered in different forms. Head and neck cancer is considered with constant radiosensitivity parameters and adenocarcinoma is considered with constant, linear, quadratic and saturation model of radiosensitivity parameters. Note that toxicity constraints on normal tissue, which are nonlinear control constraints, are also successfully incorporated in this control design.

Made available in DSpace on 2014-08-20T14:37:54Z (GMT). No. of bitstreams: 1 tese_maria_cecilia_florisbal_dame.pdf: 53923 bytes, checksum: e82c085a8b324064158221bf46a754ba (MD5) Previous issue date: 2013-12-12 / This thesis is a continuation of a research project started with a diagnosis of dermatosis mechano-bullosa in a herd of buffaloes from a farm in southern Rio Grande do Sul. After this diagnostic it was created an experimental herd where several congenital defects and / or hereditary disorders have been diagnosed during more than two decades. These diseases were studied by a research group which the author of this thesis is a member. Thus, three papers are presented: the first one is a literature review about what has been diagnosed in Brazil on congenital defects and hereditary diseases in buffalo. It was concluded that undesirable genes are widespread in the population of buffaloes in the country; the second was a study conducted in partnership with UNESP / Jaboticabal, SP and UFCG / CSTR-Patos, PB to identify the mutation in the gene that determines the oculo-cutaneous albinism in Murrah buffalo. Another paper was accomplished to describe the occurrence of malignant melanoma in two albino buffalo. Although it is not a congenital or hereditary disease it was observed only in the buffalo with oculocutaneous albinism and probably the condition is associated with a predisposition of these animals to develop tumors in the skin. / Esta tese dá continuidade a um projeto de pesquisa iniciado com o diagnóstico de dermatose mecanobolhosa em 1985 em um rebanho de búfalos pertencente a uma propriedade da zona sul do Rio Grande do Sul. A partir desse diagnóstico foi criado um rebanho experimental no qual por mais de duas décadas foram diagnosticados diversos defeitos congênitos e/ou hereditários que foram estudados por um grupo de pesquisa do qual faz parte a autora desta tese. Assim sendo, são apresentados três artigos científicos: o primeiro trata-se de uma revisão bibliográfica sobre o que foi diagnosticado no Brasil em relação a defeitos congênitos/doenças hereditárias em bubalinos concluindo-se que alguns genes indesejáveis estão disseminados na população de búfalos no País; o segundo foi um trabalho realizado em parceria com a UNESP/Jaboticabal, SP e com a UFCG/CSTR-Patos, PB que identificou a alteração no gene que determina o albinismo óculo-cutâneo em búfalos da raça Murrah. O terceiro artigo trata-se da descrição de melanoma maligno observado em dois búfalos albinos. Embora não seja um defeito congênito ou hereditário este neoplasma ocorreu somente nos búfalos com albinismo óculo-cutâneo do rebanho e acredita-se que a condição esteja associada à predisposição desses animais a desenvolverem tumores de pele.

Beside tumor cells themselves, tumors consist of many non-malignantly transformed cellular elements and an extracellular matrix. This so-called tumor microenvironment, or stroma, significantly influences the biological properties of the tumor through intercellular interactions. In this thesis I have focused on the study of tumor-associated fibroblasts in squamous cell carcinomas of the head and neck, malignant melanoma and glioblastoma. The data show the presence of cells with mesenchymal characteristics, present even in the glioblastoma stroma, which could potentially have a positive effect on proliferative activity and invasiveness of glioblastoma cells. In malignant melanoma, the presence of keratinocytes should also be considered, as they are the major cells of the epidermis influencing tumor melanocytes. The conditioned medium from UVB irradiated keratinocytes and non-irradiated fibroblasts stimulates the invasion of malignant melanoma cells. Targeting the tumor stroma may be a new direction in oncological therapy, so we have focused on the influence of synthetic polyamine on the formation of myofibroblasts, which are an active part of the population of tumor-associated fibroblasts. The tested polyamine prevents the formation of myofibroblasts but has no effect on those already formed nor on...

Daten des humanen Genomprojektes zeigen, dass ca. 8% des gesamten humanen Genoms aus retroviralen Sequenzen besteht. Der überwiegende Teil dieser Proviren ist aufgrund verschiedener Mutationen defekt. Im Gegensatz zu allen anderen HERV Proviren scheinen einige HERV-K Proviren intakt zu sein und besitzen offene Leserahmen für alle viralen Proteine. Die Familie des humanen endogenen Retrovirus K HML2 umfasst ca. 30 eng verwandte Proviren. Zusätzlich zu den Strukturproteinen Gag und Env und der Reversen Transkriptase, exprimiert HERV-K zwei regulatorische Proteine, Rec und Np9. Beide sind im Nukleus lokalisiert und tumorigene Eigenschaften bzw. eine Expression in Assoziation mit Tumorgeweben wurde nachgewiesen. Neben Zelllinien, wie die Teratokarzinomzelllinie GH und einigen Brustkrebszelllinien, für die die Expression von HERV-K mRNA und die Produktion von Viruspartikeln bekannt ist, konnte die Expression von HERV-K Proteinen und Partikeln für Melanomzellen gezeigt werden. Volllängen mRNA von HERV-K war in allen untersuchten humanen Proben nachweisbar. Gespleißtes env und rec war in 39% der Gewebe und in 38% der Melanomzelllinien exprimiert. Zusätzlich werden HERV-H, -R und -W exprimiert. Von den auf spezifische Antikörper gegen HERV-K Proteine untersuchten Seren der Melanompatienten waren 16% positiv für das transmembrane Hüllprotein, jedoch reagierte kein Serum mit Re oder Np9. Da im Zuge der Entstehung von Tumoren immer auch eine Dedifferenzierung der entarteten Zellen diskutiert wird, wurde die Expression von HERVs in undifferenzierten, embryonalen Stammzellen bestimmt. In den untersuchten embryonalen Stammzellen lässt sich Volllängen mRNA, sowie gespleißte env, rec und np9 mRNA nachweisen. Während der Differenzierung zu neuronalen Vorläuferzellen sinkt die Expression jedoch wieder auf ein mit normalen Zellen vergleichbares Niveau. Obwohl gespleißte RNA und virale Proteine von HERV-K vor allem in Tumoren und Tumorzelllinien exprimiert werden, ist deren Funktion während der Tumorentstehung noch immer ungeklärt. Auch die Bedeutung der HERV-K Expression in humanen Stammzellen ist noch unklar, insbesondere in Hinblick auf eine mögliche Tumorigenität. / In contrast to all other human endogenous retroviruses, proviruses of the human endogenous retrovirus family HERV-K have maintained open reading frames for all viral proteins. Although most proviruses are defective, structural proteins Gag and Env, the reverse transcriptase and two regulatory proteins, Rec and Np9, have been described. Rec resembles the Rev protein of HIV and tumourigenic potential was confirmed. Np9 as well is located in the nucleus and expression in association with tumour tissues was observed. Additionally to cell lines known to produce HERV-K virus particles, such as the teratocarcinoma cell line GH and breast cancer cell lines, recently melanoma cells were described to express HERV-K proteins and particles. In order to study the expression of HERV-K, -H, -R and -W, in melanoma cell lines and biopsies primer sets were used. Antisera specific for HERV-K proteins were used for immunohistochemistry and sera from melanoma patients were investigated for HERV-K specific antibodies. Full length mRNAs of all HERVs were found in all human cells. Spliced env and rec of HERV-K were detected in 39% of the melanoma biopsies and in 38% of the melanoma cell lines. Expression of HERV-K in situ was shown by immunohistochemistry. In addition, 16% of the patients sera tested showed antibodies against the HERV-K transmembrane envelope protein, but no antibodies against Np9 or Rec could be detected. A certain dedifferentiation of cells as a consequence of tumour development is discussed. Therefore the expression of HERV-K in undifferentiated embryonic stem cells was investigated. The investigated stem cells showed expression of HERV-K full length, env, rec and np9 mRNA. Although the expression decreased with differentiation to neuronal precursor cells. Even though HERV-K mRNA and proteins were expressed in a high percentage of melanomas their function in tumour development is still unclear. As well as the meaning of the HERV-K expression in embryonic stem cells, particularly for a tumourigenic potential.

No description available.