Thesis (Ph. D.)--RAND Graduate School, 2002. / Includes bibliographical references (p. 95-99).

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2015-12-16T15:00:21Z No. of bitstreams: 1 texto completo.pdf: 1241133 bytes, checksum: d5965ff51adf5b02e7d03de528c0e482 (MD5) / Made available in DSpace on 2015-12-16T15:00:21Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1241133 bytes, checksum: d5965ff51adf5b02e7d03de528c0e482 (MD5) Previous issue date: 2011-08-04 / Embora não esteja entre as neoplasias mais prevalentes é responsável pela quarta causa de morte entre os pacientes com câncer. A compreensão dos mecanismos envolvidos na gênese e progressão do melanoma é crucial para o seu tratamento. Modelos experimentais que representem as fases da progressão do melanoma ainda são raros, mas a cultura de células é uma ferramenta importante para tais investigações. Os objetivos deste trabalho foram: estabelecer um modelo de estudo do melanoma murino B16F10 por meio do estabelecimento de sublinhagens celulares que correspondem ao tumor primário e às metástases, após passagem dessas células in vivo em camundongos BALB/c; caracterizar morfologicamente por meio de microscopia óptica e eletrônica de transmissão (MET) e varredura (MEV) a linhagem B16F10 e as sublinhagens derivadas; e investigar as leis de escala que regem o crescimento das linhagens de células de melanoma. Nesse contexto, células de melanoma B16F10 foram injetadas subcutaneamente (sc.) em camundongo BALB/c e a linhagem derivada dos tumores primários foi denominada de B16F10B. A linhagem B16F10 e a sublinhagem B16F10B foram inoculadas endovenosamente (ev.) em camundongos BALB/c e as sublinhagens obtidas das metástases foram denominadas B16F10M e B16F10BM, respectivamente. A linhagem B16F10 e as sublinhagens B16F10B, -BM e -M foram analisadas em MO, MET e MEV, revelando uma grande heterogeneidade na morfologia dessas células e nos agregados por elas formados. As células B16F10 e B16F10B também revelaram à MET presença de partículas virais. Para a caracterização das leis de escala que regem os regimes de crescimento das células B16F10, -B, -BM e -M, as linhagens celulares foram cultivadas sobre lamínulas de vidro em placas de 24 poços e retiradas em intervalos de 24 horas, durante 7 dias. O número de células por agregado e o número total de agregados foram usados para determinar a função de distribuição de tamanho de agregados que sugerem uma transição nas funções de distribuição de um decaimento exponencial para um que segue lei de potência para as células B16F10B. As demais linhagens analisadas sempre exibiram um comportamento de lei de potência. A distribuição em leis de potência indica a ausência de um tamanho característico para os tamanhos dos agregados, podendo refletir na falta de mecanismos de controle da replicação celular aliada à estabilidade dos agregados. A transição no regime de crescimento exibido pelas células B16F10B sugere que o crescimento celular contínuo na cultura pode exercer forças seletivas que destroem os mecanismos de regulação, tais como a dependência de ancoragem e o crescimento de densidade-dependente. A caracterização morfológica, bem como do regime de crescimento das sublinhagens derivadas das células B16F10 mostrou que há diferenças e similaridades entre as mesmas, porém, para uma melhor caracterização dessas células, outras análises devem ser realizadas, tais como: resistência a drogas, integridade genômica e padrão de expressão de moléculas de adesão. / Melanoma is a malignant neoplasm derived from melanocytes, specialized pigmented cells that are found predominantly in the skin. Although it does not figure among the most prevalent cancer types, melanoma is responsible for the fourth leading cause of cancer patients death. Understanding of the mechanisms involved in the genesis and progression of melanoma is crucial for the cancer treatment. Experimental models that representing phases of melanoma progression are still rare, but the cell culture is an important tool for such investigations. The aims of the work were to determine a study model of murine melanoma B16F10 by the establishment of cell sublineages corresponding to the primary tumor and to the metastases, after passage of these cells in BALB/c mice; to characterize morphologically by optical microscopy (OM) and transmission electron (TEM) and scanning electron (SEM) the B16F10 lineage and sublineages derived from B16F10; to investigate the scaling laws that rule the growth of melanoma cell lines. In this context, B16F10 melanoma cells were injected subcutaneously (sc) in BALB/c mice and the subline derived from subcutaneous tumors was named B16F10B. The B16F10 lineage and the B16F10B sublineage were inoculated intravenous (iv) in BALB/c mice, and the sublineages of metastases obtained were named B16F10M and B16F10BM, respectively. The B16F10B lineage and the B16F10, -BM and -M sublineages were analyzed by OM, TEM and SEM, what revealed an extent heterogeneity in the morphology of these cells and clusters formed by them. B16F10 and B16F10B cells also disclosed to MET viral particles presence. To characterize the scaling laws that rule the growth regimes of the B16F10, -B, -BM and -M cells, the cells lineages were cultured on glass coverslips in 24-well plates and removed at intervals of 24 hours for 7 days. The number of cells per cluster and the total number of clusters were used to determine the cluster size functions. The results suggest distribution functions with a transition to an exponential decay that follows a power law for B16F10B cells. The other lineages analyzed always exhibits a power law behavior. The power law distribution indicates the absence of a characteristic size for the clusters sizes which, from the biological point of view, might reflect the lack of control mechanisms of cell replication allied to the stability of clusters. The transition in the growth regime exhibited by B16F10B cells suggests that the continued cell growth in culture may exert selective forces which destroy regulation mechanisms such as cell anchorage dependence and density-dependent growth.

This small exploratory study was designed to test the hypothesis that thin melanoma lesions contain nuclei of two similar phenotypes, in different proportions. In lesions likely to progress to metastatic disease, one of these phenotypes predominates. Histopathological sections from 18 cases of thin melanomas which did not progress to metastasis, and from 10 cases which did progress were imaged and digitized at high resolution, with a total of 2084 and 1148 nuclei, respectively, recorded. Five karyometric features were used to discriminate between nuclei from indolent and from potentially metastatic lesions. For each case, the percentage of nuclei classified by the discriminant function as having come from a potentially metastatic lesion was determined and termed as case classification criterion. Standard histopathological criteria, such as ulceration and high mitotic index, indicated in this material the need for intensive therapy for only one of the 10 participants, as compared with 7/10 identified correctly by the karyometric measure. Using a case classification criterion threshold of 40%, the overall accuracy was 86% in the test set. The proportion of nuclei of an aggressive phenotype may lend itself as an effective prognostic clue for thin melanoma lesions. The algorithm developed in this training set appears to identify those patients at high risk for metastatic disease, and demonstrates a basis for a further study to assess the utility of prognostic clues for thin melanomas.

Thesis (M.S.)--California Polytechnic State University, 2009. / Mode of access: Internet. Title from PDF title page; viewed on Jan. 7, 2010. Major professor: Dr. Lily Laiho. "Presented to the faculty of California Polytechnic State University, San Luis Obispo." "In partial fulfillment of the requirements for the degree [of] Master of Science in Biomedical Engineering." "December, 2009." Includes bibliographical references (p. 113-115).

Cutaneous melanoma remains the deadliest form of skin cancer, with a diagnosis of metastasis indicating a median survival rate of less than a year. Solar ultraviolet (UV) radiation, especially childhood sun exposure, is an important etiological risk factor of melanoma. Previous studies determined that mice selectively lacking the nuclear hormone receptor Retinoid X Receptor α in epidermal keratinocytes (RXRα[superscript ep-/-]) developed a higher number of aggressive melanocytic tumors compared to wild type mice after two-step chemical carcinogenesis, suggesting a novel role of keratinocytic nuclear receptor signalling during melanoma progression. We then discovered a progressive loss of RXRα expression in epidermal keratinocytes during melanoma progression in humans. We also investigated the contributions of CDK4[superscript R24C/R24C] and keratinocytic RXRα to influence metastatic progression in a mouse model by generating RXRα[superscript ep-/-]/CDK4[superscript R24C/R24C] bigenic mice containing an activated cyclin dependent kinase 4 (CDK4), besides lacking RXRα in epidermal keratinocytes. Those bigenic mice developed malignant melanomas that metastasized to regional lymph nodes after carcinogen exposure. Expression of several keratinocyte-derived growth factors implicated in melanomagenesis were upregulated in the skin of bigenic mice, and recruitment of RXRα was shown on the promoters of endothelin-1 (Edn1) and hepatocyte growth factor (Hgf). We then confirmed a downregulation of factors (FAS, E-cadherin and PTEN) implicated in apoptosis, invasion and survival within the melanocytic tumors. To further evaluate the paracrine role that EDN1 has on melanocyte activation, we utilized a transgenic mouse model where the gene encoding Edn1 was selectively ablated from epidermal keratinocytes using the Cre-LoxP strategy to create the EDN1[superscript ep-/-] knockout mouse line. We discovered a direct in vivo transcriptional regulation of keratinocytic Edn1 by the tumor-suppressor p53 in epidermal keratinocytes in response to UV irradiation. We also demonstrate that in vivo disruption of keratinocyte-derived EDN1 signaling alters melanocyte proliferation and decreases epidermal and dermal melanocyte populations in both normal and UV exposed mouse skin. EDN1 also has a protective role against UVR-induced DNA damage and apoptosis and similar effects on UV-induced melanocyte proliferation and DNA damage are observed in p53-null mice. Inhibition of EDN1 signaling by topical application of an EDNRB antagonist BQ788 on mouse skin also recapitulates epidermal EDN1 ablation. Furthermore, treatment of primary murine melanocytes with BQ788 abrogates signaling downstream of this receptor. Taken together, these studies demonstrate the contribution of RXRα regulated keratinocytic paracrine signaling during the cellular transformation and malignant conversion of melanocytes. Also, they establish an essential role of EDN1 in epidermal keratinocytes to mediate UV-induced melanocyte homeostasis in vivo. / Graduation date: 2013

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Malignant melanoma is a neoplasm of melanocytes. It typically originates in skin, but may metastasize to other body systems. It is a relatively common neoplasm in both humans and horses, with striking similarities across both species. Heritable genetic factors associated with melanoma have been identified in both human malignant melanoma (HMM) and equine malignant melanoma (EMM). This work investigates similarities and differences of EMM and HMM through comparative protein expression using immunohistochemical staining. Nestin, Pax-3/7, B-Raf, and SOX-10 are commonly expressed in HMM tissues. Expression of these proteins is not noted in normal human melanocytes, is associated with decreased melanocytic differentiation, and with increased proliferation leading to tumorigenesis. My findings demonstrate similar expression of these proteins in EMM. Aberrant protein expression patterns may signal underlying genetic mutations. Similar abnormal expression patterns suggest that EMM and HMM may share common genetic abnormalities. Treatment of malignant melanoma presents similar challenges in both horses and humans. In general, early stages of the disease can be successfully treated with surgical excision; however, advanced stages of EMM and HMM are often refractory. Therefore, development of novel therapies for advanced stages of melanoma is essential in both species, with the horse representing a useful model for this process. One novel therapy, frankincense oil (FO) is a resin distillate from trees of the genus Boswellia. Studies have demonstrated cytostatic and apoptotic-modulating properties of FO in various human cancer cell lines. No studies have evaluated FO as a therapy for skin neoplasms. In my work, the apoptotic properties of FO from Boswellia frereana were verified in a HMM cell line (SK-Mel-5). The cytotoxicity and therapeutic efficacy of FO were also studied by evaluating the effects of FO in cases of perianal EMM. FO was found to be consistently cytotoxic when injected directly into EMM tumors, but largely inconsequential when applied topically. FO was found to substantially decrease the size of injected masses. These findings suggest that FO could be useful for debulking large masses in late stage dermal EMM. The combined results of these studies support further investigation of EMM as a translational model for HMM. / Ph. D.

The most frequent mutation identified in both benign nevi and malignant melanoma is the constitutively activating V600E substitution of BRAF. However, how additional mutations co-operate with BRAFV600E to promote subtypes of melanoma in animals is only beginning to be understood. In this thesis, I generate and analyze zebrafish BRAFV600E melanoma models and also develop the first animal model for BRAFV600E nevus recurrence. In my first data chapter, I develop a unique animal model of nevus recurrence. In people it is not uncommon for a nevus to recur following removal, even when no pigmented nevus cells remain. The biology of how and why this happens is unknown. By partial amputation of the nevus in the zebrafish tail fin, we described both nevus regrowth, as well as nevi that do not regrow. Utilising melanin as a lineage tracer, I was able to show that recurrent nevi are repopulated from an unpigmented precursor population. This suggested that BRAFV600E nevi are supported by an undifferentiated stem cell population that is recruited to regenerate and pigment the nevus after removal. In my second data chapter, I use genetics to develop BRAFV600E zebrafish models of melanoma. In collaboration with Dr. James Lister and Professor Jeroen den Hertog, I describe three differing models of zebrafish melanoma. All three models show progression to melanoma, and in collaboration with Dr. Marie Mathers I establish that while BRAFV600E is present in all three models, co-operating mutations affect melanoma pathology. In my third data chapter, I develop tools to study the molecular differences in the BRAFV600E melanoma models. I described the optimisation of a broad range of antibodies, raised against human peptides due to the lack of reliable antibodies in the zebrafish field. I use punch core biopsies of both zebrafish and human tumours, and whole sagittal sections of juvenile zebrafish, to show specific staining throughout many organs of the developing fish. I then use some of these antibodies to analyse molecular pathways in the melanoma models.

The correlation of information obtained from in vitro investigations and in vivo experiments has frequently evaded researchers, especially in the area of tumor cell invasion and metastasis. In order to better understand and associate in vitro tumor cell invasion through basement membranes with in vivo tumor metastasis in syngeneic animal models, and the subsequent modulation of these processes, the following studies have been undertaken. Malignant murine melanoma cell lines designated B16F1 and B16F10, syngeneic to the C57BL6 mouse, a melanotic variant of the Cloudman S-91 melanoma cell line (denoted Mel-11a) with the syngeneic host being the DBA/2J mouse, and a malignant human melanoma line referenced as A375P (parental) and A375M (metastatic) were used for this dissertation project. Tumor cells were labeled with either ¹⁴C-thymidine or ¹²⁵I-deoxyuridine using previously established protocols. Radiolabeled tumor cells were introduced into the Membrane Invasion Culture System (MICS) in vitro, a system developed in our lab, and concomitantly into the lateral tail vein by injection or intracutaneously into the appropriate syngeneic host in the presence or absence of such biological response modifying agents as [Nle⁴, D-Phe⁷]-MSH, and α-MSH. The superpotent analogue of α-MSH ([Nle⁴, D-Phe⁷] -MSH) showed a proliferative and survival enhancing effect on tumor metastasis in vivo with no effect on in vitro tumor cell invasion, with similar effects demonstrated by α-MSH. The effects of these melanotropins on spontaneous metastasis formation appears to be negligible. The A375M and A375P human melanoma cells parallel their metastatic profile in vitro when assayed in MICS. In concert with these studies, the development of a control cell line, comprised of neural crest-derived melanocytes, and the study of their subsequent invasiveness in vitro were pursued. The neural crest-derived melanocytes were unable to invade the basement membranes (BM); although co-culturing neural crest cells with B16F10 melanoma cells produced an effect such that the neural crest cells did significantly invade the BMs. These studies demonstrate the ability of the MICS in vitro invasion assay to discriminate between tumor cells with differing metastatic propensities and could possibly be used in future studies to predict the effectiveness of biological response modifying agents in vivo.

No description available.