The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas with tissue microarray and immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3-mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.

No description available.

<p>Dermatopathologists need more reliable tools for analyzing biopsies of lesions that are potentially melanomas and determining the best treatment plan for the patient. Previously inaccessible, the chemical and physical properties of melanin provide insight into melanoma biochemistry. Two-color, near-infrared pump-probe microscopy of unstained, human pathology slides reveals differences in the type of melanins and the distribution of melanins between melanomas and benign nevi. Because the pump-probe response of melanin is resilient to aging, even for hundreds of millions of years, this tool could prove useful in retrospective studies to correlate melanin characteristics with patient outcome, thus eliminating the pathologist's uncertainty from the development of this classification method.</p><p>Pump-probe spectroscopy of a variety of melanin preparations including melanins with varying amounts of metal ions and toxins, those that have been photo-damaged or chemically oxidized, and melanins with a homogeneous size distribution shows that the pump-probe response is sensitive to these chemical and physical differences, not just melanin type as previously hypothesized. When sampling the response at several pump wavelengths, the specificity of this technique is derived from the absorption spectra of the underlying chromophores. Therefore, hyperspectral pump-probe microscopy of melanin could serve as an indicator of the chemical environment in a variety of biological contexts. For example, the melanin chemistry of macrophages suggests that these cells oxidize, homogenize, and compact melanin granules; whereas melanocytes produce heterogeneous melanins.</p> / Dissertation

The metastasis suppressor gene NM23-H1 has been shown to possess three enzymatic activities including nucleoside diphosphate kinase, histidine-dependent protein kinase and 3’-5’ exonuclease activity. While these properties have been demonstrated in vitro using recombinant proteins, the contribution of these activities to suppression of metastatic dissemination is unknown. Site-directed mutagenesis studies were used to identify amino acid residues which are required for proper function of each enzymatic activity associated with H1, providing a platform for studying the importance of each function on an individual basis. To assess the relevance of these activities to melanoma progression, a panel of mutants harboring selective lesions disrupting the enzymatic activities of H1 were overexpressed using stable transfection in two melanoma cell lines, WM793 (isolated from a vertical growth phase human melanoma), and the metastatic derivative cell line 1205LU. In vitro correlates of metastasis measuring motility and invasion were used in an attempt to identify the mechanism mediating H1-dependent motility suppression of cancer cells. Surprisingly, all mutants studied retained full motility suppression in this setting, suggesting that the enzymatic functions associated with H1 are not required for inhibiting cell migration. Instead, gene expression analyses conducted on the panel of stable transfectants indicate that differences in steady-state mRNA levels of genes involved in mitogen-activated protein kinase (MAPK) signaling showed significant correlations with H1 expression and motility suppression. RNAi studies have confirmed that H1-dependent modulation of the expression of two genes in particular, BRAP and IQGAP2, contribute to the observed phenotype, suggesting a novel mechanism used by NM23 to control cellular migration in human melanoma.

<p>In this dissertation, we develop pump probe spectroscopy as a method to differentiate different chemical varieties of melanin, a common biopigment, and exploit these differences to improve the accuracy of melanoma diagnosis. This method gives insight into the chemical makeup and secondary structure of melanins. Pump probe spectroscopy is implemented in a scanning laser microscope as a form of multiphoton imaging, where it is used to image biopsies of human pigmented cutaneous lesions. Melanoma diagnosis is clinically challenging: the accuracy of visual inspection by dermatologists is highly variable and heavily weighted toward false positives. Even the current gold standard of biopsy results in varying diagnoses among pathologists. Using pump probe imaging, significant chemical and morphological changes were found between melanoma and melanocytic nevi, including increased eumelanin content, chemical heterogeneity and general pigmentation. Signal processing methods revealed further differences between melanoma and melanocytic nevi on the cellular scale. Pump probe imaging directly in H&E stained biopsy samples allows integration of this technique with existing histopathology protocols. High resolution imaging found chemical heterogeneity of melanin within pigmented cells. We show that oxyhemoglobin and deoxyhemoglobin may also be differentiated by pump probe imaging. Epi mode imaging of eumelanin, pheomelanin and microvasculature is demonstrated in vivo in human xenograft mouse models of melanoma.</p> / Dissertation

Una de les estratègies terapèutiques pel tractament del càncer, consisteix en l'estimulació del sistema immunitari del pacient, de manera més o menys específica, amb l'objectiu de que es produeixi el reconeixement i destrucció de les cèl·lules tumorals. Aquesta intervenció del sistema immunitari és el que es coneix com a immunoteràpia. La immunoteràpia pot ser activa, quan s'utilitzen molècules específiques del tumor com a antígens dianes contra les quals es vol dirigir la resposta immunitària; o bé passiva, quan no s'utilitzen antígens dianes específics i l'estimulació del sistema s'aconsegueix per mitjà de citocines o altres activadors policlonals. Per a la immunoteràpia activa es poden utilitzar diverses fonts que continguin els antígens tumorals, així es pot partir de lisats de trossos de tumors, lisats o cèl·lules senceres inactivades de línies tumorals establertes o bé seqüències peptídiques d'antígens immunodominants.Per tal d'abordar aquesta estratègia terapèutica en pacients afectats de melanoma maligne, vam derivar en el laboratori línies tumorals estables. Aquest tipus de tumor, quan metastatitza, cosa que es produeix amb una elevada facilitat, no té tractaments estàndard d'elecció, i sovint les estratègies que es poden aplicar són molt tòxiques i poc efectives. Un cop caracteritzades les línies i descartat sobre tot la presència de contaminats bacterians i vírics, i en funció d'una sèrie de paràmetres fenotípics, es van seleccionar 10 línies tumorals per a composar una vacuna de cèl·lules senceres al·logèniques. Les cèl·lules abans de ser inoculades en els pacients reben un tractament d'inactivació per irradiació per motius de seguretat. Es van seleccionar un grup de pacients amb malaltia metastàsica per rebre les vacunes; situacions aprovades pel Comitè d'Ètica de l'Hospital Clínic de Barcelona. De 38 pacients estudiats s'ha pogut observar una tendència a l'augment de la supervivència, així com tres respostes complertes amb desaparició total de la malaltia i un 26 % de respostes globals. Dels paràmetres immunitaris avaluats hi ha un augment significatiu dels nivells de IL-10 en aquells pacients amb un control de la malaltia, i uns augments dels nivell de IFN-gamma i TNF-alfa.Una segona fase va ser la introducció de cèl·lules dendrítiques autòlogues dels pacients com adjuvants naturals de la vacunació. Aquestes cèl·lules són les cèl·lules presentadores d'antigen més potents descrites fins al moment, i controlen la immunitat. Malgrat que la seva proporció circulant és molt baixa, es poden obtenir a partir de monòcits i una barreja de citocines (IL-4 i GM-CSF). Aquestes cèl·lules dendrítiques obtingudes de monòcits tenen les característiques fenotípiques i funcionals de les cèl·lules dendrítiques i s'han utilitzat com a vacunes en càncer. Degut a la seva recent aplicació en humans, hi ha molts aspectes a ser optimitzats per tal de millorar l'efectivitat de la resposta antitumoral. En la tesi es descriu l'experiència de vacunar 11 pacients de melanoma amb malaltia metastàsica, que no ha respost a cap altre tractament, amb cèl·lules dendrítiques autòlogues carregades amb un lisat de les línies tumorals que composen la vacuna prèviament descrita. Dels resultats obtinguts i analitzats es pot concloure que hi ha una activació del sistema immunitari dels pacients que té com a resultat alguna regressió clínica de la malaltia en alguns pacients. El factor que és més important és que no hem observat cap efecte tòxic secundari associat a les vacunes. Per tant degut a l'activació del sistema immunitari, a les respostes clíniques observades i a la manca total de toxicitat, la immunoteràpia amb cèl·lules tumorals al·logèniques i amb cèl·lules dendrítiques és un procés segur i que es pot aplicar en estadis més inicials de la malaltia.

HIPÒTESI DE TREBALL. L'angiogènesi al melanoma maligne està influenciada per factors autocrins i paracrins, entre els que es troba el VEGF. La influència del VEGF és especialment significativa a les primeres fases del desenvolupament tumoral.La modificació de l'equilibri angiogènic actuant sobre el VEGF podria condicionar un canvi significatiu al creixement tumoral i la seva capacitat metastàtica.OBJECTIUS.1.- Valorar l'expressió de VEGF en una sèrie de melanomes malignes primaris cutanis humans de baix risc.2.- Valorar el creixement tumoral i l'angiogènesi als tumors desenvolupats en ratolins atímics inoculats ortotòpicament amb les línies de melanoma humà A375P i A375MM, coinjectades o no amb Matrigel. 3.- Avaluar la influència de la modificació genètica de l'expressió de VEGF, mitjançant la transfecció del cDNA del VEGF en sentit i antisentit a les línies de melanoma humà A375P i A375MM, als tumors desenvolupats a ratolins atímics a partir de la injecció ortotòpica d'ambdues línies així modificades.MÈTODES.1.- Melanomes humans: 53 melanomes malignes cutanis primaris. Angiogènesi: tinció amb Ulex Europaeus I. Expressió VEGF: anàlisi immunohistoquímica.2.- Expressió de VEGF a línies de melanoma humà xenoempeltades a ratolins immunodeprimits: Inoculació ortotòpica a ratolins Balb/c nude de les línies A375P i A375MM, la meitat coinjectades amb Matrigel. Angiogènesi: tinció immunohistoquímica amb Ac anti-factor VIII. Expressió VEGF: anàlisi immunohistoquímica.3.- Modificació de l'expressió de VEGF a línies de melanoma humà xenoempeltades a ratolins immunodeprimits: Construcció del cDNA del VEGF en sentit i antisentit en un vector d'expressió (pRC/CMV), transfecció mitjançant liposomes a les línies A375P i A375MM, selecció dels transfectants i inoculació ortotòpica a ratolins atímics. Extirpació de la meitat dels tumors el més aviat possible (5-6 dies), per a avaluar la malaltia en les seves fases més precoces. Per a l'altra meitat, lliure creixement, calculant les corbes de creixement i extirpant els tumors a l'assolir un volum de 1·1 cm o equivalent. Seguiment de la supervivència dels ratolins. Angiogènesi: immunotinció amb Ac anti-factor VIII.RESULTATS.1.- Melanomes humans: El VEGF ha estat present al 84,9% de melanomes avaluats.Als melanomes en fase de creixement radial (in situ), a 7 de 29 s'ha observat un grau alt d'expressió de VEGF, front 12 de 16 a melanomes amb fase vertical (p=0,002). Una vegada el melanoma era ja invasor, no s'han trobat diferències entre el grau d'expressió de VEGF i el nivell de Breslow. No s'ha trobat relació entre angiogènesi i expressió de VEGF.2.- Expressió de VEGF als tumors desenvolupats als ratolins inoculats: S'ha demostrat una major expressió de VEGF en els estadis més temprans del desenvolupament tumoral, representats aquests per aquells tumors més petits (<0,5 cm) o intervinguts més aviat (< 9 dies).3.- Efecte de la modificació de l'expressió de VEGF sobre les dues línies A375P i A375MM xenoempeltades a ratolins atímics. Transfecció cDNA del VEGF en sentit: ha incrementat significativament el creixement dels tumors a les fases inicials del desenvolupament tumoral (4 clons de 4), a la corba de creixement (2 de 4), i a la supervivència (1 de 4). Transfecció cDNA VEGF en antisentit: cap diferència repecte al control, ni a fases inicials, ni a la corba de creixement, ni a la supervivència. Angiogènesi: no diferències en la densitat vascular entre els diferents clons.CONCLUSIÓ FINAL. El VEGF és determinant a les fases inicials del desenvolupament tumoral al melanoma maligne. Més endavant perd el seu paper protagonista. Igualment, la modificació genètica de la seva expressió en un model in vivo amb línies de melanoma humà xenoempeltades a ratolins atímics té efecte només als moments inicials del creixement tumoral, perdent tota la seva influència a les fases més tardanes del desenvolupament tumoral. / We hypothesized that angiogenesis in melanoma is related to VEGF, and that the modification of the expression of VEGF could influence angiogenesis, tumour growth and metastatic capacity.First, we examined immunohistochemically the expression of VEGF in cutaneous primary melanoma. VEGF expression was graded in a scale from 0 to 3. Angiogenesis was measured with Ulex Europaeus I staining. 84,9% of tumours expressed VEGF. Vertical Growth Phase (VGP) was significatively associated with a higher intensity expression of VEGF when comparing to radial growth phase. However, once VGP had developed, there were no differences in VEGF expression between tumours. Surprisingly, VEGF expression was not related to angiogenesis.Second, in a model of human melanoma cell lines (A375P, A375MM) xenografted to nude mice, we evaluated the expression of VEGF. The most significant finding was a higher intensity expression of VEGF in tumours representing the first steps of tumour development: tumours lower than 0,5 cm in diameter and those extracted before day 9 post-inoculation demonstrated higher expression of VEGF when compared with the remainder.Third, VEGF sense and antisense cDNA were cloned to an expression vector (pRC/CMV) and transfected to A375P and A375MM melanoma cell lines with cationic liposomes; modified cell lines were xenografted to nude mice. To analyse first stages of tumour progression, half of the resultant tumours were extracted when they were minimally detectable. Growth curves of the rest of tumours were measured, and they were excised when reaching a volume of 0,4 cm3. Survival curves of mice were analysed afterwards. Angiogenesis was evaluated with Factor VIII staining.Sense VEGF transfection significatively enhanced tumour growth on initial stages of development (4 clones of 4), on growth curves (2 of 4) and on survival (1 of 4). Antisense VEGF transfection did not show any difference with control clones at any stage. Angiogenesis was not different between the clones.Conclusion: VEGF is decisive at first stages of development in melanoma. The modification of VEGF expression in an in vivo model of human melanoma cell lines xenografted to nude mice plays a role at initial stages of tumour development, losing its influence later.

Deletion of the long arm of chromosome 6 is one of the most common genetic alterations in human malignant melanoma. Recently, a reciprocal translocation between chromosomes 6q and 17p was detected in a melanoma cell line, UACC-930, using arm painting probes of 6p and 6q. Reciprocal translocation is seldom observed in solid tumors. Upon further characterization of the translocation marker using techniques such as Southern blotting, genomic library screening and DNA sequencing, a complex rearrangement including two inversions of 6q and a translocation between the inverted 6q and 17p, [der(6)inv(6)(q21q22)(q22q27)t(6;17)(q27;p13)], was detected. An NCBI blast search revealed 3 genes being interrupted by the breakpoints: prenyl diphosphate synthase subunit 2 (PDSS2) at 6q21, Parkin at 6q27 and p53 at 17p13. Down-regulation of PDSS2 was commonly observed in 59/87 (67.8%) primary melanomas, which was significantly higher than that in benign nevi (7/66, 10.6%, p<0.001), indicating the tumor-suppressive potential of PDSS2 in melanoma development. To characterize the function of PDSS2 in tumorigenesis, PDSS2 was stably transfected into a highly tumorigenic melanoma cell line, UACC-903. The tumor-suppressive function of PDSS2 was demonstrated by both in vitro and in vivo assays. The results showed that PDSS2 could inhibit tumor cell growth, decrease the colony-forming ability in soft agar, and totally abrogate the tumorigenicity of UACC-903 in nude mice. PDSS2 is the first enzyme involved in the CoQ10 biosynthesis pathway. Other studies have demonstrated PDSS2 mutations can cause severe CoQ10 deficiency and markedly reduced ATP production because of respiratory chain dysfunction. Interestingly, proteomics analysis revealed 7 out of 11 identified proteins (HSPA8, GAPDHS, TPI1, HSPA5, PGK1, ENO1, and ATP5B) differentially expressed in PDSS2-overexpressing cells were related to energy metabolism. Further studies are required to determine how PDSS2 could alter the energy supply in tumor cells. Taken together, these results support the proposal that PDSS2 is a novel tumor suppressor gene which may play an important role in the development of malignant melanoma via altering tumor metabolism.

Australia has the highest incidence of melanoma in the world. Melanocytic nevi and mutations in the CDKN2A gene are the main risk factors for the development of cutaneous melanocytic melanoma, and particularly in those of European descent. My study uses genetic epidemiological methods to investigate causes of variation in the number of melanocytic nevi and pigmentary traits such as freckles, eye colour, hair colour and skin colour collected on a sample of adolescent twins and siblings from the Brisbane Twin Nevus Study (1992-2006). Information was available for 2524 individuals from 973 families (from the first visit when the twins were aged 12 years), and from a repeat visit (two years later) for 1598 individuals from 791 families. Using the twin study design extended to siblings and parents, variance components analyses showed that the proportion of phenotypic variance explained by genetic factors ranged from 43 to 99 percent for the traits studied. The atypical nevus count and freckles showed sex differences in the magnitude of genetic and environmental effects. Genetic correlations among counts of three types of nevus (flat, raised and clinically atypical) ranged from 0.46 to 0.63. Nevus count was genetically correlated with skin colour (r=0.23). I analysed genome-wide linkage data using a total of 1190 microsatellite (STR) markers from three scans for 644 families with 1646 twins and siblings, plus genotypes for 1033 parents. These were combined with an additional 169 families with genome-wide association 100K SNP (single nucleotide polymorphism) data, where I selected a linkage analysis panel of 13,000 SNPs making a total of 3365 individuals from 811 families (each individual had more than 200 markers typed). Suggestive linkages for flat nevus count (FNC) were identified on chromosomes 2p25 and 9p21 with lod scores of 3.19 and 2.62 respectively. For raised nevus count (RNC), a suggestive QTL with a lod score of 2.20 was found on chromosome 2q37.2, and for atypical nevus count (ANC) a lod score of 2.71 was found on chromosome 7p14.1. There was suggestive evidence of linkage for freckling on chromosomes 2 and 9. Eye colour was strongly linked (lod=17.86) to chromosome 15, at the OCA2 locus. I have also carried out genetic association analyses using the 100K SNP data in 169 families, and additional fine mapping using SNPs (as well as STR markers) in the complete data set. A sample size of 169 families (461 twins) for genome-wide association data means that statistical power is low. From the 100K SNP data, the best association for total nevus count (TNC) was with SNP rs2420070, p=6.0×10-6 on chromosome 10, and included another two nearby SNPs; rs7086663, p=2.0×10-4 and rs7090904, p=1.5×10-4. These 3 SNPs also showed possible association with FNC; rs2420070, p=3.9×10-6, rs2420070, p=1.3×10-4 and rs2420070, p=8.9×10-5. For raised nevus count the top three associated SNPs were rs1885238, p=1.8×10-5 on chromosome 9; rs10503048, p=3.7×10-5 on chromosome 18 and rs4769189, p=4.0×10-5 on chromosome 13. SNP rs1412341 which is located near CDKN2A on chromosome 9, was also associated with p=2.8×10-4. There were a total 18 SNPs which showed evidence of association with atypical nevus count, the strongest signal being with rs951099 (p=3.7×10-5) on chromosome 9. In a fine-mapping dataset, I studied the association of CDKN2A SNP rs2218220 with TNC, FNC, RNC and ANC. The best SNP, rs2218220, gave p values of 2.8×10-10, 7.7×10-8, 2.7×10-12 and 9.1×10-8, respectively. A SNP, rs1800407 (R419Q) located in the OCA2 gene (chromosome 15q11.2-15q12) showed evidence of association with eye colour and particularly with blue and green eye colours, (p=1.7×10-12 and p=6.0×10-7). SNP rs12913832 from the Hect Domain and RCC1-like Domain 2 gene (HERC2) on chromosome 15q13.1, was also strongly associated with eye colour p=3.6×10-155. This SNP was associated with blue (p= 7.9×10-150) and brown (p=5.3×10-158), but not green eye colour. In addition I confirmed the association of the MC1R SNP rs1805007 and freckling (p=4.8×10-12). This SNP was also associated with FNC (p=3.5×10-8), a finding not previously described in the literature. I also carried out multi-allelic association analysis using STR markers with these traits and uncovered suggestive findings for several regions. Finally, I conducted a multivariate association analysis searching for SNPs with pleiotropic effects. The most interesting results for all types of nevi were with rs801840, p=3.5×10-5, and rs10487075, p=4.9×10-5, both on chromosome 7q21.13. Another four SNPs on chromosome 8p23.1 also showed associations, rs7009724, p=1.4×10-4, rs10503389, p=1.9×10-4, rs7832398, p=6.2×10-4 and rs7005133, p=6.9×10-4 (close to a candidate gene, MFHAS1, implicated in sarcoma risk). In conclusion I have characterised a number of definite and possible genetic factors influencing important risk factors for melanoma.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 133-173).