Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma:analysis of a clinic-based population

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

p19Arf induces p53-dependent apoptosis during Abelson virus-mediated pre-B cell transformation

The Ink4a/Arf locus encodes p16Ink4a and p19Arf and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19Arf and the p53 tumor-suppressor protein. Because Tp53 mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the Ink4a/Arf locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16Ink4a and p19Arf are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from Ink4a/Arf null mice revealed that these cells bypassed crisis. Because expression of p19Arf but not p16 Ink4a induced apoptosis in Ab-MLV-transformed pre-B cells, p19Arf appears to be responsible for these events. Consistent with the link between p19Arf and p53, Ink4a/Arf expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19Arf is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19Arf–p53 regulatory loop plays an important role in lymphoma induction.

p19ARF targets certain E2F species for degradation

p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.

Prevalence of germ-line mutations in p16, p19ARF, and CDK4 in familial melanoma: analysis of a clinic-based population.

Five to ten percent of individuals with melanoma have another affected family member, suggesting familial predisposition. Germ-line mutations in the cyclin-dependent kinase (CDK) inhibitor p16 have been reported in a subset of melanoma pedigrees, but their prevalence is unknown in more common cases of familial melanoma that do not involve large families with multiple affected members. We screened for germ-line mutations in p16 and in two other candidate melanoma genes, p19ARF and CDK4, in 33 consecutive patients treated for melanoma; these patients had at least one affected first or second degree relative (28 independent families). Five independent, definitive p16 mutations were detected (18%, 95% confidence interval: 6%, 37%), including one nonsense, one disease-associated missense, and three small deletions. No mutations were detected in CDK4. Disease-associated mutations in p19ARF, whose transcript is derived in part from an alternative codon reading frame of p16, were only detected in patients who also had mutations inactivating p16. We conclude that germ-line p16 mutations are present in a significant fraction of individuals who have melanoma and a positive family history.

Avaliação na linhagem endotelial tEnd dos efeitos diretos da transferência gênica de IFNbeta e p19arf e efeitos parácrinos mediados pela linhagem B16 transduzida pelos mesmos vetores adenovirais

A vascularização tem um papel central na progressão tumoral e representa um alvo terapêutico de grande interesse. A inibição da angiogênese tem potencial de retardar a progressão tumoral e inibir metástase. Em decorrência disto, terapias anti-angiogênicas têm demonstrado ser promissora no controle do crescimento tumoral. Segundo a literatura, interferon-? (IFN?, ativador do sistema imune inato e adaptativo) e p19Arf (supressor de tumor e parceiro funcional de p53), quando estudados individualmente, alteram a vasculatura tumoral. Nosso grupo construiu e utilizou vetores adenovirais recombinantes portadores dos cDNAs de INFbeta e p19Arf e observou que a transferência desta combinação de genes induziu morte celular e diminuiu progressão tumoral, resultados foram observados em modelos murinos de melanoma B16 de terapia genica in situ, vacina profilática e vacina terapêutica. Neste trabalho, exploramos a ideia que a combinação dos vetores adenovirais portadores de INFbeta e p19Arf proporcionam efeitos anti-angiogênicos através de seu impacto em células endoteliais. Para averiguarmos essa hipótese, células endoteliais murinas (tEnd) foram transduzidas com os vetores adenovirais, revelando que o vetor Ad-p19 confere inibição da proliferação, formação de tubos, migração e induz aumento na expressão de genes relacionados a via de p53 e morte celular. O vetor Ad-IFNbeta sozinho ou adicionado em combinação com Ad-p19, não teve impacto significante nestes ensaios. Alternativamente, a influencia indireta, ou parácrina, nas células tEnd cultivadas juntamente com as células B16 transduzidas com os vetores adenovirais também foi investigada. Quando as células B16 foram transduzidas com Ad-IFNbeta ou a co-transdução Ad-IFNbeta+Ad-p19 em co-cultura com a linhagem tEnd, houve inibição da proliferação. Não observamos efeito inibitório na tEnd da co-cultura quando as células da B16 foram transduzidas somente com Ad-p19. Seguindo o ensaio de co-cultura, produzimos meio condicionado da B16 transduzida com os vetores e aplicamos esses meios nas células tEnd. Observamos que Ad-IFN, sozinho ou em combinação com Ad-19, diminuiu a viabilidade, proliferação e levou a morte das células tEnd. Neste trabalho, constamos que inibição de células endoteliais pode ser realizada por transdução direta com Ad-19 ou quando estas células são expostas ao ambiente modulado por células tumorais transduzidas com o vetor Ad-IFNbeta. Mesmo que a transferência gênica de ambos IFNbeta e p19Arf não demonstrou ser uma abordagem superior à aplicação dos genes isolados, observamos que nossa abordagem pode ter um impacto importante na inibição da angiogênese pelas células endoteliais

Étude du rôle de Pax6 dans la gliogenèse

Les astrocytes sont des cellules gliales présentes dans le système nerveux central, qui exercent de nombreuses fonctions physiologiques essentielles et sont impliquées dans la réponse aux lésions et dans plusieurs pathologies du cerveau. Les astrocytes sont générés par les cellules de la glie radiale, les précurseurs communs de la plupart des cellules neuronales et gliales du cerveau, après le début de la production des neurones. Le passage de la neurogenèse à la gliogenèse est le résultat de mécanismes moléculaires complexes induits par des signaux intrinsèques et extrinsèques responsables du changement de propriété des précurseurs et de leur spécification. Le gène Pax6 code pour un facteur de transcription hautement conservé, impliqué dans plusieurs aspects du développement du système nerveux central, tels que la régionalisation et la neurogenèse. Il est exprimé à partir des stades les plus précoces dans les cellules neuroépithéliales (les cellules souches neurales) et dans la glie radiale, dérivant de la différenciation de ces cellules. L’objectif de cette étude est d’analyser le rôle de Pax6 dans la différenciation et dans le développement des astrocytes. À travers l’utilisation d’un modèle murin mutant nul pour Pax6, nous avons obtenu des résultats suggérant que la suppression de ce gène cause l'augmentation de la prolifération et de la capacité d'auto-renouvellement des cellules souches neurales embryonnaires. In vitro, les cellules mutantes prolifèrent de façon aberrante et sous-expriment les gènes p57Kip2, p16Ink4a, p19Arf et p21Cip1, qui inhibent la progression du le cycle cellulaire. De plus, Pax6 promeut la différenciation astrocytaire des cellules souches neurales embryonnaires et est requis pour la différenciation des astrocytes dans la moëlle épinière. Les mutants nuls pour Pax6 meurent après la naissance à cause de graves défauts développementaux dus aux fonctions essentielles de ce gène dans le développement embryonnaire de plusieurs organes. En utilisant un modèle murin conditionnel basé sur le système CRE/ loxP (hGFAP-CRE/ Pax6flox/flox) qui présente l’inactivation de Pax6 dans les cellules de la glie radiale, viable après la naissance, nous avons montré que Pax6 est impliqué dans la maturation et dans le développement post-natal des astrocytes. Le cortex cérébral des souris mutantes conditionnelles ne présente pas d’astrocytes matures à l’âge de 16 jours et une très faible quantité d’astrocytes immatures à l’âge de trois mois, suggérant que Pax6 promeut la différenciation et la maturation des astrocytes. De plus, Pax6 semble jouer un rôle même dans le processus de différenciation et de maturation de cellules gliales rétiniennes. L’étude des gènes et des mécanismes moléculaires impliqués dans la génération des astrocytes est crucial pour mieux comprendre le rôle physiologique et les altérations pathologiques des ces cellules.

Les cibles transcriptionnelles du polycomb Rae28 lors du développement de l'oeil : l'hypothèse du locus Ink4a/Arf

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Allelic imbalance studies of chromosome 9 suggest major differences in chromosomal instability among nonmelanoma skin carcinomas

CONTEXTO: Vários estudos de perda de heterozigozidade na região 9p21-p22, que abriga os genes supressores tumorais CDKN2a/p16INK4a, p19ARF e p15INK4b, têm sido realizados em uma ampla série de tumores humanos, incluindo os melanomas familiares. Perdas e ganhos em outras regiões do cromossomo 9 também têm sido observados com freqüência e podem indicar mecanismos adicionais no processo de tumorigênese dos carcinomas basocelulares da pele. OBJETIVO: Investigar o equilíbrio alélico existente na região 9p21-p22 em carcinomas basocelulares. TIPO DE ESTUDO: Análise molecular de marcadores de microssatélites em tumores e controles. LOCAL: Dois serviços de dermatologia de atendimento terciário em universidades públicas de São Paulo e o Laboratório de Genética Molecular do Câncer da Universidade Estadual de Campinas (UNICAMP), Brasil. PARTICIPANTES: Examinamos 13 casos benignos, incluindo 4 queratoses solares, 3 queratoacantomas, 3 nevos melanocíticos, 2 doenças de Bowen e 1 neurofibroma cutâneo, além de 58 tumores malignos da pele: 14 de células escamosas, 40 carcinomas basocelulares e 4 melanomas; em pacientes consecutivamente encaminhados à clínica de Dermatologia da Unicamp e que concordaram em participar do estudo. VARIÁVEIS ESTUDADAS: O tumor principal e uma porção normal de pele não-adjacente foram removidos cirurgicamente de pacientes que consecutivamente procuraram os ambulatórios de dermatologia da Universidade Estadual de Campinas (UNICAMP) e da Universidade Estadual de São Paulo (Unesp), São Paulo, por causa de lesões cutâneas. Extraímos DNA tanto de tecido tumoral como do correspondente tecido normal de cada paciente. Para amplificar regiões de repetição polimórfica de microssatélites do cromossomo 9, foram utilizados quatro pares de primers, sendo dois deles destinados à região 9p21-p22. RESULTADOS: Identificamos oito casos (20%) de desequilíbrio alélico entre os carcinomas basocelulares, sendo dois casos de perda de heterozigozidade e seis casos de instabilidade de microssatélite na região 9p21-p22. Outros marcadores também mostravam anormalidades em três destes tumores, enquanto nenhuma alteração foi detectada entre os casos benignos e nos outros tumores malignos. CONCLUSÃO: Esta dependência fenotípica sugere que existem diferenças importantes no comportamento das formas mais comuns de tumores cutâneos não-melanocíticos em relação à sua tendência para instabilidade de microssatélite no cromossomo 9. Considerando-se que os genes CDKN2a/p16INK4a, p19ARF e p15INK4b não parecem responsáveis pelas anormalidades observadas, outros genes em 9p21-p22 podem estar envolvidos na etiopatogenia e na progressão dos carcinomas basocelulares.

New molecular approaches to control rhabdomyosarcoma onset and metastasis in preclinical systems

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. The aim of this study was to identify molecular events involved in rhabdomyosarcoma onset for the development of new therapeutic approaches against specific molecular targets. BALB-p53neu mice develop pelvic rhabdomyosarcoma and combines the activation of HER-2/neu oncogene with the inactivation of an allele of p53 oncosuppressor gene. Gene expression profiling led to the identification of genes potentially involved in rhabdomyosarcoma genesis and therefore of candidate targets. The pattern of expression of p53, HER-2/neu, CDKN2A/p19ARF and IGF-2 suggested that these alterations might be involved in gender-, site- and strain-specific development of rhabdomyosarcoma. Other genes such as CDKN1A/p21 might be involved. The role of IGF-2, CDKN2A/p19ARF and CDKN1A/p21 in tumor growth was investigated with siRNA in murine rhabdomyosarcoma cells. Silencing of p19ARF and p21 induced inhibition of growth and of migration ability, indicating a possible pro-tumor and pro-metastatic role in rhabdomyosarcoma in absence of p53. In addition the autocrine IGF-2/IGF-1R loop found in early phases of cancer progression strengthens its key role in sustaining rhabdomyosarcoma growth. As rhabdomyosarcoma displays defective myogenic differentiation, a therapeutic approach aimed at enhancing myogenic differentiation of rhabdomyosarcoma cells. Forced expression of myogenin was able to restore myogenic differentiation, significantly reduced cell motility and impaired tumor growth and metastatic spread. IL-4 treatment increased rhabdomyosarcoma cell growth, decreased myogenin expression and promoted migration of cells lacking myogenin. Another approach was based on small kinase inhibitors. Agents specifically targeting members of the HER family (Lapatinib), of the IGF system (NVP-AEW541) or downstream signal transducers (NVP-BEZ235) were investigated in vitro in human rhabdomyosarcoma cell lines as therapeutic anti-tumor and anti-metastatic tools. The major effects were obtained with NVP-BEZ235 treatment that was able to strongly inhibit cell growth in vitro and showed anti-metastatic effects in vivo.

Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^

Using K5 Myc transgenic mice to delineate *Myc's apoptotic and tumorigenic pathways

The c-myc oncogene has the unusual ability to induce proliferation and apoptosis. Transgenic mice have been generated in which the expression of Myc is under the control of an epithelial-specific keratin 5 (K5) promoter. These mice have increased levels of proliferation and p53-dependent apoptosis, and are predisposed to developing spontaneous tumors in epithelial tissues. In this study, various knockout mice were bred to K5 Myc transgenic mice to identify factors involved in the aberrant apoptosis, hyperproliferation, and spontaneous tumorigenesis present in these mice. Consistent with in vitro studies, Myc-induced, p53-dependent apoptosis in transgenic epidermis was found to be partially dependent on p19ARF, a p53 regulator that inhibits mdm2. Additionally, the rate of tumorigenesis was increased when p19ARF was absent in Myc transgenic mice. Consistent with previous reports that some E2F family members may function as tumor suppressors, inactivation of either E2f1 or E2f2 was found to accelerate tumor development in the K5 Myc transgenic mice. Acceleration of tumorigenesis in the absence of E2F1 occurred despite the fact that apoptotic levels were increased in transgenic tissue and tumors null for E2f1 , whereas hyperproliferation was unaffected. In contrast, inactivation of E2f2 was found to increase hyperproliferation in the K5 Myc transgenic mice, while having no effect on apoptosis. The lack of E2f1 in the Myc transgenic mice increased the expression of several p53 transcription target genes, which may explain the increased apoptosis in these mice. In transgenic epidermis, p53 is phosphorylated at serine 18, a site of phosphorylation by ATM. Inactivation of ATM in K5 Myc transgenic mice impaired Myc-induced apoptosis, identifying ATM as having an important role in Myc-induced apoptosis. Moreover, the absence of ATM accelerates tumorigenesis in K5-expressing tissues. However, p53 accumulation and phosphorylation at serine 18 induced by Myc occurs independent of ATM. Therefore, another activity of ATM appears to be important for Myc-induced apoptosis. These findings show that acceleration of tumorigenesis in K5 Myc transgenic mice, as in the case of p53, p19ARF, E2F1, E2F2, and ATM absence, does not necessarily correlate with suppression of Myc-induced apoptosis, as seen only when p53, p19ARF or ATM was absent. ^

Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1

Expression of the DMP1 transcription factor, a cyclin D-binding Myb-like protein, induces growth arrest in mouse embryo fibroblast strains but is devoid of antiproliferative activity in primary diploid fibroblasts that lack the ARF tumor suppressor gene. DMP1 binds to a single canonical recognition site in the ARF promoter to activate gene expression, and in turn, p19ARF synthesis causes p53-dependent cell cycle arrest. Unlike genes such as Myc, adenovirus E1A, and E2F-1, which, when overexpressed, activate the ARF-p53 pathway and trigger apoptosis, DMP1, like ARF itself, does not induce programmed cell death. Therefore, apart from its recently recognized role in protecting cells from potentially oncogenic signals, ARF can be induced in response to antiproliferative stimuli that do not obligatorily lead to apoptosis.

Oncogenic ras activates the ARF-p53 pathway to suppress epithelial cell transformation

Chemically induced skin carcinomas in mice are a paradigm for epithelial neoplasia, where oncogenic ras mutations precede p53 and INK4a/ARF mutations during the progression toward malignancy. To explore the biological basis for these genetic interactions, we studied cellular responses to oncogenic ras in primary murine keratinocytes. In wild-type keratinocytes, ras induced a cell-cycle arrest that displayed some features of terminal differentiation and was accompanied by increased expression of the p19ARF, p16INK4a, and p53 tumor suppressors. In ARF-null keratinocytes, ras was unable to promote cell-cycle arrest, induce differentiation markers, or properly activate p53. Although oncogenic ras produced a substantial increase in both nucleolar and nucleoplasmic p19ARF, Mdm2 did not relocalize to the nucleolus or to nuclear bodies but remained distributed throughout the nucleoplasm. This result suggests that p19ARF can activate p53 without overtly affecting Mdm2 subcellular localization. Nevertheless, like p53-null keratinocytes, ARF-null keratinocytes were transformed by oncogenic ras and rapidly formed carcinomas in vivo. Thus, oncogenic ras can activate the ARF-p53 program to suppress epithelial cell transformation. Disruption of this program may be important during skin carcinogenesis and the development of other carcinomas.