RESUMO
Signaling centers, or organizers, regulate many aspects of embryonic morphogenesis. In the mammalian molar tooth, reiterative signaling in specialized centers called enamel knots (EKs) determines tooth patterning. Preceding the primary EK, transient epithelial thickening appears, the significance of which remains debated. Using tissue confocal fluorescence imaging with laser ablation experiments, we show that this transient thickening is an earlier signaling center, the molar initiation knot (IK), that is required for the progression of tooth development. IK cell dynamics demonstrate the hallmarks of a signaling center: cell cycle exit, condensation and eventual silencing through apoptosis. IK initiation and maturation are defined by the juxtaposition of cells with high Wnt activity to Shh-expressing non-proliferating cells, the combination of which drives the growth of the tooth bud, leading to the formation of the primary EK as an independent cell cluster. Overall, the whole development of the tooth, from initiation to patterning, is driven by the iterative use of signaling centers.
Assuntos
Dente Molar/embriologia , Dente Molar/crescimento & desenvolvimento , Odontogênese/fisiologia , Transdução de Sinais , Animais , Apoptose/fisiologia , Proteínas de Ciclo Celular/genética , Divisão Celular , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário , Células Epiteliais , Camundongos , Dente Molar/citologia , Germe de Dente/citologia , Germe de Dente/embriologiaRESUMO
Ductal growth of the mammary gland occurs in two distinct stages. The first round of branching morphogenesis occurs during embryogenesis, and the second round commences at the onset of puberty. Currently, relatively little is known about the genetic networks that control the initial phases of ductal expansion, which, unlike pubertal development, proceeds independent of hormonal input in female mice. Here we identify NF-κB downstream of the TNF-like ligand ectodysplasin (Eda) as a unique regulator of embryonic and prepubertal ductal morphogenesis. Loss of Eda, or inhibition of NF-κB, led to smaller ductal trees with fewer branches. On the other hand, overexpression of Eda caused a dramatic NF-κB-dependent phenotype in both female and male mice characterized by precocious and highly increased ductal growth and branching that correlated with enhanced cell proliferation. We have identified several putative transcriptional target genes of Eda/NF-κB, including PTHrP, Wnt10a, and Wnt10b, as well as Egf family ligands amphiregulin and epigen. We developed a mammary bud culture system that allowed us to manipulate mammary development ex vivo and found that recombinant PTHrP, Wnt3A, and Egf family ligands stimulate embryonic branching morphogenesis, suggesting that these pathways may cooperatively mediate the effects of Eda.
Assuntos
Ectodisplasinas/metabolismo , Hormônios/farmacologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Glândulas Mamárias Animais/metabolismo , Morfogênese/efeitos dos fármacos , NF-kappa B/metabolismo , Anfirregulina , Androgênios/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Família de Proteínas EGF , Desenvolvimento Embrionário/efeitos dos fármacos , Fator de Crescimento Epidérmico/metabolismo , Epigen , Epitélio/efeitos dos fármacos , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Feminino , Glicoproteínas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Ligantes , Masculino , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Transgênicos , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteínas Wnt/metabolismoRESUMO
Oncolytic adenoviruses and certain chemotherapeutics can induce autophagy and immunogenic cancer cell death. We hypothesized that the combination of oncolytic adenovirus with low-dose temozolomide (TMZ) is safe, effective, and capable of inducing antitumor immune responses. Metronomic low-dose cyclophosphamide (CP) was added to selectively reduce regulatory T-cells. Preclinically, combination therapy inhibited tumor growth, increased autophagy, and triggered immunogenic cell death as indicated by elevated calreticulin, adenosine triphosphate (ATP) release, and nuclear protein high-mobility group box-1 (HMGB1) secretion. A total of 41 combination treatments given to 17 chemotherapy-refractory cancer patients were well tolerated. We observed anti- and proinflammatory cytokine release, evidence of virus replication, and induction of neutralizing antibodies. Tumor cells showed increased autophagy post-treatment. Release of HMGB1 into serum--a possible indicator of immune response--increased in 60% of treatments, and seemed to correlate with tumor-specific T-cell responses, observed in 10/15 cases overall (P = 0.0833). Evidence of antitumor efficacy was seen in 67% of evaluable treatments with a trend for increased survival over matched controls treated with virus only. In summary, the combination of oncolytic adenovirus with low-dose TMZ and metronomic CP increased tumor cell autophagy, elicited antitumor immune responses, and showed promising safety and efficacy.
Assuntos
Adenoviridae/genética , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Dacarbazina/análogos & derivados , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Trifosfato de Adenosina/metabolismo , Adenoviridae/fisiologia , Adolescente , Adulto , Idoso , Animais , Anticorpos Neutralizantes/sangue , Calreticulina/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Criança , Terapia Combinada/métodos , Ciclofosfamida/farmacologia , Citocinas/sangue , DNA Viral/sangue , Dacarbazina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Proteína HMGB1/sangue , Proteína HMGB1/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica , Pessoa de Meia-Idade , Vírus Oncolíticos/genética , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Temozolomida , Replicação Viral , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto JovemRESUMO
Mammary glands and hair follicles develop as ectodermal organs sharing common features during embryonic morphogenesis. The molecular signals controlling the initiation and patterning of skin appendages involve the bone morphogenetic proteins and Wnt family members, which are commonly thought to serve as inhibitory and activating cues, respectively. Here, we have examined the role of the Bmp and Wnt pathway modulator Sostdc1 in mammary gland, and hair and vibrissa follicle development using Sostdc1-null mice. Contrary to previous speculations, loss of Sostdc1 did not affect pelage hair cycling. Instead, we found that Sostdc1 limits the number of developing vibrissae and other muzzle hair follicles, and the size of primary hair placodes. Sostdc1 controls also the size and shape of mammary buds. Furthermore, Sostdc1 is essential for suppression of hair follicle fate in the normally hairless nipple epidermis, but its loss also promotes the appearance of supernumerary nipple-like protrusions. Our data suggest that functions of Sostdc1 can be largely attributed to its ability to attenuate Wnt/ß-catenin signaling.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Glândulas Mamárias Animais/embriologia , Pele/embriologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Morfogenéticas Ósseas/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Cabelo/crescimento & desenvolvimento , Cabelo/metabolismo , Glândulas Mamárias Animais/metabolismo , Camundongos , Pele/metabolismo , Vibrissas/crescimento & desenvolvimento , Vibrissas/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Oncolytic adenoviruses are an emerging experimental approach for treatment of tumors refractory to available modalities. Although preclinical results have been promising, and clinical safety has been excellent, it is also apparent that tumors can become virus resistant. The resistance mechanisms acquired by advanced tumors against conventional therapies are increasingly well understood, which has allowed development of countermeasures. To study this in the context of oncolytic adenovirus, we developed two in vivo models of acquired resistance, where initially sensitive tumors eventually gain resistance and relapse. These models were used to investigate the phenomenon on RNA and protein levels using two types of analysis of microarray data, quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. Interferon (IFN) signaling pathways were found upregulated and Myxovirus resistance protein A (MxA) expression was identified as a marker correlating with resistance, while transplantation experiments suggested a role for tumor stroma in maintaining resistance. Furthermore, pathway analysis suggested potential therapeutic targets in oncolytic adenovirus-resistant cells. Improved understanding of the antiviral phenotype causing tumor recurrence is of key importance in order to improve treatment of advanced tumors with oncolytic adenoviruses. Given the similarities between mechanisms of action, this finding might be relevant for other oncolytic viruses as well.
Assuntos
Adenoviridae/fisiologia , Interferons/biossíntese , Terapia Viral Oncolítica , Animais , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante HeterólogoRESUMO
Oncolytic adenoviruses are an emerging treatment option for advanced and refractory cancer. Such patients are often treated with corticosteroids to ameliorate tumor associated symptoms. Thus, it is important to evaluate whether safety is affected by immunosuppression possibly induced by corticosteroids. Concurrent low-dose cyclophosphamide, appealing for its immunomodulatory effects, could also impact safety. In a retrospective case-control study, we evaluated the effect of systemic corticosteroid use in cancer patients receiving oncolytic virotherapy. Four treatment groups were identified: (1) oncolytic adenovirus with oral glucocorticoids, (2) virus alone, (3) virus with glucocorticoids and cyclophosphamide and (4) virus with cyclophosphamide. Adverse events, neutralizing antibody titers, viral DNA in circulation and tumor responses were evaluated. The most common adverse effects were grade 1-2 fatigue, nausea, fever and abdominal pain. Common asymptomatic findings included self-limiting grade 1-3 hyponatremia and aspartate aminotransferase increase. Safety was good and no significant differences were observed between the groups. All patients had an increase in neutralizing antibody titers post-treatment, and no trends for differences between groups were observed. There were fewer post-treatment virus genomes circulating in patients receiving glucocorticoids when compared to their control groups. Overall, glucocorticoid use in cancer patients receiving oncolytic adenovirus, with or without low-dose cyclophosphamide, seems safe.
Assuntos
Adenoviridae/genética , Glucocorticoides/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Viral Oncolítica/efeitos adversos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Embryonic development of ectodermal organs involves a very dynamic range of cellular events and, therefore, requires advanced techniques to visualize them. Ectodermal organogenesis proceeds in well-defined sequential stages mediated by tissue interactions. Different ectodermal organs feature shared morphological characteristics, which are regulated by conserved and reiterative signaling pathways. A wealth of genetic information on the expression patterns and interactions of specific signaling pathways has accumulated over the years. However, the conventional developmental biology methods have mainly relied on two-dimensional tissue histological analyses at fixed time points limiting the possibilities to follow the processes in real time on a single cell resolution. This has complicated the interpretation of cause and effect relationships and mechanisms of the successive events. Whole-mount tissue live imaging approaches are now revealing how reshaping of the epithelial sheet for the initial placodal thickening, budding morphogenesis and beyond, involve coordinated four dimensional changes in cell shapes, well-orchestrated cell movements and specific cell proliferation and apoptosis patterns. It is becoming evident that the interpretation of the reiterative morphogenic signals takes place dynamically at the cellular level. Depending on the context, location, and timing they drive different cell fate choices and cellular interactions regulating a pattern of behaviors that ultimately defines organ shapes and sizes. Here we review how new tissue models, advances in 3D and live tissue imaging techniques have brought new understanding on the cell level behaviors that contribute to the highly dynamic stages of morphogenesis in teeth, hair and related ectodermal organs during development, and in dysplasia contexts.
RESUMO
New treatment approaches are needed for hormone refractory prostate cancer. Oncolytic adenoviruses are promising anti-cancer agents, and their efficacy can be improved by combining with conventional therapies such as ionizing radiation. The aim of this study was to determine the timing of oncolytic adenovirus treatment with regard to radiation and study the mechanisms of synergy in combination treatment. Prostate cancer cells were infected with oncolytic adenoviruses, irradiated and synergy mechanisms were assessed. In vivo models of combination treatment were tested. Radiation and oncolytic viruses were synergistic when viral infection was scheduled 24 hr after irradiation. Combination of oncolytic adenovirus with radiotherapy significantly increased antitumor efficacy in vivo compared to either agent alone. Microarray analysis showed dysregulated pathways including cell cycle, mTOR and antigen processing pathways. Functional analysis showed that adenoviral infection was accompanied with degradation of proteins involved in DNA break repair. Mre11 was degraded for subsequent inactivation of Chk2-Thr68 in combination treated cells, while gammaH2AX-Ser139 was elevated implicating the persistence of DNA double strand breaks. Increased autophagocytosis was seen in combination treated cells. Combination treatment did not increase apoptosis or virus replication. The results provide evidence of the antitumor efficacy of combining oncolytic adenoviruses with irradiation as a therapeutic strategy for the treatment of prostate cancer. Further, these findings propose a molecular mechanism that may be important in radiation induced cell death, autophagy and viral cytopathic effect.
Assuntos
Autofagia , Proteínas de Ligação a DNA/antagonistas & inibidores , Terapia Viral Oncolítica , Neoplasias da Próstata/terapia , Radiação Ionizante , Adenoviridae/genética , Animais , Apoptose , Terapia Combinada , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Perfilação da Expressão Gênica , Humanos , Proteína Homóloga a MRE11 , Masculino , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , Vírus Oncolíticos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Células Tumorais Cultivadas , Replicação Viral , Irradiação Corporal Total , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
During organogenesis, cell fate specification and patterning are regulated by signaling centers, specialized clusters of morphogen-expressing cells. In many organs, initiation of development is marked by bud formation, but the cellular mechanisms involved are ill defined. Here, we use the mouse incisor tooth as a model to study budding morphogenesis. We show that a group of nonproliferative epithelial cells emerges in the early tooth primordium and identify these cells as a signaling center. Confocal live imaging of tissue explants revealed that although these cells reorganize dynamically, they do not reenter the cell cycle or contribute to the growing tooth bud. Instead, budding is driven by proliferation of the neighboring cells. We demonstrate that the activity of the ectodysplasin/Edar/nuclear factor κB pathway is restricted to the signaling center, and its inactivation leads to fewer quiescent cells and a smaller bud. These data functionally link the signaling center size to organ size and imply that the early signaling center is a prerequisite for budding morphogenesis.
Assuntos
Movimento Celular , Proliferação de Células , Células Epiteliais/fisiologia , Incisivo/embriologia , Animais , Ectodisplasinas/genética , Ectodisplasinas/metabolismo , Receptor Edar/genética , Receptor Edar/metabolismo , Células Epiteliais/metabolismo , Fase G1 , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Idade Gestacional , Incisivo/metabolismo , Camundongos Transgênicos , Microscopia Confocal , Morfogênese , NF-kappa B/genética , NF-kappa B/metabolismo , Tamanho do Órgão , Fenótipo , Transdução de SinaisRESUMO
Palmitoyl protein thioesterase 1 (PPT1) is a depalmitoylating enzyme whose deficiency leads to infantile neuronal ceroid lipofuscinosis. The disease is characterized by early loss of vision and massive neuronal death. Although PPT1 is expressed in many tissues, a deficiency of PPT1 damages neurons only in the cerebral and cerebellar cortexes and retina; other cell types remain relatively unaffected. We previously demonstrated that PPT1 is present in the synaptosomes and synaptic vesicles of neurons. To understand the crucial role of PPT1 for neuronal cells, we further investigated the expression and targeting of PPT1 in retinal, hippocampal, and cortical neurons during their maturation in culture. We found that PPT1 activity increases by neuronal maturation and is highest in retinal neuron cultures. In retinal neurons the expression of PPT1 precedes that of the synaptic vesicle protein 2 and synaptophysin, indicating a significant role for PPT1 in the early development of neuronal cells. We also found by quantitative confocal immunofluorescence microscopy that PPT1 is targeted preferably to axons in mature neurons, as indicated by its colocalization with the axonal marker microtubule-associated protein 1. In axons PPT1 is targeted specifically to axonal varicosities and presynaptic terminals, as indicated by its significant colocalization with growth-associated protein 43 and synaptophysin. Axonal localization of PPT1 was confirmed by double labeling with synaptophysin and postembedding immunoelectron microscopy. The polarized axonal targeting of PPT1 may well indicate a role for PPT1 in the exocytotic pathway of neurons.
Assuntos
Axônios/enzimologia , Córtex Cerebral/crescimento & desenvolvimento , Hipocampo/crescimento & desenvolvimento , Neurônios/enzimologia , Retina/crescimento & desenvolvimento , Tioléster Hidrolases/metabolismo , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Western Blotting , Técnicas de Cultura de Células , Córtex Cerebral/química , Córtex Cerebral/ultraestrutura , Hipocampo/química , Hipocampo/ultraestrutura , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica , Microscopia Imunoeletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/metabolismo , Retina/química , Retina/ultraestrutura , Sinaptofisina/metabolismoRESUMO
Here, we report full-length genome sequences of influenza pH1N1 viruses obtained prior to and after propagation in MDCK cells. Paired comparisons of the genomes showed that each strain acquired 1.0 to 18.8 mutations per genome per replication cycle, which corresponds to 0.5 to 5.8 mutations per virus proteome per replication cycle. Our analysis indicates that pH1N1 viruses accumulated adaptive mutations among others in response to propagation in cell culture. These results could be important for vaccine and drug-sensitivity surveillance studies, as well as for vaccine and antiviral drug development programs where cell cultures are used for influenza propagation.
RESUMO
Epithelial reorganization involves coordinated changes in cell shapes and movements. This restructuring occurs during formation of placodes, ectodermal thickenings that initiate the morphogenesis of epithelial organs including hair, mammary gland, and tooth. Signaling pathways in ectodermal placode formation are well known, but the cellular mechanisms have remained ill defined. We established imaging methodology for live visualization of embryonic skin explants during the first wave of hair placode formation. We found that the vast majority of placodal cells were nonproliferative throughout morphogenesis. We show that cell compaction and centripetal migration are the main cellular mechanisms associated with hair placode morphogenesis and that inhibition of actin remodeling suppresses placode formation. Stimulation of both ectodysplasin/NF-κB and Wnt/ß-catenin signaling increased cell motility and the number of cells committed to placodal fate. Thus, cell fate choices and morphogenetic events are controlled by the same molecular pathways, providing the framework for coordination of these two processes.
Assuntos
Movimento Celular , Proliferação de Células , Embrião de Mamíferos/citologia , Folículo Piloso/citologia , Queratinas/fisiologia , Morfogênese/fisiologia , Animais , Western Blotting , Ciclo Celular , Ectodisplasinas/fisiologia , Embrião de Mamíferos/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Folículo Piloso/embriologia , Folículo Piloso/metabolismo , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência , NF-kappa B/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
PURPOSE: In the present study, we evaluated the combination of replication-deficient adenoviruses and radiotherapy in vitro. The purpose of the present study was to analyze the mechanism of radiation-mediated upregulation of adenoviral transgene expression. METHODS AND MATERIALS: Adenoviral transgene expression (luciferase or green fluorescent protein) was studied with and without radiation in three cell lines: breast cancer M4A4-LM3, prostate cancer PC-3MM2, and lung cancer LNM35/enhanced green fluorescent protein. The effect of the radiation dose, modification of the viral capsid, and five different transgene promoters were studied. The cellular responses were studied using mass spectrometry and immunofluorescence analysis. Double strand break repair was modulated by inhibitors of heat shock protein 90, topoisomerase-I, and DNA protein kinase, and transgene expression was measured. RESULTS: We found that a wide range of radiation doses increased adenoviral transgene expression regardless of the cell line, transgene, promoter, or viral capsid modification. Treatment with adenovirus, radiation, and double strand break repair inhibitors resulted in persistence of double strand breaks and subsequent increases in adenovirus transgene expression. CONCLUSIONS: Radiation-induced enhancement of adenoviral transgene expression is linked to DNA damage recognition and repair. Radiation induces a global cellular response that results in increased production of RNA and proteins, including adenoviral transgene products. This study provides a mechanistic rationale for combining radiation with adenoviral gene delivery.
Assuntos
Adenovírus Humanos/efeitos da radiação , Reparo do DNA , Vírus Defeituosos/efeitos da radiação , Expressão Gênica/efeitos da radiação , Terapia Genética/métodos , Vetores Genéticos/efeitos da radiação , Transgenes/efeitos da radiação , Adenovírus Humanos/genética , Linhagem Celular Tumoral , Terapia Combinada/métodos , Quebras de DNA de Cadeia Dupla , Vírus Defeituosos/genética , Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Regulação Viral da Expressão Gênica/efeitos da radiação , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Luciferases/genética , Inibidores de Proteínas Quinases/farmacologia , Doses de Radiação , Inibidores da Topoisomerase I/farmacologia , Transgenes/genética , Regulação para Cima/genética , Regulação para Cima/efeitos da radiação , Replicação Viral/genética , Replicação Viral/efeitos da radiaçãoRESUMO
The continuously growing mouse incisor serves as a valuable model to study stem cell regulation during organ renewal. Epithelial stem cells are localized in the proximal end of the incisor in the labial cervical loop. Here, we show that the transcription factor Sox2 is a specific marker for these stem cells. Sox2+ cells became restricted to the labial cervical loop during tooth morphogenesis, and they contributed to the renewal of enamel-producing ameloblasts as well as all other epithelial cell lineages of the tooth. The early progeny of Sox2-positive stem cells transiently expressed the Wnt inhibitor Sfrp5. Sox2 expression was regulated by the tooth initiation marker FGF8 and specific miRNAs, suggesting a fine-tuning to maintain homeostasis of the dental epithelium. The identification of Sox2 as a marker for the dental epithelial stem cells will facilitate further studies on their lineage segregation and differentiation during tooth renewal.
Assuntos
Linhagem da Célula , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fatores de Transcrição SOXB1/biossíntese , Células-Tronco/metabolismo , Dente/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Células Epiteliais/química , Células Epiteliais/citologia , Fator 8 de Crescimento de Fibroblasto/genética , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , MicroRNAs/genética , Técnicas de Cultura de Órgãos , Fatores de Transcrição SOXB1/análise , Células-Tronco/química , Células-Tronco/citologia , Dente/citologia , Dente/embriologia , Dente/crescimento & desenvolvimentoRESUMO
BACKGROUND: Successful tumor targeting of systemically administered oncolytic adenoviruses may be hindered by interactions with blood components. MATERIALS AND METHODS: Blood distribution of oncolytic adenoviruses featuring type 5 adenovirus fiber, 5/3 capsid chimerism, or RGD-4C in the fiber knob was investigated in vitro and in patients with refractory solid tumors. RESULTS: Virus titers and prevalence in serum of patients increased over the first post-treatment week, suggesting replication. Detection of low virus loads was more sensitive in blood clots than in serum, although viral levels > 500 viral particles/mL did not differ significantly between both sample types. While adenovirus bound to erythrocytes, platelets, granulocytes, and peripheral blood mononuclear cells in vitro, the virus was mainly detectable in erythrocytes and granulocytes in cancer patients. Taken together with a temporary post-treatment decrease in thrombocyte counts, platelet activation by adenovirus and subsequent clearance seem likely to occur in humans. Fiber modifications had limited observed effect on virus distribution in blood cell compartments. Neutrophils, monocytes and cytotoxic T lymphocytes were the major leukocyte subpopulations interacting with adenoviruses. CONCLUSION: Serum and blood clots are relevant to estimate oncolytic adenovirus replication. Insight into viral interactions with blood cells may contribute to the development of new strategies for tumor delivery.
Assuntos
Adenoviridae/metabolismo , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Coagulação Sanguínea , Plaquetas/metabolismo , Eritrócitos/metabolismo , Granulócitos/metabolismo , Humanos , Monócitos/metabolismo , Neoplasias/patologia , Neutrófilos/metabolismo , Vírus Oncolíticos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Replicação ViralRESUMO
BACKGROUND: Cancer stem cells/initiating cells (CSC/CIC), are thought to exist as a small population in malignant tissues. They are resistant to conventional cancer treatments and possibly underlie post-treatment relapse. The CIC population can be targeted with capsid modified oncolytic adenoviruses. METHODOLOGY/PRINCIPAL FINDINGS: We studied the mechanisms of innate immunity to oncolytic adenovirus Ad5/3-Delta24 in conventional treatment resistant non-CIC breast cancer cells, breast cancer CD44(+)/CD24(-/low) CIC population and normal breast tissue CD44(+)/CD24(-/low) stem cells. We compared virus recognition by pattern recognition receptors for adenovirus, Toll-like receptors (TLR) 2 and 9 and virus induced type I interferon (IFN) response regulation in these cell types. We show TLR mediated virus recognition in these non-immune cell types. Normal tissue stem cells have intact type I IFN signaling. Furthermore, TLR9 and TLR2 reside constantly in recognition sites, implying constant activation. In contrast, breast cancer CD44(+)/CD24(-/low) CIC have dysregulated innate immune responses featuring dysfunctional virus recognition caused by impaired trafficking of TLR9 and cofactor MyD88 and the absence of TLR2, having a deleterious impact on TLR pattern recognition receptor signaling. Furthermore, the CIC have increased inhibitory signaling via the suppressor of cytokine signaling/Tyro3/Axl/Mer receptor tyrosine kinase (SOCS/TAM) pathway. These defects in contribute to dysfunctional induction of type I IFN response in CIC and therefore permissivity to oncolytic adenovirus. CONCLUSIONS/SIGNIFICANCE: CICs may underlie the incurable nature of relapsed or metastatic cancers and are therefore an important target regarding diagnostic and prognostic aspects as well as treatment of the disease. This study addresses the mechanisms of innate infection immunity in stem cells deepening the understanding of stem cell biology and may benefit not only virotherapy but also immunotherapy in general.
Assuntos
Adenoviridae/imunologia , Imunidade Inata/imunologia , Células-Tronco Neoplásicas/imunologia , Vírus Oncolíticos/imunologia , Adenoviridae/fisiologia , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Antígeno CD24/metabolismo , Linhagem Celular Tumoral , Feminino , Interações Hospedeiro-Patógeno/imunologia , Humanos , Receptores de Hialuronatos/metabolismo , Tolerância Imunológica/imunologia , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/virologia , Vírus Oncolíticos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismoRESUMO
PURPOSE: Radiotherapy is widely used for treatment of many tumor types, but it can damage normal tissues. It has been proposed that cancer cells can be selectively sensitized to radiation by adenovirus replication or by using radiosensitizing transgenes. Adenoviral proteins E1B55K, E4orf3, and E4orf6 play a role in radiosensitization, by targeting the Mre11, Rad50, and NBS1 complex (MRN) and inhibiting DNA double-strand break (DSB) repair. We hypothesize that combined with irradiation, these adenoviral proteins increase cell killing through the impairment of DSB repair. METHODS AND MATERIALS: We assessed the radiosensitizing/additive potential of replication-deficient adenoviruses expressing E1B55K, E4orf3, and E4orf6 proteins. Combination treatments with low-dose external photon beam radiotherapy were studied in prostate cancer (PC-3MM2 and DU-145), breast cancer (M4A4-LM3), and head and neck cancer (UT-SCC8) cell lines. We further demonstrated radiosensitizing or additive effects in mice with PC-3MM2 tumors. RESULTS: We show enhanced cell killing with adenovirus and radiation combination treatment. Co-infection with several of the viruses did not further increase cell killing, suggesting that both E4orf6 and E4orf3 are potent in MRN inhibition. Our results show that adenoviral proteins E4orf3 and E4orf6, but not E1B55K, are effective also in vivo. Enhanced cell killing was due to inhibition of DSB repair resulting in persistent double-strand DNA damage, indicated by elevated phospho-H2AX levels at 24 h after irradiation. CONCLUSIONS: This knowledge can be applied for improving the treatment of malignant tumors, such as prostate cancer, for development of more effective combination therapies and minimizing radiation doses and reducing side effects.
Assuntos
Proteínas E4 de Adenovirus/fisiologia , Adenovírus Humanos/metabolismo , Neoplasias/radioterapia , Tolerância a Radiação/fisiologia , Proteínas Virais/fisiologia , Proteínas E4 de Adenovirus/metabolismo , Adenovírus Humanos/genética , Animais , Neoplasias da Mama/radioterapia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Neoplasias de Cabeça e Pescoço/radioterapia , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/virologia , Neoplasias da Próstata/radioterapia , Dosagem Radioterapêutica , Distribuição Aleatória , Proteínas Virais/metabolismoRESUMO
PURPOSE: Twenty-one patients with cancer were treated with a single round of oncolytic adenovirus ICOVIR-7. EXPERIMENTAL DESIGN: ICOVIR-7 features an RGD-4C modification of the fiber HI-loop of serotype 5 adenovirus for enhanced entry into tumor cells. Tumor selectivity is mediated by an insulator, a modified E2F promoter, and a Rb-binding site deletion of E1A, whereas replication is optimized with E2F binding hairpins and a Kozak sequence. ICOVIR-7 doses ranged from 2 x 10(10) to 1 x 10(12) viral particles. All patients had advanced and metastatic solid tumors refractory to standard therapies. RESULTS: ICOVIR-7 treatment was well tolerated with mild to moderate fever, fatigue, elevated liver transaminases, chills, and hyponatremia. One patient had grade 3 anemia but no other serious side effects were seen. At baseline, 9 of 21 of patients had neutralizing antibody titers against the ICOVIR-7 capsid. Treatment resulted in neutralizing antibody titer induction within 4 weeks in 16 of 18 patients. No elevations of serum proinflammatory cytokine levels were detected. Viral genomes were detected in the circulation in 18 of 21 of patients after injection and 7 of 15 of the samples were positive 2 to 4 weeks later suggesting viral replication. CONCLUSIONS: Overall, objective evidence of antitumor activity was seen in 9 of 17 evaluable patients. In radiological analyses, 5 of 12 evaluable patients had stabilization or reduction in tumor size. These consisted of one partial response, two minor responses and two cases of stable disease, all occurring in patients who had progressive disease before treatment. In summary, ICOVIR-7 treatment is apparently safe, resulting in anticancer activity, and is therefore promising for further clinical testing.
Assuntos
Adenoviridae , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Adolescente , Adulto , Idoso , Anticorpos Antivirais/análise , Criança , Feminino , Humanos , Interleucinas/sangue , Masculino , Pessoa de Meia-Idade , Terapia Viral Oncolítica/efeitos adversos , Retratamento , Resultado do Tratamento , Replicação ViralRESUMO
Granulocyte macrophage colony-stimulating factor (GMCSF) can mediate antitumor effects by recruiting natural killer cells and by induction of tumor-specific cytotoxic T-cells through antigen-presenting cells. Oncolytic tumor cell-killing can produce a potent costimulatory danger signal and release of tumor epitopes for antigen-presenting cell sampling. Therefore, an oncolytic adenovirus coding for GMCSF was engineered and shown to induce tumor-specific immunity in an immunocompetent syngeneic hamster model. Subsequently, 20 patients with advanced solid tumors refractory to standard therapies were treated with Ad5-D24-GMCSF. Of the 16 radiologically evaluable patients, 2 had complete responses, 1 had a minor response, and 5 had disease stabilization. Responses were frequently seen in injected and noninjected tumors. Treatment was well tolerated and resulted in the induction of both tumor-specific and virus-specific immunity as measured by ELISPOT and pentamer analysis. This is the first time that oncolytic virus-mediated antitumor immunity has been shown in humans. Ad5-D24-GMCSF is promising for further clinical testing.
Assuntos
Adenoviridae/genética , Fator Estimulador de Colônias de Granulócitos/genética , Imunoterapia/métodos , Neoplasias/terapia , Terapia Viral Oncolítica/métodos , Adenoviridae/imunologia , Adenoviridae/metabolismo , Animais , Cricetinae , Epitopos de Linfócito T/imunologia , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/imunologia , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/imunologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/virologia , Survivina , Linfócitos T/imunologia , TransfecçãoRESUMO
Infantile neuronal ceroid lipofuscinosis (INCL) is a severe neurodegenerative disorder of children, characterized by selective death of neocortical neurons. To understand early disease mechanisms in INCL, we have studied Ppt1(Deltaex4) knock-out mouse neurons in culture and acute brain slices. Global transcript profiling showed deregulation of key neuronal functions in knock-out mice including cholesterol metabolism, neuronal maturation, and calcium homeostasis. Cholesterol metabolism showed major changes; sterol biosynthesis was enhanced and steady-state amounts of sterols were altered at the cellular level. Changes were also present in early maturation of Ppt1(Deltaex4) neurons indicated by increased proliferative capacity of neuronal stem cells. Knock-out neurons presented unaltered electrophysiological properties suggesting uncompromised synaptic function in young animals. However, knock-out neurons exhibited more efficient recovery from glutamate-induced calcium transients, possibly indicating neuroprotective activation. This study established that the neuronal deregulation in INCL is linked to neuronal maturation, lipid metabolism and calcium homeostasis.