RESUMO
Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.
Assuntos
Neoplasias , Linfócitos T , Humanos , Neoplasias/genética , Neoplasias/terapia , Imunoterapia/métodos , Organoides/patologiaRESUMO
The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDH-related PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to the classical 2D-culture of SDH-related human paragangliomas and pheochromocytomas, with the ultimate goal of identifying a viable tumour-derived cell line. PPGL tumour tissue/cells (chromaffin cells) were cultured in a variety of media formulations and supplements. Tumour explants and dissociated primary tumour cells were cultured and stained with a range of antibodies to identify markers suitable for use in human PPGL culture. We cultured 62 PPGLs, including tumours with confirmed SDHB, SDHC and SDHD variants, as well as several metastatic tumours. Testing a wide range of basic cell culture media and supplements, we noted a marked decline in chromaffin cell numbers over a 4-8 week period but the persistence of small numbers of synaptophysin/tyrosine hydroxylase-positive chromaffin cells for up to 99 weeks. In cell culture, immunohistochemical staining for chromogranin A and neuron-specific enolase was generally negative in chromaffin cells, while staining for synaptophysin and tyrosine hydroxylase was generally positive. GFAP showed the most consistent staining of type II sustentacular cells. Of the media tested, low serum or serum-free media best sustained relative chromaffin cell numbers, while lactate enhanced the survival of synaptophysin-positive cells. Synaptophysin-positive PPGL tumour cells persist in culture for long periods but show little evidence of proliferation. Synaptophysin was the most consistent cell marker for chromaffin cells and GFAP the best marker for sustentacular cells in human PPGL cultures.
Assuntos
Neoplasias das Glândulas Suprarrenais , Paraganglioma , Feocromocitoma , Neoplasias das Glândulas Suprarrenais/patologia , Cromogranina A/metabolismo , Meios de Cultura Livres de Soro , Mutação em Linhagem Germinativa , Humanos , Lactatos , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/patologia , Fosfopiruvato Hidratase/metabolismo , Succinato Desidrogenase/genética , Succinato Desidrogenase/metabolismo , Sinaptofisina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7-21 d; generation of genetically manipulated clonal organoid cultures takes 14-21 d; and organoid expansion for xenotransplantation takes >4 weeks.
Assuntos
Neoplasias da Mama/patologia , Mama/patologia , Técnicas de Cultura de Células/métodos , Técnicas Genéticas , Organoides/patologia , Transplante Heterólogo , Animais , Bancos de Espécimes Biológicos , Células Clonais , Feminino , Humanos , Camundongos , Fatores de TempoRESUMO
CONTEXT: Pheochromocytomas and paragangliomas (PPGLs) are neuroendocrine tumors in which altered central metabolism appears to be a major driver of tumorigenesis, and many PPGL genes encode proteins involved in the tricarboxylic acid (TCA) cycle. OBJECTIVE/DESIGN: While about 40% of PPGL cases carry a variant in a known gene, many cases remain unexplained. In patients with unexplained PPGL showing clear evidence of a familial burden or multiple tumors, we aimed to identify causative factors using genetic analysis of patient DNA and functional analyses of identified DNA variants in patient tumor material and engineered cell lines. PATIENTS AND SETTING: Patients with a likely familial cancer burden of pheochromocytomas and/or paragangliomas and under investigation in a clinical genetic and clinical research setting in university hospitals. RESULTS: While investigating unexplained PPGL cases, we identified a novel variant, c.1151C>T, p.(Pro384Leu), in exon 14 of the gene encoding dihydrolipoamide S-succinyltransferase (DLST), a component of the multi-enzyme complex 2-oxoglutarate dehydrogenase. Targeted sequence analysis of further unexplained cases identified a patient carrying a tumor with compound heterozygous variants in DLST, consisting of a germline variant, c.1121G>A, p.(Gly374Glu), together with a somatic missense variant identified in tumor DNA, c.1147A>G, p.(Thr383Ala), both located in exon 14. Using a range of in silico and functional assays we show that these variants are predicted to be pathogenic, profoundly impact enzyme activity, and result in DNA hypermethylation. CONCLUSIONS: The identification and functional analysis of these DLST variants further validates DLST as an additional PPGL gene involved in the TCA cycle.
Assuntos
Aciltransferases/genética , Neoplasias das Glândulas Suprarrenais/patologia , Metilação de DNA , Epigênese Genética , Mutação em Linhagem Germinativa , Paraganglioma/patologia , Feocromocitoma/patologia , Neoplasias das Glândulas Suprarrenais/genética , Adulto , Idoso , Biomarcadores/análise , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Paraganglioma/genética , Feocromocitoma/genética , Prognóstico , Adulto JovemRESUMO
Lgr6+ cells have been identified as a novel class of proliferating (Ki67+) stem cells in mouse epidermis. We investigated their response to UV exposure in Lgr6-EGFP-Ires-CreERT2/R26R-LacZ haired and hairless mice and whether they become initiating cells of UV- or chemically induced skin tumors. UV overexposure erased Lgr6+ cells (EGFP+) from the interfollicular epidermis (IFE), but - as after wounding - they apparently repopulated the IFE from the hair follicles. Under sub-sunburn chronic UV exposure, Lgr6+ cells and their progeny (LacZ+ after pulse of tamoxifen) diminished strongly in the IFE. Although the inter-tumoral IFE clearly showed Lgr6 progeny, none of the UV- or chemically induced tumors (n = 22 and 41, respectively) appeared to be clonal expansions of Lgr6+ stem cells; i.e. no Lgr6+ cells or progeny in the proliferating tumor bulk. In checking for promoter methylation we found it to occur stochastically for the EGFP-Cre cassette. Lgr6 mRNA measured by qPCR was found to be diminished in skin tumors (also in UV tumors from wt type mice). The ratio of Lgr6/Ki67 was significantly reduced, pointing at a loss of Lgr6+ cells from the proliferative pool. Our data show that Lgr6+ cells are not major tumor-initiating cells in skin carcinogenesis.
Assuntos
Epiderme/metabolismo , Receptores Acoplados a Proteínas G/genética , Neoplasias Cutâneas/genética , Células-Tronco/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/efeitos da radiação , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/efeitos da radiação , Epiderme/efeitos dos fármacos , Epiderme/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Folículo Piloso/efeitos dos fármacos , Folículo Piloso/metabolismo , Folículo Piloso/efeitos da radiação , Camundongos Pelados , Camundongos Transgênicos , Regiões Promotoras Genéticas/genética , Receptores Acoplados a Proteínas G/metabolismo , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Neoplasias Cutâneas/metabolismo , Raios UltravioletaRESUMO
Actively proliferating Lgr5+ skin stem cells are found deep in the hair follicle (HF). These cells renew the HF and drive its expansion in anagen phase. Their long residence and continuous mitotic activity make them prime candidates to transform into skin tumor-initiating cells. This was investigated by subjecting Lgr5-EGFP-Ires-CreERT2/R26R-LacZ mice (haired and hairless) to chemical and UV carcinogenic regimens. In the course of these regimens Lgr5+ cells (EGFP+) remained exclusively located in HFs, and in deep-seated cysts of hairless skin. In haired mice, progeny of Lgr5+ stem cells (LacZ+ after a pulse of tamoxifen) appeared in the interfollicular epidermis upon UV-induced sunburn and in TPA-induced hyperplasia. In hairless mice the progeny remained located in deep-seated cysts and in HF remnants. Progeny in hairless skin was only detected interfollicularly at a late stage, in between outgrowing tumors. Lgr5+ stem cells were absent in the ultimate tumor masses, and no tumor appeared to be a (clonal) expansion of Lgr5+ cells (52 tumors with tamoxifen at the start of carcinogenesis, 42 tumors with tamoxifen late during tumor outgrowth). In contrast to CD34/K15+ quiescent bulge stem cells, actively proliferating Lgr5+ stem cells do therefore not appear to be tumor drivers in experimental skin carcinogenesis.
Assuntos
Transformação Celular Neoplásica/patologia , Folículo Piloso/patologia , Receptores Acoplados a Proteínas G/metabolismo , Neoplasias Cutâneas/patologia , Células-Tronco/patologia , Animais , Epiderme/patologia , Camundongos , Camundongos Pelados , Camundongos TransgênicosRESUMO
Sunburns and especially sub-sunburn chronic UV exposure are associated with increased risk of squamous cell carcinomas (SCCs). Here we focus on a possible difference in tumor initiation from a single severe-sunburn dose (on day 1, 21 hairless mice) and from an equal dose fractionated into very low sub-sunburn doses not causing any (growth-promoting) epidermal hyperplasia (40 days daily exposure, n=20). From day 47 all mice received 12-O-Tetradecanoylphorbol-13-acetate (TPA) applications (2x/wk) for 20 weeks to promote tumor development within the lifetime of the animals. After the sub-sunburn regimen sparse DNA damage-retaining basal cells (quiescent stem cells, QSCs) remained in the non-hyperplastic epidermis. These cells were forced to divide by TPA. After discontinuation of TPA tumors regressed and disappeared in the 'sunburn group' but persisted and grew in the 'sub-sunburn group' (0.06 vs 2.50 SCCs and precursors ≥4 mm/mouse after 280 days, p=0.03). As the tumors carried no mutations in p53, H/K/N-Ras and Notch1/2, these 'usual suspects' were not involved in the UV-driven tumor initiation. Although we could not selectively eliminate QSCs (unknown phenotype) to establish causality, our data suggest that forcing specifically DNA damage-retaining QSCs to divide--with high mutagenic risk--gives rise to persisting (mainly 'in situ') skin carcinomas.
Assuntos
Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/patologia , Dano ao DNA/efeitos da radiação , Epiderme/patologia , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/toxicidade , Raios Ultravioleta/efeitos adversos , Animais , Carcinógenos/toxicidade , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/metabolismo , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Epiderme/metabolismo , Epiderme/efeitos da radiação , Feminino , Hiperplasia/etiologia , Hiperplasia/metabolismo , Hiperplasia/patologia , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Pelados , Mutação/genética , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
Mortality from colorectal cancer increases with latitude and decreases with ambient UV radiation. We investigated whether moderate UV dosages could inhibit intestinal tumor development and whether this corresponded with UV-induced vitamin D. FabplCre;Apc(15lox/+) mice, which develop intestinal tumors, and their parents were put on a vitamin D-deficient diet. Next to a control group, one group was vitamin D supplemented and another one group was daily UV irradiated from 6 weeks of age. Vitamin D statuses after 6 weeks of treatment were markedly increased: mean ± SD from 7.7 ± 1.9 in controls to 75 ± 15 nmol/l with vitamin D supplementation (no gender difference), and to 31 ± 13 nmol/l in males and 85 ± 17 nmol/l in females upon UV irradiation. The tumor load (area covered by tumors) at 7.5 months of age was significantly reduced in both the vitamin D-supplemented group (130 ± 25 mm(2), p = 0.018) and the UV-exposed group (88 ± 9 mm(2), p < 0.0005; no gender differences) compared to the control group (202 ± 23 mm(2)). No reductions in tumor numbers were found. Only UV exposure appeared to reduce progression to malignancy (p = 0.014). Our experiments clearly demonstrate for the first time an inhibitory effect of moderate UV exposure on outgrowth and malignant progression of primary intestinal tumors, which at least in part can be attributed to vitamin D.
Assuntos
Genes APC/fisiologia , Neoplasias Intestinais/patologia , Neoplasias Intestinais/prevenção & controle , Raios Ultravioleta , Vitamina D/administração & dosagem , Vitaminas/administração & dosagem , Animais , Suplementos Nutricionais , Progressão da Doença , Feminino , Neoplasias Intestinais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Immunosuppressive drugs are thought to cause the dramatically increased risk of carcinomas in sun-exposed skin of organ transplant recipients. These drugs differ in local effects on skin. We investigated whether this local impact is predictive of skin cancer risk and may thus provide guidance on minimizing the risk. Immunosuppressants (azathioprine, cyclosporine, tacrolimus, mycophenolate mofetil, and rapamycin) were assessed on altering the UV induction of apoptosis in human skin models and of p53 mutant cell clones (putative tumor precursors) and ensuing skin carcinomas (with mutant p53) in the skin of hairless mice. Rapamycin was found to increase apoptosis (three-fold), whereas cyclosporine decreased apoptosis (three-fold). Correspondingly, a 1.5- to five-fold reduction (P = 0.07) or a two- to three-fold increase (P < 0.001) was found in cell clusters overexpressing mutant p53 in chronically UV-exposed skin of mice that had been fed rapamycin or cyclosporine, respectively. Deep sequencing showed, however, that the allelic frequency (â¼5%) of the hotspot mutations in p53 (codons 270 and 275) remained unaffected. The majority of cells with mutated p53 seemed not to overexpress the mutated protein. Unexpectedly, none of the immunosuppressants admixed in high dosages to the diet accelerated tumor development, and cyclosporine even delayed tumor onset by approximately 15% (P < 0.01). Thus, in contrast to earlier findings, the frequency of p53-mutant cells was not predictive of the incidence of skin carcinoma. Moreover, the lack of any accelerative effect on tumor development suggests that immunosuppressive medication is not the sole cause of the dramatic increase in skin cancer risk in organ transplant recipients.
Assuntos
Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Dieta , Imunossupressores/farmacologia , Neoplasias Induzidas por Radiação/patologia , Neoplasias Cutâneas/patologia , Animais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/genética , Transformação Celular Neoplásica/efeitos da radiação , Células Cultivadas , Células Clonais/metabolismo , Células Clonais/patologia , Células Clonais/efeitos da radiação , Progressão da Doença , Feminino , Genes p53 , Humanos , Imunossupressores/administração & dosagem , Imunossupressores/efeitos adversos , Masculino , Camundongos , Camundongos Pelados , Proteínas Mutantes/fisiologia , Mutação/fisiologia , Neoplasias Induzidas por Radiação/genética , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Raios UltravioletaRESUMO
Sézary syndrome (SS) is an aggressive cutaneous T-cell lymphoma with CD4+ tumor cells localized in the skin, lymph nodes and peripheral blood. Characteristic molecular aberrancies in SS have been identified; however, paucity of functional models severely hampered the translation of these observations into pathogenic mechanisms, and subsequent validation of novel therapeutic targets. We therefore developed a mouse model for SS using intrahepatic injection of SS cells in newborn immunodeficient RAG2(-/-) γc(-/-) mice that are completely devoid of T-, B- and NK-cell activity. Injection of the SS cell line SeAx led to long-term and reproducible systemic repopulation of the mice. Injection of mice with the SS cell line HuT-78 led to the death of the mice owing to massive growth of internal tumors. Four weeks after injection of primary SS cells, human CD3+ T cells could be tracked back in the liver, peripheral blood, lymph nodes, spleen and skin of the mice, although the engraftment rate varied when using cells from different patients. In conclusion, we demonstrate that injection of SS cell lines or primary cells in newborn RAG2(-/-) γc(-/-) mice results in long-term systemic repopulation of the mice, thereby providing a novel mouse model for Sézary syndrome.
Assuntos
Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Síndrome de Sézary/patologia , Neoplasias Cutâneas/patologia , Animais , Linfócitos T CD4-Positivos/transplante , Humanos , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Antígeno Ki-67/metabolismo , Linfonodos/metabolismo , Camundongos , Camundongos Knockout , Camundongos SCID , Transplante de Neoplasias , Síndrome de Sézary/imunologia , Síndrome de Sézary/metabolismo , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/metabolismoRESUMO
Skin cancer is the most prevalent cancer worldwide and is primarily caused by chronic UV exposure. Here, we describe the topical field-directed treatment of SKH1/hr mice with UVB-damaged skin with ingenol mebutate, a new topical drug shown to be effective for the treatment of actinic keratosis (AK). Application of 0.05% ingenol mebutate gel to photo-damaged skin resulted in a ≈70% reduction in the number of skin lesions that subsequently emerged compared with placebo treatment. Ingenol mebutate treatment also reduced the number of mutant p53 keratinocyte patches by ≈70%. The treatment resulted in epidermal cell death, acute inflammation, recruitment of neutrophils, hemorrhage, and eschar formation, all of which resolved over several weeks. Ingenol mebutate field-directed treatment might thus find utility in the removal of subclinical precancerous cells from UV-damaged skin. Field-directed treatment may be particularly suitable for patients who have AKs surrounded by UV-damaged skin.
Assuntos
Diterpenos/uso terapêutico , Queratinócitos/metabolismo , Queratinócitos/patologia , Ceratose Actínica/tratamento farmacológico , Lesões Pré-Cancerosas/tratamento farmacológico , Pele/efeitos da radiação , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta/efeitos adversos , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/prevenção & controle , Modelos Animais de Doenças , Diterpenos/farmacologia , Relação Dose-Resposta a Droga , Queratinócitos/efeitos dos fármacos , Ceratose Actínica/patologia , Masculino , Camundongos , Camundongos Pelados , Mutação/genética , Neoplasias Induzidas por Radiação/prevenção & controle , Lesões Pré-Cancerosas/patologia , Pele/efeitos dos fármacos , Pele/patologia , Neoplasias Cutâneas/prevenção & controle , Proteína Supressora de Tumor p53/genéticaRESUMO
The induction of skin cancer involves both mutagenic and proliferative responses of the epidermis to ultraviolet (UV) light. It is believed that tumor initiation requires the mutagenic replication of damaged DNA by translesion synthesis (TLS) pathways. The mechanistic basis for the induction of proliferation, providing tumor promotion, is poorly understood. Here, we have investigated the role of TLS in the initiation and promotion of skin carcinogenesis, using a sensitive nucleotide excision repair-deficient mouse model that carries a hypomorphic allele of the error-prone TLS gene Rev1. Despite a defect in UV-induced mutagenesis, skin carcinogenesis was accelerated in these mice. This paradoxical phenotype was caused by the induction of inflammatory hyperplasia of the mutant skin that provides strong tumor promotion. The induction of hyperplasia was associated with mild and transient replicational stress of the UV-damaged genome, triggering DNA damage signaling and senescence. The concomitant expression of Interleukin-6 (IL-6) is in agreement with an executive role for IL-6 and possibly other cytokines in the autocrine induction of senescence and the paracrine induction of inflammatory hyperplasia. In conclusion, error-prone TLS suppresses tumor-promoting activities of UV light, thereby controlling skin carcinogenesis.
Assuntos
Dano ao DNA , Replicação do DNA , Alelos , Animais , Reparo do DNA , Interleucina-6/genética , Interleucina-6/fisiologia , Camundongos , Modelos Animais , Neoplasias Induzidas por Radiação/genética , Neoplasias Cutâneas/genética , Raios UltravioletaRESUMO
Skin carcinomas are the most common cancers in fair-skinned populations of North West European descent. The risk is closely related to sun (UV) exposure and susceptibility to sunburn. Induction of squamous cell carcinomas (SCCs) in the skin of hairless mice by daily UVB exposure appears to emulate the genesis of these tumors in humans quite well. The carcinomas, and the UVB signature mutations that they carry in their p53 genes, can be linked most specifically to the induction of cyclobutane pyrimidine dimers (CPDs). The wavelength dependence of the induction of carcinomas parallels that of CPD induction over the UVB and UVA2 spectral regions. Microscopic clusters of cells overexpressing p53 with UVB signature mutations ("p53 patches") can be detected in the interfollicular epidermis long before the skin tumors arise. DNA repair--more precisely nucleotide excision repair--is a crucial line of defense against UV-induced p53 patches and skin carcinomas. Although chemoprevention of UV carcinogenesis, e.g. with difluoromethylornithine, may be successful by inhibiting the outgrowth of tumors, it may be better to counter the initial steps in tumor development. As the p53 patches appear to be potential precursors of SCCs, regression of p53 patches in unexposed skin should lower subsequent development of SCCs. However, "holoclonal" p53 patches might persist. Ablation of the interfollicular epidermis would be expected to abrogate development of SCC, and negation of this expectation [Faurschou A. et al., Exp. Dermatol. 2007;16:485-489] would indicate that SCCs stem from deep-seated cells in the hair follicles. Careful examination of archival material showed that although most small p53 patches arise interfollicularly, some may actually arise high up in a follicle, in the infundibulum.
Assuntos
Dano ao DNA , Mutação , Neoplasias Induzidas por Radiação , Proteína Supressora de Tumor p53/genética , Raios Ultravioleta , Animais , Humanos , CamundongosRESUMO
Immunosuppressive medication in renal transplant recipients (RTR) strongly increases the risk of cancers on sun-exposed skin. This increased risk was considered an inevitable collateral effect of immunosuppression, because UV-induced carcinomas in mice were found to be highly antigenic. Here, we posed the question whether immunosuppression also increases the frequency of p53-mutant foci ('p53 patches'), putative microscopic precursors of squamous cell carcinomas. As the majority of RTR was kept on azathioprine for most of the time, we investigated whether this drug could increase UV-induced p53 patches by immunosuppression. As azathioprine can impair UV-damaged DNA repair under certain conditions, we also investigated whether DNA repair was affected. Archive material of RTR and immunocompetent patients (ICP), as well as azathioprine-administered hairless mice were examined for p53 patches. DNA repair was investigated by ascertaining the effect of azathioprine on unscheduled DNA synthesis (UDS) in UV-irradiated human keratinocytes. P53 patches were more prevalent in RTR than in ICP in normal skin adjacent to carcinomas (P = 0.02), in spite of a lower mean age in the RTR (52 vs 63 years, P = 0.001), but we found no increase in UV-induced p53 patches in mice that were immunosuppressed by azathioprine. We found a significant reduction in DNA repair activity in keratinocytes treated with azathioprine (P = 0.011). UV-induced UDS in humans is dominated by repair of cyclobutane pyrimidine dimers, and these DNA lesions can lead to 'UV-signature' mutations in the P53 gene, giving rise to p53 patches.
Assuntos
Azatioprina/efeitos adversos , Carcinoma de Células Escamosas/genética , Reparo do DNA/efeitos dos fármacos , Epiderme/metabolismo , Genes p53 , Imunossupressores/efeitos adversos , Neoplasias Cutâneas/genética , Adulto , Idoso , Animais , Carcinoma de Células Escamosas/induzido quimicamente , Transformação Celular Neoplásica/genética , Células Clonais/metabolismo , Epiderme/efeitos da radiação , Feminino , Humanos , Imunocompetência/genética , Hospedeiro Imunocomprometido/genética , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Transplante de Rim , Masculino , Camundongos , Camundongos Pelados , Pessoa de Meia-Idade , Mutação , Neoplasias Cutâneas/induzido quimicamente , Raios Ultravioleta/efeitos adversosRESUMO
UV-damaged DNA-binding protein (UV-DDB) is essential for global genome repair (GGR) of UV-induced cyclobutane pyrimidine dimers (CPD). Unlike human cells, rodent epidermal cells are deficient in GGR of CPDs and express a subunit of UV-DDB, DDB2, at a low level. In this study, we generated mice (K14-DDB2) ectopically expressing mouse DDB2 at elevated levels. Enhanced expression of DDB2 both delayed the onset of squamous cell carcinoma and decreased the number of tumors per mouse in chronically UV-B light-exposed hairless mice. Enhanced expression of DDB2 improved repair of both CPDs and pyrimidine(6-4)pyrimidone photoproducts (6-4PP) in dermal fibroblasts. However, GGR of CPDs in K14-DDB2 mice did not reach the level of efficiency of human cells, suggesting that another repair protein may become rate limiting when DDB2 is abundantly present. To complement these studies, we generated mice in which the DDB2 gene was disrupted. DDB2-/- and DDB2+/- mice were found to be hypersensitive to UV-induced skin carcinogenesis. On the cellular level, we detected a delay in the repair of 6-4PPs in DDB2-/- dermal fibroblasts. Neither the absence nor the enhanced expression of DDB2 affected the levels of UV-induced apoptosis in epidermal keratinocytes or cultured dermal fibroblasts. Our results show an important role for DDB2 in the protection against UV-induced cancer and indicate that this protection is most likely mediated by accelerating the repair of photolesions.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Neoplasias Cutâneas/prevenção & controle , Animais , Apoptose/fisiologia , Apoptose/efeitos da radiação , Reparo do DNA , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Masculino , Camundongos , Camundongos Pelados , Camundongos Transgênicos , Oligopeptídeos , Peptídeos/genética , Peptídeos/metabolismo , Dímeros de Pirimidina/metabolismo , Tolerância a Radiação/fisiologia , Pele/citologia , Pele/metabolismo , Pele/efeitos da radiação , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Raios UltravioletaRESUMO
Clusters of p53 immunopositive epidermal keratinocytes (so-called p53 patches, clones or foci) are found in sun or ultraviolet (UV) light-exposed skin. We investigated to what extent these p53 patches are genuine precursors of skin carcinomas in chronically irradiated hairless (SKH1) mice. The mutation spectra of exons 5-8 of the p53 gene of laser-micro-dissected mutant p53 patches and carcinomas were therefore compared. The mutations we found were mainly UV-signature mutations (C-->T and CC-->TT at dipyrimidine sites) located at known hotspots. No significant differences were found between both spectra, indicating that all p53 patches harbour mutations with which they could progress to carcinomas. To examine whether these p53 patches can be used as tumour risk indicators, we made an extensive comparison of the induction kinetics of these patches and carcinomas in genetically modified mice with various defects in nucleotide excision repair (NER), i.e. xeroderma pigmentosum A (Xpa), Xpc and Cockayne syndrome B (Csb) and wild-type mice. In this aforementioned order, the mouse strains developed both p53 patches and carcinomas in the course of daily exposure to 40 J/m(2) UV. Hence, the order in which the NER-deficient mice developed patches was predictive of the order in which they developed tumours. The induction kinetics of the patches in Xpc-deficient mice differed notably from the others: there was a stationary phase (days 13-41) where the numbers were limited to 5-10 patches per mouse before an explosive increase which ran parallel to the other groups. The chance that a p53 patch progresses to carcinoma is relatively small (estimated at 1 out of 8300-40,000/individual when the first tumour appears), but our results are strongly indicative of a causal relationship between p53 patches and carcinomas.
Assuntos
Reparo do DNA/fisiologia , Genes p53/genética , Mutação/genética , Neoplasias Induzidas por Radiação/etiologia , Neoplasias Cutâneas/etiologia , Pele/metabolismo , Pele/efeitos da radiação , Animais , Síndrome de Cockayne , Cruzamentos Genéticos , DNA Helicases/genética , DNA Helicases/fisiologia , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Relação Dose-Resposta à Radiação , Feminino , Heterozigoto , Cinética , Masculino , Camundongos , Camundongos Pelados , Camundongos Knockout , Neoplasias Induzidas por Radiação/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose , Fatores de Risco , Neoplasias Cutâneas/metabolismo , Luz Solar , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/fisiologiaRESUMO
BACKGROUND: The high and steadily increasing incidence of ultraviolet-B (UV-B)-induced skin cancer is a problem recognized worldwide. UV introduces different types of damage into the DNA, notably cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs). If unrepaired, these photolesions can give rise to cell death, mutation induction, and onset of carcinogenic events, but the relative contribution of CPDs and 6-4PPs to these biological consequences of UV exposure is hardly known. Because placental mammals have undergone an evolutionary loss of photolyases, repair enzymes that directly split CPDs and 6-4PPs into the respective monomers in a light-dependent and lesion-specific manner, they can only repair UV-induced DNA damage by the elaborate nucleotide excision repair pathway. RESULTS: To assess the relative contribution of CPDs and 6-4PPs to the detrimental effects of UV light, we generated transgenic mice that ubiquitously express CPD-photolyase, 6-4PP-photolyase, or both, thereby allowing rapid light-dependent repair of CPDs and/or 6-4PPs in the skin. We show that the vast majority of (semi)acute responses in the UV-exposed skin (i.e., sunburn, apoptosis, hyperplasia, and mutation induction) can be ascribed to CPDs. Moreover, CPD-photolyase mice, in contrast to 6-4PP-photolyase mice, exhibit superior resistance to sunlight-induced tumorigenesis. CONCLUSIONS: Our data unequivocally identify CPDs as the principal cause of nonmelanoma skin cancer and provide genetic evidence that CPD-photolyase enzymes can be employed as effective tools to combat skin cancer.
Assuntos
Carcinoma/etiologia , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Desoxirribodipirimidina Fotoliase/metabolismo , Dímeros de Pirimidina/metabolismo , Neoplasias Cutâneas/etiologia , Raios Ultravioleta , Animais , Apoptose/efeitos da radiação , Carcinoma/prevenção & controle , Linhagem Celular , Desoxirribodipirimidina Fotoliase/genética , Expressão Gênica , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/prevenção & controleRESUMO
Overexposure to short- and long-wave ultraviolet radiations (UVB, UVA) may contribute to melanoma development through combined genotoxic and mitogenic effects in melanocytes. This study compares the impact of UVA-1 versus UVB, and single versus fractionated exposures on melanocyte proliferation in hairless SKH-2 mice. A single erythemal dose was compared with an equal dose fractionated over 8 d, and dose-dependency was studied. Proliferation (Ki-67 positive-sign) in melanocytes (melanoma antigen recognized by T-cells-1 positive or micropthalmia transcription factor positive) was ascertained in double-labeled skin sections. Single erythemal UVB exposures caused a delayed, dose-dependent increase of melanocyte proliferation. The highest, 17-fold, increase (from 0.05% to 0.8% of melanocytes) occurred 4 d after UVB exposure, without any detectable effect on overall melanocyte numbers. Correspondingly, DNA repair-deficient xeroderma pigmentosum A (Xpa) mice proved exquisitely sensitive to melanocyte proliferation induction by UVB exposure. No discernable effects were measured from fractionated suberythemal UVB exposures, or from any UVA-1 exposure regimen. Hence, melanocyte proliferation appears to be most efficiently induced by a single UVB overexposure. Moreover, the ineffectiveness of UVA-1 radiation and the enhanced sensitivity of Xpa mice point at pyrimidine dimers as causative DNA lesions. Consequently, murine nevi and melanoma are expected to be most effectively induced by intermittent UVB overexposures.
Assuntos
Epiderme/patologia , Epiderme/efeitos da radiação , Melanócitos/patologia , Melanócitos/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Divisão Celular/efeitos da radiação , Proteínas de Ligação a DNA/genética , Eritema , Hiperplasia , Camundongos , Camundongos Pelados , Camundongos Knockout , Dímeros de Pirimidina/efeitos da radiação , Doses de Radiação , Timidina , Proteína de Xeroderma Pigmentoso Grupo ARESUMO
Nucleotide excision repair (NER), cell cycle regulation and apoptosis are major defence mechanisms against the carcinogenic effects of UVB radiation. NER eliminates UVB-induced DNA photolesions via two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). In a previous study, we found UVB-induced accumulation of tetraploid (4N) keratinocytes in the epidermis of Xpc(-/-) mice (no GGR), but not in Xpa(-/-) (no TCR and no GGR) or in wild-type (WT) mice. We inferred that this arrest in Xpc(-/-) mice is caused by erroneous replication past photolesions, leading to 'compound lesions' known to be recognised by mismatch repair (MMR). MMR-induced futile cycles of breakage and resynthesis at sites of compound lesions may then sustain a cell cycle arrest. The present experiments with Xpc(-/-)Msh2(-/-) mice and derived keratinocytes show that the MMR protein Msh2 indeed plays a role in the generation of the UVB-induced arrested cells: a Msh2-deficiency lowered significantly the percentage of arrested cells in vivo (40-50%) and in vitro (30-40%). Analysis of calyculin A (CA)-induced premature chromosome condensation (PCC) of cultured Xpc(-/-) keratinocytes showed that the delayed arrest occurred in late S phase rather than in G(2)-phase. Taken together, the results indicate that in mouse epidermis and cultured keratinocytes, the MMR protein Msh2 plays a role in the UVB-induced S-phase arrest. This indicates that MMR plays a role in the UVB-induced S-phase arrest. Alternatively, Msh2 may have a more direct signalling function.
Assuntos
Pareamento Incorreto de Bases/genética , Ciclo Celular/efeitos da radiação , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas/genética , Animais , Bromodesoxiuridina , Ciclo Celular/genética , Citometria de Fluxo , Queratinócitos/fisiologia , Toxinas Marinhas , Camundongos , Camundongos Mutantes , Proteína 2 Homóloga a MutS , Oxazóis , Raios UltravioletaRESUMO
Xeroderma pigmentosum (XP) patients are deficient in nucleotide excision repair (NER) because of mutations in one of the genes coding for NER enzymes. This results predominantly in high frequency of UV-induced skin tumors at an early age; the most severe phenotype is found in patients of complementation group A (XPA). However, in a subset of these XPA patients no skin tumors appear, even at advanced age. Fibroblasts of this subset of patients are not capable of raising UV-induced enhanced reactivation (ER) of viruses and up-regulation of ornithine decarboxylase (ODC). We hypothesized that prevention of ODC induction would protect NER-deficient patients from cancer. To simulate the situation in XPA patients, we used a hairless Xpa knockout mouse model and down-regulated the ODC activity by difluoromethylornithine (DFMO) administered in the drinking water. The DFMO treatment significantly suppressed UV-induced carcinogenesis. In a crossover study, we additionally found that discontinuation of the DFMO treatment resulted in a rapid appearance of skin tumors, up to levels found in mice not treated with DFMO. Late-stage DFMO treatment significantly reduced the number of carcinomas by a factor of 2-3, and it appeared to select for carcinomas with high ODC activity. These results indicate that DFMO suppresses the outgrowth but not the initiation of UV-induced tumors. The DFMO treatment reduced the tumor load but did not offer the Xpa knockout mice full protection against UV carcinogenesis.