RESUMO
XIAP (X-linked inhibitor of apoptosis) deficiency is a rare inborn error of immunity. XIAP deficiency causes hyperinflammatory disease manifestations due to dysregulated TNF (tumor necrosis factor)-receptor signaling and NLRP3 (NOD- [nucleotide-binding oligomerization domain], LRR- [leucine-rich repeat] and pyrin domain-containing protein 3) inflammasome function. Safe and effective long-term treatments are needed and are especially important to help prevent the need for high-risk allogeneic hematopoietic cell transplantation. Here we evaluated inflammasome inhibitors as potential therapeutics with a focus on the natural flavonoid antioxidant quercetin. Bone marrow (BM)-derived macrophages were derived from XIAP-deficient or wild-type (WT) mice. Human monocytes were obtained from control or XIAP-deficient patients. Cells were stimulated with TLR (Toll-like receptor) agonists or TNF-α ± inhibitors or quercetin. For in vivo lipopolysaccharide (LPS) challenge experiments, XIAP-deficient or WT mice were fed mouse chow ± supplemental quercetin (50 mg/kg per day exposure) for 7 days followed by a challenge with 10 ng/kg LPS. IL-1ß (interleukin-1ß) and IL-18 were measured by ELISA (enzyme-linked immunosorbent assay). In murine studies, quercetin prevented IL-1ß secretion from XIAP knockout cells following TLR agonists or TNF-α stimulation (P < .05) and strongly reduced constitutive production of IL-18 by both WT and XIAP-deficient cells (P < .05). At 4 hours after in vivo LPS challenge, blood levels of IL-1ß and IL-18 were significantly decreased in mice that had received quercetin-supplemented chow (P < .05). In experiments using human cells, quercetin greatly reduced IL-1ß secretion by monocytes following TNF-α stimulation (P < .05). Our data suggest that quercetin may be an effective natural therapeutic for the prevention of XIAP deficiency-associated hyperinflammation. Clinical trials, including careful pharmacokinetic and pharmacodynamic studies to ensure that effective levels of quercetin can be obtained, are warranted.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Proteínas Inibidoras de Apoptose , Interleucina-18 , Interleucina-1beta , Lipopolissacarídeos/farmacologia , Transtornos Linfoproliferativos , Camundongos , Quercetina/farmacologia , Quercetina/uso terapêutico , Fator de Necrose Tumoral alfa , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
BACKGROUND: We hypothesized that α4ß7 integrin expression on effector memory T cells (TEMs) would be elevated in pediatric hematopoietic stem cell transplant (HSCT) patients before and at diagnosis of acute gastrointestinal graft-versus-host disease (GI GVHD) symptoms compared to patients without GVHD, and that clinical blockade of α4ß7 integrin with vedolizumab would be effective in pediatric GI GVHD. METHODS: We analyzed surface expression of α4ß7 integrin on T cells from 48 pediatric allogeneic HSCT recipients from our biorepository with known clinical outcomes as follows: acute GI GVHD (n = 22), isolated skin GVHD (n = 12), and no GVHD (n = 14). T-cell analyses were performed 1 week before and at GVHD diagnosis in patients with GVHD, and day +30 after HSCT in patients without GVHD. We describe clinical outcomes of seven additional patients, different from above-described 48 patients, who received vedolizumab (anti-α4ß7 integrin antibody) for the treatment of steroid-refractory acute GI GVHD. RESULTS: Expression of α4ß7 integrin on CD8+ TEMs was upregulated in patients with GI GVHD compared to the no GI GVHD (skin GVHD + no GVHD) group 1 week prior to clinical symptoms (p = .02) and at acute GI GVHD diagnosis (p = .05). Four of seven treated patients with clinical steroid-refractory acute GI GVHD were evaluable for response to vedolizumab. One patient had a complete response at day +28, while two had a partial response, and one had no response. No adverse effects directly attributable to vedolizumab were observed. CONCLUSION: Our data suggest a rationale for the blockade of α4ß7 integrin for acute GI GVHD management in children.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Criança , Doença Enxerto-Hospedeiro/tratamento farmacológico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Integrinas , Células T de Memória , Esteroides , Adulto JovemRESUMO
Hemophagocytic lymphohistiocytosis (HLH) is a fatal disorder of immune hyperactivation that has been described as a cytokine storm. Sepsis due to known or suspected infection has also been viewed as a cytokine storm. Although clinical similarities between these syndromes suggest similar immunopathology and may create diagnostic uncertainty, distinguishing them is critical as treatments are widely divergent. We examined T-cell profiles from children with either HLH or sepsis and found that HLH is characterized by acute T-cell activation, in clear contrast to sepsis. Activated T cells in patients with HLH were characterized as CD38high/HLA-DR+ effector cells, with activation of CD8+ T cells being most pronounced. Activated T cells were type 1 polarized, proliferative, and displayed evidence of recent and persistent activation. Circulating activated T cells appeared to be broadly characteristic of HLH, as they were seen in children with and without genetic lesions or identifiable infections and resolved with conventional treatment of HLH. Furthermore, we observed even greater activation and type 1 polarization in tissue-infiltrating T cells, described here for the first time in a series of patients with HLH. Finally, we observed that a threshold of >7% CD38high/HLA-DR+ cells among CD8+ T cells had strong positive and negative predictive value for distinguishing HLH from early sepsis or healthy controls. We conclude that the cytokine storm of HLH is marked by distinctive T-cell activation whereas early sepsis is not, and that these 2 syndromes can be readily distinguished by T-cell phenotypes.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Linfócitos T CD8-Positivos/imunologia , Síndrome da Liberação de Citocina/diagnóstico , Antígenos HLA-DR/metabolismo , Ativação Linfocitária/imunologia , Linfo-Histiocitose Hemofagocítica/diagnóstico , Glicoproteínas de Membrana/metabolismo , Sepse/diagnóstico , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/patologia , Diagnóstico Diferencial , Feminino , Humanos , Lactente , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/patologia , Masculino , Sepse/imunologia , Sepse/patologia , Adulto JovemRESUMO
We have previously reported that a peripheral blood absolute CD38brightCD8+ effector memory T cell (TEM) population expansion of >35 cells/µL predicts the development of acute graft-versus-host disease (GVHD). We hypothesized that these T cells are activated, proliferating, and cytotoxic trafficking cells that are not a response to viral reactivation and may be involved in acute GVHD. We characterized peripheral blood T cell populations at the time of maximum CD38brightCD8+ TEM expansion in patients from our originally reported pediatric allogeneic hematopoietic cell transplantation recipient cohort. Samples were incubated with fluorochrome-conjugated antibodies directed against CD3, CD8, CD38, HLA-DR (T cell activation), Ki-67 (T cell proliferation), granzyme B (marker of cytotoxic T cells), CLA (skin trafficking), CCR5 (visceral trafficking), and CXCR6 (liver trafficking). We also incubated samples with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) peptide pools and measured IFN-γ production by flow cytometry and performed EBV and CMV tetramer staining. Higher median proportions of cell expression of HLA-DR, Ki-67, granzyme B, CLA, CCR5, and CXCR6 were observed for CD38brightCD8+ T cells compared with CD38nonbrightCD8+ T cells in patients with acute GVHD (Pâ¯< .05) but not in patients without acute GVHD (P not significant). No IFN-γ production was observed after incubation with CMV and EBV peptide pools. EBV-specific tetramer populations of 6.85% and 3.17% were detected in 2 patients with acute GVHD, whereas a CMV-specific tetramer population of 3.77% was detected in 1 patient with acute GVHD. No EBV- or CMV-specific tetramer populations were detected in any patient without acute GVHD. We conclude that CD38brightCD8+ T cells associated with the development of acute GVHD are activated, proliferating, and cytotoxic trafficking cells that do not appear to respond to CMV or EBV reactivation. Further studies are needed to determine whether these cells are directly involved in acute GVHD pathogenesis.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , Linfócitos T CD8-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Ativação Linfocitária , Glicoproteínas de Membrana/imunologia , Adolescente , Adulto , Aloenxertos , Linfócitos T CD8-Positivos/patologia , Criança , Pré-Escolar , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Doença Enxerto-Hospedeiro/patologia , Antígenos HLA-DR/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Lactente , MasculinoRESUMO
Mutations in the LRBA gene (encoding the lipopolysaccharide-responsive and beige-like anchor protein) cause a syndrome of autoimmunity, lymphoproliferation, and humoral immune deficiency. The biological role of LRBA in immunologic disease is unknown. We found that patients with LRBA deficiency manifested a dramatic and sustained improvement in response to abatacept, a CTLA4 (cytotoxic T lymphocyte antigen-4)-immunoglobulin fusion drug. Clinical responses and homology of LRBA to proteins controlling intracellular trafficking led us to hypothesize that it regulates CTLA4, a potent inhibitory immune receptor. We found that LRBA colocalized with CTLA4 in endosomal vesicles and that LRBA deficiency or knockdown increased CTLA4 turnover, which resulted in reduced levels of CTLA4 protein in FoxP3(+) regulatory and activated conventional T cells. In LRBA-deficient cells, inhibition of lysosome degradation with chloroquine prevented CTLA4 loss. These findings elucidate a mechanism for CTLA4 trafficking and control of immune responses and suggest therapies for diseases involving the CTLA4 pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doenças Autoimunes/tratamento farmacológico , Antígeno CTLA-4/deficiência , Imunodeficiência de Variável Comum/tratamento farmacológico , Imunoconjugados/uso terapêutico , Abatacepte , Proteínas Adaptadoras de Transdução de Sinal/genética , Adolescente , Doenças Autoimunes/metabolismo , Antígeno CTLA-4/genética , Criança , Cloroquina/farmacologia , Imunodeficiência de Variável Comum/metabolismo , Endossomos/metabolismo , Feminino , Fatores de Transcrição Forkhead/análise , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/metabolismo , Ativação Linfocitária , Lisossomos/metabolismo , Masculino , Proteólise , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Adulto JovemRESUMO
Acute graft-versus-host disease (aGVHD) is mediated by allogeneic T cell responses. We hypothesized that increases of peripheral blood-activated CD8+ effector memory T (TEM) cells would be observed after hematopoietic stem cell transplantation (HSCT) before onset of aGVHD symptoms. Blood was collected twice weekly after HSCT for 7 weeks in 49 consecutive pediatric and adult HSCT recipients. Samples were incubated with fluorochrome-conjugated antibodies against CD45, CD3, CD8, CD38, CD45RA, and CCR7 and analyzed using flow cytometry. TEM cells were defined as CD3+ CD8+ CCR7- CD45RA(-) lymphocytes. CD38 expression was used as a marker of T cell activation. Patients were followed for 100 days for development of aGVHD. Twenty-three patients developed grade 1 to 4 aGVHD at a median of 37 days (range, 15 to 79 days) after HCST. Absolute CD38 bright CD8+ TEM of > 35 cells/µL predicted aGVHD at a median of 8 days (range, 1 to 34) before aGVHD onset with a sensitivity of 82.6% and specificity of 91.6%. The cumulative incidence of aGVHD was 90% in patients with absolute CD38 bright CD8+ TEM >35 cells/µL and 15% in patients without (P < .0001). Quantification of CD38 bright CD8+ TEM cells may predict aGVHD in children and young adult HSCT recipients.
Assuntos
ADP-Ribosil Ciclase 1/imunologia , Linfócitos T CD8-Positivos/imunologia , Imunodeficiência de Variável Comum/terapia , Doença Enxerto-Hospedeiro/diagnóstico , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Glicoproteínas de Membrana/imunologia , Condicionamento Pré-Transplante , ADP-Ribosil Ciclase 1/genética , Doença Aguda , Adolescente , Adulto , Biomarcadores/análise , Linfócitos T CD8-Positivos/patologia , Criança , Pré-Escolar , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/patologia , Feminino , Expressão Gênica , Doença Enxerto-Hospedeiro/tratamento farmacológico , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Teste de Histocompatibilidade , Humanos , Memória Imunológica , Imunofenotipagem , Imunossupressores/uso terapêutico , Lactente , Ativação Linfocitária , Masculino , Glicoproteínas de Membrana/genética , Agonistas Mieloablativos/uso terapêutico , Transplante HomólogoRESUMO
Previously, we have shown that statistical synergism between amino acid variants in thyroglobulin (Tg) and specific HLA-DR3 pocket sequence signatures conferred a high risk for autoimmune thyroid disease (AITD). Therefore, we hypothesized that this statistical synergism mirrors a biochemical interaction between Tg peptides and HLA-DR3, which is key to the pathoetiology of AITD. To test this hypothesis, we designed a recombinant HLA-DR3 expression system that was used to express HLA-DR molecules harboring either AITD susceptibility or resistance DR pocket sequences. Next, we biochemically generated the potential Tg peptidic repertoire available to HLA-DR3 by separately treating 20 purified human thyroglobulin samples with cathepsins B, D, or L, lysosomal proteases that are involved in antigen processing and thyroid biology. Sequences of the cathepsin-generated peptides were then determined by matrix-assisted laser desorption ionization time-of-flight-mass spectroscopy, and algorithmic means were employed to identify putative AITD-susceptible HLA-DR3 binders. From four predicted peptides, we identified two novel peptides that bound strongly and specifically to both recombinant AITD-susceptible HLA-DR3 protein and HLA-DR3 molecules expressed on stably transfected cells. Intriguingly, the HLA-DR3-binding peptides we identified had a marked preference for the AITD-susceptibility DR signatures and not to those signatures that were AITD-protective. Structural analyses demonstrated the profound influence that the pocket signatures have on the interaction of HLA-DR molecules with Tg peptides. Our study suggests that interactions between Tg and discrete HLA-DR pocket signatures contribute to the initiation of AITD.
Assuntos
Regulação da Expressão Gênica , Antígeno HLA-DR3/metabolismo , Proteínas Recombinantes/química , Algoritmos , Animais , Doenças Autoimunes , Catepsinas/química , Linhagem Celular , Células HeLa , Antígenos de Histocompatibilidade Classe II , Humanos , Peptídeos/química , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tireoglobulina/química , Doenças da Glândula Tireoide/imunologia , Glândula Tireoide/metabolismoRESUMO
Whether terminal differentiation/stratum corneum formation of keratinocytes (KCs) represents a form of programmed cell death, utilizing mediators of classical apoptosis, is unclear. Apoptosis, an evolutionarily conserved death process, is comprised of extrinsic and intrinsic pathways, which converge using caspase 3. To define upstream and downstream caspases involved in terminal differentiation, we utilized human epidermal equivalents (EEs). Using submerged cultures comprised of human KCs, EEs were sequentially analyzed before and after being raised to an air/liquid (A/L) interface at 3-24 h intervals. At each time point, EEs were analyzed morphologically and for specific enzyme activity to distinguish different initiator (caspases 1, 2, 8, 9) and effector caspases (3, 6, 7). Terminal differentiation began at 6-8 h, as defined by stratum corneum with loricirin expression and completed at 18-24 h producing an epidermis resembling normal skin. Enzyme activity for caspases 1, 2, 3, 6, 7, 8, and 9 (but not 4, 5) was enhanced (>two-fold nmol/mg/h) at 3-6 h compared with submerged cultures. Processing of caspase 14 occurred at 18 h, and cleaved caspase 14 was increased at 24 h. Activated caspase 3-positive and terminal deoxynucleotidyl transferase-mediated nick end labeling-positive KCs were identified in EEs at 3-6 h corresponding to initiation sites of terminal differentiation. Addition of caspase inhibitors reduced levels of involucrin and loricrin in EEs raised to an A/L interface. We conclude caspases function as important death effectors strategically positioned at intersection of intrinsic and extrinsic pathways in KCs undergoing stratum corneum formation.
Assuntos
Apoptose/fisiologia , Caspases/fisiologia , Epiderme/fisiologia , Western Blotting , Caspases/metabolismo , Diferenciação Celular/fisiologia , Ativação Enzimática , Humanos , Técnicas Imunoenzimáticas , Queratinócitos/fisiologia , Modelos BiológicosRESUMO
BACKGROUND: Keratinocytes (KCs) in healthy skin only undergo death following differentiation to produce stratum corneum. By contrast, in inflammatory pathological conditions featuring type I (IFN-alpha) and type II (IFN-gamma) interferons KCs undergo premature apoptosis. OBJECTIVE: To define apoptotic susceptibility of KCs, response to interferons was examined. Since molecular cross-talk occurs between interferons and p53, potential mechanistic roles for p53 in KC apoptosis were investigated. METHODS: Knock down of p53 was performed, and apoptotic response to addition of interferons was assessed using FACS and by staining for activated caspase 3 and TUNEL. Elucidation of death pathway was accomplished by using a dominant negative death receptor construct and a neutralizing TRAIL antibody. RESULTS: Reduction in p53 levels in KCs by siRNA treatment enhanced, rather than reduced, apoptotic responses to IFN-alpha plus IFN-gamma. In an immortalized human KC cell line (HaCaT cells with both p53 alleles mutated) enhanced apoptotic susceptibility to interferon exposure was also observed. The mechanism for this enhanced apoptosis involved induction of TRAIL and its interaction with death receptors, as blocking the death receptor pathway using dominant negative FADD, or by addition of neutralizing antibody against TRAIL, reduced the apoptotic response to IFN-alpha and IFN-gamma. CONCLUSION: These results indicate IFN-alpha plus IFN-gamma triggers apoptosis independent of p53 in HaCaT cells, and also demonstrate an unexpected survival role for p53 in human KCs as regards apoptotic responsiveness to cytokines such as IFN-alpha and IFN-gamma involving activation of TRAIL-related death receptors. Strategies enhancing p53 regulated survival proteins in KCs may be of therapeutic benefit in skin disorders characterized by activated immunocytes triggering premature KC apoptosis.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/fisiologia , Queratinócitos/citologia , Queratinócitos/fisiologia , Glicoproteínas de Membrana/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica , Humanos , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Glicoproteínas de Membrana/genética , RNA Interferente Pequeno , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Pele/citologia , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/genéticaRESUMO
Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.
Assuntos
Ácidos Borônicos/farmacologia , Melanoma/tratamento farmacológico , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Pirazinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Bortezomib , Feminino , Humanos , Melanócitos/citologia , Melanócitos/efeitos dos fármacos , Melanoma/enzimologia , Melanoma/patologia , Camundongos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Mieloma Múltiplo/enzimologia , Mieloma Múltiplo/patologia , Oligonucleotídeos Antissenso/genética , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ultraviolet (UV) light exposure is a common cause of epithelial-derived skin cancers, and the epidermal response to UV-light has been extensively studied using both mouse models and cultured human keratinocytes (KCs). Elimination of cells with UV-induced DNA damage via apoptosis provides a powerful mechanism to minimize retention or expansion of genetically abnormal cells. This cell editing function has largely been ascribed to the biological role of the p53 tumor suppressor gene, as mutations or deletions involving p53 have been linked to skin cancer development. Rather than introducing mutations, or using cells with complete loss of wild-type p53, we used an siRNA-based approach to knockdown, but not eliminate, p53 levels in primary cultures of human KCs followed by UV-irradiation. Surprisingly, when p53 levels were reduced by 50-80% the apoptosis induced by exposure to UV-light was accelerated and markedly enhanced (two- to three- fold) compared to control siRNA treated KCs. The p53 siRNA treated KCs were characterized by elevated E2F-1 levels accompanied by accelerated elimination of the Mcl-1 and Bcl-x(L) antiapoptotic proteins, as well as enhanced Bax oligomerization. Forced overexpression of either Mcl-1 or Bcl-x(L) reduced the UV-light enhanced apoptotic response in p53 siRNA treated KCs. We conclude that p53 not only can provide proapoptotic signals but also regulates a survival pathway influencing Mcl-1 and Bcl-x(L) levels. This overlooked survival function of p53 may explain previous paradoxical responses noted by investigators using p53 heterozygous and knockout mouse models, and opens up the possibility that not all liaisons within the cell involving p53 necessarily represent fatal attractions.
Assuntos
Apoptose , Queratinócitos/fisiologia , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/fisiologia , Raios Ultravioleta/efeitos adversos , Animais , Apoptose/efeitos da radiação , Sobrevivência Celular , Dano ao DNA , Regulação para Baixo , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Interferência de RNA , Regulação para Cima , Proteína bcl-XAssuntos
Apoptose , Queratinócitos/citologia , Animais , Divisão Celular , Células Cultivadas , Humanos , Camundongos , Especificidade da EspécieRESUMO
It has been established that Id proteins can block the basic helix-loop-helix (HLH) transcription factors, thereby impacting the onset of senescence in keratinocytes, as well as influencing tumorigenesis involving squamous cell carcinomas. However, the ability of Id-1 to influence the immunologic response of epithelial cells to cytokines implicated in cutaneous oncology such as gamma interferon (IFN-gamma) has not been determined. Using a whole population of human keratinocytes infected with a retrovirus to induce over-expression of Id-1, the influence on early differentiation of rapidly proliferating keratinocytes was assessed, as was the response to IFN-gamma. While induction of involucrin, a marker of early differentiation, was not altered in Id-1 overexpressing keratinocytes, the IFN-gamma mediated increase in intercellular adhesion molecule-1 (ICAM-1) and HLA-DR was reduced. No change in constitutive or inducible levels of MHC class I antigen, CD95 (Fas antigen) or LFA-3 (CD58) was observed in this system. Immunostaining and Western blot analysis revealed over-expression of Id-1 in basal cell carcinomas (BCCs). These tumors not only strongly and diffusely expressed Id-1, but were also characterized by reduced ICAM-1 and HLA-DR expression. Thus, dysregulated Id-1 may not only contribute to delaying the senescence program in keratinocytes, it may also contribute to the escape of the relatively undifferentiated tumor cells in BCC from immune surveillance.
Assuntos
Carcinoma Basocelular/imunologia , Queratinócitos/imunologia , Proteínas Repressoras , Neoplasias Cutâneas/imunologia , Fatores de Transcrição/imunologia , Antineoplásicos/farmacologia , Diferenciação Celular , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/imunologia , Antígenos HLA-DR/genética , Humanos , Proteína 1 Inibidora de Diferenciação , Molécula 1 de Adesão Intercelular/genética , Interferon gama/farmacologia , Queratinócitos/citologia , Fatores de Transcrição/genética , Transfecção , Células Tumorais CultivadasRESUMO
During malignant transformation in skin, epidermal keratinocytes (KCs) frequently acquire the capacity to by-pass cellular senescence, a response that normally limits their unrestricted proliferation. Despite growing interest in the role for senescence during aging of skin and cutaneous carcinogenesis, little is known regarding regulation of three proteins encoded by the INK4a/ARF locus (p12, p14(ARF), p16) in KCs. In this study, several molecular pathways are explored using cultured KCs and KCs freshly isolated from psoriatic plaques. p16 and p14(ARF) are predominantly expressed spontaneously when foreskin-derived early-passage KCs undergo confluency-induced premature senescence. Induction of p14(ARF) on confluency occurred with low E2F-1 levels. Suspension of KCs in methylcellulose induced p12 expression. Addition of various cytokines (interferon-gamma, tumor necrosis factor-alpha) or a phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] only induced p16, but not p14(ARF). Confluent KCs up-regulated Ras activity and the downstream signaling involving ERK. Addition of MAPK inhibitor blocked cytokine and TPA-induced p16 expression. Confluency and interferon-gamma induced premature senescence and p16 expression was linked to induction of the transcription factor Egr-1. KCs derived from chronic psoriatic plaques were characterized by enhanced p16, p14(ARF), and p12 expression accompanied by elevated Egr-1 levels. These results demonstrate that multiple and highly divergent stimuli can trigger the senescent checkpoint in human KCs with differential regulation of p16, p14(ARF), and p12. Although abnormal mitogenic signaling by oncogenic Ras is generally cited as being responsible for induction of premature senescence, our findings indicate that a broader perspective is warranted, to include confluency and cytokine-/TPA-induced pathways for KCs.
Assuntos
Senescência Celular , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Genes cdc , Proteínas Imediatamente Precoces , Queratinócitos/metabolismo , Psoríase/metabolismo , Proteína Supressora de Tumor p14ARF/biossíntese , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Células Cultivadas , Senescência Celular/fisiologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Citocinas/farmacologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genes cdc/fisiologia , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Metilcelulose/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Psoríase/genética , Psoríase/patologia , Valores de Referência , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transfecção , Proteína Supressora de Tumor p14ARF/genética , Proteínas ras/metabolismoRESUMO
Properly regulated keratinocyte cell death is fundamentally important to maintain structural integrity and homeostatic function of epidermis. Moreover, from an oncological perspective, therapeutic approaches selectively targeting apoptosis of malignant cell types while sparing normal keratinocytes in surrounding skin is desirable. Apo2Ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) has been observed to preferentially induce cytopathic effects on transformed/malignant cell types compared with their non-neoplastic counterparts. In this report, two different biologically active preparations of Apo2L/TRAIL, a non-tagged version, NT-Apo2L/TRAIL, and a leucine zipper fusion protein, LZ-Apo2L/TRAIL, were examined for their ability to trigger apoptosis in normal human keratinocytes, and in an immortalized cell line (HaCaT cells). Differences between these preparations were observed, including: NT-Apo2L/TRAIL induced less keratinocyte apoptosis compared with LZ-Apo2L/TRAIL; NT-Apo2L/TRAIL also induced less apoptosis of HaCaT cells compared with LZ-Apo2L/TRAIL; LZ-Apo2L/TRAIL but not NT-Apo2L/TRAIL induced cytotoxic effects when keratinocytes became growth arrested due to undergoing spontaneous replicative senescence--a biological state previously observed to be resistant to UV-light-induced apoptosis. Similarities between preparations included: an enhanced ability for both Apo2L/TRAIL preparations to kill a greater relative percentage of HaCaT cells compared with keratinocytes; enhanced cytotoxicity towards keratinocytes that had their NF-B activity inhibited; a dependence of both Apo2L/TRAIL preparations on FADD and caspase activation; triggering of the same caspase cascades including caspase 8 and 3; and an ability to induce apoptosis even when HaCaT cells and keratinocytes were transduced to overexpress either Bcl-2 or Bcl-x(L) (survival factors that reduce susceptibility to UV-light-induced apoptosis). These results indicate that while both preparations of Apo2L/TRAIL possess biological activity, there are important differences as regards their ability to induce apoptosis in normal and immortalized keratinocytes. Moreover, the death receptor pathway triggered by LZ-Apo2L/TRAIL can overcome the apoptotic resistance normally observed in response to UV-light mediated by Bcl-2/Bcl-x(L), as well as by the state of cellular senescence. Unraveling the molecular basis for these differential biological effects may reveal a new strategic role for these death receptor/ligands linked to apoptosis in maintaining the dynamic balance of keratinocyte proliferation, differentiation, and cell death necessary to achieve a homeostatic thickness and function of normal skin. In addition, it may be possible to utilize these Apo2L/TRAIL preparations for the treatment of various sun-induced skin cancers as they can differentially trigger apoptosis of transformed keratinocytes, or keratinocytes with abnormal NF-kappaB signaling, while sparing adjacent normal keratinocytes.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/fisiologia , Glicoproteínas de Membrana/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Reguladoras de Apoptose , Proteínas de Transporte/fisiologia , Caspases/metabolismo , Divisão Celular , Linhagem Celular Transformada/efeitos dos fármacos , Células Cultivadas , Senescência Celular/fisiologia , Proteína de Domínio de Morte Associada a Fas , Humanos , Queratinócitos/citologia , NF-kappa B/fisiologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Proteínas Recombinantes de Fusão , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF , Proteína bcl-XRESUMO
The carcinogenic effects of sunlight in human epidermis may be thwarted by either: transient growth arrest and repair of DNA photodamage in keratinocytes (KCs); elimination of KCs with damaged DNA via apoptosis; or by stimulating a senescence switch whereby KCs become irreversibly growth arrested. Using normal human skin organ cultures and living epidermal equivalents, we demonstrate that in the proliferative basal layer, removal of KCs via apoptosis had a rapid onset (beginning within 2 h) following UV-light exposure generating progressively greater numbers of KCs with thymine dimers as the dose of UV-light was increased; involved induction of Apaf-1, activation of caspase-3, and was dependent on p53 activation as addition of a p53 chemical inhibitor blocked the apoptotic response. Suprabasal layer KCs underwent apoptosis at much later time points (>8 h). KCs in the basal layer repaired DNA damage more rapidly than KCs in suprabasal layers. Steady state levels of p53 increased in irradiated cells, and the increase was accompanied by phosphorylation of serine 9 and serine 15, but not serine 6 residues. By contrast, cultured KCs undergoing spontaneous replicative senescence were resistant to UV-induced apoptosis. Senescent KCs constitutively contained low levels of p53, which were neither increased nor phosphorylated or acetylated after UV-exposure and possessed minimal DNA binding activity, indicative of functional inactivation. Furthermore, treatment of senescent KCs with DNA damaging agent adriamycin did not result in activation of latent p53 or apoptosis. When KCs within psoriatic plaques were examined, they resembled senescent KCs in that they expressed p53, which was not phosphorylated or acetylated. Thus, UV-light induces DNA damage in human epidermal KCs triggering p53 activation, and subsequent apoptosis involving distinct cell layers and kinetics. However, the lack of p53 activation as seen in senescent KCs and psoriatic plaques, is associated with a relative resistance of KCs to UV-induced apoptosis. In conclusion, the sensitivity and resistance of KCs to apoptosis depends not only on the location within various layers of epidermis and levels of p53, but may also involve p53 activation via post-translational modifications.
Assuntos
Queratinócitos/patologia , Psoríase/patologia , Proteína Supressora de Tumor p53/fisiologia , Raios Ultravioleta/efeitos adversos , Caspase 3 , Caspases/metabolismo , Divisão Celular , Senescência Celular , Dano ao DNA , Reparo do DNA , Relação Dose-Resposta à Radiação , Células Epidérmicas , Epiderme/patologia , Regulação da Expressão Gênica , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Fosforilação , Processamento de Proteína Pós-Traducional , Psoríase/genética , Dímeros de Pirimidina , RNA Mensageiro/biossínteseRESUMO
OBJECTIVE: It has been suggested that keratinocyte (KC) stem cells reside at the epicenter of a clonal population of cells. To estimate the territory or surface area covered by a single stem-cell-derived KC population in human skin, clonal skin maps were created from 3 healthy adult women and from normal skin of a psoriatic patient. DESIGN: Two hundred fifty-eight punch biopsy samples of various sizes (ranging from 2 to 8 mm in diameter) were analyzed for clonality employing X chromosome inactivation patterns at the human androgen receptor gene (HUMARA) locus. DNA was isolated and clonality established by significant decrease of either maternal or paternal X chromosome band patterns following restriction enzyme digestion, polymerase chain reaction amplification, and gel electrophoresis. RESULTS: Fifty-three (41%) of 128 two-mm biopsies were clonal, whereas only 6 (14%) of 43 three-mm, 5 (14%) of 36 four-mm, and 3 (8%) of 35 five-mm biopsies revealed a clonal population of KCs. By contrast, in 5 different biopsies from a psoriatic patient, including 4- or 5-mm sizes, all but 1 were clonal; even an 8-mm biopsy contained a clonal population of KCs. Mantel-Haenszel chi(2) analysis revealed a P value of.001, reflecting a strong trend in probability for presence of a single clone of KCs as related to size of the biopsy sample. By sequentially analyzing 30 contiguous 2-mm biopsy samples within a given strip of skin, 10 clonal domain changes, as reflected in maternal versus paternal switches, were observed. CONCLUSIONS: These results provide direct evidence of a clonal population of KCs in normal and psoriatic lesion-free skin, and indicate that a clonal epidermal unit of KCs frequently can be detected in small biopsies (2 mm), but that in normal skin sampling, overlapping clones are apparently present in larger (ie, 4-5-mm) biopsies, producing nonclonal patterns. The clonal domain of progeny in normal skin has a rather limited territorial boundary (2 mm in diameter). However, in lesion-free skin from a psoriatic patient, there may be clonal expansion of KCs due to perturbation in epidermopoiesis and/or stem cell distribution.