RESUMO
Two major chaperones, calreticulin (CRT) and binding immunoglobulin protein (GRP78/BiP) dependent on their location, have immunoregulatory or anti-inflammatory functions respectively. CRT induces pro-inflammatory cytokines, dendritic cell (DC) maturation and activates cytotoxic T cells against tumours. By contrast, GRP78/BiP induces anti-inflammatory cytokines, inhibits DC maturation and heightens T-regulatory cell responses. These latter functions rebalance immune homeostasis in inflammatory diseases, such as rheumatoid arthritis. Both chaperones are therapeutically relevant agents acting primarily on monocytes/DCs. Endogenous exposure of CRT on cancer cell surfaces acts as an 'eat-me' signal and facilitates improved elimination of stressed and dying tumour cells by DCs. Therefore, therapeutics that promote endogenous CRT translocation to the cell surface can improve the removal of cancer cells. However, infused recombinant CRT dampens this cancer cell eradication by binding directly to the DCs. Low levels of endogenous BiP appear as a surface biomarker of endoplasmic reticulum (ER) stress in some types of tumour cells, a reflection of cells undergoing proliferation, in which resulting hypoxia and nutrient deprivation perturb ER homeostasis triggering the unfolded protein response, leading to increased expression of GRP78/BiP and altered cellular location. Conversely, infusion of an analogue of GRP78/BiP (IRL201805) can lead to long-term immune resetting and restoration of immune homeostasis. The therapeutic potential of both chaperones relies on them being relocated from their intracellular ER environment. Ongoing clinical trials are employing therapeutic interventions to either enhance endogenous cell surface CRT or infuse IRL201805, thereby triggering several disease-relevant immune responses leading to a beneficial clinical outcome.
Assuntos
Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico , Humanos , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Transporte/metabolismo , Citocinas/metabolismo , Anti-InflamatóriosRESUMO
BACKGROUND: There is a great unmet need for advanced therapies that provide rapid, robust, and sustained disease control for patients with ulcerative colitis. We assessed the efficacy and safety of upadacitinib, an oral selective Janus kinase 1 inhibitor, as induction and maintenance therapy in patients with moderately to severely active ulcerative colitis. METHODS: This phase 3, multicentre, randomised, double-blind, placebo-controlled clinical programme consisted of two replicate induction studies (U-ACHIEVE induction [UC1] and U-ACCOMPLISH [UC2]) and a single maintenance study (U-ACHIEVE maintenance [UC3]). The studies were conducted across Europe, North and South America, Australasia, Africa, and the Asia-Pacific region at 199 clinical centres in 39 countries (UC1), 204 clinical centres in 40 countries (UC2), and 195 clinical centres in 35 countries (UC3). Patients aged 16-75 years with moderately to severely active ulcerative colitis (Adapted Mayo score 5-9; endoscopic subscore 2 or 3) for at least 90 days were randomly assigned (2:1) to oral upadacitinib 45 mg once daily or placebo for 8 weeks (induction studies). Patients who achieved clinical response following 8-week upadacitinib induction were re-randomly assigned (1:1:1) to upadacitinib 15 mg, upadacitinib 30 mg, or placebo for 52 weeks (maintenance study). All patients were randomly assigned using web-based interactive response technology. The primary endpoints were clinical remission per Adapted Mayo score at week 8 (induction) and week 52 (maintenance). The efficacy analyses in the two induction studies were based on the intent-to-treat population, which included all randomised patients who received at least one dose of treatment. In the maintenance study, the primary efficacy analyses reported in this manuscript were based on the first 450 (planned) clinical responders to 8-week induction therapy with upadacitinib 45 mg once daily. The safety analysis population in the induction studies consisted of all randomised patients who received at least one dose of treatment; in the maintenance study, this population included all patients who received at least one dose of treatment as part of the primary analysis population. These studies are registered at ClinicalTrials.gov, NCT02819635 (U-ACHIEVE) and NCT03653026 (U-ACCOMPLISH). FINDINGS: Between Oct 23, 2018, and Sept 7, 2020, 474 patients were randomly assigned to upadacitinib 45 mg once daily (n=319) or placebo (n=155) in UC1. Between Dec 6, 2018, and Jan 14, 2021, 522 patients were randomly assigned to upadacitinib 45 mg once daily (n=345) or placebo (n=177) in UC2. In UC3, a total of 451 patients (21 from the phase 2b study, 278 from UC1, and 152 from UC2) who achieved a clinical response after 8 weeks of upadacitinib induction treatment were randomly assigned again to upadacitinib 15 mg (n=148), upadacitinib 30 mg (n=154), and placebo (n=149) in the primary analysis population. Statistically significantly more patients achieved clinical remission with upadacitinib 45 mg (83 [26%] of 319 patients in UC1 and 114 [34%] of 341 patients in UC2) than in the placebo group (seven [5%] of 154 patients in UC1 and seven [4%] of 174 patients; p<0·0001; adjusted treatment difference 21·6% [95% CI 15·8-27·4] for UC1 and 29·0% [23·2-34·7] for UC2). In the maintenance study, clinical remission was achieved by statistically significantly more patients receiving upadacitinib (15 mg 63 [42%] of 148; 30 mg 80 [52%] of 154) than those receiving placebo (18 [12%] of 149; p<0·0001; adjusted treatment difference 30·7% [21·7-39·8] for upadacitinib 15 mg vs placebo and 39·0% [29·7-48·2] for upadacitinib 30 mg vs placebo). The most commonly reported adverse events in UC1 were nasopharyngitis (15 [5%] of 319 in the upadacitinib 45 mg group vs six [4%] of 155 in the placebo group), creatine phosphokinase elevation (15 [4%] vs three [2%]), and acne (15 [5%] vs one [1%]). In UC2, the most frequently reported adverse event was acne (24 [7%] of 344 in the upadacitinib 45 mg group vs three [2%] of 177 in the placebo group). In both induction studies, serious adverse events and adverse events leading to discontinuation of treatment were less frequent in the upadacitinib 45 mg group than in the placebo group (serious adverse events eight [3%] vs nine (6%) in UC1 and 11 [3%] vs eight [5%] in UC2; adverse events leading to discontinuation six [2%] vs 14 [9%] in UC1 and six [2%] vs nine [5%] in UC2). In UC3, the most frequently reported adverse events (≥5%) were worsening of ulcerative colitis (19 [13%] of 148 in the upadacitinib 15 mg group vs 11 [7%] of 154 in the upadacitinib 30 mg group vs 45 [30%] of 149 in the placebo group), nasopharyngitis (18 [12%] vs 22 [14%] vs 15 [10%]), creatine phosphokinase elevation (nine [6%] vs 13 [8%] vs three [2%]), arthralgia (nine [6%] vs five [3%] vs 15 [10%]), and upper respiratory tract infection (seven [5%] vs nine [6%] vs six [4%]). The proportion of serious adverse events (ten [7%] vs nine [6%] vs 19 [13%]) and adverse events leading to discontinuation (six [4%] vs ten [6%] vs 17 [11%]) was lower in both upadacitinib groups than in the placebo group. Events of cancer, adjudicated major adverse cardiac events, or venous thromboembolism were reported infrequently. There were no treatment-related deaths. INTERPRETATION: Upadacitinib demonstrated a positive efficacy and safety profile and could be an effective treatment option for patients with moderately to severely active ulcerative colitis. FUNDING: AbbVie.
Assuntos
Acne Vulgar , Colite Ulcerativa , Nasofaringite , Colite Ulcerativa/tratamento farmacológico , Creatina Quinase , Método Duplo-Cego , Compostos Heterocíclicos com 3 Anéis , Humanos , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
BACKGROUND: Higher residual anatomic disease was associated with increased mortality in a recent randomized controlled trial of revascularization after ST-elevation myocardial infarction (STEMI). Less is known about the impact of residual disease post-STEMI in race-ethnic minorities. METHODS: Synergy Between Percutaneous Coronary Intervention With Taxus and Cardiac Surgery (SYNTAX)- II (SS-II) score is an established scoring method for anatomic disease and prevalent co-morbidities to describe patient complexity. We evaluated residual (r) SS-II in 165 patients from a single center urban US registry (n = 1208) presenting for primary percutaneous coronary intervention of STEMI and treated for 3-vessel or left main and any combination of 0, 1, 2 or 3-vessel disease. RESULTS: The median age was 62 years (IQR 52-70), 29.1% women, 44.9% Hispanic/Latino and 19.4% non-Hispanic Black. Over median of 4.9 years (IQR 2.9-6.3), higher rSS-II was associated with increased death [hazard ratio 2.46 per SD increment in log rSS-II (~five-fold increment on the original scale) 95% CI 1.51, 3.99], death or all-cause readmission (hazard ratio 1.37 per SD increment in log rSS-II 95% CI, 1.11-1.70) and death or cardiovascular disease readmission (hazard ratio 1.46 per SD increment in log rSS-II 95% CI, 1.14-1.88). rSS-II was higher in older women with more co-morbidities, but not different by race-ethnicity. CONCLUSIONS: In summary, higher rSS-II was associated with long-term outcomes post-STEMI in a prospective urban, minority cohort, suggesting a potential role for risk stratification with this measure in a non-trial setting.
Assuntos
Intervenção Coronária Percutânea , Infarto do Miocárdio com Supradesnível do Segmento ST , Idoso , Angiografia Coronária , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Intervenção Coronária Percutânea/efeitos adversos , Estudos Prospectivos , Sistema de Registros , Fatores de Risco , Infarto do Miocárdio com Supradesnível do Segmento ST/diagnóstico , Infarto do Miocárdio com Supradesnível do Segmento ST/cirurgia , Resultado do TratamentoRESUMO
Faithful chromosome segregation maintains chromosomal stability as errors in this process contribute to chromosomal instability (CIN), which has been observed in many diseases including cancer. Epigenetic regulation of kinetochore proteins such as Cse4 (CENP-A in humans) plays a critical role in high-fidelity chromosome segregation. Here we show that Cse4 is a substrate of evolutionarily conserved Cdc7 kinase, and that Cdc7-mediated phosphorylation of Cse4 prevents CIN. We determined that Cdc7 phosphorylates Cse4 in vitro and interacts with Cse4 in vivo in a cell cycle-dependent manner. Cdc7 is required for kinetochore integrity as reduced levels of CEN-associated Cse4, a faster exchange of Cse4 at the metaphase kinetochores, and defects in chromosome segregation, are observed in a cdc7-7 strain. Phosphorylation of Cse4 by Cdc7 is important for cell survival as constitutive association of a kinase-dead variant of Cdc7 (cdc7-kd) with Cse4 at the kinetochore leads to growth defects. Moreover, phospho-deficient mutations of Cse4 for consensus Cdc7 target sites contribute to CIN phenotype. In summary, our results have defined a role for Cdc7-mediated phosphorylation of Cse4 in faithful chromosome segregation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/fisiologia , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Cromatina/metabolismo , Instabilidade Cromossômica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/fisiologia , Cromossomos/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Epigênese Genética , Histonas/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4 We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4 We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4 Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1 Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Proteína Centromérica A , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Serina-Treonina Quinases , Proteólise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , UbiquitinaçãoRESUMO
CDC7-DBF4 kinase (DDK) initiates DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has diverse and apparently conflicting roles in the replication checkpoint response in various organisms, but the underlying mechanisms are far from settled. We show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1 and phosphorylates EXO1 in vitro. EXO1 is also required for nascent strand degradation following exposure to HU, so DDK might regulate EXO1 directly. Lastly, sublethal DDK inhibition causes various mitotic abnormalities, which is consistent with a checkpoint deficiency. In summary, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Dimetil Sulfóxido/farmacologia , Etoposídeo/farmacologia , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Mitose/efeitos dos fármacos , Piperidonas/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinonas/farmacologia , Pirróis/farmacologia , Transdução de SinaisRESUMO
DBF4-dependent kinase (DDK) is a two-subunit kinase required for initiating DNA replication at individual origins and is composed of CDC7 kinase and its regulatory subunit DBF4. Both subunits are highly expressed in many diverse tumor cell lines and primary tumors, and this is correlated with poor prognosis. Inhibiting DDK causes apoptosis of tumor cells, but not normal cells, through a largely unknown mechanism. Firstly, to understand why DDK is often overexpressed in tumors, we identified gene expression signatures that correlate with DDK high- and DDK low-expressing lung adenocarcinomas. We found that increased DDK expression is highly correlated with inactivation of RB1-E2F and p53 tumor suppressor pathways. Both CDC7 and DBF4 promoters bind E2F, suggesting that increased E2F activity in RB1 mutant cancers promotes increased DDK expression. Surprisingly, increased DDK expression levels are also correlated with both increased chemoresistance and genome-wide mutation frequencies. Our data further suggest that high DDK levels directly promote elevated mutation frequencies. Secondly, we performed an RNAi screen to investigate how DDK inhibition causes apoptosis of tumor cells. We identified 23 kinases and phosphatases required for apoptosis when DDK is inhibited. These hits include checkpoint genes, G2/M cell cycle regulators, and known tumor suppressors leading to the hypothesis that inhibiting mitotic progression can protect against DDKi-induced apoptosis. Characterization of one novel hit, the LATS2 tumor suppressor, suggests that it promotes apoptosis independently of the upstream MST1/2 kinases in the Hippo signaling pathway.
Assuntos
Adenocarcinoma/genética , Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Proteínas Serina-Treonina Quinases/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Apoptose/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Fatores de Transcrição E2F/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/genética , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Fosforilação , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteínas de Ligação a Retinoblastoma/genética , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The number of identified monogenic causes of childhood-onset autoimmunity due to nodal and extranodal lymphoproliferation has increased. These pathogenic genetic variants provide the potential for pathway-specific treatment. Novel variants also require pathway-specific verification. In this report, we describe a 14-year-old patient with a novel variant in STAT3. We report clinical and laboratory findings that support STAT3 p.G419R as a novel pathogenic STAT3 gain-of-function variant.
RESUMO
To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant 'Cdc6-E224Q' promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Replicação do DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Saccharomyces cerevisiae/fisiologiaRESUMO
Sinus of Valsalva aneurysm (SOVA), a congenital or acquired cardiac defect that is present in roughly 0.09% of the general population, often presents as an incidental finding during cardiac imaging. Although an echocardiogram is the standard imaging technique for such findings, cardiac computed tomography angiography (CCTA) has been increasingly utilized. If SOVA is diagnosed, CCTA is also a useful test for patients who are at low to intermediate risk for coronary artery disease (CAD) prior to surgical repair. CCTA can accurately rule out CAD, obviating the need for invasive angiography in most cases, which may be more risky in SOVA patients because their coronaries may be more difficult to engage and their aortic root may be more prone to injury. Although surgery has previously been the treatment of choice, transcatheter techniques have added to the spectrum of nonsurgical alternatives for repair. We report here 4 incidental SOVA cases and review the current literature.
Assuntos
Aneurisma Aórtico/terapia , Seio Aórtico/cirurgia , Procedimentos Cirúrgicos Vasculares , Adulto , Idoso , Aneurisma Aórtico/diagnóstico , Aneurisma Aórtico/cirurgia , Aortografia/métodos , Dilatação Patológica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada Multidetectores , Seio Aórtico/diagnóstico por imagem , Seio Aórtico/patologia , Resultado do TratamentoRESUMO
Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC50 values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of â¼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral/efeitos dos fármacos , Piperidonas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirimidinonas/farmacologia , Pirróis/farmacologia , Apoptose/efeitos dos fármacos , Disponibilidade Biológica , Proliferação de Células/efeitos dos fármacos , Reposicionamento de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Humanos , Pirimidinonas/farmacocinéticaRESUMO
Several gene-deficiency models promote the development of innate CD8(+) T cells that have diverse T cell antigen receptors (TCRs) but have a memory phenotype and rapidly produce cytokines. We demonstrate here that similar cells developed in mice deficient in the transcription factor KLF2. However, this was not due to intrinsic deficiency in KLF2 but instead was due to interleukin 4 (IL-4) produced by an expanded population of T cells expressing the transcription factor PLZF. The development of innate CD8(+) T cells in mice deficient in the tyrosine kinase Itk and coactivator CBP was also attributable to this IL-4-dependent mechanism. Finally, we show that the same mechanism drove the differentiation of innate CD8(+) T cells in BALB/c mice. Our findings identify a previously unknown mechanism of regulation of CD8(+) T cells via the production of IL-4 by PLZF(+) T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Interleucina-4/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Processos de Crescimento Celular/imunologia , Quimera , Regulação da Expressão Gênica , Imunidade Inata/imunologia , Memória Imunológica , Imunofenotipagem , Interleucina-4/biossíntese , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteína com Dedos de Zinco da Leucemia PromielocíticaRESUMO
gammadelta T cells are generated in the thymus and traffic to secondary lymphoid organs and epithelial surfaces, where they regulate immune responses. alphabeta T cells require sphingosine 1-phosphate receptor type 1 (S1P(1)) and CD62L for thymic emigration and circulation through secondary lymphoid organs. Both of these genes are regulated by the transcription factor Krüppel-like factor 2 (KLF2) in conventional alphabeta T cells. It is unclear if gammadelta T cells use similar mechanisms. In this study, we show that thymic gammadelta T cells express S1P(1) and that it is regulated by KLF2. Furthermore, KLF2 and S1P(1)-deficient gammadelta T cells accumulate in the thymus and fail to populate the secondary lymphoid organs or gut, in contrast to the expectation from published work. Interestingly, KLF2 but not S1P(1) deficiency led to the expansion of a usually rare population of CD4(+) promyelocytic leukemia zinc finger(+) "gammadelta NKT" cells. Thus, KLF2 is critically important for the homeostasis and trafficking of gammadelta T cells.
Assuntos
Quimiotaxia de Leucócito/imunologia , Homeostase/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Subpopulações de Linfócitos T/citologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Separação Celular , Quimiotaxia de Leucócito/genética , Citometria de Fluxo , Técnicas de Introdução de Genes , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Lisoesfingolipídeo/imunologia , Receptores de Lisoesfingolipídeo/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Cdc7 is a conserved serine/threonine kinase essential for the initiation of DNA replication, likely by activating the MCM DNA helicase at the G(1-) to S-phase transition. Cdc7 kinase activity requires association with its regulatory subunit Dbf4/activator of S-phase kinase. Cdc7-Dbf4 is also downstream of the conserved Ataxia telangectasia and RAD3-related kinase that responds to stalled replication forks or DNA damage. In this study, we found that Cdc7 protein was very low or undetectable in normal tissues and cell lines but had increased expression in approximately 50% of the 62 human tumor cell lines we examined. Most cell lines with increased Cdc7 protein levels also had increased Dbf4 abundance, and some tumor cell lines had extra copies of the DBF4 gene. A high expression of Cdc7 protein was also detected in primary breast, colon, and lung tumors but not in the matched normal tissues. We also found a high correlation between p53 loss and increased CDC7 and DBF4 expression in primary breast cancers (P = 3.6 x 10(-9) and 1.8 x 10(-10), respectively) and in the cancer cell lines we studied. Therefore, increased Cdc7-Dbf4 abundance may be a common occurrence in human malignancies.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Ciclo Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , HumanosRESUMO
RATIONALE: Birt-Hogg-Dubé syndrome (BHDS) is an autosomal, dominantly inherited genodermatosis that predisposes to fibrofolliculomas, kidney neoplasms, lung cysts, and spontaneous pneumothorax. OBJECTIVES: We evaluated 198 patients from 89 families with BHDS to characterize the risk factors for pneumothorax and genotype-pulmonary associations. METHODS: Helical computed tomography scans of the chest were used to screen for pulmonary abnormalities. BHD mutation data were used for genotype-pulmonary associations. We examined the relationship of pneumothorax with categorical parameters (sex, smoking history, and lung cysts) and continuous parameters (number of cysts, lung cyst volume, and largest cyst diameter and volume). Logistic regression analyses were used to identify the risk factors associated with pneumothorax. MEASUREMENTS AND MAIN RESULTS: Twenty-four percent (48/198) of patients with BHDS had a history of pneumothorax. The presence of lung cysts was significantly associated with pneumothorax (p = 0.006). Total lung cyst volume, largest cyst diameter and volume, and every parameter related to the number of lung cysts were significantly associated (p < 0.0001) with pneumothorax. A logistic regression analysis showed that only the total number of cysts in the right parenchymal lower lobe and the total number of cysts located on the pleural surface in the right middle lobe were needed to classify a patient as to whether or not he or she was likely to have a pneumothorax. Exon location of the BHD mutation was associated with the numbers of cysts (p = 0.0002). CONCLUSIONS: This study indicates that patients with BHDS have a significant association between lung cysts and spontaneous pneumothorax.
Assuntos
Cisto Broncogênico/epidemiologia , Pneumotórax/epidemiologia , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Dermatopatias/genética , Proteínas Supressoras de Tumor/genética , Cisto Broncogênico/diagnóstico , Cisto Broncogênico/genética , Análise Mutacional de DNA , Éxons , Feminino , Ligação Genética , Testes Genéticos , Genótipo , Humanos , Masculino , Mutação , Fenótipo , Pneumotórax/diagnóstico , Pneumotórax/genética , Fatores de Risco , Dermatopatias/epidemiologia , SíndromeRESUMO
The HRPT2 (hereditary hyperparathyroidism type 2) tumor suppressor gene encodes a ubiquitously expressed 531 amino acid protein termed parafibromin. Inactivation of parafibromin predisposes one to the development of HPT-JT syndrome. To date, the role of parafibromin in tumorigenesis is largely unknown. Here, we report that parafibromin is a nuclear protein that possesses anti-proliferative properties. We show that overexpression of parafibromin inhibits colony formation and cellular proliferation, and induces cell cycle arrest in the G1 phase. Moreover, HPT-JT syndrome-derived mutations in HRPT2 behave in a dominant-negative manner by abolishing the ability of parafibromin to suppress cell proliferation. These findings suggest that parafibromin has a critical role in cell growth, and mutations in HRPT2 can directly inhibit this role.
Assuntos
Fase G1/efeitos dos fármacos , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Supressoras de Tumor/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Mutação/genética , Neoplasias/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Nearly 15 years have elapsed since the US Food and Drug Administration last approved a major new hematopoietic cytokine. Promiscuous binding to multiple receptors, or to receptors expressed by multiple tissues, reduces growth factor specificity and promotes side effects. Here we show that hematopoiesis can be differentially regulated using receptors rather than ligands. Conditional derivatives of both fibroblast growth factor receptor-1 (F36VFGFR1) and the thrombopoietin receptor (F36VMpl) induced a sustained expansion of mouse marrow cells ex vivo, and erythroid cells in vivo. Only F36VFGFR1 could support the ex vivo expansion of short-term repopulating hematopoietic stem cells (HSCs), the ex vivo survival of long-term repopulating HSCs, and the prolonged in vivo expansion of granulocytes, monocytes, and platelets. Only F36VMpl induced a response sufficiently rapid to accelerate recovery from radiation-induced anemia. These results establish receptors as a new class of hematopoietic regulators possessing activities unobtainable with growth factors.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Trombopoetina/metabolismo , Substituição de Aminoácidos , Anemia/etiologia , Anemia/genética , Anemia/metabolismo , Anemia/terapia , Animais , Células da Medula Óssea , Sobrevivência Celular/genética , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Feminino , Terapia Genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Leucócitos/citologia , Leucócitos/metabolismo , Camundongos , Camundongos Mutantes , Mutação de Sentido Incorreto , Lesões Experimentais por Radiação/genética , Lesões Experimentais por Radiação/mortalidade , Lesões Experimentais por Radiação/terapia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores de Trombopoetina/genética , Fatores de Tempo , Transdução Genética , Quimeras de Transplante/genética , Quimeras de Transplante/metabolismo , Estados Unidos , United States Food and Drug AdministrationRESUMO
Mammalian Kruppel-like transcription factors are implicated in regulating terminal differentiation of several tissue types. Deficiency in Kruppel-like factor (KLF) 2 (also known as LKLF) leads to a massive loss of the peripheral T-cell pool, suggesting KLF2 regulates T-cell quiescence and survival. Here we show, however, that KLF2 is essential for T-cell trafficking. KLF2-deficient (Klf2-/-) thymocytes show impaired expression of several receptors required for thymocyte emigration and peripheral trafficking, including the sphingosine-1-phosphate (S1P) receptor S1P1, CD62L and beta7 integrin. Furthermore, KLF2 both binds and transactivates the promoter for S1P1--a receptor that is critical for thymocyte egress and recirculation through peripheral lymphoid organs. Our findings suggest that KLF2 serves to license mature T cells for trafficking from the thymus and recirculation through secondary lymphoid tissues.
Assuntos
Movimento Celular , Fatores de Transcrição Kruppel-Like/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Timo/citologia , Transferência Adotiva , Animais , Linhagem Celular Tumoral , Quimera/metabolismo , Feto , Humanos , Células Jurkat , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Fígado/embriologia , Camundongos , Regiões Promotoras Genéticas/genética , Receptores de Lisoesfingolipídeo/genética , Linfócitos T/transplante , Ativação TranscricionalRESUMO
Dbf4p is an essential regulatory subunit of the Cdc7p kinase required for the initiation of DNA replication. Cdc7p and Dbf4p orthologs have also been shown to function in the response to DNA damage. A previous Dbf4p multiple sequence alignment identified a conserved approximately 40-residue N-terminal region with similarity to the BRCA1 C-terminal (BRCT) motif called "motif N." BRCT motifs encode approximately 100-amino-acid domains involved in the DNA damage response. We have identified an expanded and conserved approximately 100-residue N-terminal region of Dbf4p that includes motif N but is capable of encoding a single BRCT-like domain. Dbf4p orthologs diverge from the BRCT motif at the C terminus but may encode a similar secondary structure in this region. We have therefore called this the BRCT and DBF4 similarity (BRDF) motif. The principal role of this Dbf4p motif was in the response to replication fork (RF) arrest; however, it was not required for cell cycle progression, activation of Cdc7p kinase activity, or interaction with the origin recognition complex (ORC) postulated to recruit Cdc7p-Dbf4p to origins. Rad53p likely directly phosphorylated Dbf4p in response to RF arrest and Dbf4p was required for Rad53p abundance. Rad53p and Dbf4p therefore cooperated to coordinate a robust cellular response to RF arrest.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA/genética , DNA Fúngico/biossíntese , DNA Fúngico/química , DNA Fúngico/genética , Genes Fúngicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Homologia de Sequência de AminoácidosRESUMO
Cell-based therapies have potential widespread applications in clinical medicine, and methods for controlling the fate of transplanted cells are needed. We have previously described a means for directing the growth of genetically modified cells in vivo using a derivative of the thrombopoietin receptor, mpl, that is reversibly activated by a drug called a chemical inducer of dimerization (CID). Since Jak2 participates in signaling from a number of different cytokine receptors (including mpl), we tested whether direct activation of the JH1 domain of Jak2 would broaden the repertoire of hematopoietic lineages responsive to the CID. While the engineered Jak2 induced a significant rise in genetically modified red cells, as we have observed previously with mpl, it lacked mpl's ability to expand genetically modified platelets and failed to expand genetically modified granulocytes, B cells, or T cells. These findings identify a signaling molecule other than mpl that can function as a cell growth switch in vivo and demonstrate that signaling molecules used for in vivo selection need not be confined to receptors. The erythroid-restricted growth response suggests that CID-activated Jak2 may be well suited to gene therapy applications in sickle cell anemia or beta-thalassemia.