RESUMEN
Multiple myeloma (MM) is an incurable B-cell malignancy often accompanied by profound immunodeficiency. Lenalidomide (Len) is an immunomodulatory drug that exerts promising therapeutic effects on MM through the immune system. However, predictive markers related to the effects of Len treatment are not fully understood. This study aimed to identify candidate biomarkers for predicting the clinical efficacy of Len and dexamethasone (Ld) therapy through a comprehensive analysis of serum cytokines. The levels of 48 cytokines in the serum of patients with MM just before Ld therapy (n = 77), at the time of best response (n = 56), and at disease progression (n = 49) were measured and evaluated. Patients with high IL-18 and M-CSF levels showed significantly shorter progression-free survival and overall survival (OS). In contrast, patients with high PDGF-BB levels had longer survival. Moreover, low levels of G-CSF, IL-7, IL-8, and SDF-1α were associated with shorter OS after Ld therapy. During Ld therapy, pro-inflammatory cytokines such as IL-2Rα, IL-18, and TNF-α were decreased, while IFN-γ was increased. IL-4 and IL-6 levels increased during disease progression. In conclusion, this study provides a better understanding of the association between cytokines and the efficacy of Ld therapy as well as the unique changes in cytokines related to inflammatory and immune responses during Ld therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Citocinas , Dexametasona , Lenalidomida , Mieloma Múltiple , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/sangre , Mieloma Múltiple/mortalidad , Lenalidomida/uso terapéutico , Dexametasona/uso terapéutico , Citocinas/sangre , Masculino , Femenino , Persona de Mediana Edad , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Adulto , Anciano de 80 o más Años , Progresión de la Enfermedad , Biomarcadores de Tumor/sangreRESUMEN
We previously reported the Marimo cell line, which was established from the bone marrow cells of a patient with essential thrombocythemia (ET) at the last stage after transformation to acute myeloid leukemia (AML). This cell line is widely used for the biological analysis of ET because it harbors CALR mutation. However, genetic processes during disease progression in the original patient were not analyzed. We sequentially analyzed the genetic status in the original patient samples during disease progression. The ET clone had already acquired CALR and MPL mutations, and TP53 and NRAS mutations affected the disease progression from ET to AML in this patient. Particularly, the variant allele frequency of the NRAS mutation increased along with the disease progression after transformation, and the NRAS-mutated clone selectively proliferated in vitro, resulting in the establishment of the Marimo cell line. Although CALR and MPL mutations co-existed, MPL was not expressed in Marimo cells or any clinical samples. Furthermore, mitogen-activated protein kinase (MAPK) but not the JAK2-STAT pathway was activated. These results collectively indicate that MAPK activation is mainly associated with the proliferation ability of Marimo cells.
Asunto(s)
Calreticulina , Evolución Clonal , Leucemia Mieloide Aguda , Mutación , Receptores de Trombopoyetina , Trombocitemia Esencial , Humanos , Trombocitemia Esencial/genética , Trombocitemia Esencial/patología , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Calreticulina/genética , Calreticulina/metabolismo , Receptores de Trombopoyetina/genética , Evolución Clonal/genética , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , GTP Fosfohidrolasas/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Masculino , Progresión de la Enfermedad , Femenino , Línea Celular Tumoral , Anciano , Persona de Mediana EdadRESUMEN
The Escherichia coli (E. coli)-based protein synthesis using recombinant elements (PURE) system is a cell-free protein synthesis system reconstituted from purified factors essential for E. coli translation. The PURE system is widely used for basic and synthetic biology applications. One of the major challenges associated with the PURE system is that the protein yield of the system varies depending on the protein. Studies have reported that the efficiency of translation is significantly affected by nucleotide and amino acid sequences, especially in the N-terminal region. Here, we investigated the inherent effect of various N-terminal sequences on protein synthesis using the PURE system. We found that a single amino acid substitution in the N-terminal region significantly altered translation efficiency in the PURE system, especially at low temperatures. This result gives us useful suggestions for the expression of the protein of interest in vitro and in vivo.
Asunto(s)
Escherichia coli , Biosíntesis de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Aminoácidos/metabolismo , Sistema Libre de Células , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Frío , Temperatura , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/biosíntesisRESUMEN
Ribosomes polymerize nascent peptides through repeated inter-subunit rearrangements between the classic and hybrid states. The peptidyl-tRNA, the intermediate species during translation elongation, stabilizes the translating ribosome to ensure robust continuity of elongation. However, the translation of acidic residue-rich sequences destabilizes the ribosome, leading to a stochastic premature translation cessation termed intrinsic ribosome destabilization (IRD), which is still ill-defined. Here, we dissect the molecular mechanisms underlying IRD in Escherichia coli. Reconstitution of the IRD event reveals that (1) the prolonged ribosome stalling enhances IRD-mediated translation discontinuation, (2) IRD depends on temperature, (3) the destabilized 70S ribosome complex is not necessarily split, and (4) the destabilized ribosome is subjected to peptidyl-tRNA hydrolase-mediated hydrolysis of the peptidyl-tRNA without subunit splitting or recycling factors-mediated subunit splitting. Collectively, our data indicate that the translation of acidic-rich sequences alters the conformation of the 70S ribosome to an aberrant state that allows the noncanonical premature termination.
Asunto(s)
Proteínas de Escherichia coli , Biosíntesis de Proteínas , Péptidos/metabolismo , Ribosomas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismoRESUMEN
The N-terminal modification of nascent proteins, such as acetylation and myristoylation, is one of the most abundant post-translational modifications. To analyze the function of the modification, it is important to compare the modified and unmodified proteins under defined conditions. However, it is technically difficult to prepare unmodified proteins because cell-based systems contain endogenous modification systems. In this study, we developed a cell-free method to conduct N-terminal acetylation and myristoylation of nascent proteins in vitro using a reconstituted cell-free protein synthesis system (PURE system). Proteins synthesized using the PURE system were successfully acetylated or myristoylated in a single-cell-free mixture in the presence of modifying enzymes. Furthermore, we performed protein myristoylation in giant vesicles, which resulted in their partial localization to the membrane. Our PURE-system-based strategy is useful for the controlled synthesis of post-translationally modified proteins.
Asunto(s)
Biosíntesis de Proteínas , Proteínas , Proteínas/metabolismo , Ácido Mirístico/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
To realize nucleic acid-targeting photodynamic therapy, a photosensitizer should be attached at the optimal position on a complementary oligonucleotide, where a guanine photooxidation is maximized. Here we show the photooxidation of 22 DNA duplexes with varied lengths between a 1O2-generating biphenyl photosensitizer attached at a midchain thymine in a strand and the single guanine reactant in the other strand. The best photooxidation efficiencies are achieved at 9, 10, and 21 base intervals, which coincides with the pitch of 10.5 base pairs per turn in a DNA duplex. The low efficiencies for near and far guanines are due to quenching of the biphenyl by guanine and dilution of 1O2 by diffusion, respectively. The 1O2-diffusion mapping along DNA duplex provides clues to the development of efficient and selective photosensitizer agents for nucleic acid-targeting photodynamic therapy, as well as an experimental demonstration of diffusion of a particle along cylindrical surface in molecular level.
Asunto(s)
Guanina , Fármacos Fotosensibilizantes , Conformación de Ácido Nucleico , ADNRESUMEN
POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.
Asunto(s)
Mieloma Múltiple , Síndrome POEMS , Humanos , Síndrome POEMS/diagnóstico , Síndrome POEMS/etiología , Síndrome POEMS/patología , Células Plasmáticas/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Análisis de la Célula Individual , Cadenas lambda de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/metabolismo , Células Clonales/patología , Mieloma Múltiple/patología , Aminoácidos/metabolismoRESUMEN
We have previously developed a glucose-linked biphenyl photosensitizer that can pass through glucose transporters, aiming for cancer-selective photodynamic therapy (PDT). Its small size (MW: 435) will allow oral administration and a fast clearance avoiding photosensitivity. However, its fluorescence efficiency was quite low, causing difficulty in monitoring cellular uptake. We thus synthesized a series of monosaccharide-linked biphenyl derivatives with a sulfur atom replacing an oxygen atom, in search of a photosensitizer with a brighter fluorescence. Among them, 4'-nitrobiphenyl thioglucoside showed a fluorescence emission extending to near infra-red region with a strength three times greater than that of the previous compound. This compound was found to have a higher 1O2-producing efficiency (ΦΔ: 0.75) than the previous compound (ΦΔ: 0.65). The thioglucoside indicated a significant photodamaging effect (IC50: 250 µM) against cancer cells. Although the galactose and mannose analogs exerted similar photodamaging effects, they were moderately toxic in the dark at a concentration of 300 µM. The thioglucoside and thiomannoside were at least partially uptaken through glucose transporters as demonstrated by inhibition with cytochalasin B, whereas no inhibition was observed for the galactoside. The behavior of d-glucose toward the cellular uptakes of these photosensitizers was bipolar: inhibitory at a low concentration and recovery or acceleratory at a higher concentration. These results indicate that 4'-nitrobiphenyl thioglucoside is the smallest (MW: 393) cancer-targeting photosensitizer with a trackable fluorescence property.
Asunto(s)
Neoplasias , Fotoquimioterapia , Colorantes , Glucosa , Humanos , Ligandos , Neoplasias/tratamiento farmacológico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , TioglucósidosRESUMEN
Werner syndrome (WS) is a progeroid syndrome caused by mutations in the WRN gene, which encodes the RecQ type DNA helicase for the unwinding of unusual DNA structures and is implicated in DNA replication, DNA repair, and telomere maintenance. patients with WS are prone to develop malignant neoplasms, including hematological malignancies. However, the pathogenesis of WS-associated hematological malignancies remains uncharacterized. Here we investigated the somatic gene mutations in WS-associated myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Whole-exome sequencing (WES) of 4 patients with WS with MDS/AML revealed that all patients had somatic mutations in TP53 but no other recurrent mutations in MDS/AML. TP53 mutations were identified at low allele frequencies at more than one year before the MDS/AML stage. All 4 patients had complex chromosomal abnormalities including those that involved TP53. Targeted sequencing of nine patients with WS without apparent blood abnormalities did not detect recurrent mutations in MDS/AML except for a PPM1D mutation. These results suggest that patients with WS are apt to acquire TP53 mutations and/or chromosomal abnormalities involving TP53, rather than other MDS/AML-related mutations. TP53 mutations are frequently associated with prior exposure to chemotherapy; however, all four patients with WS with TP53 mutations/deletions had not received any prior chemotherapy, suggesting a pathogenic link between WRN mutations and p53 insufficiency. These results indicate that WS hematopoietic stem cells with WRN insufficiency acquire competitive fitness by inactivating p53, which may cause complex chromosomal abnormalities and the subsequent development of myeloid malignancies. These findings promote our understanding of the pathogenesis of myeloid malignancies associated with progeria.
Asunto(s)
Neoplasias Hematológicas , Leucemia Mieloide Aguda , Trastornos Mieloproliferativos , Progeria , Síndrome de Werner , Aberraciones Cromosómicas , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Mutación , Prevalencia , Progeria/genética , Proteína p53 Supresora de Tumor/genética , Síndrome de Werner/complicaciones , Síndrome de Werner/genéticaRESUMEN
Attempts to create complex molecular systems that mimic parts of cellular systems using a bottom-up approach have become important in the field of biology. Among various molecular systems, in vitro protein synthesis inside lipid vesicles (liposomes), which we refer to as the artificial cell, has become an attractive system because it possesses two fundamental features of living cells: central dogma, and compartmentalization. Here, we investigated the effect of altering the amount or concentration of four constituents of the artificial cell consisting of a commercially available reconstituted in vitro transcription-translation (IVTT) system. As this IVTT system is available worldwide, the results will be useful to the scientific community when shared, unlike those from a lab-made IVTT system. We succeeded in revealing the effect and trend of altering each parameter and identified a suitable condition for preparing liposomes that are unilamellar and can synthesize proteins equally as well as the original IVTT system. Because the commercially available reconstituted IVTT system is an important standardization tool and the constituents can be adjusted as desired, our results will be useful for the bottom-up creation of more complex molecular systems.
Asunto(s)
Células Artificiales , Liposomas , ProteínasRESUMEN
Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.
Asunto(s)
Proteínas de Escherichia coli , Proteínas Ribosómicas , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph-) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.
Asunto(s)
Isocitrato Deshidrogenasa/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras , Enfermedad Aguda , Adolescente , Adulto , Factor de Transcripción CDX2/genética , Factor de Transcripción CDX2/metabolismo , Niño , Humanos , Isocitrato Deshidrogenasa/metabolismo , Persona de Mediana Edad , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Pronóstico , Transcriptoma , Adulto JovenRESUMEN
Monogalactosyldiacylglycerol (MGDG), the most abundant lipid in thylakoid membranes, is involved in photosynthesis and chloroplast development. MGDG lipase has an important role in lipid remodeling in Chlamydomonas reinhardtii. However, the process related to turnover of the lysogalactolipid that results from MGDG degradation, monogalactosylmonoacylglycerol (MGMG), remains to be clarified. Here we identified a homolog of Arabidopsis thaliana lysophosphatidylcholine acyltransferase (LPCAT) and characterized two independent knockdown (KD) alleles in C. reinhardtii. The enzyme designated as C. reinhardtiiLysolipid Acyltransferase 1 (CrLAT1) has a conserved membrane-bound O-acyl transferase domain. LPCAT from Arabidopsis has a key role in deacylation of phosphatidylcholine (PC). Chlamydomonas reinhardtii, however, lacks PC, and thus we hypothesized that CrLAT1 has some other important function in major lipid flow in this organism. In the CrLAT1 KD mutants, the amount of MGMG was increased, but triacylglycerols (TAGs) were decreased. The proportion of more saturated 18:1 (9) MGDG was lower in the KD mutants than in their parental strain, CC-4533. In contrast, the proportion of MGMG has decreased in the CrLAT1 overexpression (OE) mutants, and the proportion of 18:1 (9) MGDG was higher in the OE mutants than in the empty vector control cells. Thus, CrLAT1 is involved in the recycling of MGDG in the chloroplast and maintains lipid homeostasis in C. reinhardtii.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Galactolípidos/metabolismo , Homeostasis , Metabolismo de los Lípidos , Tilacoides/metabolismoRESUMEN
Although next-generation sequencing-based panel testing is well practiced in the field of cancer medicine for the identification of target molecules in solid tumors, the clinical utility and clinical issues surrounding panel testing in hematological malignancies have yet to be fully evaluated. We conducted a multicenter prospective clinical sequencing study to verify the feasibility of a panel test for hematological tumors, including acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B-cell lymphoma. Out of 96 eligible patients, 79 patients (82%) showed potentially actionable findings, based on the clinical sequencing assays. We identified that genetic alterations with a strong clinical significance were found at a higher frequency in terms of diagnosis (n = 60; 63%) and prognosis (n = 61; 64%) than in terms of therapy (n = 8; 8%). Three patients who harbored a germline mutation in either DDX41 (n = 2) or BRCA2 (n = 1) were provided with genetic counseling. At 6 mo after sequencing, clinical actions based on the diagnostic (n = 5) or prognostic (n = 3) findings were reported, but no patients were enrolled in a clinical trial or received targeted therapies based on the sequencing results. These results suggest that panel testing for hematological malignancies would be feasible given the availability of useful diagnostic and prognostic information. This study is registered with the UMIN Clinical Trial Registry (UMIN000029879, multiple myeloma; UMIN000031343, adult acute myeloid leukemia; UMIN000033144, diffuse large B-cell lymphoma; and UMIN000034243, childhood leukemia).
Asunto(s)
Biomarcadores de Tumor , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Biología Computacional/métodos , Femenino , Estudios de Asociación Genética/métodos , Pruebas Genéticas , Mutación de Línea Germinal , Neoplasias Hematológicas/terapia , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Previous genomic studies have revealed the genomic landscape of myeloma cells. Although some of the genomic abnormalities shown are believed to be correlated to the molecular pathogenesis of multiple myeloma and/or clinical outcome, these correlations are not fully understood. The aim of this study is to elucidate the correlation between genomic abnormalities and clinical characteristics by targeted capture sequencing in the Japanese multiple myeloma cohort. We analysed 154 patients with newly diagnosed multiple myeloma. The analysis revealed that the study cohort consisted of a less frequent hyperdiploid subtype (37·0%) with relatively high frequencies of KRAS mutation (36·4%) and IGH-CCND1 translocation (26·6%) compared with previous reports. Moreover, our targeted capture sequencing strategy was able to detect rare IGH-associated chromosomal translocations, such as IGH-CCND2 and IGH-MAFA. Interestingly, all 10 patients harboured MAX mutations accompanied by 14q23 deletion. The patients with del(17p) exhibited an unfavourable clinical outcome, and the presence of KRAS mutation was associated with shorter survival in patients with multiple myeloma, harbouring IGH-CCND1. Thus, our study provides a detailed landscape of genomic abnormalities, which may have potential clinical application for patients with multiple myeloma.
Asunto(s)
Deleción Cromosómica , Cromosomas Humanos Par 14/genética , Mieloma Múltiple/genética , Proteínas de Neoplasias/genética , Síndrome de Smith-Magenis/genética , Adulto , Cromosomas Humanos Par 17/genética , Femenino , Humanos , Japón , Masculino , Persona de Mediana EdadRESUMEN
In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/metabolismo , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Transcripción Genética , Proteínas Bacterianas/genética , Escherichia coli/genética , Mutación , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Proteínas Ribosómicas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/genéticaRESUMEN
We developed new DNA triplexes that contain four base triads T-A·T, A-ψ·CBr, G-PIC·YO, and C-G·Py+, where CBr, YO, Py, ψ, and PIC are 5-bromocytosine, 5-methyl-4-pyrimidone, 2-aminopyridine, the aglycons of deoxypseudouridine, and deoxypseudoisocytidine, respectively. DNA duplex incorporating T-A, A-ψ, G-PIC, and C-G, and triplex forming oligonucleotide incorporating T, CBr, YO, and Py formed the triplex as evaluated by Tm measurements. The triplex formation was successfully applied to the inhibition of transcription of the DNA duplex incorporating T7-promoter sequence modified by the above modified bases.
Asunto(s)
ADN/química , Oligonucleótidos/química , Aminopiridinas/química , Secuencia de Bases , Citosina/análogos & derivados , Citosina/química , Enlace de Hidrógeno , Modelos Moleculares , Conformación de Ácido Nucleico , Transición de Fase , Regiones Promotoras Genéticas , Temperatura de TransiciónRESUMEN
In this study, we designed 5'-amino-5'-deoxy-5'-hydroxymethylthymidine as a new oligonucleotide modification with an amino group directly attached to the 5'-carbon atom. We successfully synthesized two isomers of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine via dihydroxylation of the 5'-vinyl group incorporated into 5'-deoxy-5'-C-methenylthymidine derivative. Moreover, it was found that the nuclease resistance, binding selectivity to single-stranded RNA, and triplex-forming ability of an oligonucleotide containing RT residues of the new compound were higher than those of the unmodified oligonucleotide.
Asunto(s)
Oligonucleótidos/síntesis química , Timidina/química , Modelos Moleculares , Conformación de Ácido Nucleico , Oligonucleótidos/química , Timidina/análogos & derivadosRESUMEN
Cereblon (CRBN) is a target for immunomodulatory drugs. This study investigated the prognostic value of the expression of CRBN-pathway genes on the clinical relevance of lenalidomide (Len) treatment and evaluated the levels of CRBN-binding proteins and mutations in these genes after Len treatment. Forty-eight primary multiple myeloma cells were collected prior to treatment with Len and dexamethasone (Ld) and 25 paired samples were obtained post-Ld therapy. These tumor cells were used to determine the expression and mutated forms of the CRBN-pathway genes. Following normalization with CRBN levels, there was a significantly reduced IKZF1/CRBN ratio in samples that responded poorly to Ld therapy. Moreover, patients with low ratios of IKZF1/CRBN showed a significantly shorter progression-free survival (PFS) and overall survival (OS) than those with higher ratios. However, patients with high ratios of KPNA2/CRBN showed a significantly shorter PFS and OS than patients with lower ratios. Of the 25 paired samples analyzed, most samples showed a reduction in the expression of CRBN and an increase in IKZF1 gene expression. No mutations were observed in CRBN, IKZF1, or CUL4A genes in the post-Ld samples. In conclusion, a decreased expression of IKZF1 and increased expression of KPNA2 compared to that of CRBN mRNA predicts poor outcomes of Ld therapy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Factor de Transcripción Ikaros/genética , Lenalidomida/administración & dosificación , Mieloma Múltiple/tratamiento farmacológico , alfa Carioferinas/genética , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Proteínas Cullin/genética , Dexametasona/administración & dosificación , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunomodulación , Lenalidomida/efectos adversos , Masculino , Metilación , Persona de Mediana Edad , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Mutación , Pronóstico , Supervivencia sin Progresión , Ubiquitina-Proteína LigasasRESUMEN
α-Galactosylceramide (α-GalCer) is recognized by the CD1d proteins on antigen-presenting cells at the ceramide moiety and the galactose moiety is presented to iNKT cells, which stimulates the immune responses. However, the immune suppression by repeated injections of α-GalCer has discouraged its development as an anti-cancer agent. To overcome the shortcoming by spatiotemporal restriction of its exposure, we synthesized the photochromic azobenzene-incorporated analogues and tested the photo-immunoregulation effect in its binding to CD1d. FACS analyses indicated that some of these analogues enhanced the affinity to CD1d on photo-irradiation by about 20%. A docking simulation suggests that the photochromic molecule should be bulkier for a clearer discrimination between on and off states.