RESUMEN
BACKGROUND: Leukemia is a cancer of blood and bone marrow cells, causing about 300,000 deaths worldwide. Photodynamic therapy (PDT) is a promising alternative for the treatment of malignant tumors. KillerRed is a genetically encoded red fluorescent protein photosensitizer (PS). In this study, we aimed to investigate the effects of KillerRed-mediated PDT on chronic myelogenous leukemia K562 cells, acute monocytic leukemia NB4 cells, and acute monocytic leukemia THP1 cells. METHODS: KillerRed was expressed in Escherichia coli cells, purified by Q-Sepharose column, and confirmed by western-blotting. The PDT effect on cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8). Cell apoptosis was determined by PE Annexin V/7-AAD staining and flow cytometry. The distribution of KillerRed in leukemia cells was detected by confocal laser scanning microscopy (CLSM) and western-blotting. The ROS generation was measured by flow cytometry. RESULTS: Pure KillerRed was obtained with a yield of about 37 mg per liter of bacterial cells. KillerRed photodynamic inactivated the leukemia cells in a concentration-dependent manner, but exhibited no obvious dark toxicity. PDT mediated by KillerRed could also induce apoptotic response (mainly early apoptosis) in the three cell lines. The CLSM imaging indicated that KillerRed was distributed within the cytoplasm and nuclei of leukemia cells, causing damages to the cytoplasm and leaving the nuclear envelope intact during light irradiation. KillerRed distributed both in the cytosol and nuclei was confirmed by western blotting, and ROS significantly increased in PDT treated cells compared to the cells treated with KillerRed alone. CONCLUSIONS: Our studies demonstrated that KillerRed-mediated PDT could effectively inactivate K562, NB4, and THP1 leukemia cells and trigger cell apoptosis, and it has potential to be used individually or complementally, in the treatment of leukemia.
Asunto(s)
Leucemia/tratamiento farmacológico , Proteínas Luminiscentes , Fotoquimioterapia , Fármacos Fotosensibilizantes , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Leucemia/metabolismo , Proteínas Luminiscentes/aislamiento & purificación , Proteínas Luminiscentes/metabolismo , Fármacos Fotosensibilizantes/aislamiento & purificación , Fármacos Fotosensibilizantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína Fluorescente RojaRESUMEN
The variable region of murine immunoglobulin heavy chain (Igh) is assembled by sequential DH -JH and VH -DJH recombination. The accessibility of the Igh locus determines the order of rearrangement. Because of the large number of VH genes and the lack of a suitable model, the epigenetic modifications of VH genes after DJH recombination have not previously been characterized. Here, we employed two v-Abl pro-B cell lines, in which the Igh locus is in germline and DJH -recombined configurations, respectively. The DJH junction displays the characteristics of a recombination centre, such as high levels of activation-associated histone modifications and recombination-activating gene protein (RAG) binding in DJH -rearranged pro-B cells, which extend the recombination centre model proposed for the germline Igh locus. The different domains of the VH region have distinct epigenetic characteristics after DJH recombination. Distal VH genes have higher levels of active histone modifications, germline transcription and Pax5 binding, and good quality recombination signal sequences. Proximal VH genes are relatively close to the DJH recombination centre, which partially compensates for the low levels of the above active epigenetic modifications. DJH recombination centre might serve as a cis-acting element to regulate the accessibility of the VH region. Furthermore, we demonstrate that RAG weakly binds to functional VH genes, which is the first detailed assessment of RAG dynamic binding to VH genes. We provide a way for VH -DJH recombination in which the VH gene is brought into close proximity with the DJH recombination centre for RAG binding by a Pax5-dependent chromosomal compaction event, and held in this position for subsequent cleavage and VH -DJH joining.
Asunto(s)
Epigénesis Genética , Reordenamiento Génico de Linfocito B , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Células Precursoras de Linfocitos B/inmunología , Acetilación , Animales , Línea Celular Transformada , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Genes abl , Células HEK293 , Histonas/metabolismo , Proteínas de Homeodominio/inmunología , Proteínas de Homeodominio/metabolismo , Humanos , Región de Unión de la Inmunoglobulina/genética , Región de Unión de la Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/inmunología , Región Variable de Inmunoglobulina/metabolismo , Metilación , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Células Precursoras de Linfocitos B/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Transcripción GenéticaRESUMEN
Toxoplasma gondii pathogen is a threat to human health that results in economic burden. Unfortunately, there are very few high-efficiency and low-toxicity drugs for toxoplasmosis in the clinic. (+)-Usnic acid derived from lichen species has been reported to have anti-inflammatory, antibacterial, anti-parasitology, and even anti-cancer activities. Herein, the systematic effect of (+)-usnic acid and (+)-usnic acid-liposome on toxoplasma were studied in vitro and in vivo. The viability of toxoplasma tachyzoite was assayed with trypan blue and Giemsa staining; while the invasive capability of tachyzoite to cardiofibroblasts was detected using Giemsa staining. The survival time of mice and the changes in tachyzoite ultrastructure were studied in vivo. The results showed that (+)-usnic acid inhibited the viability of tachyzoite; pretreatment with (+)-usnic acid significantly decreased the invasion of tachyzoite to cardiofibroblasts in vitro; (+)-usnic acid and (+)-usnic acid-liposome extensively prolonged the survival time of mice about 90.9% and 117%, respectively; and improved the ultrastructural changes of tachyzoite, especially in dense granules, rhoptries, endoplasmic reticulum, mitochondria and other membrane organelles. In summary, these results demonstrate that (+)-usnic acid and (+)-usnic acid-liposome with low toxicity have an inhibitory effect on the viability of toxoplasma tachyzoite, and mainly destructed membrane organelles which are connected with the virulence of toxoplasma. These findings provide the basis for further study and development of usnic acid as a potential agent for treating toxoplasmosis.
Asunto(s)
Antiprotozoarios/farmacología , Benzofuranos/farmacología , Toxoplasma/efectos de los fármacos , Animales , Antiprotozoarios/administración & dosificación , Benzofuranos/administración & dosificación , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/parasitología , Liposomas , Masculino , Ratones , Microscopía Electrónica de Transmisión , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/parasitología , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Toxoplasma/ultraestructura , Toxoplasmosis Animal/tratamiento farmacológico , Usnea/químicaRESUMEN
The evolutionary conservation of non-core RAG regions suggests significant roles that might involve quantitative or qualitative alterations in RAG activity. Off-target V(D)J recombination contributes to lymphomagenesis and is exacerbated by RAG2' C-terminus absence in Tp53-/- mice thymic lymphomas. However, the genomic stability effects of non-core regions from both Rag1c/c and Rag2c/c in BCR-ABL1+ B-lymphoblastic leukemia (BCR-ABL1+ B-ALL), the characteristics, and mechanisms of non-core regions in suppressing off-target V(D)J recombination remain unclear. Here, we established three mouse models of BCR-ABL1+ B-ALL in mice expressing full-length RAG (Ragf/f), core RAG1 (Rag1c/c), and core RAG2 (Rag2c/c). The Ragc/c (Rag1c/c and Rag2c/c) leukemia cells exhibited greater malignant tumor characteristics compared to Ragf/f cells. Additionally, Ragc/c cells showed higher frequency of off-target V(D)J recombination and oncogenic mutations than Ragf/f. We also revealed decreased RAG cleavage accuracy in Ragc/c cells and a smaller recombinant size in Rag1c/c cells, which could potentially exacerbate off-target V(D)J recombination in Ragc/c cells. In conclusion, these findings indicate that the non-core RAG regions, particularly the non-core region of RAG1, play a significant role in preserving V(D)J recombination precision and genomic stability in BCR-ABL1+ B-ALL.
Asunto(s)
Proteínas de Unión al ADN , Proteínas de Fusión bcr-abl , Proteínas de Homeodominio , Recombinación V(D)J , Animales , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Recombinación V(D)J/genética , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Ratones , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Carcinogénesis/genética , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismoRESUMEN
The BCL6 proto-oncogene encodes a transcriptional repressor, which is required for germinal centers (GCs) formation and lymphomagenesis. Previous studies have been reported that the constitutive expression of BCL6 leads to diffuse large B cell lymphoma (DLBCL) through activation-induced cytidine deaminase (AID) mediated chromosomal translocations and mutations. However, other DLBCLs (45%) without structural variants were characterized by abnormally high level of BCL6 expression through an unknown mechanism. Herein, we report that deficiency in AID or methyltransferase 1 (DNMT1) triggers high level of BCL6 expression. AID-DNMT1 complex binds to -0.4â¯kb -0â¯kb region of BCL6 promoter and contributes to generate BCL6 methylation which results in inhibition of BCL6 expression. The proteasome pathway inhibitor MG132 induces accumulation of AID and DNMT1, causes decreased BCL6 expression, and leads to cell apoptosis and tumor growth inhibition in DLBCL cell xenograft mice. These findings propose mechanistic insight into an alternative cofactor role of AID in assisting DNMT1 to maintain BCL6 methylation, thus suppress BCL6 transcription in DLBCL. This novel mechanism will provide a new drug selection in the therapeutic approach to DLBCL in the future.
Asunto(s)
Citidina Desaminasa/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN , Regulación Leucémica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Animales , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Genes Reporteros , Humanos , Linfoma de Células B Grandes Difuso/patología , Ratones , Modelos Biológicos , Regiones Promotoras Genéticas , Unión Proteica , Proto-Oncogenes MasRESUMEN
Breakpoint cluster region (BCR)Abelson murine leukemia (ABL)1+ acute Blymphoblastic leukemia (BALL) is a disease associated with a dismal prognosis and a high incidence of central nervous system (CNS) metastasis. However, BCRABL1+ BALL with CNS infiltration has not been previously characterized, at least to the best of our knowledge. In the present study, a murine model of BCRABL1+ BALL with CNS metastasis was established using retroviral transduction. The vast majority of BCRABL1+ leukemic cells were found to be immature B cells with a variable proportion of proB and preB populations. The present results indicated that the BCRABL1+ Bleukemic cells expressed high levels integrin subunit alpha 6 (Itga6) and Lselectin adhesion molecules, and have an intrinsic ability to disseminate and accumulate in CNS tissues, predominantly in meninges. On the whole, these results provide an approach for addressing the mechanisms of BCRABL1+ BALL with CNS metastasis and may guide the development of novel therapeutic strategies.
Asunto(s)
Neoplasias del Sistema Nervioso Central/secundario , Modelos Animales de Enfermedad , Proteínas de Fusión bcr-abl/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Animales , Trasplante de Médula Ósea , Neoplasias del Sistema Nervioso Central/metabolismo , Neoplasias del Sistema Nervioso Central/terapia , Femenino , Proteínas de Fusión bcr-abl/genética , Integrinas/metabolismo , Selectina L/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapiaRESUMEN
Acute myeloid leukemia (AML) is a heterogeneous hematopoietic disorder initiated from a small subset of leukemia stem cell (LSC), which presents unrestricted self-renewal and proliferation. Long non-coding RNA HOTAIR is abundantly expressed and plays oncogenic roles in solid cancer and AML. However, whether HOTAIR regulates the self-renewal of LSC is largely unknown. Here, we reported that the expression of HOTAIR was increased in LSC than in normal hematological stem and progenitor cells (HSPCs). HOTAIR inhibition by short hairpin RNAs (shRNAs) decreased colony formation in leukemia cell lines and primary AML blasts. We then investigated the role of HOTAIR in leukemia in vivo. HOTAIR knockdown extends the survival time in U937-transplanted NSG mice. Furthermore, HOTAIR knockdown reduced infiltration of leukemic blasts, decreased frequency of LSC, and prolonged overall survival in MLL-AF9-induced murine leukemia, suggesting that HOTAIR is required for the maintenance of AML. Mechanistically, HOTAIR inhibited p15 expression through zeste homolog 2 (EZH2)-enrolled tri-methylation of Lys 27 of histone H3 (H3K27me3) in p15 promoter. In addition, p15 partially reversed the decrease of colony and proliferation induced by HOTAIR knockdown, suggesting that p15 plays an important role in the leukemogenesis by HOTAIR. In conclusion, our study suggests that HOTAIR facilitates leukemogenesis by enhancing self-renewal of LSC. HOTAIR might be a potential target for anti-LSC therapy.
Asunto(s)
Autorrenovación de las Células/fisiología , Transformación Celular Neoplásica/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Silenciador del Gen , Código de Histonas/genética , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/citología , ARN Largo no Codificante/fisiología , ARN Neoplásico/fisiología , Animales , Trasplante de Médula Ósea , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/fisiología , Proteína Potenciadora del Homólogo Zeste 2/fisiología , Regulación Leucémica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Terapia Molecular Dirigida , Proteínas de Fusión Oncogénica/genética , Regiones Promotoras Genéticas , Ensayo de Tumor de Célula Madre , Células U937RESUMEN
Toxoplasma gondii pathogen is a threat to human health that results in economic burden. Unfortunately, there are very few high-efficiency and low-toxicity drugs for toxoplasmosis in the clinic. (+)-Usnic acid derived from lichen species has been reported to have anti-inflammatory, antibacterial, anti-parasitology, and even anti-cancer activities. In associated with the published article "Effects of (+)-Usnic Acid and (+)-Usnic Acid-Liposome on Toxoplasma gondii" [1], this dataset article provided the detailed information of experimental designing, methods, features as well as the raw data of (+)-usnic acid and (+)-usnic acid-liposome on toxoplasma in vivo and vitro. (+)-Usnic acid may be a potential agent for treating toxoplasmosis.