RESUMO
ABSTRACT: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis and limited treatment options. Programmed cell death ligand 1(PD-L1) is recognized to be involved in the pathobiology of ATLL. However, what molecules control PD-L1 expression and whether genetic or pharmacological intervention might modify PD-L1 expression in ATLL cells are still unknown. To comprehend the regulatory mechanisms of PD-L1 expression in ATLL cells, we performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening in this work. In ATLL cells, we discovered that the neddylation-associated genes NEDD8, NAE1, UBA3, and CUL3 negatively regulated PD-L1 expression, whereas STAT3 positively did so. We verified, in line with the genetic results, that treatment with the JAK1/2 inhibitor ruxolitinib or the neddylation pathway inhibitor pevonedistat resulted in a decrease in PD-L1 expression in ATLL cells or an increase in it, respectively. It is significant that these results held true regardless of whether ATLL cells had the PD-L1 3' structural variant, a known genetic anomaly that promotes PD-L1 overexpression in certain patients with primary ATLL. Pevonedistat alone showed cytotoxicity for ATLL cells, but compared with each single modality, pevonedistat improved the cytotoxic effects of the anti-PD-L1 monoclonal antibody avelumab and chimeric antigen receptor (CAR) T cells targeting PD-L1 in vitro. As a result, our work provided insight into a portion of the complex regulatory mechanisms governing PD-L1 expression in ATLL cells and demonstrated the in vitro preliminary preclinical efficacy of PD-L1-directed immunotherapies by using pevonedistat to upregulate PD-L1 in ATLL cells.
Assuntos
Ciclopentanos , Leucemia-Linfoma de Células T do Adulto , Linfoma , Pirimidinas , Adulto , Humanos , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Antígeno B7-H1/metabolismo , Linfoma/genéticaRESUMO
Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.
Assuntos
Leucemia-Linfoma de Células T do Adulto , Linfoma de Células T Periférico , Adulto , Humanos , Antígeno CD48/genética , Antígeno CD48/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Leucemia-Linfoma de Células T do Adulto/genética , Linfoma de Células T Periférico/genética , Células Matadoras NaturaisRESUMO
Adult T-cell leukemia/lymphoma (ATLL) is an aggressive T-cell malignancy with a poor prognosis with current therapy. Here we report genome-wide CRISPR-Cas9 screening of ATLL models, which identified CDK6, CCND2, BATF3, JUNB, STAT3, and IL10RB as genes that are essential for the proliferation and/or survival of ATLL cells. As a single agent, the CDK6 inhibitor palbociclib induced cell cycle arrest and apoptosis in ATLL models with wild-type TP53. ATLL models that had inactivated TP53 genetically were relatively resistant to palbociclib owing to compensatory CDK2 activity, and this resistance could be reversed by APR-246, a small molecule activator of mutant TP53. The CRISPR-Cas9 screen further highlighted the dependence of ATLL cells on mTORC1 signaling. Treatment of ATLL cells with palbociclib in combination with mTORC1 inhibitors was synergistically toxic irrespective of the TP53 status. This work defines CDK6 as a novel therapeutic target for ATLL and supports the clinical evaluation of palbociclib in combination with mTORC1 inhibitors in this recalcitrant malignancy.
Assuntos
Leucemia-Linfoma de Células T do Adulto , Linfoma , Adulto , Apoptose/genética , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Humanos , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transdução de SinaisRESUMO
T cells infected with human T-cell leukemia virus type 1 (HTLV-1) transform into malignant/leukemic cells and develop adult T-cell leukemia (ATL) after a long latency period. The tax (transactivator from the X-gene region) and HBZ (HTLV-1 bZIP factor) genes of HTLV-1 play crucial roles in the development of ATL. The process and mechanism by which HTLV-1-infected T cells acquire malignancy and develop ATL remain to be elucidated. Constitutive expression of interleukin-2 (IL-2) receptor α-chain (IL-2Rα/CD25), induced by the tax and HBZ genes of HTLV-1, on ATL cells implicates the involvement of IL-2/IL-2R pathway in the growth and development of ATL cells in vivo. However, the leukemic cells in the majority of ATL patients appeared unresponsive to IL-2, raising controversies on the role of this pathway for the growth of ATL cells in vivo. Here, we report the establishment of 32 IL-2-dependent T-cell lines infected with HTLV-1 from 26 ATL patients, including eight leukemic cell lines derived from five ATL patients, while no T-cell lines were established without IL-2. We have shown that the IL-2-dependent ATL cell lines evolved into IL-2-independent/-unresponsive growth phase, resembling ATL cells in vivo. Moreover, the IL-2-dependent non-leukemic T-cell lines infected with HTLV-1 acquired IL-2-independency and turned into tumor-producing cancer cells as with the ATL cell lines. HTLV-1-infected T cells in vivo could survive and proliferate depending on IL-2 that was produced in vivo by the HTLV-1-infected T cells of ATL patients and patients with HTLV-1-associated diseases and, acts as a physiological molecule to regulate T-cell growth. These results suggest that ATL cells develop among the HTLV-1-infected T cells growing dependently on IL-2 and that most of the circulating ATL cells progressed to become less responsive to IL-2, acquiring the ability to proliferate without IL-2.
RESUMO
Adult T cell leukemia/lymphoma (ATLL) is a frequently incurable disease associated with the human lymphotropic virus type I (HTLV-I). RNAi screening of ATLL lines revealed that their proliferation depends on BATF3 and IRF4, which cooperatively drive ATLL-specific gene expression. HBZ, the only HTLV-I encoded transcription factor that is expressed in all ATLL cases, binds to an ATLL-specific BATF3 super-enhancer and thereby regulates the expression of BATF3 and its downstream targets, including MYC. Inhibitors of bromodomain-and-extra-terminal-domain (BET) chromatin proteins collapsed the transcriptional network directed by HBZ and BATF3, and were consequently toxic for ATLL cell lines, patient samples, and xenografts. Our study demonstrates that the HTLV-I oncogenic retrovirus exploits a regulatory module that can be attacked therapeutically with BET inhibitors.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Redes Reguladoras de Genes , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Fatores Reguladores de Interferon/genética , Leucemia-Linfoma de Células T do Adulto/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Linhagem Celular Tumoral , Genes myc , Humanos , Camundongos , Proteínas/antagonistas & inibidores , Proteínas dos Retroviridae/fisiologiaRESUMO
With the emerging success of treating CD19 expressing B cell malignancies with ex vivo modified, autologous T cells that express CD19-directed chimeric antigen receptors (CAR), there is intense interest in expanding this evolving technology to develop effective modalities to treat other malignancies including solid tumors. Exploiting this approach to develop a therapeutic modality for T cell malignancies for which the available regimens are neither curative, nor confer long term survival we generated a lentivirus-based CAR gene transfer system to target the chemokine receptor CCR4 that is over-expressed in a spectrum of T cell malignancies as well as in CD4+ CD25+ Foxp3+ T regulatory cells that accumulate in the tumor microenvironment constituting a barrier against anti-tumor immunity. Ex vivo modified, donor-derived T cells that expressed CCR4 directed CAR displayed antigen-dependent potent cytotoxicity against patient-derived cell lines representing ATL, CTCL, ALCL and a subset of HDL. Furthermore, these CAR T cells also eradicated leukemia in a mouse xenograft model of ATL illustrating the potential utility of this modality in the treatment of a wide spectrum of T cell malignancies.
Assuntos
Neoplasias Hematológicas , Receptores de Antígenos de Linfócitos T , Receptores CCR4/antagonistas & inibidores , Linfócitos T Reguladores/imunologia , Animais , Linhagem Celular Tumoral , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Leucemia de Células T/genética , Leucemia de Células T/imunologia , Leucemia de Células T/patologia , Leucemia de Células T/terapia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores CCR4/genética , Receptores CCR4/imunologia , Linfócitos T Reguladores/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Citoesqueleto/metabolismo , Células HEK293 , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos SCID , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adult T-cell leukemia (ATL) develops in individuals infected with human T-cell lymphotropic virus-1 (HTLV-1). Presently there is no curative therapy for ATL. HTLV-1-encoded protein Tax (transactivator from the X-gene region) up-regulates Bcl-xL (B-cell lymphoma-extra large) expression and activates interleukin-2 (IL-2), IL-9, and IL-15 autocrine/paracrine systems, resulting in amplified JAK/STAT signaling. Inhibition of JAK signaling reduces cytokine-dependent ex vivo proliferation of peripheral blood mononuclear cells (PBMCs) from ATL patients in smoldering/chronic stages. Currently, two JAK inhibitors are approved for human use. In this study, we examined activity of multiple JAK inhibitors in ATL cell lines. The selective JAK inhibitor ruxolitinib was examined in a high-throughput matrix screen combined with >450 potential therapeutic agents, and Bcl-2/Bcl-xL inhibitor navitoclax was identified as a strong candidate for multicomponent therapy. The combination was noted to strongly activate BAX (Bcl-2-associated X protein), effect mitochondrial depolarization, and increase caspase 3/7 activities that lead to cleavage of PARP (poly ADP ribose polymerase) and Mcl-1 (myeloid cell leukemia 1). Ruxolitinib and navitoclax independently demonstrated modest antitumor efficacy, whereas the combination dramatically lowered tumor burden and prolonged survival in an ATL murine model. This combination strongly blocked ex vivo proliferation of five ATL patients' PBMCs. These studies provide support for a therapeutic trial in patients with smoldering/chronic ATL using a drug combination that inhibits JAK signaling and antiapoptotic protein Bcl-xL.
Assuntos
Interleucina-2/metabolismo , Janus Quinases/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Fatores de Transcrição STAT/metabolismo , Proteína bcl-X/metabolismo , Compostos de Anilina/farmacologia , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Citometria de Fluxo , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Janus Quinases/antagonistas & inibidores , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Nitrilas , Pirazóis/farmacologia , Pirimidinas , Fatores de Transcrição STAT/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína bcl-X/antagonistas & inibidoresRESUMO
BACKGROUND: Adult T-cell leukemia/lymphoma (ATL) is an aggressive malignancy of CD4+CD25+ lymphocytes caused by human T-cell lymphotropic virus type 1. While much progress has been made in understanding the mechanisms of cellular dysregulation, the prognosis for aggressive ATL still remains poor. Therefore, new therapeutic approaches need to be developed. RESULTS: Previously, we demonstrated that the viral protein Tax inactivates p53 in HTLV-1-infected T-cells. Here we show that 9-aminoacridine (9AA) through p53 reactivation and NF-κB inhibition has selective toxicity for infected leukemic cells independent of their p53 status. We further demonstrate that 9AA activates caspase-3/7 resulting in PARP cleavage. Next we investigated the efficacy of 9AA in the MET-1 ATL model. Alone, 9AA did not cause significant drops in surrogate tumor markers, soluble IL-2Rα or ß2-micorglobulin (ß2µ) levels with only a slight increase in survival of MET-1-bearing mice. However, in combination with Campath-1H, 9AA treatment resulted in low soluble IL-2Rα and ß2µ levels at 2 and 4 weeks. Consistent with reduced tumor cell burden, combination treatment significantly increased survival of MET-1-bearing mice compared to mice treated with either drug alone. Splenic cells isolated from 9AA or combination treated mice showed increased p53 protein levels and transcriptional activity. Consistent with increased tumor suppressor activity, we found increased PARP-1 cleavage in 9AA and combination treated cells. CONCLUSION: Our results indicate that targeting reactivation of p53 and inhibition of NF-κB with acridine-derivatives in combination with other chemotherapeutics could result in increased efficacy and selective killing of tumor cells.
Assuntos
Aminacrina/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Alemtuzumab , Animais , Caspase 3/genética , Caspase 7/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Células Jurkat , Leucemia-Linfoma de Células T do Adulto/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B/genética , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/genética , Linfócitos T/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Forty years have passed since our initial description of peculiar cases of adult-onset leukemia with abnormal cells having multi-convoluted nuclei and T cell properties, frequent in the southern regions of Japan in the early 1970s. Retrospectively, the study of adult T cell leukemia (ATL) and the related virus HTLV-I was a forerunner for all of human retrovirology, in which AIDS and the related retrovirus HIV were identified a few years later in the 1980s. Using the anti-TAC monoclonal antibody generated by the late Takashi Uchiyama during his stay in T. A. Waldmann's laboratory in NIH Bethesda, a cDNA encoding IL-2Rα chain was cloned by our group in Kyoto and by Waldmann's group in Bethesda. Abnormal IL-2Rα chain expression and the IL-2 dependency of ATL cell lines greatly contributed to the study of leukemogenesis of ATL. A new soluble factor named ADF/ATL-derived factor was also detected in ATL cell lines. After years of study, ADF proved to be a first human counterpart of thiol-related oxido-reductase thioredoxin/TRX, which opened the field of redox regulation of cell signaling involved in a variety of diseases. Close interaction among Drs. Kimishige Ishizaka, Kiyoshi Takastuki and T. A. Waldmanns before ATL and HTLV-I study was an essential base for our initiation of ATL research with Takashi Uchiyama and many other colleagues.
Assuntos
Vírus Linfotrópico T Tipo 1 Humano/genética , Leucemia-Linfoma de Células T do Adulto/virologia , Anticorpos Monoclonais/imunologia , Técnicas de Cultura de Células , Clonagem Molecular , Citocinas/genética , DNA Complementar/genética , História do Século XX , Vírus Linfotrópico T Tipo 1 Humano/imunologia , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Japão , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/história , Proteínas de Neoplasias/genética , Oxirredução , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Linfócitos T/imunologia , Tiorredoxinas , Estados UnidosRESUMO
Glucocorticoid (GC) is widely used for therapeutic purposes in immunological and hematological disorders. Annexin A1 (ANXA1/lipocortin-1/lipomodulin), a GC-inducible molecule, was regarded as a vital anti-inflammatory mediator of GC. Thioredoxin binding protein-2 (TBP-2/VDUP1/TXNIP), a regulator of redox reactions, cell growth and lipid metabolism, was also reportedly induced by GC. HTLV-I infected T cells undergo the transition from the IL-2 dependent to IL-2 independent growth during the long-term culture in vitro. We found that these T cells responded to GC with growth arrest and apoptosis in the IL-2 dependent growth stage, whereas they failed to respond to GC after their growth had shifted into the IL-2 independent stage. Here we employed these T cell lines and studied the roles of ANXA1 and TBP-2 in mediating GC-induced apoptosis. In GC-sensitive T cells, ANXA1 expression was negligible and unaffected by GC treatment, whereas TBP-2 was expressed and induced by GC treatment. In GC-resistant T cells, however, ANXA1 was highly expressed regardless of GC treatment and promoted cellular proliferation. In contrast, TBP-2 expression was lost and could not mediate the GC-induced apoptosis. In conclusion, these results suggest that TBP-2, but not ANXA1, is directly involved in the switching of GC sensitivity and GC resistance in HTLV-I infected T cell lines, whereas ANXA1 may be a biomarker indicative of the advanced stage of the transformation.
Assuntos
Anexina A1/metabolismo , Proteínas de Transporte/metabolismo , Transformação Celular Viral , Glucocorticoides/farmacologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/virologia , Anexina A1/genética , Apoptose , Proteínas de Transporte/genética , Linhagem Celular Transformada , Proliferação de Células , Regulação da Expressão Gênica , Glucocorticoides/metabolismo , Humanos , Interleucina-2/metabolismo , Leucemia-Linfoma de Células T do Adulto , Ativação Linfocitária , Linfócitos T/metabolismo , Linfócitos T/fisiologiaRESUMO
Thioredoxin-1 (TRX) plays important roles in cellular signaling by controlling the redox state of cysteine residues in target proteins. TRX is released in response to oxidative stress and shows various biologic functions from the extracellular environment. However, the mechanism by which extracellular TRX transduces the signal into the cells remains unclear. Here we report that the cysteine modification at the active site of TRX promotes the internalization of TRX into the cells. TRX-C35S, in which the cysteine at residue 35 of the active site was replaced with serine, was internalized more effectively than wild-type TRX in human T-cell leukemia virus-transformed T cells. TRX-C35S bound rapidly to the cell surface and was internalized into the cells dependent on lipid rafts in the plasma membrane. This process was inhibited by wild-type TRX, reducing reagents such as dithiothreitol, and methyl-beta-cyclodextrin, which disrupts lipid rafts. Moreover, the internalized TRX-C35S binds to endogenous TRX, resulting in the generation of intracellular reactive oxygen species (ROS) and enhanced cis-diamine-dichloroplatinum (II) (CDDP)-induced apoptosis via a ROS-mediated pathway involving apoptosis signal-regulating kinase-1 (ASK-1) activation. These findings suggest that the cysteine at the active site of TRX plays a key role in the internalization and signal transduction of extracellular TRX into the cells.
Assuntos
Microdomínios da Membrana/fisiologia , Tiorredoxinas/metabolismo , Substituição de Aminoácidos , Apoptose , Cisteína , Humanos , Células Jurkat , Modelos Biológicos , Proteínas Recombinantes/metabolismo , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/genéticaRESUMO
Laeverin/aminopeptidase Q (APQ) is a cell surface protein specifically expressed on human embryo-derived extravillous trophoblasts that invades the uterus during placentation. The cDNA cloning of Laeverin/APQ revealed that the sequence encodes a protein with 990 amino acid residues, and Laeverin/APQ contains the HEXXHX(18)E gluzincin motif, which is characteristic of the M1 family of aminopeptidases, although the exopeptidase motif of the family, GAMEN, is uniquely substituted for the HAMEN sequence. In this study, we expressed a recombinant human Laeverin/APQ using a baculovirus expression system, purified to homogeneity, and characterized its enzymatic properties. It was found that Laeverin/APQ had a broad substrate specificity toward synthetic substrate, although it showed a preference for Leu-4-methylcoumaryl-7-amide. Searching natural substrates, we found that Laeverin/APQ was able to cleave the N-terminal amino acid of several peptides such as angiotensin III, kisspeptin-10, and endokinin C, which are abundantly expressed in the placenta. In contrast to the case with other M1 aminopeptidases, bestatin inhibited the aminopeptidase activity of Laeverin/APQ much more effectively than other known aminopeptidase inhibitors. These results indicate that Laeverin/APQ is a novel bestatin-sensitive leucine aminopeptidase and suggest that the enzyme plays important roles in human placentation by regulating biological activity of key peptides at the embryo-maternal interface.
Assuntos
Antígenos CD13/metabolismo , Leucina/análogos & derivados , Metaloproteases/metabolismo , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/metabolismo , Trofoblastos/enzimologia , Motivos de Aminoácidos , Animais , Baculoviridae , Antígenos CD13/antagonistas & inibidores , Antígenos CD13/química , DNA Complementar/genética , DNA Complementar/metabolismo , Feminino , Expressão Gênica , Humanos , Leucina/farmacologia , Metaloproteases/química , Metaloproteases/genética , Placentação/fisiologia , Gravidez , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por Substrato/efeitos dos fármacosRESUMO
BACKGROUND: Human T-cell leukemia virus type I (HTLV-I) causes adult T-cell leukemia (ATL) after a long latent period. Among accessory genes encoded by HTLV-I, the tax gene is thought to play a central role in oncogenesis. However, Tax expression is disrupted by several mechanims including genetic changes of the tax gene, deletion/hypermethylation of 5'-LTR. To clarify the role of epigenetic changes, we analyzed DNA methylation and histone modification in the whole HTLV-I provirus genome. RESULTS: The gag, pol and env genes of HTLV-I provirus were more methylated than pX region, whereas methylation of 5'-LTR was variable and 3'-LTR was not methylated at all. In ATL cell lines, complete DNA methylation of 5'-LTR was associated with transcriptional silencing of viral genes. HTLV-I provirus was more methylated in primary ATL cells than in carrier state, indicating the association with disease progression. In seroconvertors, DNA methylation was already observed in internal sequences of provirus just after seroconversion. Taken together, it is speculated that DNA methylation first occurs in the gag, pol and env regions and then extends in the 5' and 3' directions in vivo, and when 5'-LTR becomes methylated, viral transcription is silenced. Analysis of histone modification in the HTLV-I provirus showed that the methylated provirus was associated with hypoacetylation. However, the tax gene transcript could not be detected in fresh ATL cells regardless of hyperacetylated histone H3 in 5'-LTR. The transcription rapidly recovered after in vitro culture in such ATL cells. CONCLUSION: These results showed that epigenetic changes of provirus facilitated ATL cells to evade host immune system by suppressing viral gene transcription. In addition, this study shows the presence of another reversible mechanism that suppresses the tax gene transcription without DNA methylation and hypoacetylated histone.
Assuntos
Epigênese Genética , Inativação Gênica , Vírus Linfotrópico T Tipo 1 Humano/genética , Transcrição Gênica , Portador Sadio/virologia , Linhagem Celular , Metilação de DNA , Histonas/metabolismo , Humanos , Leucemia-Linfoma de Células T do Adulto/virologia , Provírus/genética , Sequências Repetidas TerminaisRESUMO
During human placentation, the invasion of extravillous trophoblasts (EVTs) into maternal decidual tissues, especially toward maternal spiral arteries, is considered an essential process for subsequent normal fetal development. However, the precise regulatory mechanisms to induce EVT invasion toward arteries and/or to protect EVTs from further invasion have not been well understood. Recently, we found that two cell surface peptidases, dipeptidyl peptidase IV (DPPIV) and carboxypeptidase-M (CP-M,) are differentially expressed on EVTs. DPPIV expression was mainly observed on EVTs that had already ceased invasion. CP-M was detected on migrating EVTs including endovascular trophoblasts in the maternal arteries. The enzymatic inhibition of these peptidases affected the invasive property of choriocarcinoma-derived cell lines, BeWo and JEG3 cells. In addition, a chemokine, RANTES, that is one of the substrates for DPPIV, enhanced invasion of EVTs isolated from primary villous explant culture and its receptor, CCR1, was specifically expressed on migrating EVTs toward maternal arteries. Furthermore, a novel membrane-bound cell surface peptidase, named laeverin, was found to be specifically expressed on EVTs that had almost ceased invasion. These findings suggest that membrane-bound peptidases are important factors regulating EVT invasion during early placentation in humans.
Assuntos
Dipeptidil Peptidase 4/fisiologia , Metaloendopeptidases/fisiologia , Placentação/fisiologia , Trofoblastos/fisiologia , Sequência de Aminoácidos , Quimiocina CCL5/fisiologia , Feminino , Proteínas Ligadas por GPI , Humanos , Metaloproteases/fisiologia , Dados de Sequência Molecular , Gravidez , Receptores CCR1 , Receptores de Quimiocinas/fisiologia , Útero/fisiologiaRESUMO
We have recently shown constitutive IkappaB kinase (IKK) activation and aberrant p52 expression in adult T cell leukemia (ATL) cells that do not express human T cell leukemia virus type I (HTLV-I) Tax, but the mechanism of IKK activation in these cells has remained unknown. Here, we demonstrate distinct regulation of IKK activity in ATL and HTLV-I-transformed T cells in response to protein synthesis inhibition or arsenite treatment. Protein synthesis inhibition for 4 h by cycloheximide (CHX) barely affects IKK activity in Tax-positive HTLV-I-transformed cells, while it diminishes IKK activity in Tax-negative ATL cells. Treatment of ATL cells with a proteasome inhibitor MG132 prior to protein synthesis inhibition reverses the inhibitory effect of CHX, and MG132 alone greatly enhances IKK activity. In addition, treatment of HTLV-I-transformed cells with arsenite for 1 h results in down-regulation of IKK activity without affecting Tax expression, while 8 h of arsenite treatment does not impair IKK activity in ATL cells. These results indicate that a labile protein sensitive to proteasome-dependent degradation governs IKK activation in ATL cells, and suggest a molecular mechanism of IKK activation in ATL cells distinct from that in HTLV-I-transformed T cells.
Assuntos
Transformação Celular Viral/fisiologia , Leucemia-Linfoma de Células T do Adulto/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arsenitos/farmacologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Transformação Celular Viral/efeitos dos fármacos , Cicloeximida/antagonistas & inibidores , Cicloeximida/farmacologia , Inibidores Enzimáticos/farmacologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Humanos , Quinase I-kappa B , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leupeptinas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/biossíntese , Inibidores da Síntese de Proteínas/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Naturally occurring CD25(+)CD4(+) regulatory T cells are engaged in the maintenance of immunological self-tolerance and down-regulation of various immune responses. Recent studies with mice showed that Foxp3, which encodes the transcription factor Scurfin, is a master regulatory gene for the development and function of CD25(+)CD4(+) regulatory T cells. Here we examined the role of FOXP3 in human CD25(+)CD4(+) regulatory T cells. The FOXP3 gene and its protein product were preferentially expressed in peripheral CD25(+)CD4(+) T cells, in particular CD25(+)CD45RO(+)CD4(+) T cells in normal individuals and, interestingly, in some human T cell leukemia virus type 1-infected T cell lines, which constitutively express CD25. TCR stimulation of CD25(-)CD45RO(-)CD4(+) naive T cells failed to elicit FOXP3 expression at the gene or protein level. Ex vivo retroviral gene transfer of FOXP3, on the other hand, converted peripheral CD25(-)CD45RO(-)CD4(+) naive T cells into a regulatory T cell phenotype similar to CD25(+)CD4(+) regulatory T cells. For example, FOXP3-transduced T cells exhibited impaired proliferation and production of cytokines including IL-2 and IL-10 upon TCR stimulation, up-regulated the expression of regulatory T cell-associated molecules such as CD25 and CTL-associated antigen-4 and suppressed in vitro proliferation of other T cells in a cell-cell contact-dependent manner. Thus, human FOXP3 is a crucial regulatory gene for the development and function of CD25(+)CD4(+) regulatory T cells, and can be used as their reliable marker. Furthermore, regulatory T cells de novo produced from normal naive T cells by FOXP3 transduction can be instrumental for treatment of autoimmune/inflammatory diseases and negative control of various immune responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/imunologia , Tolerância Imunológica , Receptores de Interleucina-2/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/terapia , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fatores de Transcrição Forkhead , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Terapia Genética , Humanos , Transdução GenéticaRESUMO
Adult T-cell leukemia (ATL) is a fatal T-cell malignancy that arises long after infection with human T-cell leukemia virus type I (HTLV-I). We reported previously that nuclear factor-kappaB (NF-kappaB) was constitutively activated in ATL cells, although expression of the viral proteins was barely detectable, including Tax, which was known to persistently activate NF-kappaB. Here we demonstrate that ATL cells that do not express detectable Tax protein exhibit constitutive IkappaB kinase (IKK) activity. Transfection studies revealed that a dominant-negative form of IKK1, and not of IKK2 or NF-kappaB essential modulator (NEMO), suppressed constitutive NF-kappaB activity in ATL cells. This IKK activity was accompanied by elevated expression of p52, suggesting that the recently described noncanonical pathway of NF-kappaB activation operates in ATL cells. We finally show that specific inhibition of NF-kappaB by a super-repressor form of IkappaBalpha (SR-IkappaBalpha) in HTLV-I-infected T cells results in cell death regardless of Tax expression, providing definitive evidence of an essential role for NF-kappaB in the survival of ATL cells. In conclusion, the IKK complex is constitutively activated in ATL cells through a cellular mechanism distinct from that of Tax-mediated IKK activation. Further elucidation of this cellular mechanism should contribute to establishing a rationale for treatment of ATL.
Assuntos
Produtos do Gene tax/metabolismo , Leucemia-Linfoma de Células T do Adulto/enzimologia , Leucemia-Linfoma de Células T do Adulto/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Morte Celular , Linhagem Celular Tumoral , Ativação Enzimática , Humanos , Quinase I-kappa B , Leucemia-Linfoma de Células T do Adulto/genética , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Repressoras/genética , Fatores de Transcrição/metabolismoRESUMO
To clarify the status of tax gene, we analyzed human T-cell leukemia virus type-I (HTLV-I) associated cell lines and fresh adult T-cell leukemia (ATL) cells. We compared 2 types of HTLV-I associated cell lines: one was derived from leukemic cells (leukemic cell line) and the other from nonleukemic cells (nonleukemic cell line). Although all nonleukemic cell lines expressed Tax, it could not be detected in 3 of 5 leukemic cell lines, in which nonsense mutation or deletion (60 bp) of tax genes, and DNA methylation in 5'-LTR were identified as the responsible changes. We found such genetic changes of the tax gene in 5 of 47 fresh ATL cases (11%). The tax gene transcripts could be detected in 14 of 41 fresh ATL cases (34%) by RT-PCR. In ATL cases with genetic changes that could not produce Tax protein, the tax gene was frequently transcribed, suggesting that such cells do not need the transcriptional silencing. Although DNA methylation of 5'-LTR was detected in the fresh ATL cases (19 of 28 cases; 68%), the complete methylation associated with transcriptional silencing was observed only in 4 cases. Since partial methylation could not silence the transcription, and the tax gene transcription was not detected in 27 of 41 cases (66%), the epigenetic change(s) other than DNA methylation is considered to play an important role in the silencing.
Assuntos
Códon sem Sentido , Metilação de DNA , Genes pX/genética , Leucemia-Linfoma de Células T do Adulto/genética , Acetilação , Deleção de Genes , Inativação Gênica , Infecções por HTLV-I/genética , Infecções por HTLV-I/virologia , Histonas/metabolismo , Leucemia-Linfoma de Células T do Adulto/metabolismo , Provírus , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sequências Repetidas Terminais/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Human T-cell leukemia virus type I (HTLV-I) is the causative agent of adult T-cell leukemia (ATL). However, the low incidence of ATL among HTLV-I-infected carriers, together with a long latent period, suggests that multiple host-viral events are involved in the progression of HTLV-I-dependent transformation and subsequent development of ATL. Human thioredoxin (TRX) is a redox active protein highly expressed in HTLV-I-transformed cell lines, whereas the TRX-binding protein-2/vitamin D3 up-regulated protein 1 (TBP-2/VDUP1) was recently identified as a negative regulator of TRX. We report here that expression of TBP-2 is lost in HTLV-I-positive, interleukin-2-independent T-cell lines but maintained in HTLV-I-positive, interleukin-2-dependent T-cell lines, as well as HTLV-I-negative T-cell lines. Ectopic overexpression of TBP-2 in HTLV-I-positive T cells resulted in growth suppression. In the TBP-2-overexpressing cells, a G1 arrest was observed in association with an increase of p16 expression and reduction of retinoblastoma phosphorylation. The results suggest that TBP-2 plays a crucial role in the growth regulation of T cells and that the loss of TBP-2 expression in HTLV-I-infected T cells is one of the key events involved in the multistep progression of ATL leukemogenesis.