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1.
Biochim Biophys Acta ; 1849(9): 1198-208, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26187004

RESUMO

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, ß and γ, arise from the human gene. We now show that DMP1α, ß and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1ß and γ did not activate the ARF promoter, whereas only ß resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1ß reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1ß- and γ-isoforms share domains necessary for the inhibitory function of the ß-isoform. That DMP1ß may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/ß in the nucleus. Cells stably expressing DMP1ß, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and ß-ratios are tightly regulated in hematopoietic cells and DMP1ß antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.


Assuntos
Proliferação de Células/fisiologia , Regulação da Expressão Gênica/fisiologia , Isoformas de Proteínas/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologia , Proteína Supressora de Tumor p14ARF/genética , Animais , Linhagem Celular , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Isoformas de Proteínas/genética , Splicing de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética
2.
Cancers (Basel) ; 16(8)2024 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-38672672

RESUMO

MYCN amplification (MNA) and disruption of tumor suppressor microRNA (TSmiR) function are key drivers of poor outcomes in neuroblastoma (NB). While MYCN and TSmiRs regulate glucose metabolism, their role in de novo fatty acid synthesis (FAS) and unsaturated FAS (UFAS) remains poorly understood. Here, we show that FAS and UFAS (U/FAS) genes FASN, ELOVL6, SCD, FADS2, and FADS1 are upregulated in high-risk (HR) NB and that their expression is associated with lower overall survival. RNA-Seq analysis of human NB cell lines revealed parallel U/FAS gene expression patterns. Consistent with this, we found that NB-related TSmiRs were predicted to target these genes extensively. We further observed that both MYC and MYCN upregulated U/FAS pathway genes while suppressing TSmiR host gene expression, suggesting a possible U/FAS regulatory network between MYCN and TSmiRs in NB. NB cells are high in de novo synthesized omega 9 (ω9) unsaturated fatty acids and low in both ω6 and ω3, suggesting a means for NB to limit cell-autonomous immune stimulation and reactive oxygen species (ROS)-driven apoptosis from ω6 and ω3 unsaturated fatty acid derivatives, respectively. We propose a model in which MYCN and TSmiRs regulate U/FAS and play an important role in NB pathology, with implications for other MYC family-driven cancers.

3.
Mol Cancer Ther ; 7(3): 688-97, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18319335

RESUMO

The efficiency of chemotherapeutic treatments in cancer patients is often impaired by the acquisition of drug resistance. Cancer cells develop drug resistance through dysregulation of one or more genes or cellular pathways. To isolate efficient regulators of drug resistance in tumor cells, we have adopted a genome-wide scanning approach based on the screening of large libraries of artificial transcription factors (ATFs) made of three and six randomly assembled zinc finger domains. Zinc finger libraries were linked to a VP64 activation domain and delivered into a paclitaxel-sensitive tumor cell line. Following drug treatment, several ATFs were isolated that promoted drug resistance. One of these ATFs, 3ZF-1-VP, promoted paclitaxel resistance in cell lines having mutated or inactivated p53, such as MDA-MB-435 and Kaposi's sarcoma cell lines. 3ZF-1-VP also induced strong resistance to etoposide, vincristine, and cisplatinum. Linkage of a repression domain to the selected ATF resulted in enhanced sensitivity to multiple drugs, particularly vincristine, cisplatinum, and 5-fluorouracil. Small interfering RNA-mediated inhibition of p53 revealed that 3ZF-1-VP activated both p53-dependent and p53-independent mechanisms to promote survival, whereas other ATF required intact p53. Real-time expression analysis and DNA microarrays showed that several ATFs up-regulated targets of p53, such as the cyclin-dependent kinase inhibitor p21(WAF1/CIP1), and genes participating in the p14(ARF)-MDM2-p53 tumor suppressor pathway, such as hDMP1. Thus, ATF can be used to map genes and pathways involved in drug resistance phenotypes and have potential as novel therapeutic agents to inhibit drug resistance.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição/farmacologia , Sequência de Bases , Primers do DNA , Células HeLa , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Retrovirology ; 1: 38, 2004 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-15555065

RESUMO

BACKGROUND: The protease inhibitor, TL-3, demonstrated broad efficacy in vitro against FIV, HIV and SIV (simian immunodeficiency virus), and exhibited very strong protective effects on early neurologic alterations in the CNS of FIV-PPR infected cats. In this study, we analyzed TL-3 efficacy using a highly pathogenic FIV-C isolate, which causes a severe acute phase immunodeficiency syndrome, with high early mortality rates. RESULTS: Twenty cats were infected with uncloned FIV-C and half were treated with TL-3 while the other half were left untreated. Two uninfected cats were used as controls. The general health and the immunological and virological status of the animals was monitored for eight weeks following infection. All infected animals became viremic independent of TL-3 treatment and seven of 20 FIV-C infected animals developed severe immunodepletive disease in conjunction with significantly (p < or = 0.05) higher viral RNA loads as compared to asymptomatic animals. A marked and progressive increase in CD8+ T lymphocytes in animals surviving acute phase infection was noted, which was not evident in symptomatic animals (p < or = 0.05). Average viral loads were lower in TL-3 treated animals and of the 6 animals requiring euthanasia, four were from the untreated cohort. At eight weeks post infection, half of the TL-3 treated animals and only one of six untreated animals had viral loads below detection limits. Analysis of protease genes in TL-3 treated animals with higher than average viral loads revealed sequence variations relative to wild type protease. In particular, one mutant, D105G, imparted 5-fold resistance against TL-3 relative to wild type protease. CONCLUSIONS: The findings indicate that the protease inhibitor, TL-3, when administered orally as a monotherapy, did not prevent viremia in cats infected with high dose FIV-C. However, the modest lowering of viral loads with TL-3 treatment, the greater survival rate in symptomatic animals of the treated cohort, and the lower average viral load in TL-3 treated animals at eight weeks post infection is indicative of a therapeutic effect of the compound on virus infection.


Assuntos
Síndrome de Imunodeficiência Adquirida Felina/transmissão , Vírus da Imunodeficiência Felina/patogenicidade , Inibidores de Proteases/uso terapêutico , Animais , Tronco Encefálico/fisiopatologia , Gatos , Estudos de Coortes , Potenciais Evocados Auditivos/fisiologia , Síndrome de Imunodeficiência Adquirida Felina/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida Felina/fisiopatologia , Feminino , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Vírus da Imunodeficiência Felina/isolamento & purificação , Carga Viral , Redução de Peso/efeitos dos fármacos
5.
Curr Drug Targets Infect Disord ; 3(4): 295-309, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14754431

RESUMO

A global effort has been undertaken to control human immunodeficiency virus (HIV) though the development of vaccines and pharmacologics. Current FDA approved pharmacological inhibitors target two of the three viral enzymes critical to replication and maturation of infectious viral particles: reverse transcriptase (RT) and protease (Pr). Although combination therapies targeting RT and Pr have significantly reduced AIDS related morbidity and mortality, resistance to individual inhibitors is a growing concern. Currently, there are six protease inhibitors in clinical use. These inhibitors target the active site of protease using peptidomimetic transition state analogs based on natural substrates. However, treatment failures arise as a lack of compliance due to HIV-inhibitor pharmacokinetics, toxicity, and tolerance. This allows reduced HIV-inhibitor pressure, increased viral replication, and the emergence of drug resistant mutations. Continued use of protease inhibitors in the face of incomplete viral suppression may result in HIV-1 escape mutants not only being resistant to the protease inhibitor used, but to all clinically available protease inhibitors. Thus, new broad-based protease inhibitors are needed to control the emerging multi-drug, cross-resistant HIV-1. Moreover, given the emergence of cross-resistant HIV-1, there is a need to target novel protease structural sites to reduce the risk of multi-drug cross-resistance. In this review, we discuss the resistance to protease inhibitors and the rationale for new strategies towards drug design for suppressing protease activity. We focus on the structure and function relationship and the influence that drug resistance mutants exert on the evolution of HIV-1 protease.


Assuntos
Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/farmacologia , HIV-1/fisiologia , Sequência de Aminoácidos , Farmacorresistência Viral Múltipla/genética , Infecções por HIV/virologia , Inibidores da Protease de HIV/química , HIV-1/enzimologia , HIV-1/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
6.
J Virol ; 80(24): 12032-40, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17035327

RESUMO

Dominant epitope-specific CD8(+) T-lymphocyte responses play a central role in controlling viral spread. We explored the basis for the development of this focused immune response in simian immunodeficiency virus (SIV)- and simian-human immunodeficiency virus (SHIV)-infected rhesus monkeys through the use of two dominant (p11C and p199RY) and two subdominant (p68A and p56A) epitopes. Using real-time PCR to quantitate T-cell receptor (TCR) variable region beta (Vbeta) family usage, we show that CD8(+) T-lymphocyte populations specific for dominant epitopes are characterized by a diverse Vbeta repertoire, whereas those specific for subdominant epitopes employ a dramatically more focused Vbeta repertoire. We also demonstrate that dominant epitope-specific CD8(+) T lymphocytes employ TCRs with multiple CDR3 lengths, whereas subdominant epitope-specific cells employ TCRs with a more restricted CDR3 length. Thus, the relative dominance of an epitope-specific CD8(+) T-lymphocyte response reflects the clonal diversity of that response. These findings suggest that the limited clonal repertoire of subdominant epitope-specific CD8(+) T-lymphocyte populations may limit the ability of these epitope-specific T-lymphocyte populations to expand and therefore limit the ability of these cell populations to contribute to the control of viral replication.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Imunidade Celular/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Vírus da Imunodeficiência Símia/imunologia , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Cromatografia Líquida de Alta Pressão , Primers do DNA , DNA Complementar/genética , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , Citometria de Fluxo , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Macaca mulatta , Dados de Sequência Molecular , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sequência de DNA
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