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1.
Technol Cancer Res Treat ; 20: 15330338211011965, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34013821

RESUMO

Prostate cancer is the second leading cause of cancer death in men. Its current treatment includes various physical and chemical approaches for the localized and advanced prostate cancer [e.g. metastatic castrate resistant prostate cancer (mCRPC)]. Although many new drugs are now available for prostate cancer, none is suitable for local treatment that can reduce adverse effects often associated with the current physical treatment. Of the drugs approved by FDA for mCRPC, the best mean improvement in overall survival is only about 4.8 months. Therefore, there is a need for improved treatment approaches for prostate cancer, especially drug-resistant cancer.Ultrasound therapy represents a useful new physical approach for the drug-resistant cancer treatment by facilitating the entry of the related chemotherapy drug into the target cancer cells. There are two versions of ultrasound: High Intensity Focused Ultrasound (HIFU) and Low Intensity Pulsed Ultrasound (LIPUS). HIFU has been a promising treatment option for prostate cancer due to its noninvasiveness and various biological effects on cancer tissue. It has been approved for the treatment of cancer and in recent years there have been numerous findings suggesting HIFU can reduce cancer cell viability and possibly reverse the spread of cancerous tumors. LIPUS is currently being studied as an alternative treatment option for prostate cancer. Preliminary studies have found LIPUS to reduce cancer cell viability without the side effects seen in HIFU. Reversible cell membrane damage caused by LIPUS could allow increased uptake of anticancer drugs, enhancing cytotoxicity and death of cancer cells. In this way, a low dose of anticancer drug is more effective toward cancer cells while there is less damage to normal cells. The combination of LIPUS with certain chemotherapeutic agents can be an exciting physical-chemical combination therapy for prostate cancer. This review will focus on this topic as well as the clinical use of HIFU to provide an understanding of their current use and future potential role for prostate cancer therapy.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ablação por Ultrassom Focalizado de Alta Intensidade/métodos , Neoplasias da Próstata/terapia , Terapia Combinada , Humanos , Masculino , Prognóstico , Neoplasias da Próstata/patologia
2.
Front Cell Dev Biol ; 8: 504, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32656213

RESUMO

Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncover novel mechanosensory dependent cellular processes. Since it is non-invasive, it holds great promise for future therapeutic applications in patients used either alone or as a complement to boost existing treatments. For example, FUS stimulation causes invasive but not non-invasive cancer cell lines to exhibit marked activation of calcium signaling pathways. Here, we identify the membrane channel PANNEXIN1 (PANX1) as a mediator for activation of calcium signaling in invasive cancer cells. Knockdown of PANX1 decreases calcium signaling in invasive cells, while PANX1 overexpression enhances calcium elevations in non-invasive cancer cells. We demonstrate that FUS may directly stimulate mechanosensory PANX1 localized in endoplasmic reticulum to evoke calcium release from internal stores. This process does not depend on mechanosensory stimulus transduction through an intact cytoskeleton and does not depend on plasma membrane localized PANX1. Plasma membrane localized PANX1, however, plays a different role in mediating the spread of intercellular calcium waves via ATP release. Additionally, we show that FUS stimulation evokes cytokine/chemokine release from invasive cancer cells, suggesting that FUS could be an important new adjuvant treatment to improve cancer immunotherapy.

3.
Cells ; 9(5)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32375298

RESUMO

In glucose-stimulated insulin secretion (GSIS) of pancreatic ß-cells, the rise of free cytosolic Ca2+ concentration through voltage-gated calcium channels (VGCCs) triggers the exocytosis of insulin-containing granules. Recently, mechanically induced insulin secretion pathways were also reported, which utilize free cytosolic Ca2+ ions as a direct regulator of exocytosis. In this study, we aimed to investigate intracellular Ca2+ responses on the HIT-T15 pancreatic ß-cell line upon low-intensity pulsed ultrasound (LIPUS) stimulation and found that ultrasound induces two distinct types of intracellular Ca2+ oscillation, fast-irregular and slow-periodic, from otherwise resting cells. Both Ca2+ patterns depend on the purinergic signaling activated by the rise of extracellular ATP or ADP concentration upon ultrasound stimulation, which facilitates the release through mechanosensitive hemichannels on the plasma membrane. Further study demonstrated that two subtypes of purinergic receptors, P2X and P2Y, are working in a competitive manner depending on the level of glucose in the cell media. The findings can serve as an essential groundwork providing an underlying mechanism for the development of a new therapeutic approach for diabetic conditions with further validation.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Espaço Intracelular/metabolismo , Ultrassom , Animais , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Cricetinae , Modelos Biológicos , Receptores Purinérgicos/metabolismo
4.
Ann Biomed Eng ; 46(1): 48-59, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29086222

RESUMO

In recent years, ultrasound has gained attention in new biological applications due to its ability to induce specific biological responses at the cellular level. Although the biophysical mechanisms underlying the interaction between ultrasound and cells are not fully understood, many agree on a pivotal role of Ca2+ signaling through mechanotransduction pathways. Because Ca2+ regulates a vast range of downstream cellular processes, a better understanding of how ultrasound influences Ca2+ signaling could lead to new applications for ultrasound. In this study, we investigated the mechanism of ultrasound-induced Ca2+ mobilization in human mesenchymal stem cells using 47 MHz focused ultrasound to stimulate single cells at low intensities (~ 110 mW/cm2). We found that ultrasound exposure triggers opening of connexin 43 hemichannels on the plasma membrane, causing release of ATP into the extracellular space. That ATP then binds to G-protein-coupled P2Y1 purinergic receptors on the membrane, in turn activating phospholipase C, which evokes production of inositol trisphosphate and release of Ca2+ from intracellular stores.


Assuntos
Cálcio/metabolismo , Conexina 43/metabolismo , Células-Tronco Mesenquimais/efeitos da radiação , Ondas Ultrassônicas , Sobrevivência Celular , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/metabolismo
5.
Front Oncol ; 7: 161, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28824873

RESUMO

Cancer cells undergo a number of biophysical changes as they transform from an indolent to an aggressive state. These changes, which include altered mechanical and electrical properties, can reveal important diagnostic information about disease status. Here, we introduce a high-throughput, functional technique for assessing cancer cell invasion potential, which works by probing for the mechanically excitable phenotype exhibited by invasive cancer cells. Cells are labeled with fluorescent calcium dye and imaged during stimulation with low-intensity focused ultrasound, a non-contact mechanical stimulus. We show that cells located at the focus of the stimulus exhibit calcium elevation for invasive prostate (PC-3 and DU-145) and bladder (T24/83) cancer cell lines, but not for non-invasive cell lines (BPH-1, PNT1A, and RT112/84). In invasive cells, ultrasound stimulation initiates a calcium wave that propagates from the cells at the transducer focus to other cells, over distances greater than 1 mm. We demonstrate that this wave is mediated by extracellular signaling molecules and can be abolished through inhibition of transient receptor potential channels and inositol trisphosphate receptors, implicating these proteins in the mechanotransduction process. If validated clinically, our technology could provide a means to assess tumor invasion potential in cytology specimens, which is not currently possible. It may therefore have applications in diseases such as bladder cancer, where cytologic diagnosis of tumor invasion could improve clinical decision-making.

6.
Sci Rep ; 5: 11207, 2015 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-26053433

RESUMO

RE1-Silencing Transcription factor (REST) has a well-established role in regulating transcription of genes important for neuronal development. Its role in cancer, though significant, is less well understood. We show that REST downregulation in weakly invasive MCF-7 breast cancer cells converts them to a more invasive phenotype, while REST overexpression in highly invasive MDA-MB-231 cells suppresses invasiveness. Surprisingly, the mechanism responsible for these phenotypic changes does not depend directly on the transcriptional function of REST protein. Instead, it is driven by previously unstudied mid-size (30-200 nt) non-coding RNAs (ncRNAs) derived from the first exon of an alternatively spliced REST transcript: REST-003. We show that processing of REST-003 into ncRNAs is controlled by an uncharacterized serine/arginine repeat-related protein, SRRM3. SRRM3 expression may be under REST-mediated transcriptional control, as it increases following REST downregulation. The SRRM3-dependent regulation of REST-003 processing into ncRNAs has many similarities to recently described promoter-associated small RNA-like processes. Targeting ncRNAs that control invasiveness could lead to new therapeutic approaches to limit breast cancer metastasis.


Assuntos
Neoplasias da Mama/genética , Invasividade Neoplásica/genética , Proteínas/genética , RNA não Traduzido/genética , Proteínas Repressoras/genética , Processamento Alternativo/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Humanos , Células MCF-7 , Invasividade Neoplásica/patologia , Metástase Neoplásica/genética , Interferência de RNA , RNA Interferente Pequeno/genética
7.
Radiat Oncol ; 8: 203, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23961877

RESUMO

BACKGROUND: Neoadjuvant chemoradiation therapy followed by curative surgery has gained acceptance as the therapy of choice in locally advanced rectal cancer. However, deterioration of anorectal function after long-course neoadjuvant chemoradiation therapy combined with surgery for rectal cancer is poorly defined. The aim of this study was to evaluate the physiological and clinical change of anorectal function after neoadjuvant chemoradiation therapy for rectal cancer. METHODS: We analyzed 30 patients on whom preoperative anorectal manometry data were available both before and after chemoradiation from October 2010 to September 2011. All patients underwent long-course neoadjuvant chemoradiation therapy. We compared manometric parameters between before and after neoadjuvant chemoradiation therapy. RESULTS: Of 30 patients, 20 were males and 10 females. The mean age was 64.9 ± 9.9 years (range, 48-82). Before nCRT, the rectal compliance was higher in patients with ulceroinfiltrative type (P = 0.035) and greater involvement of luminal circumference (P = 0.017). However, there was the tendency of increased rectal sensory threshold for desire to defecate when the patient had decreased circumferential ratio of the tumor (P = 0.099), down-graded T stage (P = 0.016), or reduced tumor volume (P = 0.063) after neoadjuvant chemoradiation. CONCLUSIONS: Neoadjuvant chemoradiation therapy did not significantly impair overall sphincter function before radical operation. The relationship between tumor response of chemoradiation and sensory threshold for desire to defecate may suggest that neoadjuvant chemoradiation may be helpful for defecatory function as well as local disease control, at least in the short-term period after the radiation in locally advanced rectal cancer patients.


Assuntos
Adenocarcinoma/terapia , Quimiorradioterapia/efeitos adversos , Terapia Neoadjuvante/efeitos adversos , Neoplasias Retais/terapia , Idoso , Idoso de 80 Anos ou mais , Canal Anal/efeitos dos fármacos , Canal Anal/efeitos da radiação , Complacência (Medida de Distensibilidade) , Feminino , Humanos , Masculino , Manometria , Pessoa de Meia-Idade , Projetos Piloto , Reto/efeitos dos fármacos , Reto/efeitos da radiação , Limiar Sensorial/efeitos dos fármacos , Limiar Sensorial/efeitos da radiação
8.
Biotechnol Bioeng ; 110(10): 2697-705, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23568761

RESUMO

In this article, we investigate the application of contactless high frequency ultrasound microbeam stimulation (HFUMS) for determining the invasion potential of breast cancer cells. In breast cancer patients, the finding of tumor metastasis significantly worsens the clinical prognosis. Thus, early determination of the potential of a tumor for invasion and metastasis would significantly impact decisions about aggressiveness of cancer treatment. Recent work suggests that invasive breast cancer cells (MDA-MB-231), but not weakly invasive breast cancer cells (MCF-7, SKBR3, and BT-474), display a number of neuronal characteristics, including expression of voltage-gated sodium channels. Since sodium channels are often co-expressed with calcium channels, this prompted us to test whether single-cell stimulation by a highly focused ultrasound microbeam would trigger Ca(2+) elevation, especially in highly invasive breast cancer cells. To calibrate the diameter of the microbeam ultrasound produced by a 200-MHz single element LiNbO3 transducer, we focused the beam on a wire target and performed a pulse-echo test. The width of the beam was ∼17 µm, appropriate for single cell stimulation. Membrane-permeant fluorescent Ca(2+) indicators were utilized to monitor Ca(2+) changes in the cells due to HFUMS. The cell response index (CRI), which is a composite parameter reflecting both Ca(2+) elevation and the fraction of responding cells elicited by HFUMS, was much greater in highly invasive breast cancer cells than in the weakly invasive breast cancer cells. The CRI of MDA-MB-231 cells depended on peak-to-peak amplitude of the voltage driving the transducer. These results suggest that HFUMS may serve as a novel tool to determine the invasion potential of breast cancer cells, and with further refinement may offer a rapid test for invasiveness of tumor biopsies in situ.


Assuntos
Neoplasias da Mama , Espaço Intracelular , Invasividade Neoplásica , Imagem Óptica/métodos , Som , Antineoplásicos/farmacologia , Neoplasias da Mama/química , Neoplasias da Mama/metabolismo , Cálcio/análise , Cálcio/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Espaço Intracelular/química , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Espaço Intracelular/efeitos da radiação , Paclitaxel/farmacologia
9.
J Neurophysiol ; 109(7): 1979-88, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23343890

RESUMO

Epiretinal implants for the blind are designed to stimulate surviving retinal neurons, thus bypassing the diseased photoreceptor layer. Single-unit or multielectrode recordings from isolated animal retina are commonly used to inform the design of these implants. However, such electrical recordings provide limited information about the spatial patterns of retinal activation. Calcium imaging overcomes this limitation, as imaging enables high spatial resolution mapping of retinal ganglion cell (RGC) activity as well as simultaneous recording from hundreds of RGCs. Prior experiments in amphibian retina have demonstrated proof of principle, yet experiments in mammalian retina have been hindered by the inability to load calcium indicators into mature mammalian RGCs. Here, we report a method for labeling the majority of ganglion cells in adult rat retina with genetically encoded calcium indicators, specifically GCaMP3 and GCaMP5G. Intravitreal injection of an adeno-associated viral vector targets ∼85% of ganglion cells with high specificity. Because of the large fluorescence signals provided by the GCaMP sensors, we can now for the first time visualize the response of the retina to electrical stimulation in real-time. Imaging transduced retinas mounted on multielectrode arrays reveals how stimulus pulse shape can dramatically affect the spatial extent of RGC activation, which has clear implications in prosthetic applications. Our method can be easily adapted to work with other fluorescent indicator proteins in both wild-type and transgenic mammals.


Assuntos
Cálcio/metabolismo , Optogenética , Células Ganglionares da Retina/fisiologia , Potenciais de Ação , Animais , Proteínas de Ligação ao Cálcio/genética , Dependovirus/genética , Estimulação Elétrica , Microscopia de Fluorescência , Ratos , Ratos Long-Evans , Células Ganglionares da Retina/metabolismo
10.
PLoS One ; 7(4): e35521, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22530041

RESUMO

Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use of a novel "indirect" dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-producing (INS(+)) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS(+) phenotype. Our strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of INS(+) and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem cells.


Assuntos
Linhagem Celular Tumoral , Genes Reporter , Células Secretoras de Insulina/metabolismo , Cálcio/metabolismo , Regulação Neoplásica da Expressão Gênica , Ordem dos Genes , Vetores Genéticos , Humanos , Insulina/genética , Insulinoma , Ativação do Canal Iônico , Fenótipo , Regiões Promotoras Genéticas
11.
Biochem Biophys Res Commun ; 377(2): 434-440, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-18930031

RESUMO

Embryonic stem cells (ESCs) and embryonal carcinoma cells (ECCs) possess the remarkable property of self-renewal and differentiation potency. They are model preparations for investigating the underlying mechanisms of "stemness". microRNAs are recently discovered small noncoding RNAs with a broad spectrum of functions, especially in control of development. Here, we show that miR-302b indirectly regulates expression of the pluripotent stem cell marker Oct4, and it directly regulates expression of Cyclin D2 protein, a developmental regulator during gastrulation. Using loss-of function and gain-of function approaches, we demonstrate that functional miR-302b is necessary to maintain stem cell self-renewal and inhibit neuronal differentiation of human ECCs. During retinoic acid-induced neuronal differentiation, Cyclin D2 protein but not mRNA expression is strongly increased, concurrent with the down-regulation of miR-302b and Oct4. Our results suggest that miR-302b plays an important role in maintaining the pluripotency of ECCs and probably ESCs, by post-transcriptional regulation of Cyclin D2 expression.


Assuntos
Ciclinas/biossíntese , Células-Tronco de Carcinoma Embrionário/metabolismo , MicroRNAs/fisiologia , Células-Tronco Pluripotentes/metabolismo , Biossíntese de Proteínas , Diferenciação Celular , Linhagem Celular Tumoral , Ciclina D2 , Ciclinas/genética , Células-Tronco de Carcinoma Embrionário/citologia , Regulação da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Pluripotentes/citologia , Biossíntese de Proteínas/genética , RNA Mensageiro/biossíntese , Ativação Transcricional
12.
RNA ; 14(9): 1823-33, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18697923

RESUMO

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.


Assuntos
Interferência de RNA , RNA Catalítico/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Nuclear Pequeno/genética , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Catalítico/genética , RNA Interferente Pequeno/genética
13.
J Biol Chem ; 280(17): 16949-54, 2005 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-15722335

RESUMO

Small interfering RNA (siRNA) duplexes induce the specific cleavage of target RNAs in mammalian cells. Their involvement in down-regulation of gene expression is termed RNA interference (RNAi). It is widely believed that RNAi predominates in the cytoplasm. We report here the co-existence of cytoplasmic and nuclear RNAi phenomena in primary human myotonic dystrophy type 1 (DM1) cells by targeting myotonic dystrophy protein kinase (DMPK) mRNAs. Heterozygote DM1 myoblasts from a human DM1 fetus produce a nuclear retained mutant DMPK transcript with large CUG repeats ( approximately 3,200) from one allele of the DMPK gene and a wild type transcript with 18 CUG repeats, thus providing for both a nuclear and cytoplasmic expression profile to be evaluated. We demonstrate here for the first time down-regulation of the endogenous nuclear retained mutant DMPK mRNAs targeted with lentivirus-delivered short hairpin RNAs (shRNAs). This nuclear RNAi(-like) phenomenon was not observed when synthetic siRNAs were delivered by cationic lipids, suggesting either a link between processing of the shRNA and nuclear import or a separate pathway for processing shRNAs in the nuclei. Our observation of simultaneous RNAi on both cytoplasmic and nuclear retained DMPK has important implications for post-transcriptional gene regulation in both compartments of mammalian cells.


Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Distrofia Miotônica/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Ativo do Núcleo Celular , Alelos , Sequência de Bases , Sítios de Ligação , Northern Blotting , Western Blotting , Diferenciação Celular , Linhagem Celular , Clonagem Molecular , Regulação para Baixo , Heterozigoto , Humanos , Lentivirus/genética , Microscopia de Fluorescência , Modelos Genéticos , Dados de Sequência Molecular , Músculo Esquelético/citologia , Miotonina Proteína Quinase , Interferência de RNA , Processamento Pós-Transcricional do RNA , RNA Catalítico/química , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Frações Subcelulares , Transfecção
14.
Virus Res ; 102(1): 53-8, 2004 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15068880

RESUMO

Small interfering RNAs (siRNAs) have been shown to direct sequence-specific inhibition of gene expression in mammalian cells. siRNAs are RNA duplexes of 21-23 nucleotides (nts) with approximately 2nt 3' overhangs that can induce degradation of their homologous target mRNAs without interferon responses in mammalian cells. The degradation of the target occurs at the post-transcriptional level, meaning a post-transcriptional gene silencing (PTGS) mechanism called as RNA interference (RNAi). RNAi has emerged as an efficient method to inhibit gene expression in mammalian cells with increasingly successful cases of knockdown of many specific genes. Recent works have shown that the use of RNAi could inhibit HIV-1 replication by targeting viral or cellular genes. RNAi can be considered as a gene-specific therapeutic option for controlling HIV-1 replication. However, the control of HIV-1 replication has become complex because of the limited effectiveness of existing anti-HIV-1 agents and the high speed mutation rate of the HIV-1 genome. Careful assessments are required for the potential of RNAi as a gene therapy approach for controlling HIV-1 replication. This review will discuss the status of the science using RNAi for controlling HIV-1 replication and will describe possible problems for therapeutic applications of RNAi-mediated technologies for HIV-1 behind this novel mechanism.


Assuntos
HIV-1/fisiologia , Interferência de RNA , Replicação Viral , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/terapia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
15.
Mol Ther ; 8(2): 196-206, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12907142

RESUMO

A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.


Assuntos
DNA Polimerase III/metabolismo , Terapia Genética/métodos , Vetores Genéticos/genética , Infecções por HIV/genética , Infecções por HIV/terapia , Lentivirus/genética , RNA Interferente Pequeno/metabolismo , Sequência de Bases , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , DNA Polimerase III/genética , Expressão Gênica , Repetição Terminal Longa de HIV/genética , Humanos , Regiões Promotoras Genéticas/genética , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Interferente Pequeno/genética , Receptores CCR5/biossíntese , Receptores CCR5/genética , Linfócitos T/metabolismo , Linfócitos T/virologia
16.
Mol Ther ; 8(1): 62-71, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12842429

RESUMO

The phenomenon of RNA interference mediated by small interfering RNAs (siRNAs) is a potent gene-silencing mechanism. A number of recent studies demonstrated inhibition of HIV-1 replication in cultured cells using this approach. To make further progress and harness this technology for HIV-1 gene therapy in a stem cell setting, in vivo studies using primary hematopoietic cells are needed. Using an HIV-based lentiviral vector we introduced an anti-Rev siRNA construct into CD34(+) hematopoietic progenitor cells. The siRNA-transduced progenitor cells were allowed to mature into macrophages in vitro and T cells in vivo in SCID-hu mouse thy/liv grafts. Phenotypically normal T cells and macrophages displaying characteristic surface markers were obtained. In vitro HIV-1 challenge of the siRNA-expressing macrophages and T cells with macrophage-tropic and T-cell-tropic HIV-1, respectively, showed marked viral resistance. These experiments demonstrate the utility of siRNAs delivered into hematopoietic stem cells via lentiviral vectors for future in vivo applications.


Assuntos
Antígenos CD34/biossíntese , Terapia Genética/métodos , Infecções por HIV/terapia , HIV-1/metabolismo , Lentivirus/genética , Macrófagos/metabolismo , RNA Interferente Pequeno/genética , Linfócitos T/metabolismo , Animais , Diferenciação Celular , Separação Celular , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Genes Reporter , Vetores Genéticos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Fígado/embriologia , Camundongos , Camundongos SCID , Modelos Genéticos , Fenótipo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Timo/citologia , Fatores de Tempo , Transcrição Gênica
17.
Mol Ther ; 7(6): 811-6, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12788655

RESUMO

The EWS/Fli-1 fusion gene encodes an oncogenic fusion protein. The fusion is a product of the translocation t(11;22) (q24;q12), which is detected in 85% of Ewing sarcoma and primitive neuroectodermal tumor cells. Utilizing intracellularly expressed 21- to 23-nucleotide small interfering RNAs (siRNAs) targeting the EWS/Fli-1 fusion transcript in an Ewing sarcoma cell line, we achieved a greater than 80% reduction in the EWS/Fli-1 transcript. The reduction in transcript levels was accompanied by growth inhibition of an Ewing cell line. In addition to quantitating the reduction of the fusion transcript, we carefully monitored reduction of the endogenous EWS and Fli-1 mRNAs as well. One of the two siRNAs targeted to the fusion transcript also partially downregulated the Fli-1 mRNA, further potentiating the growth inhibition. These results highlight both the power of siRNAs and the potential side reactions that need to be carefully monitored. In addition, these results provide the first demonstration of expressed siRNAs downregulating an oncogenic fusion transcript. The results and observations from these studies should prove useful in targeting other fusion transcripts characteristic of sarcomas and erythroleukemias.


Assuntos
Neoplasias Ósseas/terapia , DNA Polimerase III/genética , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/uso terapêutico , Sarcoma de Ewing/terapia , Fatores de Transcrição/genética , Neoplasias Ósseas/genética , DNA Polimerase III/metabolismo , Regulação para Baixo , Humanos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1 , Proteína EWS de Ligação a RNA , Sarcoma de Ewing/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas
18.
Mol Ther ; 7(5 Pt 1): 670-80, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12718910

RESUMO

Myotonic dystrophy type 1 (DM1) is caused by an unstable CTG expansion in the 3' untranslated region (3'UTR) of the myotonic dystrophy protein kinase gene (DMPK). Transcripts from this altered gene harbor large CUG expansions that are retained in the nucleus of DM1 cells and form foci. It is believed that the formation of these foci is closely linked to DM1 muscle pathogenesis. Here we investigated the possibility of using a nuclear-retained hammerhead ribozyme expressed from a modified tRNAmeti promoter to target and cleave mutant transcripts of DMPK. Accessible ribozyme target sites were identified in the 3'UTR of the DMPK mRNA and a hammerhead ribozyme was designed to cut the most accessible site. Utilizing this system, we have achieved 50 and 63% reductions, respectively, of the normal and CUG expanded repeat-containing transcripts. We also observed a significant reduction in the number of DMPK mRNA-containing nuclear foci in human DM1 myoblasts. Reduction of mutant DMPK mRNA and nuclear foci also corroborates with partial restoration of insulin receptor isoform B expression in DM1 myoblasts. These studies demonstrate for the first time intracellular ribozyme-mediated cleavage of nuclear-retained mutant DMPK mRNAs, providing a potential gene therapy agent for the treatment of myotonic dystrophy.


Assuntos
Núcleo Celular , Mioblastos/metabolismo , Distrofia Miotônica/genética , Proteínas Serina-Treonina Quinases/genética , RNA Catalítico/genética , Expansão das Repetições de Trinucleotídeos , Regiões 3' não Traduzidas/genética , Pareamento de Bases , Northern Blotting , Diferenciação Celular , Primers do DNA/química , Primers do DNA/genética , Feminino , Feto/enzimologia , Humanos , Lactente , Mutação , Miotonina Proteína Quinase , Gravidez , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , RNA Catalítico/farmacologia , RNA Mensageiro/metabolismo , Receptor de Insulina/metabolismo
19.
Mol Ther ; 6(6): 770-82, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12498773

RESUMO

Effective suppression of HIV-1 replication requires inhibition of critical viral target molecules. Tat and Rev are indispensable regulatory factors for HIV-1 replication, whereas Env mediates virus entry by direct interaction with surface receptor CD4 and coreceptor CCR5 or CXCR4. Anti-HIV-1 tat-rev and env ribozymes and Rev aptamers were previously demonstrated to provide relatively long-term protection against HIV-1 infection in vitro. However, further improvements in these constructs for clinical application in a stem-cell-based gene therapy setting requires in vivo characterization. Toward this end, we introduced these constructs into CD34(+) hematopoietic progenitor cells by retrovirus-mediated gene transduction. Ribozyme- and aptamer-transduced CD34(+) cells differentiated normally into multiple lineages of erythroid and myeloid progenies in a colony-forming unit assay. Macrophages that differentiated from the transduced CD34(+) cells expressed anti-tat-rev and -env ribozymes and Rev aptamers and displayed their normal characteristic surface markers CD14, CD4, and CCR5. Using the SCID-hu mouse in vivo human thymopoiesis model, we demonstrated that ribozyme- and aptamer-transduced CD34(+) cells retained their normal capacity to reconstitute human fetal thymus and liver tissue (thy/liv) grafts. Reconstitution by ribozyme- and aptamer-transduced CD34(+) cells reached levels of up to 87% based on HLA surface marker staining. Differentiated thymocytes derived from reconstituted grafts expressed anti-tat-rev and -env ribozymes and Rev aptamers and showed significant resistance to HIV-1 infection upon challenge. Analysis of reconstituted thymocytes by hybridization revealed an average of 0.4 to 2 copies of vector sequences per cell. Southern analysis of proviral integration junctions in progeny thymocytes demonstrated that the human thy/liv grafts were reconstituted by a few primitive hematopoietic stem cells. These results highlight the utility of RNA-based anti-HIV-1 gene therapeutic approaches and their preclinical testing in a surrogate animal model harboring human tissue.


Assuntos
Modelos Animais de Doenças , Terapia Genética/métodos , Infecções por HIV/genética , Infecções por HIV/terapia , RNA Catalítico/uso terapêutico , Animais , Antígenos CD34/metabolismo , Sequência de Bases , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Citocinas/farmacologia , Regulação Viral da Expressão Gênica , Produtos do Gene rev/genética , Produtos do Gene tat/genética , HIV-1/genética , HIV-1/fisiologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/virologia , Humanos , Transplante de Fígado , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos SCID , Mitógenos/farmacologia , RNA Catalítico/genética , RNA Catalítico/metabolismo , Receptores CXCR4/metabolismo , Timo/citologia , Timo/embriologia , Timo/imunologia , Timo/virologia , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência Humana
20.
Nat Biotechnol ; 20(5): 500-5, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11981565

RESUMO

RNA interference (RNAi) is the process of sequence-specific, posttranscriptional gene silencing in animals and plants initiated by double-stranded (ds) RNA that is homologous to the silenced gene. This technology has usually involved injection or transfection of dsRNA in model nonvertebrate organisms. The longer dsRNAs are processed into short (19 25 nucleotides) small interfering RNAs (siRNAs) by a ribonucleotide protein complex that includes an RNAse III related nuclease (Dicer), a helicase family member, and possibly a kinase and an RNA-dependent RNA polymerase (RdRP). In mammalian cells it is known that dsRNA 30 base pairs or longer can trigger interferon responses that are intrinsically sequence-nonspecific, thus limiting the application of RNAi as an experimental and therapeutic agent. Duplexes of 21-nucleotide siRNAs with short 3' overhangs, however, can mediate RNAi in a sequence-specific manner in cultured mammalian cells. One limitation in the use of siRNA as a therapeutic reagent in vertebrate cells is that short, highly defined RNAs need to be delivered to target cells--a feat thus far only accomplished by the use of synthetic, duplex RNAs delivered exogenously to cells. In this report, we describe a mammalian Pol III promoter system capable of expressing functional double-stranded siRNAs following transfection into human cells. In the case of the 293 cells cotransfected with the HIV-1 pNL4-3 proviral DNA and the siRNA-producing constructs, we were able to achieve up to 4 logs of inhibition of expression from the HIV-1 DNA.


Assuntos
Produtos do Gene rev/metabolismo , Técnicas Genéticas , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Sequência de Bases , Northern Blotting , Linhagem Celular , Humanos , Metabolismo dos Lipídeos , Modelos Genéticos , Dados de Sequência Molecular , Biossíntese de Proteínas , RNA de Cadeia Dupla , RNA Interferente Pequeno , Fatores de Tempo , Transfecção
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