Analytical Biochemistry 388 (2009) 164–166
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Notes & Tips
Disrupting actin filaments promotes efficient transfection of a leukemia cell line using cell adhesive protein-embedded carbonate apatite particles K. Kutsuzawa a,b, S. Tada a, S. Hossain a, K. Fukuda a,b, K. Maruyama b, Y. Akiyama b, T. Akaike a, E.H. Chowdhury c,* a
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan Shizuoka Cancer Center Research Institute, Sunto-gun, Shizuoka 411-8777, Japan c School of Medicine, International Medical University (IMU), Bukit Jalil 57000, Kuala Lumpur, Malaysia b
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Article history: Received 18 November 2008 Available online 10 February 2009 Keywords: Protein Kinase C Actin filament Gene delivery Nanoparticles Extracellular matrix
a b s t r a c t Tumor cells such as leukemia and lymphoma cells are obvious and attractive targets for gene therapy. Gene transfer and expression for cytokine and immunomodulatory molecules in various kinds of tumor cells have been shown to mediate tumor regression and antimetastatic effects. Moreover, genetically modified leukemia cells expressing costimulatory molecules or cytokines are likely to have significant therapeutic roles for patients with leukemia. One of the major hurdles to the successful implementation of these promising approaches is the lack of a suitable nanocarrier for transgene delivery and expression in a safe and effective manner. Recently, we reported on the development of a safe, efficient nanocarrier system of carbonate apatite that can assist both intracellular delivery and release of DNA, leading to very high level of transgene expression in cancer and primary cells. However, its efficiency in human lymphocytes is poor. We show here that nanocrystals of carbonate apatite, when electrostatically associated with fibronectin and/or E-cadherin-Fc, accelerated transgene delivery in a human T leukemia cell line (Jurkat). Moreover, transgene expression efficiency could be enhanced dramatically with the cell adhesive protein-embedded particles finally up to 150 times by selectively disrupting the actin filaments. Ó 2009 Elsevier Inc. All rights reserved.
Tumor cells such as leukemia and lymphoma cells are obvious and attractive targets for gene therapy. Gene transfer and expression for cytokine and immunomodulatory molecules in various kinds of tumor cells have been shown to mediate tumor regression and antimetastatic effects [1–4]. Moreover, genetically modified leukemia cells expressing costimulatory molecules or cytokines are likely to have significant therapeutic roles for patients with leukemia [5,6]. One major hurdle to the successful implementation of these promising approaches is the lack of a suitable nanocarrier for transgene delivery and expression in a safe and effective manner. Although virus-based systems enhance delivery efficiency, recombinant viral treatment has been associated with complications that result from highly evolved and complex viral biology and/or host– parasite interactions [7]. On the other hand, currently nonviral methods for transfection of hematopoietic cells [2] remain poorly efficient. Recently, we developed an efficient DNA delivery and expression system based on pH-sensitive inorganic nanocrystals of carbonate apatite with capability of effective intracellular delivery
* Corresponding author. Fax: +60 45 924 5879. E-mail address:
[email protected] (E.H. Chowdhury). 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.02.006
and release of associated DNA molecules, leading to very high level of transgene expression in cancer and primary cells [8,9]. However, the efficiency of transfection in human lymphocytes is relatively poor. Here we show that nanocrystals of carbonate apatite, when electrostatically associated with naturally occurring fibronectin and/or chimeric E-cadherin-Fc, accelerated transgene delivery in a human T leukemia cell line (Jurkat). Moreover, transgene expression efficiency could be enhanced dramatically with the cell adhesive protein-embedded particles finally up to 150 times by selectively disrupting actin filaments. Jurkat cells were grown in RPMI 1640 medium (Gibco BRL) containing 10% fetal bovine serum (FBS)1 and seeded at 5 105 cells per well into a 24-well plate the day before transfection. Then 3 ll of 1 M CaCl2 was mixed with 2 lg of plasmid DNA in 1 ml of fresh serum-free HCO3 -buffered medium (pH 7.5, Dulbecco’s modified Eagle’s medium [DMEM], Gibco-BRL) and incubated for 30 min at 37 °C for complete generation of DNA/carbonate apatite particles. For generation of cell adhesive protein-embedded carbonate apatite particles, fibronectin and E-cadherin-Fc proteins were added, either
1 Abbreviations used: FBS, fetal bovine serum; DMEM, Dulbecco’s modified Eagle’s medium; PBS, phosphate-buffered saline; RLU, relative light units; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DMSO, dimethyl sulfoxide.
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Fig. 1. Comparison of luciferase expression among differentially formulated apatite particles in the presence or absence of actin-disrupting agent. Particles were prepared by the addition of 3 ll of 1 M CaCl2, 2 lg of luciferase plasmid DNA, and 2 lg of fibronectin and/or E-cadherin-Fc (E-cad-Fc) to 1 ml of bicarbonate-buffered DMEM and incubation for 30 min at 37 °C. Jurkat cells were incubated with the generated particles for 2 days in the absence or presence of 0, 5, 50, and 500 nM cytochalasin D, followed by quantitation of luciferase expression. Transfection efficiency was normalized after estimation of total proteins in cell lysate.
Fig. 2. Assessment of cell viability after incubation with differentially formulated apatite particles in the presence or absence of cytochalasin D (CytD). Jurkat cells were transfected in the same way as described in the Fig. 1 legend, and MTT solution (5 mg/ml) was added to each well, followed by incubation for 4 h. Then 0.5 ml of DMSO was added following the removal of the medium. After dissolving crystals and incubating for 5 min at 37 °C, absorbance was measured in a microplate reader at 570 nm with a reference wavelength of 630 nm. E-cad-Fc, Ecadherin-Fc.
alone or together to a final concentration of 2 lg/ml, to Ca2+ and DNA-containing DMEM, followed by incubation at 37 °C for 30 min. Medium with generated cell adhesive protein-associated or -nonassociated DNA-containing particles was added with 10% FBS into the cells in each well. After a continuous 2-day incubation, the medium was removed and the cells were washed with phosphate-buffered saline (PBS)( ) before being lysed. Luciferase gene expression was monitored by using a commercial kit (Promega) and photon counting (TD-20/20 Luminometer, Turner BioSystems). Each transfection experiment was done in triplicate, and transfection efficiency was expressed as mean relative light units (RLU) per milligram of cell proteins. Jurkat cells were transfected according to the aforementioned protocol, and 30 ll of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/ml) was added to each well, followed by incubation for 4 h. Then 0.5 ml of dimethyl sulfoxide (DMSO) was added following removal of the medium. After dissolving crystals and incubating for 5 min at 37 °C, absorbance was measured in a microplate reader at 570 nm with a reference wavelength of 630 nm. T cell expresses on its membrane a4b1 and a5b1 integrins, which can bind fibronectin during lymphocyte adhesion and migration from the vascular compartment to the injured tissues [10]. Moreover, aEb7 integrin on some T cells can interact with epithelial E-cadherin for tissue-specific retention of lymphocytes [11]. Therefore, we aimed to functionalize the surface of DNA-associated nanocrystals with fibronectin and E-cadherin-Fc for transgene delivery through integrin-mediated endocytosis [12,13]. As shown in Fig. 1 and Table 1, luciferase expression in Jurkat cells was significantly lower after delivery of luciferase gene-containing plasmid DNA with the help of carbonate apatite particles. However, a sixfold enhancement in transgene expression was observed following delivery with fibronectin-embedded particles.
Transgene expression could also be increased up to three and six times with the particles complexed with E-cadherin and fibronectin/E-cadherin, respectively. Because lymphocytes possess two different types of integrins (a4b1 and a5b1) that are able to bind fibronectin [10], particles with electrostatically associated fibronectin and fibronectin/E-cadherin-Fc could recognize either of the two receptors for efficient endocytosis in Jurkat cells, leading to high transgene expression. However, particles with adsorbed E-cadherin-Fc showed lower transfection efficiency, probably owing to their low-affinity interactions with the cell membrane integrins (aEb7) or inefficient endocytosis at the subsequent step. On the other hand, a dramatic enhancement in transgene expression could be observed after treatment with cytochalasin D, a selective actin-disrupting agent (Fig. 1). Particles coated with either fibronectin or fibronectin/E-cadherin resulted in nearly 90 to 100 times more efficient luciferase expression in the presence of 500 nM cytochalasin D than did particles in the absence of the agent. A robust increase in expression (150 times) was observed with the E-cadherin-embedded particles following treatment with cytochalasin D. Because an increased dose of cytochalasin D can increase mobility (diffusion) of lymphocyte-specific integrins in the cell membrane by depolymerizing the actin filaments and releasing the integrins from cytoskeletal constraints [14], the endocytosis of the particles, preferably those coated with cell adhesive proteins, might be enhanced profoundly for uptake of particleassociated DNA, leading to a high level of protein expression. However, higher protein expression in the presence of the actin-disrupting agent was accompanied by less viability of the cells, as observed by MTT assay (Fig. 2). In summary, we have established an efficient nonviral method based on cell adhesive protein-anchored inorganic nanocrystals for transfecting a human T leukemia cell line having potential applications in the treatment of leukemia.
Table 1 Transfection efficiency for differentially formulated apatite particles in the presence or absence of actin-disrupting agent. Transfection efficiency (RLU/mg of proteins) Particle Particle + fibronectin Particle + E-cadherin-Fc Particle + fibronectin + E-cadherin-Fc
(5.6 ± 1.8) 103 (3.7 ± 2.9) 104 (1.5 ± 1.2) 104 (3.4 ± 1.5) 104 0 nM
(3.7 ± 1.3) 104 (l.3 ± 0.59) 105 (3.0 ± (0.69) 105 (3.7 ± 1.l) 105 5 nM
Concentrations of cytochalasin D maintained in particle suspension during incubation with cells.
(4.4 ± 1.1) 104 (1.7 ± 0.62) 105 (8.9 ± 3.1) 104 (1.2 ± 0.32) 105 50 nM
(1.0 ± 0.16) 105 (4.9 ± 0.87) I05 (8.3 ± 3.2) 105 (5.4 ± 1.1) 105 500 nM
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Acknowledgments This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT, Japan) program, ‘‘Promotion of Environmental Improvement for Independence of Young Researchers,” under the Special Coordination Funds for Promoting Science and Technology.
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