Novel non-viral method for transfection of primary leukemia cells and cell lines
© Schakowski et al; licensee BioMed Central Ltd. 2004
Received: 26 September 2003
Accepted: 12 January 2004
Published: 12 January 2004
Tumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods.
The aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions.
Using this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP.
The non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy.
Keywordsleukemia gene transfer green fluorescent protein nucleofection gene gun
Leukemia cells are obvious and attractive targets for gene transfer since these cells are potentially susceptible to immunotherapeutic strategies. Recently, cytokine gene transfer and expression of immunomodulatory genes in various kinds of tumor cells have been shown to mediate tumor regression and antimetastatic effects in several animal models . Many leukemic entities respond to a treatment with interferon-alpha . Therefore, gene transfer of various cytokine genes such as interleukin-2 (IL-2), IL-7 and IL-12 has been envisaged [3, 4]. Despite of expressing MHC molecules, leukemia cells are ineffective antigen presenting cells (APC) . Often leukemic cells are unable to stimulate T cells because they lack expression of important co-stimulatory molecules . The use of vectors expressing co-stimulatory molecules or cytokines and the use of genetically modified cells for therapeutic purposes are likely to have a significant role for patients with leukemia in the future [6, 7]. To date, only a few strategies applicable to the therapy of these diseases have reached the point of clinical trial [8, 9].
The availability of molecular genetic technology has opened up a large range of potential strategies for the treatment of leukemia. Hematological malignancies have several features that make them particularly amenable to gene transfer approaches. The neoplastic cells circulate in the blood, so that large numbers of tumor cells can be harvested and sorted for ex vivo manipulation. The efficiency of transduction can easily be monitored in vitro and simple blood tests can be used to monitor expression of the transgene or changes in bystander effects following gene transfer. Finally, normal host cells that infiltrate the tumor can be found in the blood, making them accessible for isolation and analysis. Therapeutic approaches using ex vivo immunological modification of malignant cells have not been widely investigated in hematological malignancies. One of the prerequesites to these applications is an appropriate vector that can achieve high efficiency gene transfer in leukemic cells, without major cytotoxicity. Adenoviral vectors are able to transduce a wide range of cells , however there are only little data concerning their ability to transduce hematopoetic cells [11–13]. Recent reports have described successful gene transfer with adenoviral vectors into chronic myeloid leukemia cells (CML) after preactivation of the target cells . With the use of an adenoviral vector, containing a modified fiber protein, an increased gene transfer in acute myeloid leukemic (AML) cells could be shown . Although virus based systems enhance delivery efficiency, recombinant viral based treatments have been associated with complications that result from highly evolved and complex viral biology and / or host parasites interactions . The future of gene therapy requires the development of efficient and nontoxic delivery mechanisms. However, at the present non-viral methods concerning the transfection of hematopoetic cells [17–19] remain poorly efficient.
Oliver Zelphati et al. tested seven commercially available transfection reagents, and he found out that all tested reagents were inefficient for delivery charged molecules into hematopoetic cell lines and primary AML blasts . Very little data on cell viability and transfection efficiency of primary leukemic cells could be provided, most of the researchers using enzymatic bulk assays or a PCR analysis to detect reporter gene expression.
Studies in our laboratory aimed developing an efficient non-viral DNA delivery system for transfection of leukemia cells. We have analyzed several methods of gene delivery into this cell type. To compare non-viral and viral techniques, leukemia cells were transfected with an adenoviral vector expressing the reporter gene green fluorescent protein (GFP). Electroporation as general approach to the introduction of macromolecules into cells was used as a non-viral method. In addition, gene gun, a helium gas pressure-driven device, that delivers gold microparticles coated with plasmid DNA directly into cells, and a novel electroporation based technique called nucleofection  were used.
Here, we describe a new transfection protocol accomplishing highly efficient gene transfer to human chronic and acute myeloid leukemia (AML) cell lines and into primary AML cells derived from three patients.
Primary leukemia cells
Three untreated AML patients were included in the present study. AML cells were isolated from peripheral blood by Ficoll-Paque density centrifugation (Lymphoprep, Nycomed, Oslo, Norway). Cells were cultured in complete RPMI 1640 with Glutamax (GIBCO, Berlin, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS) (PAA, Cölbe, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin (Seromed, Berlin, Germany), and 25 mM Hepes (hydroxyethylpiperazine ethane sulfonic acid, GIBCO).
Patient 1 was a 71-year-old man with FAB M1 classification (acute myelocytic leukemia). Immunophenotyping for this patient was not done. Patient 2 was a 43-year-old woman with TdT-positive FAB M5b classification (acute monoblastic leukemia) with the following immunophenotype: CD13 (78%), CD14 (66%), CD15 (72%), CD33 (73%), and CD64 (77%). Patient 3 was a 63-year-old woman with FAB M4 EO classification (acute myelomonocytic leukemia) with the following immunophenotype: CD13 (89%), CD14 (15%), CD15 (13%), CD33 (24%) and CD64 (12%).
The following cell lines were analyzed: K562 (human chronic myeloid leukemia cell line) and HL60 (human acute myeloid leukemia cell line), both obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). The cell lines were grown in complete RPMI 1640 with Glutamax supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin, and were kept in a humid incubator with 5% CO2 at 37°C. Virus propagation was performed in the Ad5 E1-transformed human embryonic retina cell line 911 . This cell line was grown in Dulbecco's modified eagle medium (DMEM, GIBCO) supplemented with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin.
Adenovirus preparation and infection of leukemic cells
Transfection efficacy was determined with the GFP expressing adenovirus pQB-AdBM5GFP (E1 and E3 deleted replication defective Adenovirus type 5, Quantum Biotechnologies INC., Montreal, Canada). This adenovirus contains a cytomegalovirus (CMV) promotor. Viral stocks were generated as described before  and purified by CsCl2 centrifugation . Plaque assays were essentially performed as described by Graham and Prevec . The titer of the Ad-GFP was 5 × 109 plaque forming units (pfu)/ml. For the adenoviral transfection we used our protocol for transfection of lymphoma cells published recently . In brief, adenoviral transfections with double CsCl2 purified Ad-GFP of K562 cells were carried out in 24-well plates with 5 × 105cells in 50 μl phosphate-buffered saline (PBS) with 1 mM MgCl2/1% horse serum (HS), at an MOI of 200. After 2 hours of incubation at 37°C, 5% CO2, 1 ml of complete culture medium was added to the cells. Because no visible toxic effect in comparison to the controls (only PBS +1 mM MgCl2/1% HS) were observed, it was not necessary to remove the virus.
Expression plasmid for eGFP
The plasmid pMGV was described before  and was obtained from Mologen (Berlin, Germany). The vector contains a CMV enhancer promotor sequence from the immediate early gene of the human cytomegalovirus, the GFP open reading frame from A. victoria, a simian virus (SV)-40 polyadenylation signal, a self-replication-origin (ori p) and a gene for ampicillin resistance.
The plasmid used for transfection was prepared with the Qiagen EndoFree plasmid kit following the manufactures instructions (Qiagen, Hilden, Germany). This kit removes more than 99% of contaminating endotoxin.
Electroporation of leukemia cells
K562 and HL60 cells were transfected by electroporation at various conditions using the electroporation system easyject plus (Eurogentec, Seraing, Belgium). In brief, 5 × 106 cells were suspended in 500 μl complete RPMI medium, mixed with 30 μg of pMGV-plasmid in a 4 mm electroporation cuvette, and incubated on ice for 10 min. After electroporation with a single pulse (electrical parameters for K562 between 270 volt, 1050 μF and 300 volt, 1800 μF, 99 Ω, and for HL60 electroporation condition differs between 1050 and 1800 μF, 200 – 450 V (in 50 V steps), 99 Ω.) the cells were transferred into complete RPMI medium at a density of 1 × 106cells per ml.
Nucleofection of cells
Primary AML cells, K562 and HL60 cells were transfected by nucleofection with the optimised conditions by using the Nucleofector system from amaxa GmbH (Cologne, Germany). The Nucleofector technology is a highly efficient non-viral gene transfer method for most primary cells and for hard-to-transfect cell lines [25–27]. This technology is based on the long-known method of electroporation, which has now been significantly improved. Cell-type specific combinations of electrical current and solutions make the technology unique in its ability to transfer polyanionic macromolecules directly into the nucleus. Thus, cells with limited potential to divide, like many medically highly relevant primary cells, are made accessible for efficient gene transfer. The condition for each cell type have been optimised by using the manufactures' guidelines.
After centrifugation 5 × 105 (cell lines) or 1 × 106 (primary cells) cells were suspended in 100 μl prewarmed Nucleofector Solution Kit R (K562, HL60) or Nucleofector Solution Kit T (primary AML cells), containing 10 μg of pMGV-plasmid in a 2 mm electroporation cuvette (amaxa GmbH, Cologne; Germany). The Nucleofector Kits are cell type specific solutions and commercial available for different cell types (amaxa GmbH). The samples were kept in the cuvette only for the time of the pulse. The leukemic cell line K562 was transfected with the electrical setting P-13 and T-02, the HL60 cell line with electrical setting T-01 and S-11. For primary cells we used the electrical setting U-15 and S-04. After nucleofection with optimized programs the cells were transferred immediately into prewarmed complete RPMI medium.
Particle bombardement of leukemic cells (Gene gun)
Here we used a Biolistic PDS-1000/He unit (BioRad; Munich, Germany). Gold particles (1.0 μm, ABCR) were washed twice in 70% ethanol followed by washing twice in aqua dest. and were concentrated at 60 mg/ml. 6.3 mg gold particles (0.9 mg/macrocarrier) and 42 μg plasmid-DNA were mixed by pipetting (total volume: 56 μl). Afterwards, 504 μl isopropanol was added drop by drop during vortexing the gold/DNA suspension. 7 macrocarriers (BioRad) were overlayed with 80 μl gold/DNA/isopropanol suspension and air-dried. Transfection was performed by 20 Hg below atmospheric pressure, 2200 or 1550 psi (rupture disks) at different positions.
1 × 108 cells were transferred to transwell dishes (Corning costar, 3.0 μm pore size). Short before transfection supernatant was removed by pipetting. After transfection cells was harvest, resuspended in medium and cultured in cell culture flask. 24 hours after transfection expression of transgene was measured by flow cytometry.
Growth curves after transfection
Cell viability was determinated by trypan blue exclusion. PBS was used for control transfection. Non-transfected and GFP transfected cells were counted after adenoviral infection, nucleofection, and particle bombardment 24 – 72 hours after transfection.
Immunofluorescence and flow cytometric studies
Leukemia cells (5 × 105 cells) were washed with PBS and stained with 10 μl monoclonal CD80-FITC and CD86-PE antibody (Pharmingen, Heidelberg, Germany) in a total volume of 50 μl for 15 minutes. Isotype-matched antibodies were used as controls. Stained cells were washed with PBS/1% BSA and subsequently analyzed using an Epics XL flow cytometry system (Coulter-Immunotech, Hamburg, Germany). Background staining using irrelevant antibodies was less than 2%. 105 cells were analyzed for each sample.
To analyze the percentage of GFP positive cells we measured 5 × 105 cells. Cells were washed with PBS, resuspended in 1 ml PBS and 10 μg/ml propidiumiodid (PI) was added immediately before flow cytometric analysis. Lymphocytes were gated based on their scatter profile and cells were evaluated for GFP expression. The transfection efficiency was determined 24 hours up to 72 hours posttransfection. Nucleofected primary cells were also measured after four hours.
Expression of co-stimulatory molecules
In order to elucidate the possibility of co-stimulatory molecules-mediated gene therapy for leukemia cells we analyzed the expression of CD80 and CD86. We determined the expression of these receptors on the cell surface of primary AML cells and leukemic cell lines. The primary cells derived from three AML patients did not express CD80 receptors and only a low level of CD86 (4.5 +/- 1.6 %). The leukemic cell lines K562 and HL60 did not express CD80 receptors as determined by immunophenotyping and FACS analysis. In contrast expression of CD86 was found on the cell surface of K562 (72.4+/-5.1%) and HL60 (76.5+/- 0.5%) cells.
Adenoviral gene transfer
Transfection of leukemia cells using electroporation
Comparison of various methods in the transfection efficiency of leukemic cells. Transfections were performed with an expression plasmid for eGFP or adenoviral GFP expressing vector as described in materials and methods. 24 hours after transfections cells were harvested and assayed by flow cytometry. Ad-GFP transfected cells were analyzed 72 hours after transduction. Results of three separate experiments are presented (ND, not done).
Adenoviral gene transfer (MOI 200) GFP-pos. cells [%]
Electroporation GFP-pos. cells [%]
Gene gun GFP-pos. cells [%]
Nucleofection GFP-pos. cells [%]
primary AML cells
60.3 +/- 9.7
49 +/- 4
15.5 +/- 3.5
1.5 +/- 0.5
74.7 +/- 8.0
30.2 +/- 5.6
3.0 +/- 1
49.0 +/- 9.7
Transfection of leukemia cells using gene gun
We transfected HL60 and K562 cells by gene gun technique. Various parameters were examined. Here we used 2200 or 1550 psi (rupture disks) at different positions (second, third, and fourth position). The transfection efficiency was determined and quantified by the expression of the encoding plasmid pMGV after 24 and 48 hours posttransfection. In summary, the transfection rates were 3.0+/-1% for HL60 and 1.5+/-0.5% for K562 cells (Table 1).
Transfection of leukemic cells by nucleofection
Growth curves of transfected leukemia cell lines
Chemotherapy and allogeneic bone marrow transplantation (BMT) are the conventional treatment strategies for acute myelogenous leukemia (AML) [32–35]. Complete remissions can be achieved in the majority of patients, but disease recurrence remains a frequent subsequent of treatment failure. For example, in patients whose AML blasts bear complex chromosomal mutations the risk of leukemia relapse is very high, and in AML patients over 60 years of age the five year survival rate after established treatment regimens is below 20% . Unfortunately, therapeutic options for patients with recurrent leukemia are still limited and the prognosis is poor . Second marrow transplants from the same donor may be considered for patients with disease relapse after BMT, but the mortality, treatment-related morbidity and risk of further relapse are high [38, 39].
Alternative or additional treatment strategies are provided by immunotherapeutic approaches. Successful employment of donor lymphocyte infusions (DLI) in patients with relapsed chronic myelogenous leukemia (CML) after allogeneic BMT gave reason for the application to acute leukemia patients, but the treatment turned out to be far less effective [40–43]. In other cases of refractory or relapsed AML, infusions of anti-CD33 antibody-conjugated antitumor agents have been successfully used . Furthermore, gene therapy has emerged as a promising approach to provide new treatment options.
Leukemia cells are considered as suitable targets for gene therapy. Cytogenetic studies of leukemia cells have identified mutations, chromosomal aberrations the failure of expression of co-stimulatory molecules [1, 5, 9]. Co-stimulatory molecules such as CD80 (B7.1) and CD86 (B7.2) that are necessary to bind CD28 on T-cells, to maintain production of IL-2 after initial T cells activation, have been shown to be lacking in acute leukemic cells, resulting in T-cell anergy . The lack of expression of CD80 could also be detected in the leukemic cell lines used here. Vectors expressing co-stimulatory molecules or cytokines have been suggested for gene therapy strategies [1, 3]. Adenovirus based vectors can be used for targeted gene transfer to AML  and after stimulation of the target cells CML and B-CLL could be efficiently transfected by adenoviral vectors. Similar results could be obtained by the use of primary cells . We previously demonstrated efficient gene transfer in Burkitt lymphoma (BL) cell lines and primary lymphoma cells after transfection with adenoviral vectors  and could here show similar results in transfection efficiency of the leukemic cell line K562 by use of adenoviral vectors. Recent reports have described the successful gene transfer in the cell line HL60 and primary cells derived from AML patients up to 100% of positive cells after adenoviral gene transfer . Although viral vectors induce long term, high gene expression, phenomena such as the possibility of creating recombination competent adenovirus (RCA) induction of the host immune response are cutting back the use of viral delivery.
Since the efficiency of non-viral gene transfer by naked DNA is lower than that of viral delivery, both chemical and physical techniques have been used to increase the efficiency of DNA uptake and expression. The physical gene transfer approaches allow DNA to penetrate directly the cell membrane and bypass endosomes / lysosomes, thus avoiding enzymatic degradation. The DNA may also be delivered directly to the nucleus by gene gun, electroporation and novel electroporation based technique called nucleofection. Physical gene transfer methods, unlike viral vectors, do not require cell type specific receptors, are safe, highly reproducible and time saving. Due to low transfection efficiency most investigations were made with established cell lines after stable transfection and selection . Even many transfection reagents which show high gene transfer efficiency in common adherent cell lines are not suitable to transfect establish blood cell lines or primary leukemia cells from patients. All samples showed a transfection rate of below 5% positive cells . There is no data concerning efficient non viral gene transfer into primary leukemic cells with gene gun or standard electroporation.
The use of electrotransfer for DNA delivery to eukaryotic cells in vitro has been well known and widely used in basic research. However, it is only recently that electric fields have been used to enhance DNA transfer to animal cells in vivo, and this is known as DNA electrotransfer or in vivo DNA electroporation. This is especially useful to transfect whole tissues or tumors. As well as exciting applications in developmental biology, in vivo DNA electrotransfer is also being used to transfer genes to skeletal muscle and drive expression of therapeutically active proteins and to examine exogenous gene and protein function in normal adult cells situated within the complex environment of a tissue and organ system in vivo . However, the use of in vivo electroporation has just begun and so far nothing has been published of in vivo transfection of cells of the blood system.
Here, we established an optimized non-viral gene delivery into leukemic cells as a first step towards a gene therapy approach. We compared the efficiency of adenoviral mediated gene transfer with the efficiency obtained by electroporation, particle bombardment and nucleofection into leukemic cells in vitro. Using established human leukemic cell lines we have analyzed the standard techniques with the novel nucleofection technique. We have examined different electrical programs with the new nucleofector advice to determine the effects on the transfection efficiency and viability of the cells.
In this study we have shown that the novel non-viral transfection technique called nucleofection is an efficient way to transfect not only AML cells lines but also primary AML cells. Here, we achieved transfection efficiency for primary cells from three AML patients up to 75 % with low toxicity after 24 hours (< 20 %). After 72 hours the toxicity inside the lymphocyte gate increased up to 40–45 % (non-nucleofected primary cells 25 %). However, the ability of nucleofection to mediate gene transfer into non-dividing cells and the feasibility to transfect a high range of cells makes it attractive for gene delivery in vitro. Nucleofection does only very rarely result in nuclear integration of the transgene. Loss of gene expression during propagation of cells is most likely to be due to loss of the transgene rather than due to loss of transgene expression. In terms of cell numbers, K562 and HL60 cells cease proliferation 72 hrs after nucleofection. It is expected that these cells finally enter cell death. This, however, makes the procedure less suitable for biochemical or pharmacological studies due to severe cell damage during nucleoporation. However, in immunotherapeutic approaches, sustained gene transfer is not essential. Thus, the transient gene expression achieved by use of nucleofection is an available tool for gene therapy.
The ability to efficiently manipulate gene expression in leukemia using non-viral methods should facilitate the functional characterization of pathways affecting lymphocytes physiology. In conclusion, we present a protocol of a new gene transfer method leading to highly efficient gene transfer in primary leukemic cells and established cell lines without major toxicity and low risk of insertional mutagenesis or induction of the host immune response. This protocol should have an important impact on the use of hematopoetic cells in cancer gene therapy protocols.
FS, PB, MM and AM carried out the studies, BS was clinically involved and helped in carrying out the studies, ISW participated in the design of the study and its coordination. All authors read and approved the final manuscript.
List of abbreviations
acute myeloid leukemia
antigen presenting cell
bone marrow transplantation
chronic myelogenous leukemia
donor lymphocyte infusion
fetal calf serum
green fluorescent protein
This work was kindly supported by a generous grant from the H. W. & J. Hector-Stiftung, Weinheim, Germany and from the Deutsche José Carreras Leukaemie-Stiftung e.V., Munich, Germany.
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