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 22. 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.

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 11 and purified by CsCl₂ centrifugation 22. Plaque assays were essentially performed as described by Graham and Prevec 23. 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 11. 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.

The plasmid pMGV was described before 24 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.

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.

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 252627. 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.

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.

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.

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.

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.

In preliminary experiments using the leukemic cell line K562, we observed that these cells could be successfully transfected with an adenoviral vector expressing the reporter gene GFP 28. At an MOI of 200, 49+/-4% of the cells showed a positive GFP signal after 72 hours in flow cytometric analysis (Figure 1a). These results were in accordance with previous reports 141529, which showed that leukemic cells could be efficiently transfected with adenoviral vectors. Roddie and coworkers showed high adenoviral transduction efficiency in three of four leukemia cell lines but not in HL60 30.

To compare the new nucleofection technology (nucleofector, amaxa) with the standard electroporation techniques (easyject plus, BioRad), we transfected K562 cells with pMGV with various electrical parameters as described in the literature 171831. Transfection efficiency was determined 24, 48, and 72 hours after electroporation by flow cytometric and fluorescence microscopical analysis. For K562 cells the transfection efficiency was 15.5+/-3.5% (Table 1). Cell viability determined by trypan blue exclusion, was markedly impaired by electroporation (data not shown). These results are in accordance with previous reports 1718. For HL60 cells maximum transfection efficiency was 30.2+/-5.6% (1050 μF, 450 V, 99Ω) with high toxicity 66.2+/-6.8% (Table 1).

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).

With the aim of developing a gene therapy protocol for leukemia, we were interested in identifying the most effective non-viral method of DNA delivery into primary leukemia cells and leukemia cell lines. Several approaches have been developed to enhance the efficiency of non-viral gene transfer via naked DNA including gene gun and electroporation. Here, we tested a novel electroporation based technique called nucleofection. This electroporation based technique combines cell type specific solutions with mild conditions which guarantee high efficiency and low cell death rates. Physical approach allow DNA to penetrate directly the cell membrane and bypass endosomes / lysosomes, thus avoiding enzymatic degradation. The DNA may also be directly delivered to the nucleus by nucleofection. Transfection efficiency was determined 24 hours after nucleofection by flow cytometric assays. Figure 2 shows the flow cytometry analysis of the three primary AML cells 24 hours after nucleofection. Cells were pulsed with two different programs (U15, S04) and show transfection relative efficiencies up to 71,5% (+/- 1.8) (program S04) with low toxicity (5,4% +/-0.2). Flow cytometric analysis four hours after nucleofection showed same transfection efficiencies like the 24 hours measurement (data not shown) The transfection data of the primary AML cells of three patients and flow cytometric analysis are shown in figure 2a and 2b. Figures 1b (K562 cells) and 1c (HL60 cells) show the primary data of one representative experiment with the optimized electrical parameters. The shaded histogram represents the background fluorescence of transfected cells without DNA, positive transfected cells express intracellular green fluorescent protein. Various electrical parameters were assayed to optimize the transfection efficiency of the leukemic cell lines K562 and HL60. Figure 3 demonstrates the two optimized programs for each cell line (K562, HL60) and shows the absolute percentage of GFP positive cells and the relative efficiency of the transfected gated cells. Viability was determined by PI staining. By balancing survival rate and transfection efficiency optimal program for K562 resulted in a population of 76.1 +/- 2.3% positive viable cells in the gate. The program T02 showed low toxicity with 34.0 +/- 14.6% dead cells 24 hours after nucleofection. Gene transfer into leukemic HL60 cell line showed relative efficiencies ranging from 33.4 +/-14.9 to 49.0 +/- 9.7%. Due to the higher percentage of dead cells (49.6 +/- 21.5 up to 63.0 +/- 16%) the relative efficiency was lower in comparison with K562 cells. These results demonstrated a high efficient gene transfer with the nucleofection technique. The time course of GFP transgene expression after 72 hours showed a constant expression of GFP in the cell line K562 (relative efficiency: 74.7 +/- 8%, program T02). The transgene expression in HL60 cells decreased to 24.3 +/- 9.1% after 72 hours (program T01). Fourteen days after transfection the percentage of transgene expressing cells decreased to 3%. These results indicate that nucleofection mediated gene expression in cells was transient.

Cell counts were determined 24, 48 and 72 hours after transfection. Following transfection with various parameters cell numbers differed from untransfected and control transfected leukemia cells (Fig. 4). In the case of HL60 cells the viability of transfected cells decreased rapidly (Fig. 4b). As shown in figure 4a nucleofection of K562 cells with different electrical parameters revealed a small reduction of cell number over a three day period. Control transfected cells (without DNA) showed only a reduction of cell number in comparison to non-transfected cells (1.3 × 10⁶ cells/ml versus 9.8 × 10⁵ cells/ml). The cell proliferation of transfected HL60 cells was strongly retarded (untransfected cells after 72 hours 1.9 × 10⁶ cells/ml versus transfected cells 4 × 10⁴ cells/ml), the non-transfected control cells proliferated continuously. Transfection of the HL60 cells lines with nucleofection resulted in a stagnation of cell growth. After 72 hours cell lines continued to proliferate however at a slower rate than the control (data not shown).