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Toxicogenomics of Non-viral Vectors for Gene Therapy: AMicroarray Study of Lipofectin- and Oligofectamine-induced
Gene Expression Changes in Human Epithelial Cells
YADOLLAH OMIDIa, ANDREW J. HOLLINSa, MUSTAPHA BENBOUBETRAa, ROSS DRAYTONa, IBRAHIM F. BENTERb andSAGHIR AKHTARa,*
aCentre for Genome-based Therapeutics, The Welsh School of Pharmacy, Cardiff University, Redwood Building,King Edward VII Avenue, Cardiff CF10 3XF, UK
bDepartment of Pharmacology, Kuwait University, Kuwait
(Received 3 October 2003)
Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery ofgenes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs.However, little is known about the impact of these delivery systems on global gene expression in targetcells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have usedmicroarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles ofhuman A431 epithelial cells. Using the manufacturer’s recommended CL concentrations routinely usedfor gene delivery, cDNA microarray expression profiling revealed marked changes in the expression ofseveral genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrayshousing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cellsresulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstreamfunctional consequences of CL-induced gene expression alterations led to an increased tendency ofcells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect wasgreater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient toinduce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay.These data highlight the fact that inadvertent gene expression changes can be induced by the deliveryformulation alone and that these may, ultimately, have important safety implications for the use of thesenon-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be takeninto consideration in gene therapy or gene silencing experiments using CL formulations where theymay potentially mask or interfere with the desired genotype and/or phenotype end-points.
Keywords: Cationic lipids; Gene delivery; Gene silencing; Gene expression profiling; siRNA;Antisense
INTRODUCTION
Gene-based therapies such as gene therapy, antisense
oligonucleotides, ribozymes, DNAzymes and RNA-
interference all represent promising therapeutic para-
digms for future healthcare in the post-genomic era (for
reviews see Akhtar et al., 2000; Somia and Verma,
2000; Zallen, 2000; Hughes et al., 2001; McManus and
Sharp, 2002; Dykxhoorn et al., 2003; Kerr, 2003).
Gene-based therapies require acceptable and efficient
delivery along with minimal toxicity and maximum
patient safety. These issues are important for both ex
vivo, where cells undergo gene therapy in culture prior
to implantation into the patient, or in vivo gene therapy
where nucleic acids are administered directly to
the patient to effect the desired gene change. In either
approach, ideally only the therapy-intended gene
expression changes should occur. However, this is not
always the case. For example, viral vectors are known
to be efficient delivery systems for nucleic acids but
can also induce toxic responses including inadvertent
gene expression changes following random integration
into the host genome (Tarahovsky and Ivanitsky, 1998;
Pleyer et al., 2001; Audouy et al., 2002; Whitehouse,
2003). Hence, several non-viral vector systems have
been developed for gene and oligonucleotide delivery.
Of these the cationic lipid (CL) formulations that can
readily condense DNA into complexes termed
ISSN 1061-186X print/ISSN 1029-2330 online q 2003 Taylor & Francis Ltd
DOI: 10.1080/10611860310001636908
*Corresponding author. Tel.: þ44-29-2087-6309. Fax: þ44-29-2087-4149. E-mail: saghirakhtar@cf.ac.uk
Journal of Drug Targeting, 2003 Vol. 11 (6), pp. 311–323
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lipoplexes have been the most widely studied for ex vivo
and in vivo gene therapy approaches (for reviews see
Juliano and Akhtar, 1992; Akhtar et al., 2000; Aissaoui
et al., 2002; Hirko et al., 2003).
CL can be classified into several categories based on
their structure, size and charge (Pedroso de Lima et al.,
2001). Due to their high delivery efficiency, particular
interest has been given to the positively charged CL or
liposome formulations for DNA transfer (Pedroso de Lima
et al., 2001). These reagents form unilamellar liposomes
bearing positive charges which interact spontaneously
with polyanionic DNA to form lipoplexes (Duzgunes
et al., 2003). The lipid-DNA lipoplex is thought to enter
cells via adsorptive endocytosis and, by mechanisms not
fully understood as yet, release nucleic acids out of the
endosomal/lysosomal compartments with the net effect of
yielding high uptake and intracellular delivery of genes
and oligonucleotides (Zelphati and Szoka, 1996; Pedroso
de Lima et al., 2001).
Despite the enhanced delivery and the resultant
improved biological activity of nucleic acids delivered
with CLs, they can often, but not always (e.g. Canonico
et al., 1994), result in cellular toxicity (Campbell, 1983;
Filion and Phillips, 1997; Scheule et al., 1997; Nagahiro
et al., 2000; Pedroso de Lima et al., 2001). However, little
is known about the impact of CL delivery systems on global
gene expression in target cells. In a previous study aimed at
screening the downstream changes in gene expression
following treatment of cells with antisense oligonucleo-
tides targeting the epidermal growth factor receptor, we
became aware of non-target gene expression changes that
may have been due to the Lipofectin-delivery system used
(Petch et al., 2003). Here, in an attempt to study the geno-
compatibility and toxicogenomics of CL formulations in
target cells, we have used gene expression microarrays to
compare the effect of two commercially available CL
formulations, Lipofectin and Oligofectamine, on gene
expression in human A431 epithelial cells.
MATERIALS AND METHODS
Materials
Total RNA isolation kit Trireagente, Ethidum Bromide,
isopropanol, chloroform, formaldehyde and low melting
point agarose (LMPA) were from Sigma (Poole, UK).
CL (Lipofectin and Oligogectamne), Dulbecco’s modi-
fied Eagle’s medium (DMEM) containing 25 mM
HEPES, fetal bovine serum (FBS), penicillin G,
streptomycin, L-glutamine 200 mM ( £ 100), first strand
PCR buffer ( £ 5), Moloney Murine Leukaemia Virus
reverse transcription (MMLV-rt), dithiotheritol (DTT)
and RNase/DNase free ddH2O were purchased from
Invitrogen, (Paisley, UK). The deoxynucleotide triphos-
phate monomers (dNTPs) and random hexamer
primers (pdN6) were from Amersham Biosciences
(Amersham, Bucks, UK). The cyanine fluorescent dyes
(Cy3 and Cy5) were obtained from Amersham Life
Science (Little Chalfont, UK). Tissue culture treated
multi-well plates and flasks were obtained from Corning
Costar (High Wycombe, UK). RNasine was from
Promega (Southampton, UK) and Taq polymerase from
Qiagen Ltd (Crawley, UK). The dual-window Comet
slide was obtained from Trevigen (Gaithersburg, MD).
Annexin V-FITC apoptosis detection kit was obtained
from Oncogene Research Products, (San Diego, CA,
USA). Human epidermoid carcinoma A431 cell line was
purchase from ECACC, (Salisbury, UK).
Cell Culture
A431 cells were seeded at 5 £ 104 cells/cm2 into either 6- or
96-well plates and were subjected to a standard transfection
protocol. Briefly, following the manufacturer protocol, CL
(Lipofectin or Oligofectamine) was prepared initially at a
concentration of 0.2mg/ml in serum-free medium (SFM)
and then diluted, after 30–45 min incubation at room
temperature, to 0.045mg/ml in SFM. Cells, at the 40–50%
confluency, were washed twice with SFM, exposed to CL
mixture (0.8 ml/well for 6 well plates) and incubated at
378C for 4 h. Cells were replenished with normal growth
medium, incubated at 378C for 24 h, and were then used for
gene expression or other assays.
Total RNA Isolation
Total RNA was isolated, from CL treated and untreated
A431 cells, using TRIREAGENTe according to the
manufacturer’s (Sigma) protocol. Briefly, cells were lysed
directly on the culture dish where the medium was
discarded and 1 ml of the TRIREAGENTe per 10 cm2 of
culture area was added. The homogenous lysate was then
transferred to a RNase/DNase free 1.5 ml micro-
centrifuge tube and 0.2 ml of chloroform per 1 ml of
TRIREAGENTe was added, mixed and kept for 5 min at
room temperature. After centrifugation, 12,000g for
15 min at 48C, using Eppendorf Centrifuge 5417R,
(Eppendorf, Hamburg, Germany), the upper aqueous
phase of three separated phases was mixed with 0.5 ml of
isopropanol, kept for 5 min at room temperature, and then
centrifuged at 12,000g for 10 min at 48C and the RNA was
recovered as a precipitate. RNA pellet was washed twice
with 1 ml of 75% ethanol, spun at 10,000g for 5 min, then
dried and dissolved in 50ml DNase/RNase free water.
Quantification and purity of extracted total RNA were
assessed by UV spectrophotometry scans (Ultraspec 3100
pro, Amersham, Bucks, UK) and by electrophoresis of a
sample on 1% denaturing agarose gel followed by UV
visualisation on the Gel Doc 1000 gel documentation
system (BioRad, Hertfordshire, UK).
The cDNA Microarray Labelling Procedure
The aminoallyl (aa)-cDNA labelling was undertaken
using a modified version of the TIGR group protocol
(Hegde et al., 2000). Briefly, a 100 mM aa-dUTP was
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prepared by dissolving 1 mg in 19.1ml 0.1 M KPO4
buffer, pH 7.5. The (50 £ ) labelling mix of aminoallyl-
dNTP (aa-dNTP) containing 2:3 ratio of aa-dUTP to dTTP
with a final concentration of 25 mM of each dNTPs,
(i.e. dATP, 25 mM dCTP, 25 mM dGTP, 15 mM dTTP,
10 mM aa-dUTP). Ten micrograms of total RNA was
mixed with 6mg of Random Hexamer primers in a final
volume of 25ml. The mixture was incubated at 708C for
10 min, then placed on ice for 3 min. In the RT reaction
6ml of 5 £ first strand buffer, 3ml of 0.1 M DTT, 0.6ml
50 £ aa-dNTP mix and 2ml SuperScript II RT (200 U/ml)
were added to RNA/pdN6 mix and incubated at 428C for
6 h in a PCR thermal cycler (Techne Flexigene,
Cambridge, UK). The remaining total RNA was
hydrolysed in 10ml of 1 M NaOH and 10ml of 0.5 M
EDTA at 658C for 15 min with subsequent neutralisation
with 10ml of 1 M HCl.
To purify the aa-cDNA from unincorporated aa-dUTP
and free amines prior to conjugation with NHS-ester Cy
dyes, a Qiagen PCR purification kit was used. This
purification step prevented Cy dyes coupling to free amine
groups in the reaction. The aa-cDNA product was mixed
with 350ml (5 £ reaction volume) binding buffer PB,
transferred to QIAquick column and centrifuged for 1 min
at 12,000g for 1 min. To maximize aa-cDNA binding,
eluate was re-spun using same column. Washing steps
(twice) were carried out with 500ml Tris-free phosphate
wash buffer (5 mM KPO4, pH 8.0 and 80% EtOH)
spinning at 12,000g for 45 s. Column was then transferred
to a fresh tube and 30ml 4 mM KPO4, pH 8.5 was added.
Column was incubated at room temperature for 1 min and
centrifuged at 12,000g for 1 min to elute the aa-cDNA. To
maximize the aa-cDNA recovery an additional 30ml
4 mM KPO4, pH 8.5 was introduced to column, incubated
for 1 min, and centrifuged to elute with to a total volume
of 60ml. Finally, the labelled sample was dried using a
speed vacuum lyophiliser (DNA120 SpeedVac, Thermo-
Savant, Thermo Life Science, NY, USA). The aa-cDNA
was quantified using UV spectrophotometry scans
(Ultraspec 3100 pro, Amersham, Bucks, UK) prior to
conjugation with NHS-ester Cy dyes. Normally 10mg
total RNA in such reactions should provide 4–6mg
aa-cDNA after QIAquick purification.
The aminoallyl-labelled cDNA was resuspended in 5ml
of 0.1 M carbonate buffer, pH 9.0. For covalent coupling
of the dye-ester to the aminoallyl-labelled cDNA, 5ml of
3 mM stock solution of fluorescent cyanine dye was added
and incubated in the dark at room temperature for 1 h.
Thirty five microlitres of 100 mM NaOAc pH 5.2 was then
added to reaction. Uncoupled dye was removed prior to
hybridisation using QIAquick PCR purification kit.
Elution was performed twice in 30ml EB for a total of
60ml as described above. Cy3 or Cy5 coupled cDNA
was quantified using UV spectrophotometry (Ultraspec
3100 pro, Amersham, Bucks, UK). Coupled aa-cDNA
with Cy-dye was finally dried using a speed vacuum.
Approximately 150–200 pmol of dye incorporation per
sample (aa-cDNA product of 10mg total RNA) and a ratio
of 40–50 nucleotides/dye molecules yielded optimal
hybridisation results.
Hybridisation of Cy-dye Coupled cDNA
MWG-Biotech human starter arrays (housing 200 gene
spots) were prepared with Gene Framesw 1 day prior to
hybridisation following MWG protocol (Milton Keynes,
UK). The Gene Frames were used because gas-tight
sealing system can withstand temperatures of up to 978C
and prevents reagent loss due to evaporation, thus
improving the hybridisation reliability. Hybridisation
was performed following MWG-Biotech protocol with
slight modifications. Briefly, dried Cy-dye coupled cDNA
was dissolved in 60ml of MWG hybridisation solution.
A measured quantity of 1.5–2mg of each probe was
mixed in one tube in a final volume of 120ml salt-based
hybridisation buffer and incubated at 958C for 3 min. The
combined probes were incubated on ice for 3 min, then
introduced to one end of the Gene frame after removing
the thin polyester backing sheet from the frame. The
polyester cover slip was then carefully placed over the
Gene Frame. The arrays were incubated at 428C for
18–24 h in a shaking water bath. Following hybridisation,
the array slides were carefully removed from the
hybridisation chamber and washed with wash buffer A
(2 £ SSC, 0.1% SDS), wash buffer B (1 £ SSC), and
wash buffer C (0.5 £ SSC), 4 times in each wash, 2 min
each time. The array slide was briefly rinsed with
distilled water, centrifuged at 1600g for 5 min using
Avanti J-25 cetrifuge, Beckman Coulter (California,
USA). Arrays were scanned for Cy3 (green laser
532 nm; filter FM570-10) and Cy5 (red laser 635 nm;
filter FM665-12) at gain 35, resolution normal (10mm)
and line average 1 using Affymetrix 428 Array Scanner
(California, USA). In some experiments the actual Cy-dye
used for labelling treated versus control cDNA samples
was interchanged (referred to as “Dye-flipping” exper-
iments).
Analysis of cDNA Microarrays
The microarray data analysis packages, ImaGene and
GeneSight, from BioDiscovery (California, USA) were
used for gene expression profiling analyses. In this study
data for each gene were typically reported as an
“expression ratio” or as the base 2 logarithm of the
expression ratio of experiment to control. Normalisation
was fulfilled based on the total intensity by dividing mean
value of each spot by mean values of entire slide. Genes
were assumed to be up- or down-regulated if they revealed
an expression ratio of .2 and ,0.5 (or .1 and ,21 for
log2 transformed data), respectively.
One way analysis of variance (ANOVA) followed by
multiple comparison test (post-hoc) and/or unpaired two-
tailed t-test were used with an assumption of p value less
than 0.05 for significant differences using GraphPad prism
software.
TOXICOGENOMICS OF NON-VIRAL VECTORS 313
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Semi-quantitative RT-PCR
Standard RT-PCR reactions were performed using the
following primers: b-actin (forward: 50-GGC ATG GGT
CAG AAG GAT T-30 and reverse: 50-GGG GTG TTG
AAG GTC TCA AA-30, 254 bp); proteasomeb4 (forward:
50-CCA GGA CAG TTT TAC CGC AT-30 and reverse:
50-CAA GCA TGT CCA CAT AAC CG-30, 469 bp);
annexin 2 (forward: 50-CTC TAC ACC CCC AAG TGC
AT-30 and reverse: 50-TGA GAG AGT CCT CGT CGG
TT-30, 335 bp). The primers were designed using internet-
based primer design program Primer3 (http://
www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/).
The PCR thermocycling program was as follows:
denaturation at 948C for 30 s, annealing at 57–648C for
45 s and extension at 728C for 45 s through a total of 30
cycles. The PCR recipe was (for 25ml reaction): 8.5ml
RNase/DNase free ddH2O, 2.5ml Taq 10 £ buffer
containing MgCl2 (15 mM), 1ml MgCl2 (25 mM), 2ml
dNTPs (10mM), 2ml cDNA (100 ng/1ml), 0.02–0.5ml
forward and reverse primers (10 pmol/1 ml), and
0.1ml Qiagen Taq polymerase enzyme (5 Units/1ml).
The PCR products were electrophoresed through a 1.2%
agarose gel in the presence of 1mg ethidium bromide/1 ml
gel and visualised under UV light. As positive controls,
mouse brain and kidney total RNA were used whereas for
negative controls, RT-PCR reactions were performed
without the desired cDNA and/or primer. The density of
expressed bands was measured using the GS-700
densitometer (Bio-Rad Molecular analysis software,
Hemel, Hempstead, UK). The density of each spot was
subjected to local background density subtraction and
normalised to the density of the house keeping gene,
b-actin. The ratio of treated over untreated were
performed for two independent replicates.
Annexin V-FITC Flow Cytometric Assay for Apoptosis
Flow cytometry (fluorescence-activated cell sorting or
FACS) analysis was performed using annexin V-FITC to
detect the induction of early and/or late apoptosis in
CL-treated A431 cells as described (Zhang et al., 1997;
Peng et al., 2002). Briefly, CL-treated cells, untreated
control cells or, as a positive control for apoptosis, cells
treated with 5% DMSO were stored on ice following
removal of culture media and gently washed once with
PBS. Cells were then detached by gentle trypsinisation
using 0.5 ml 0.5 £ trypsin, and resuspended in the original
media to include detached cells in the suspension for
analysis. Approximately, 1 £ 106 cells were exposed to
10ml media binding reagent and 1.25ml Annexin V-FITC
and incubated in the dark for 15 min at room temperature.
Cell suspension was centrifuged at 1000g for 5 min, the
supernatant was discarded and the cells resuspended in
0.5 ml ice-cold 1 £ binding buffer. Finally, 10ml
propidium iodide was added and the cells were transferred
to FACS tubes (Fahrenheit, UK) and analysed immedi-
ately. Cell associated fluorescence distributions were
obtained from 20,000 events per cell sample through
a FL1 band-pass filter (FITC-Annexin V binding) and FL2
band-pass filter (Propidium iodide) using a FACScalibar
flow cytometer (BD Biosciences, Oxford, UK). The
fluorescence of cell populations was analysed using
validated analysis software, WinMDI 2.8 (http://facs.
scripps.edu/).
Assessment of DNA Damage by COMET Assay
COMETassays on Lipofectin-treated cells were performed
as described previously (Singh et al., 1988), with slight
modifications. In brief, a suspension of cells (5 £
105 cells/ml) in LMPA was prepared. A desired small
amount of the suspension was added into a dual-window
COMET slide (Trevigen, Gaithersburg, MD), such that
each window contained approximately 5–10 £ 105 cells
suspended in 75ml LMPA. Slides were then immersed
overnight in lysis solution (2.5 M NaCl, 100 mM
Na2EDTA, 10 mM Tris, 10% (v/v) DMSO, 1% Triton X-
100—pH 10). Slides were then exposed in an alkaline
solution (300 mM NaOH, 1 mM EDTA—pH . 13) for
30 min. The slides were placed for electrophoresis, using
alkaline solution, at 2 V/cm for 20 min. In order to
minimise the risk of LMPA lifting from the slide, all the
solutions were kept at 48C during the assay. Following
electrophoresis, the slides were neutralised using the
supplied neutralising buffer (Ikzus, Genoa), before being
fixed in ethanol and air dried. Slides were stained with
FLUO-Plus DNA stain (Ikzus, Genoa) and visualised on a
fluorescent microscope. Image data were analysed using
CASP software (Konca, 2003). To quantify the DNA
damage, olive tail moment (OTM, i.e. %DNA £ distance
of centre of gravity of DNA) and/or tail moment (TM, i.e.
%DNA £ tail length) of 30–50 cells from each window of
each slide were analysed. Replicates, 3–4, were performed
for all experiments including Lipofectin treated, SFM as
negative control and H2O2 treated (100mM) cells, the latter
serving as a positive control for induced DNA damage.
RESULTS
Lipofectin and Oligofectamine Induce Gene
Expression in A431 Cells as Shown by Microarray-based Gene Expression Profiling
In our previous studies with microarray-based gene
expression profiling (e.g. Petch et al., 2003), we learned
that the data handling of the large amount of information
generated was more manageable with low-density arrays.
Thus, in this study we used low-density cDNA
microarrays housing 200 gene spots containing 160
different genes that were replicated twice on the same
glass slide to yield a total of 400 gene spots per slide.
Human A431 epithelial cells treated with either Lipofectin
or Oligofectamine (at the recommended concentrations
routinely used for nucleic acid delivery) for 4 h were then
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grown in serum-containing medium for a total period of
24 h. Gene expression profiles of CL-treated cells were
compared to those of control cells that had been handled in
exactly the same manner except that no lipids were added
during the 4 h treatment period.
Figure 1 shows typical results from microarrays
hybridised with cy3- or cy-5 labelled cDNA from either
Lipofectin (left hand panels A to D) or Oligofectamine
(right hand panels E to H) treated A431 cells. Images
from their respective experiments for the untreated
control cell samples are also shown (panels B and G).
The top panels A and E show hybridisation intensity of
gene spots from a section of the microarray housing 4
grids of 50 gene spots each. A magnified image of the
selected grid is given in the middle panels B–C and F–G
for treated and untreated samples. Finally, in the lower
panels (D and H), gene expression images from treated
and untreated cells are superimposed. The high
reproducibility of the control spots can be observed by
noting the equal expression intensity of the control gene,
FIGURE 1 Representative cDNA microarrays showing the intensity of hybridisation for Lipofectin (LF)—or Oligofectamine (OF)-treated and untreatedsamples. Panels A and E show 200 gene spots (i.e. 4 £ 50 grids) for LF- and OF-treated cells, respectively. Panels B–C and F–G is the selected 50-genegrid for Cy3 or Cy5 labelled cDNA of treated or untreated samples. Panels D and H show superimposed Cy-dyes for treated and untreated samples.The white arrow heads represent the internal control spots of ubiquitin showing the equal intensities of this control protein within arrays.
TOXICOGENOMICS OF NON-VIRAL VECTORS 315
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ubiquitin, as marked by white arrowheads in all the
arrays shown. The expression of ubiquitin and several
other control gene spots were used as quality assurance
markers. Furthermore, in panels B, F, C and G, the
treated and controls for the two CL formulations are
shown with the Cy-dyes “flipped” to highlight the
reproducibility of our hybridisations as the ubiquitin
control highlighted gave equal intensities of spots
irrespective of the dye used in these experiments.
To achieve this the number of dye molecules per
nucleotide were carefully controlled during the labelling
procedure as described in the “Methods” section.
Arrays showing non-equal intensity or variable intensity
of control gene spots in replicates on the same slide or
between slides in dye-flipping experiments were nor-
mally rejected.
Figure 2 shows a scatter plot of all the genes represented
on the array for either Lipofectin or Oligofectamine-
treated cells. The gene spots highlighted in solid circles
represent those that change their expression by 2-fold or
more in the treated versus untreated controls. It can be
seen that a greater number of genes are highlighted for
Oligofecatmine compared to Lipofectin treatment with
a greater number of these genes falling into the range of
4-fold or greater change in expression. The exact identity
of these genes are given in Tables I and II.
Genes from different functional categories showed
expression changes in A431 epithelial cells upon treatment
of CL formulations. Of the genes represented on our array,
Lipofectin initiated up-regulation of the replication protein
a1 (rpa1) as shown in Table I, whereas Oligofectamine
induced the over-expression of 24 genes (Table II),
including genes involved in apoptosis such as bcl2-related
protein a1 (bcl2a1), Caspase 8 isoform c (casp8), heat
shock protein 70 (hsp70) and heat shock 60 kDa protein 1,
chaperonin (hspd1). Lipofectin triggered suppression of
some genes (Table I), e.g. endothelin receptor type b
isoform 2 (ednrb), Ribosomal protein 16 (rp16) and
endothelin receptor type b, isoform 1 (ednrb), where
Oligofectamine inhibited the expression of the genes
annexin a2 (anxa2), retinoid x receptor alpha (rxra) and
s100 calcium-binding protein a8 (s100a8).
Figure 3 represents gene expression data for
Oligofectamine as a single linkage Hierarchical Cluster-
ing Plot as performed on the GeneSight software. The
algorithm used subjects the expression intensity ratio of
treated versus untreated samples to single-linkage
Hierarchical clustering using Euclidean distance metric
analyses in order to arrange each gene with its related
group members exhibiting a similar ratio of change in
expression (Panel A). Gene-expression families were
colour coded for comparison, i.e. the over-expressed and
FIGURE 2 Scatter plots of gene expression changes induced by CL in A431 cells. Data represent Log2 transformed gene expression valuesfor Lipofectin (panel A) and Oligofectamine (panel B). The reference lines shown indicate the intensity ratios of genes exhibiting no changes (0),under-expression by 2-fold (21) or 4-fold (22) or over-expression by 2-fold (1) or 4-fold (2).
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TABLE I Lipofectin-induced gene expression changes in A431 epithelial cells
Accession No Gene function and annotation Ratio (T:UT) Changes
NM_002945 Replication protein a1 (70 kd); rpa1 2.02 +NM_007209 Ribosomal protein l35; rpl35 1.65 NCNM_005319 H1 histone family, member 2; h1f2 1.64 NCNM_033158 Hyaluronoglucosaminidase 2; hyal2 1.64 NCNM_021103 Thymosin beta tmsb10. 1.60 NCNM_021065 H2a histone family, member g; h2afg 1.56 NCNM_006086 Tubulin, beta, 4; tubb4 1.39 NCNM_004537 Nucleosome assembly protein 1-like 1; nap1l1 1.10 NCNM_001943 Desmoglein 2 preproprotein; dsg2 1.07 NCNM_002690 Polymerase (dna directed), beta; polb 1.04 NCNM_006716 Activator of s phase kinase; ask 1.02 NCNM_006013 Ribosomal protein l10; rpl10 1.02 NC– b-actin 1.00 NCNM_002623 Prefoldin 4; pfdn4 1.00 NCNM_006087 Tubulin, beta, 5; tubb5 0.96 NCNM_002796 Proteasome (prosome, macropain) subunit, beta type, 4; psmb4 0.96 NCNM_004039 Annexin a2; anxa2 0.94 NCNM_004383 C-src tyrosine kinase; csk 0.67 NCXM_016685 Similar to activator of s phase kinase (h. sapiens); loc143173 0.45 –NM_002416 Monokine induced by gamma interferon; mig 0.44 –NM_002064 Glutaredoxin (thioltransferase); glrx 0.36 –NM_004417 Dual specificity phosphatase 1; dusp1 0.31 –NM_001870 Mast cell carboxypeptidase a3 precursor; cpa3 0.23 –NM_006890 Carcinoembryonic antigen-related cell adhesion molecule 7; ceacam7 0.23 –NM_003991 Endothelin receptor type b isoform 2; ednrb 0.23 –NM_000970 Ribosomal protein l6; rpl6 0.21 –NM_000115 Endothelin receptor type b, isoform 1; ednrb 0.17 –
Ratio of treated: untreated cells is given and genes that are overexpressed (þ), underexpressed (2 ) or unchanged (NC) are indicated.
TABLE II Oligofectamine-induced gene expression changes in A431 epithelial cells
Accession No Gene function and annotation Ratio (T:UT) Changes
NM_004417 Dual specificity phosphatase 1; dusp1 3.69 +NM_001801 Cysteine dioxygenase, type i; cdo1 3.64 +XM_016685 Similar to activator of s phase kinase (h. sapiens); loc143173 3.11 +NM_002467 v-myc Myelocytomatosis viral oncogene homolog (avian); myc 2.97 +NM_004049 bcl2-Related protein a1; bcl2a1 2.93 +NM_002424 Matrix metalloproteinase 8 preproprotein; mmp8 2.90 +NM_033356 Caspase 8, isoform c; casp8 2.89 +NM_003991 Endothelin receptor type b isoform 2; ednrb 2.63 +NM_003875 Guanine monophosphate synthetase; gmps 2.52 +NM_003195 Transcription elongation factor a (sii), 2; tcea2 2.46 +NM_002698 Pou domain, class 2, transcription factor 2; pou2f2 2.46 +NM_002623 Prefoldin 4; pfdn4 2.39 +NM_033158 Hyaluronoglucosaminidase 2; hyal2 2.28 +L12723 Heat shock protein 70; hsp70 2.22 +NM_000245 Met proto-oncogene precursor; met 2.21 +NM_006716 Activator of s phase kinase; ask 2.20 +NM_006235 Pou domain, class 2, associating factor 1; pou2af1 2.18 +NM_001983 Excision repair cross-complementing rodent repair deficiency, ercc1 2.16 +NM_000994 Ribosomal protein l32; rpl32 2.11 +NM_004094 Eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kd); eif2s1 2.10 +NM_000698 Arachidonate 5-lipoxygenase; alox5 2.09 +NM_002849 Protein tyrosine phosphatase, receptor type, r; ptprr 2.03 +NM_002156 Heat shock 60 kd protein 1 (chaperonin); hspd1 2.02 +NM_021067 Kiaa0186 gene product; kiaa0186 2.01 +NM_004383 c-src tyrosine kinase; csk 1.94 NC– b-actin 1.08 NCNM_001943 Desmoglein 2 preproprotein; dsg2 1.04 NCNM_001469 Thyroid autoantigen 70 kd (ku antigen); g22p1 1.03 NCNM_001274 chk1 checkpoint homolog (s. pombe); chek1 1.00 NCNM_002796 Proteasome (prosome, macropain) subunit, beta type, 4; psmb4 0.70 NCNM_006087 Tubulin, beta, 5; tubb5 0.57 NCNM_005566 Ldha 0.54 –NM_004039 Annexin a2; anxa2 0.50 –NM_002957 Retinoid £ receptor, alpha; rxra 0.49 –
Ratio of treated: untreated cells is given and genes that are overexpressed (þ), underexpressed (2 ) or unchanged (NC) are indicated.
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under-expressed genes appear in red and green,
respectively, with the relative ratio reflected by the
intensity of the colour. Example genes from these
different expression families are magnified into groups
and single genes of interest highlighted (Panel B). The
purpose of such clustering is that this methodology
allows classification of genes by their relative expression
ratios and thus allows any patterns to be easily
visualised. For instance, in the examples given in
Panel B it can be seen that Oligofectamine treatment of
epithelial cells resulted in overexpression (red bars) of
several genes linked with apoptosis including casp8,
bcl2a1 and hsp70.
Semi-quantitative Reverse Transcriptase Polymerase
Chain Reaction (RT-PCR)
The gene expression data obtained with cDNA micro-
arrays was validated by semi-quantitative RT-PCR.
Figure 4 shows the expression of b-actin, proteasome
beta 4 and annexin 2 as example genes. The mRNA
expression of these genes for untreated controls and CL
treated samples of A431 cells (panel A), and their
normalised ratio represented as an histogram (panel B).
The expression ratio of Proteasome beta 4 and annexin 2
relative to actin as obtained by RT-PCR correlated well
and exhibited similar trends to those obtained by cDNA
microarray analysis (see Tables I and II).
Impact of CL-induced Gene Changes on Apoptosis and
DNA Damage
In an attempt to understand the functional consequences
of the gene changes induced by Oligofectamine and
Lipofectin in A431 cells we investigated their effect on the
apoptosis and DNA damage status of these cells especially
as some of the genes altered were known to be associated
with programmed cell death. Figure 5 shows that
CL-treatment of A431 cells significantly altered the
FIGURE 3 Single-linkage hierarchical clustering of gene expression induced by Oligofectamine in A431 cells. Gene-expression families were colourcoded for comparison, i.e. the over-expressed or under-expressed genes appear in red or green, respectively, with the relative ratio reflected by theintensity of the colour (see Panel A inset). Panel A is the clustered representation for entire array. Panel B is the selected examples of genes in the clustersof over-expressed, no changes and under-expressed gene families.
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apoptosis status of these cells as assessed by annexin
V-FITC flow cytometry analyses. Treatment of cells with
5% DMSO, as a positive inducer of apoptosis, led to a
marked increase in the proportion of cells entering early
(e.g. indicative of membrane damage) and late (e.g.
indicative of membrane and DNA damage) apoptosis can
be observed in Panels B and E. Lipofectin treatment
induced a significant increase in the number of late
apoptotic cells and a non-significant increase in early
apoptotic cells (( p . 0.05); Panels C and E) whereas
Oligofectamine induced a significant increase in the
number of cells undergoing early apoptosis and a non-
significant increase in cells in late apoptosis (( p . 0.05);
Panels D and E). These results confirm that CL treatment
leads to an altered apoptosis status of cells.
Focusing on Lipofectin, the most widely used of the CL
lipids, we next examined its effect on inducing DNA
damage. Using the same concentration of Lipofectin that
induced the above mentioned gene expression changes and
the enhanced entry of cells to late apoptosis (shown above)
we used the single cell electrophoresis (the so called
COMET) assay to assess the degree of DNA damage. This
assay is widely used for the study of DNA damage in cells
(Singh et al., 1988; Schindewolf et al., 2000).
Figure 6 shows the typical results obtained with the
COMET assay of A431 cells treated with Lipofectin or
hydrogen peroxide, H2O2, a positive control that is known
to induce DNA damage. Panel C shows the typical diffuse
migration (in the shape of an astronomical comet) of
damaged DNA around each of the single cells during
electrophoresis of samples treated with the positive control.
In contrast, statistical analysis showed no significant
( p . 0.05) difference between untreated and Lipofectin-
treated A431 cells (Fig. 6) implying that the observed gene
expression changes induced by this CL were not associated
with a marked increase in DNA damage.
DISCUSSION
The use of viral vectors for ex vivo and in vivo gene
therapy typically results in high transfection efficiencies
but this advantage is often offset by important safety
concerns relating to immunological and toxicological
responses in cells, animals and human patients (for
reviews see Somia and Verma, 2000; Zallen, 2000).
Indeed, recent gene therapy trials were halted due to
unexplained toxicity surrounding the death of a patient
FIGURE 4 Semi-quantitative RT-PCR for b-actin, annexin2 and Proteasome beta 4. Panel A shows the expression of the amplified fragments in 1%agarose gel in the presence of ethidium bromide. Panel B presents histograms of the relative expressions of these genes normalised to b-actin andrepresents a ratio of treated over untreated. Asterisk represents a significant ( p , 0.05) gene expression change.
TOXICOGENOMICS OF NON-VIRAL VECTORS 319
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undergoing ornithine transcarbamylase gene therapy in a
Phase 1 clinical trial using second generation adenoviral
vectors (Somia and Verma, 2000). Non-viral vectors,
though often less efficient as gene transfection agents,
have been investigated as potentially safer alternatives
for the delivery of genes. Of these the CL lipid
formulations are the widely used. By virtue of their net
positive charge, which is important for binding to the
nucleic acid and association with the negatively charged
cell membrane to achieve high uptake and transfection
efficiency, these CL formulations can also be cytotoxic
(Filion and Phillips, 1997; Pedrosa de Lima et al., 2001).
However, little is known about the extent or nature of the
gene changes induced by CL in target cells. Here, we
report on the toxicogenomics and geno-compatibility of
these lipids in human epithelial cells by studying
FIGURE 5 The influence of Lipofectin- or Oligofectamine-induced gene expression changes on apoptosis in A431 cells using flow cytometric AnnexinV-FITC apoptosis assay. Panels A–D show A431 cell population for untreated (control), DMSO (5%) Lipofectin-treated (LF) and Oligofectamine-treated (OF) cells, respectively. In each panel, C, N, EA and LA represent abbreviations for control, necrotic, early apoptotic and late apoptotic,respectively. Panel E represents a histogram of the relative proportion of the cell population undergoing EA or LA. Significant differences ( p , 0.05)are as follow: ‡LA vs EA in LF-treated cells; † EA vs LA in OF-treated cells; *EA of OF-treated vs EA of untreated cells; **EA of vs LA inOF-treated cells.
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the CL-induced gene expression changes using cDNA
microarrays.
At concentrations routinely used to obtain
efficient delivery of genes, oligonucleotides and gene
silencing siRNAs, we found that both Lipofectin and
Oligofectamine-induced marked gene changes in human
epithelial cells (Figs. 1–3; Tables I and II). In the case
of Oligofectamine, this CL elicited the expression of up to
27 genes representing 16% of the genes on the microarray.
The affected genes were functionally known to be
involved in various cellular processes, including cell
proliferation, differentiation and programmed cell death
(apoptosis). Of the genes involved in apoptosis, these
lipids triggered the over- or under-expression of some
important genes such as bcl2-related protein a1 (bcl2a1),
caspase 8, isoform c (casp8), heat shock protein 70
(hsp70) and 60 (hspd1), annexin a2 (anxa2) and tubulin
beta 5 (tubb5). For example, the Oligofectamine-induced
up-regulation of caspase-8 suggests that this CL initiates
the activation of procaspases and caspases, a process that
would normally result in activation of a series of apoptotic
signalling cascades involving, amongst others, the
electron carrier protein cytochrome C, adaptor protein
Apaf-1, Bcl-2 family, p53 and various transcription factors
(Sedlakova et al., 1999; Kanduc et al., 2002). However, as
we only used low-density microarrays (for ease of data
handling) in the present study, many of the downstream
genes were not represented on our array. Thus, the use of
higher density or more focused custom arrays housing all
apoptotic genes may be more useful to explore the exact
nature of these changes. Further studies using such arrays
are planned.
The gene expression changes observed for CL treated
cells ranged in magnitude from more than 5-fold under-
expressed to almost 4-fold over-expressed (see Tables I
and II). This degree of change was similar to that observed
for downstream signalling molecules when A431 cells
were treated with an inhibitor of epidermal growth factor
receptor expression (Petch et al., 2003). Thus, these
changes are reflective of what might happen during
normal signalling processes such as those following
switching on or off of receptor signalling cascades as
exemplified by EGFR. Furthermore, the observed gene
changes were reproducibly seen on replicate arrays even
during dye-flipping experiments. Thus, these findings
coupled with independent validation of the array data with
RT-PCR analyses further confirmed the observed gene
changes upon CL treatment of A431 cells were real
changes that may have important functional consequences
in cells.
In an attempt to study the functional consequences of
the altered gene expression arising from CL treatment,
we examined the effects of Lipofectin or Oligofectamine
on apoptosis and DNA damage in epithelial cells.
FIGURE 6 The single cell gel (SCG) electrophoresis (COMETassay) for DNA damage detection induced by CL in A431 cells. Lipofectin-treated cells(2mg/cm2 of cultured cells) showed no significant difference compare to untreated, whereas the positive control hydrogen peroxide-treated cells(100mM) revealed the characteristic comet around cells indicative of DNA damage (Panel A–C). Panels E shows the expressed DNA damagerepresented in the olive tail moment (OTM, i.e. %DNA £ distance of centre of gravity of DNA).
TOXICOGENOMICS OF NON-VIRAL VECTORS 321
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Consistent with the nature of the gene changes described
above, increased apoptosis was observed in cells treated
with the lipids compared to untreated cells (Fig. 5). In the
case of Oligofectamine, this involved an increased number
of cells in early apoptosis whereas for Lipofectin this was
associated with an increased population of cells in late
apoptosis. However, the latter did not appear to involve
extensive DNA damage (Fig. 6) further implying that
apoptosis in Lipofectin-treated cells was associated with
mostly membrane damage and not with extensive DNA
scission.
Of interest, our data suggested that gene changes, and
thus, geno-toxicity, elicited by the CL formulations were
lipid-dependent as no extensive overlap was observed in
those genes exhibiting expression changes upon either
Lipofectin or Oligofectamine treatment. However, this
may simply be reflective of the low number of genes
present on the arrays used; the likelihood of finding
common gene changes would be greater for high density
arrays. Alternatively, it may be a function of the
responsiveness of these cells to the lipids used. Indeed,
it is well known that the transfection capability of these
CL formulations is both cell type and lipid dependent
(for reviews see Akhtar et al., 2000; Hirko et al., 2003),
thus it is not surprising they have markedly different
effects on the gene expression profile of a given cell type.
Also for example, Filion and Phillips (1997) reported high
toxicity of some CL in phagocytic cells (macrophages and
U937 cells), but not in non-phagocytic T lymphocytes.
Thus, for a detailed characterisation of the toxicogenomics
of these lipid delivery systems, it is, therefore, likely that
gene expression patterns/profiles will need to be
determined in many cell types of interest.
In addition, CL formulations may also affect the
outcome of gene therapy experiments. In studies with
antisense nucleic acids, siRNA or plasmid DNA where
often only a single desired genetic change is sought, it is
possible that gene changes induced by the lipid delivery
system itself could exacerbate, attenuate or even mask the
desired effects of the nucleic acid. The net effect is likely
to be determined by many factors but will include the
relative magnitude of the over- or under-expression of the
genes of interest. Potentially, this could be of particular
concern where the gene targeted by the nucleic acid is the
same as the one directly affected by the CL formulation
used. Furthermore, the presence of nucleic acid in the CL
formulation may modulate the induced gene expression
changes and thus needs to be determined in such studies.
In conclusion, our studies highlight the fact that
inadvertent gene expression changes can be induced by
the CL delivery formulation itself and that these
alterations in gene expression can lead to downstream
functional consequences such as increased apoptosis.
Thus, gene expression changes that, ultimately, lead to
changes in cell phenotype will have important safety
implications for the use of these non-viral vectors in gene-
based therapies. Also, the induced or inadvertent gene
expression changes should be taken into consideration
in gene therapy or gene silencing experiments using
CL formulations where they may potentially mask
or interfere with the desired genotype and/or phenotype
end-points.
References
Aissaoui, A., Oudrhiri, N., Petit, L., Hauchecorne, M., Kan, E., Sainlos,M., Julia, S., Navarro, J., Vigneron, J.P., Lehn, J.M. and Lehn, P.(2002) “Progress in gene delivery by cationic lipids: guanidinium-cholesterol-based systems as an example”, Curr. Drug Target. 3,1–16.
Akhtar, S., Hughes, M.D., Khan, A., Bibby, M., Hussain, M., Nawaz, Q.,Double, J. and Sayyed, P. (2000) “The delivery of antisensetherapeutics”, Adv. Drug Deliv. Rev. 44, 3–21.
Audouy, S.A., de Leij, L.F., Hoekstra, D. and Molema, G. (2002) “In vivocharacteristics of cationic liposomes as delivery vectors for genetherapy”, Pharm. Res. 19, 1599–1605.
Campbell, P.I. (1983) “Toxicity of some charged lipids used in liposomepreparations”, Cytobios 37, 21–26.
Canonico, A.E., Plitman, J.D., Conary, J.T., Meyrick, B.O. and Brigham,K.L. (1994) “No lung toxicity after repeated aerosol or intravenousdelivery of plasmid-cationic liposome complexes”, J. Appl. Physiol.77, 415–419.
Duzgunes, N., De Ilarduya, C.T., Simoes, S., Zhdanov, R.I., Konopka, K.and Pedroso de Lima, M.C. (2003) “Cationic liposomes for genedelivery: novel cationic lipids and enhancement by proteins andpeptides”, Curr. Med. Chem. 10, 1213–1220.
Dykxhoorn, D.M., Novian, C.D. and Sharp, P.A. (2003) “Killing themessenger: short RNAs that silence expression”, Nat. Rev. 4,257–467.
Filion, M.C. and Phillips, N.C. (1997) “Toxicity and immunomodulatoryactivity of liposomal vectors formulated with cationic lipids towardimmune effector cells”, Biochim. Biophys. Acta 1329, 345–356.
Hegde, P., Qi, R., Abernathy, K., Gay, C., Dharap, S., Gaspard, R.,Hughes, J.E., Snesrud, E., Lee, N. and Quackenbush, J. (2000)“A concise guide to cDNA microarray analysis”, Biotechniques 29,548–554, See also page 556.
Hirko, A., Tang, F. and Hughes, J.A. (2003) “Cationic lipid vectors forplasmid DNA delivery”, Curr. Med. Chem. 10, 1185–1193.
Hughes, M.D., Hussain, M., Nawaz, Q., Petch, A.K., Sayyed, P. andAkhtar, S. (2001) “Cellular delivery of antisense oligonucleotides andribozymes”, Drug Discov. Today 6(6), 303–315.
Juliano, R.L. and Akhtar, S. (1992) “Liposomes as a drug deliverysystem for antisense oligonucleotides”, Antisense Res. Dev. 2,165–176.
Kanduc, D., Mittelman, A., Serpico, R., Sinigaglia, E., Sinha, A.A.,Natale, C., Santacroce, R., Di Corcia, M.G., Lucchese, A., Dini, L.,Pani, P., Santacroce, S., Simone, S., Bucci, R. and Farber, E. (2002)“Cell death: apoptosis versus necrosis (review)”, Int. J. Oncol. 21,165–170.
Kerr, D. (2003) “Nature reviews cancer, clinical development of genetherapy for colorectal cancer”, Nat. Rev. Cancer 3(8), 615–622.
Konca, K., Lankoff, A., Banasik, A., Lisowska, H., Kuszewski, T.,Gozdz, S., Koza, Z. and Wojcik, A. (2003) “A cross-platform publicdomain PC image-analysis program for the comet assay”, Mutat Res534, 15–20.
McManus, M.T. and Sharp, P.A. (2002) “Gene silencing in mammals bysmall interfering RNAs”, Nat. Rev. 3, 737–747.
Nagahiro, I., Mora, B.N., Boasquevisque, C.H., Scheule, R.K. andPatterson, G.A. (2000) “Toxicity of cationic liposome-DNA complexin lung isografts”, Transplantation 69, 1802–1805.
Pedroso de Lima, M.C., Simoes, S., Pires, P., Faneca, H. andDuzgunes, N. (2001) “Cationic lipid-DNA complexes in genedelivery: from biophysics to biological applications”, Adv. DrugDeliv. Rev. 47, 277–294.
Peng, L., Jiang, H. and Bradely, C. (2002) “Detection of B lymphomacells undergoing apoptosis by Annexin-V assay”, Chin. Med. Sci. J.17, 17–21.
Petch, A.K., Sohail, M., Hughes, M.D., Benter, I., Darling, J.,Southern, E.M. and Akhtar, S. (2003) “Messenger RNA expressionprofiling of genes involved in epidermal growth factor receptorsignalling in human cancer cells treated with scanning array-designed antisense oligonucleotides”, Biochem. Pharmacol. 66,819–830.
Y. OMIDI et al.322
Jour
nal o
f D
rug
Tar
getin
g D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y U
nive
rsita
et Z
ueri
ch o
n 07
/12/
14Fo
r pe
rson
al u
se o
nly.
Pleyer, U., Groth, D., Hinz, B., Keil, O., Bertelmann, E., Rieck, P. andReszka, R. (2001) “Efficiency and toxicity of liposome-mediated gene transfer to corneal endothelial cells”, Exp. Eye Res.73, 1–7.
Scheule, R.K., St George, J.A., Bagley, R.G., Marshall, J., Kaplan, J.M.,Akita, G.Y., Wang, K.X., Lee, E.R., Harris, D.J., Jiang, C., Yew, N.S.,Smith, A.E. and Cheng, S.H. (1997) “Basis of pulmonary toxicityassociated with cationic lipid-mediated gene transfer to themammalian lung”, Hum. Gene. Ther. 8, 689–707.
Schindewolf, C., Lobenwein, K., Trinczek, K., Gomolka, M.,Soewarto, D., Fella, C., Pargent, W., Singh, N., Jung, T. and Hrabede Angelis, M. (2000) “Comet assay as a tool to screen for mousemodels with inherited radiation sensitivity”, Mamm. Genome 11,552–554.
Sedlakova, A., Kohut, A. and Kalina, I. (1999) “Biochemical changes inapoptosis and methods for their determination (review)”, Cesk.Fysiol. 48, 107–118.
Singh, N.P., McCoy, M.T., Tice, R.R. and Schneider, E.L. (1988) “A simpletechnique for quantitation of low levels of DNA damage in individualcells”, Exp. Cell Res. 175, 184–191.
Somia, N. and Verma, I.M. (2000) “Gene therapy: trials and tribulations”,Nat. Rev. Genet. 1, 91–99.
Tarahovsky, Y.S. and Ivanitsky, G.R. (1998) “Liposomes in gene therapy.Structural polymorphism of lipids and effectiveness of genedelivery”, Biochemistry (Mosc. 63, 607–618.
Whitehouse, A. (2003) “Herpesvirus saimiri: a potential gene deliveryvector (Review)”, Int. J. Mol. Med. 11, 139–148.
Zallen, D.T. (2000) “US gene therapy in crisis”, Trend Genet. 16, 272–275.Zelphati, O. and Szoka, F.C. Jr. (1996) “Mechanism of oligonucleotide
release from cationic liposomes”, Proc. Natl Acad. Sci. USA 93(21),11493–11498.
Zhang, G., Gurtu, V., Kain, S.R. and Yan, G. (1997) “Early detection ofapoptosis using a fluorescent conjugate of annexin V”, Biotechniques23, 525–531.
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