Báo cáo khoa học: Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells pdf

Transfection with 4-hydroxynonenal-metabolizing glutathione
S
-transferase isozymes leads to phenotypic transformation
and immortalization of adherent cells
Rajendra Sharma
1,
*, David Brown
1,
*, Sanjay Awasthi
2
, Yusong Yang
1
, Abha Sharma
1
, Brad Patrick
1
,
Manjit K. Saini
1
, Sharda P. Singh
3
, Piotr Zimniak
3
, Shivendra V. Singh
4
and Yogesh C. Awasthi
1
1
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, TX, USA;
2
Department of Chemistry and Biochemistry, University of Texas at Arlington, TX, USA;

3
Department of Pharmacology
and Toxicology and Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences,
and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA;
4
Department of Pharmacology,
University of Pittsburgh Cancer Center, PA, USA
4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end
products of lipid peroxidation, has been shown to induce
apoptosis in a variety of cell lines. It appears to modulate
signaling processes in more than one way because it has been
suggested to have a role in signaling for differentiation and
proliferation. We show for the first time that incorporation
of 4-HNE-metabolizing glutathione S-transferase (GST)
isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and
CCL-75, by either cDNA transfection or microinjection of
active enzyme, leads to their transformation. The dramatic
phenotypic changes due to the incorporation of hGSTA4-4
include rounding of cells and anchorage-independent rapid
proliferation of immortalized, rounded, and smaller cells.
Incorporation of the inactive mutant of hGSTA4-4 (Y212F)
in cells by either microinjection or transfection does not
cause transformation, suggesting that the activity of
hGSTA4-4 toward 4-HNE is required for transformation.
This is further confirmed by the fact that mouse and Dro-
sophila GST isozymes (mGSTA4-4 and DmGSTD1-1),
which have high activity toward 4-HNE and subsequent
depletion of 4-HNE, cause transformation whereas human
GST isozymes hGSTP1-1 and hGSTA1-1, with minimal
activity toward 4-HNE, do not cause transformation. In

cells overexpressing active hGSTA4-4, expression of trans-
forming growth factor b1, cyclin-dependent kinase 2, pro-
tein kinase C bII and extracellular signal regulated kinase is
upregulated, whereas expression of p53 is downregulated.
These studies suggest that alterations in 4-HNE homeostasis
can profoundly affect cell-cycle signaling events.
Keywords: 4-hydroxy-2-trans-nonenal; glutathione S-trans-
ferase; lipid peroxidation; oxidative stress; transformation.
Oxidative stress causes generation of reactive oxygen
species, which leads to the onset of lipid peroxidation [1].
4-Hydroxynonenal (4-HNE) is one of the end products of
this process [2]. In recent years there has been an increasing
interest in the role of 4-HNE in signaling mechanisms
[3–12]. There are reports suggesting that 4-HNE can cause
cell cycle arrest [2], apoptosis [3,6,7,12], differentiation [12]
or proliferation [11,12] in different cell types in a concen-
tration-dependent manner. These seemingly opposite effects
of 4-HNE on cell cycle signaling (e.g. cell cycle arrest and
apoptosis vs. proliferation) are intriguing. If 4-HNE does
indeed differentially affect signal transduction in a concen-
tration-dependent manner, the regulation of 4-HNE homeo-
stasis may be important for cell cycle signaling. It is
inherently difficult to characterize the functional conse-
quences of changes in intracellular 4-HNE concentration
because 4-HNE is formed by lipid peroxidation, mostly an
uncontrolled nonenzymatic process. In this study, we
circumvented this problem by regulating 4-HNE concen-
tration through its metabolism, and investigated the effect
of altered 4-HNE homeostasis on proliferation and cell
cycle signaling in two different adherent cell lines.

To test the hypothesis that 4-HNE may be a determinant
in cell cycle regulation, we stably transfected the human lens
epithelial cell line (HLE B-3) with cDNA for human
glutathione S-transferase (GST, EC 2.5.1.18) isozyme
hGSTA4-4. This isozyme conjugates GSH to 4-HNE with
high efficiency [13], and cells overexpressing it, or similar
enzymes [14], have lower steady-state levels of 4-HNE [12].
In accordance with accepted convention [15], we refer to the
gene and the dimeric enzyme as hGSTA4 and hGSTA4-4,
Correspondence to Y. C. Awasthi, 551 Basic Science Building,
Department of Human Biological Chemistry and Genetics, University
of Texas Medical Branch, Galveston, TX 77555-0647, USA.
Fax: + 1 409 772 6603, Tel.: + 1 409 772 2735,
E-mail:
Abbreviations: 4-HNE, 4-hydroxy-2-trans-nonenal; GST, glutathione
S-transferase; HLE B-3, human lens epithelial cell; CCL-75, human
lung fibroblast cell; JNK, c-Jun N-terminal kinase; OG-dextran,
Oregon green 488-dextran; GFP, green fluorescent protein; eGFP,
enhanced green fluorescent protein; GS-HNE, glutathione conjugate
of 4-HNE.
Enzyme: glutathione S-transferase (GST; EC 2.5.1.18).
*These authors contributed equally to this work.
(Received 22 January 2004, revised 24 February 2004,
accepted 2 March 2004)
Eur. J. Biochem. 271, 1690–1701 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04067.x
respectively. Surprisingly, the clonal lines of HLE B-3/
hGSTA4 transfectants overexpressing enzymatically active
hGSTA4-4 acquired a transformed phenotype with time.
We then examined whether an adherent cell line other than
HLE B-3 would also be affected by hGSTA4 transfection

and exhibit a similar transformed phenotype. Furthermore,
to correlate specifically the effects of hGSTA4 transfection
with the increased metabolism and depletion of 4-HNE, we
investigated the effect of transfection with mutant hGSTA4
devoid of GST activity towards 4-HNE. Finally, we
compared the effect of microinjection of different GST
isozymes from several species into HLE B-3 cells to rule out
nonspecific effects of GST overexpression of active or
mutant hGSTA4-4 protein. The results show that lowering
intracellular levels of 4-HNE by incorporation of active
hGSTA4-4, by either transfection or microinjection, led to
phenotypic transformation of attached cells into rounded,
smaller cells which acquired immortality and grew rapidly in
an anchorage-independent manner.
Experimental procedures
Cell culture
HLE B-3 cells were a gift from U. P. Andley (Department
of Ophthalmology and Visual Sciences, Washington Uni-
versity at St Louis, MO, USA). The cells were received on
passage no. 14 and were maintained in minimal essential
medium containing 20% fetal bovine serum and
50 lgÆmL
)1
gentamicin at 37 °Cin5%CO
2
.Humanlung
fibroblast cell line, CCL-75, obtained from ATCC (Man-
assas, VA, USA) was maintained in minimal essential
medium containing 10% fetal bovine serum, 1 m
M

sodium
pyruvate and 10 m
M
nonessential amino acids.
Antibodies
Polyclonal antibodies were developed against recombinant
hGSTA4-4 in chicken as described previously [16]. All other
antibodies were from commercial sources.
Preparation of recombinant hGSTA4-4 and other
GST isozymes
hGSTA4-4 was expressed in Escherichia coli and purified as
described previously [16]. The purity of the enzyme was
confirmed by SDS/PAGE; a single band at 26 kDa was
recognized by hGSTA4-4 antibodies on Western blots.
Activity of the purified enzyme using 1-chloro-2,4-dinitro-
benzene and 4-HNE as substrates was measured as
described previously [6]. Methods for preparation of
recombinant GST isozymes mGSTA4-4 [14], Drosophila
melanogaster DmGSTD1-1 [17], hGSTA1-1 [18] and
hGSTP1-1 [19] have been described previously.
Preparation of hGSTA4-4 eukaryotic expression
constructs
The hGSTA4 ORF was amplified by PCR from the bacterial
expression vector pET-30a[+]/hGSTA4, and subcloned into
the pTarget-T mammalian expression vector (Promega).
The hGSTA4 insert was confirmed by sequencing.
Transfection of HLE B-3 cells with p-Target-hGSTA4
expression vector
HLE B-3 cells (2 · 10
5

) at passage no. 18 were plated in
60 mm dishes in complete growth medium. When the cells
reached nearly 80% confluency, the medium was changed,
and the cells were transfected 3–4 h later with 6 lg plasmid
using the Profection mammalian transfection kit (Promega)
according to the manufacturer’s protocol. After 4 h, the
cells were treated with 10% dimethyl sulfamethoxazole in
minimal essential medium for 30 s. After dimethyl sulfa-
methoxazole shock, the cells were allowed to recover in
complete growth medium for 48 h. Stable transfectants
were selected in 200 lgÆmL
)1
G418 by the dilution method
in 96 well plates. Wells containing single cells were marked,
and growth in these wells was monitored daily. Expression
of hGSTA4-4 protein was ascertained by Western blot
analysis.
Site-directed mutagenesis of hGSTA4-4
The Y212F mutation was introduced in both the bacterial
and the mammalian hGSTA4-4 expression vectors using
the Quickchange site-directed mutagenesis kit (Stratagene,
La Jolla, CA, USA) with the mutagenic sense primer
5¢-CCTGATGAATT
TTCGTGAGAACCGT (mutation
underlined) and the complementary antisense primer. In
this paper, hGSTA4-4(Y212F) is referred to as mut-
hGSTA4-4.
Immunohistochemical localization studies
Immunofluorescence studies on adherent HLE B-3 and
CCL-75 cells (wild-type, empty-vector-transfected and

mut-hGSTA4-transfected) were carried out by seeding
1 · 10
4
cells on to coverslips. Next day, the coverslips with
attached cells were washed in NaCl/P
i
(pH 7.0) three times
(5mineach)andfixedin4%paraformaldehydesolution
prepared in NaCl/P
i
(pH7.4)for15minatroom
temperature. The fixed cells were washed three times with
NaCl/P
i
, permeabilized in cold methanol ()20 °C) for
30 s, treated with sodium borohydride (0.5 mgÆmL
)1
)for
15 min to reduce aldehyde groups, and washed three times
with NaCl/P
i
(5 min each). The cells were then incubated
with blocking buffer (1% BSA + 1% goat serum in
NaCl/P
i
) for 2 h at room temperature in a humidified
chamber, and incubated with primary antibodies against
hGSTA4-4 developed in chicken (1 : 200 dilution pre-
paredin1%BSAinNaCl/P
i

) overnight at 4 °C. Cells
were washed three times in NaCl/P
i
and then incubated
with Alexa fluor 488 fluorescein isothiocyanate-conjugated
anti-chicken secondary IgG (Molecular Probes; 1 : 200,
diluted in 1% BSA in NaCl/P
i
) for 2 h at room
temperature in a humidified chamber. Cells were washed
three times with NaCl/P
i
, mounted on slides with 50%
glycerol in NaCl/P
i
, and visualized under a fluorescence
microscope (Nikon Eclipse 600). The cells treated with
preimmune chicken IgY were used as negative controls.
Slides for suspension culture of hGSTA4-transfected and
transformed HLE B-3 cells were prepared by centrifu-
ging the cells on polylysine-coated slides in a cytospin at
28 g.
Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur. J. Biochem. 271) 1691
In situ
detection of apoptosis
To detect cells undergoing apoptosis during the course of
microinjection experiments, we performed immunolocali-
zation of cleaved caspase-3 by using monoclonal antibod-
ies against cleaved caspase-3. After cytospinning the cells
at 28 g for5min,thecellswerefixedin4%paraformal-

dehyde (15 min) and washed three times in NaCl/P
i
.The
cells were permeabilized by incubation in 0.1% Triton
X-100 for 2 min, washed with NaCl/P
i
,treatedwith
blocking buffer for 2 h at room temperature in a
humidified chamber, and then incubated with cleaved
caspase-3 IgG (1 : 100 dilution prepared in 1% BSA)
overnight at 4 °C. Cells were washed three times in NaCl/
P
i
and then incubated with mouse tetramethyl rhodamine
isothiocyanate-conjugated secondary antibodies (1 : 500)
for 2 h. After the cells had been washed and mounted as
described above, the expression of cleaved caspase-3, a
marker of apoptosis, was ascertained by observing the
cells under a fluorescence microscope.
Determination of intracellular levels of malondialdehyde
and 4-HNE
Lipid peroxide levels as determined by malondialdehyde
and 4-HNE concentrations in hGSTA4-transfected and
control HLE B-3 cells were determined using the Biotech
LPO-586
TM
kit (Oxis International, Portland, OR, USA)
according to the manufacturer’s protocol as described
previously [6].
SDS/PAGE and Western blot analysis

For checking the expression of hGSTA4-4 by Western blot
analysis, cells (1 · 10
6
) were lysed in 10 m
M
potassium
phosphate buffer, pH 7.0, containing 1.4 m
M
2-mercapto-
ethanol, sonicated on ice for 30 s, and centrifuged at
28 000 g for 30 min. Buffer-soluble proteins (25 lg) present
in the supernatants were mixed with Laemmeli’s sample
buffer [20] and loaded in the wells of gels containing 12%
polyacrylamide. Proteins resolved on SDS/polyacrylamide
gels were transferred to nitrocellulose or poly(vinylidene
difluoride) membranes, and the blots probed by using
hGSTA4-4 antibodies developed in chicken as primary
antibodies, and secondary antibodies as horseradish per-
oxide-conjugated anti-chicken IgG developed in goat. Blots
were developed by West Pico-chemiluminescence’s reagent
(Pierce). To check the expression of p53, transforming
growth factor b1, cyclin-dependent kinase 2 and protein
kinase C bII proteins in HLE B-3 cells, Western blot
analyses were performed by preparing whole cell extracts in
RIPA buffer [20 m
M
Tris/HCl, pH 7.4, 150 m
M
NaCl, 1%
Nonidet P40, 1 m

M
EDTA, 1 m
M
NaF, 1 m
M
sodium
vanadate, 1 m
M
phenylmethanesulfonyl fluoride and pro-
tease inhibitor cocktail (Sigma Chemical Co)]. For these
analyses extracts containing 100 lg protein were used for
each sample.
Cell growth analysis
The growth kinetics of HLE B-3 cells and their transfect-
ants was measured both by manual cell count using a
hemocytometer (after trypsinization in the case of adherent
cells) and by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-
zolium bromide analysis as described previously [18].
Assay of soft agar colony formation
This was performed as described previously [21]. Briefly,
5 000 cells per dish, mixed in 0.35% agarose/complete
medium, were seeded on to 0.7% agarose/complete medium
bottom layer. The Petri dishes were incubated at 37 °Cand
a drop of medium was added every 3 days. Four weeks
later, cells were stained with 0.5% crystal violet (Sigma) in
20% methanol for 2 h, and colonies were counted under a
microscope.
Microinjection of cells
Protein sample preparation. Immediately before injection,
the recombinant hGSTA4-4 protein (wild-type or mutant)

was dialyzed against injection buffer (114 m
M
KCl, 0.5 m
M
K
2
HPO
4
and 5.5 m
M
KH
2
PO
4
, pH 7.4) for 10 min, and
brought to a concentration of 2 mgÆmL
)1
with injection
buffer and a 5 mgÆmL
)1
stock solution of Oregon Green
488-dextran (OG-dextran; 70 kDa; Molecular Probes),
bringing the injection samples to an OG-dextran concen-
tration of 0.4 mgÆmL
)1
, a concentration used in previous
studies that had no effect on cell viability and proliferation
[22]. The samples were then centrifuged at 10 000 g for
10 min to remove large aggregates. All steps of the sample
preparation were performed at 4 °C, and the samples kept

on ice until injected into cells. Samples of recombinant
mGSTA4-4, DmGSTD1-1, hGSTA1-1 and hGSTP1-1
used for microinjection were prepared in an identical
manner.
DNA sample preparation. Both wild-type- and mut-
hGSTA4-4 expression vectors were brought to a concentra-
tion of 20 copies per 5 fL with injection buffer when injected
as individual samples. We had previously determined that
optimal expression occurred with this concentration of
DNA [22]. For the experiments in which mGSTA4-4
expression vectors were coinjected with the green fluorescent
protein (GFP) expression vector, all coinjected vectors were
brought to a concentration of 40 copies per 5 fL with
injection buffer. Just before injection, coinjected samples
were mixed 1 : 1 bringing the coinjected vectors to a
concentration of 20 copies of each vector per 5 fL. Just
before injection of the vectors into cells, all samples were
dialyzed against injection buffer for 10 min, and then
centrifuged at 10 000 g for 10 min at room temperature.
Glass-needle-mediated microinjection of proteins and
DNA expression vectors. HLE B-3 and CCL-75 cells were
maintained as monolayer cultures as described above. For
the experiments performed in this study, HLE B-3 and
CCL-75 cells were used at passage 18. On the day before
each experiment, 2 · 10
4
cells were plated in 35 mm
2
tissue
culture dishes (Corning) containing 1.5 mL medium. Before

plating of the cells, circles were etched into each of the dishes
to facilitate subsequent identification of injected cells.
Injection needles were pulled from borosilicate capillaries
using a Flaming/Brown Micropipette Puller, model P-97
1692 R. Sharma et al.(Eur. J. Biochem. 271) Ó FEBS 2004
(Sutter Instrument Co., Novato, CA, USA) with a range of
outer tip diameters of 2.5–3 lm, as determined by scanning
electron microscopy [23]. Phase contrast microscopy was
used to visualize the injection procedure using an Olympus
Corp. (Melville, NY, USA) IX70 inverted microscope
equipped with a temperature-controlled stage kept at 37 °C.
The cells were injected with either  5 fL sample containing
protein (cytoplasmic injections, with OG-dextran as the
marker of the injected cells) or sample containing DNA
(nuclear injections, with GFP as the marker of the injected
cells), using the electronically interfaced Eppendorf Micro-
manipulator (model 5171) and Transjector (model 5246) as
described previously [23]. All injections were performed
manually, with each injection sample being injected into
75 cells per dish per experiment. All experiments were
repeated two or more times. Only cells within an etched
boundary were injected, to allow easy localization of the
injected cells.
Single-cell assay of post-injection viability and GS-
induced cell rounding or apoptosis. Fluorescence micros-
copy (IX70 inverted microscope) was used to identify
injected cells. The percentage post-cytoplasmic and post-
nuclear injection viabilities were determined for both
HLE B-3 and CCL-75 cells by calculating: (number of
fluorescent cells 24 h after injection/75) · 100. Viabilities

were determined from cells coinjected with either mut-
hGSTA4-4 protein and OG-dextran or the mut-hGSTA4-4
expression vector expressing the mutant form of hGSTA4
(Y212F) and the GFP expression vector, or with fluorescent
markers alone. At 24 h after the injection, any cells killed by
the injection procedure were lifted off the dish leaving only
the injected cells that survived the injection. Such cells were
flat and attached to the dish as shown in Fig. 2. Stratagene
enhanced GFP (eGFP) and OG-dextran fluorescent mark-
ers of injected cells as well as mutant hGSTA4-4 protein had
no effect on post-injection viabilities. The mean viability
after nuclear injection into HLE B-3 and CCL-75 cells
ranged from 40% to 70%. To determine the effect of wild-
type and mutant forms of GST on injected cells, all
surviving HLE B-3 and CCL-75 cells were scored at 24 and
48 h and 24, 48, and 72 h, respectively, as being either flat,
round or apoptotic. The mean percentage of the injected
cells showing the above morphologies was calculated with
data from three or more experiments for each injection
sample at each time point.
Results
Effect of transfection of HLE B-3 cells with
hGSTA4
The HLE B-3 cell line was originally developed after
infection with adenovirus (Ad12-SV40) [24] and is referred
to here as WT-HLE B-3. These cells have been reported to
be relatively resistant to oxidative stress [25], grow in
monolayers (Fig. 1A, a) with a population doubling time of
48–52 h, and become senescent after 76 population dou-
blings [24]. Keeping this in view, we used WT-HLE B-3 cells

with low passage numbers (passages 18–20) for these
studies. WT-HLE B-3 cells were transfected with a eukary-
otic expression vector containing hGSTA4 cDNA, and three
clones overexpressing hGSTA4-4, designated C4, D7 and
E1, were selected. Initially, hGSTA4-transfected cells grew
normally in monolayers (Fig. 1A, b) with a doubling time
identical with that of empty-vector-transfected cells. How-
ever, four weeks after transfection (two passages) during
their clonal selection in medium containing G418, cells
stopped proliferating and some began to enlarge (Fig. 1A,
c). Even though the growth medium was changed every
72 h, the cells remained in a quiescent state for the next four
weeks. Eight weeks after transfection, cells originating from
clones C4, D7 and E1 started to transform their shape, as
was apparent from the characteristic budding of round cells
from giant cells. A typical example of this transformation is
showninFig.1A,d. The transformed round cells becoming
anchorage-independent (Fig. 1A, e) continued to express
higher levels of hGSTA4-4 (Fig. 1B, a-p and a-f), and had
lower levels of 4-HNE (Fig. 1C). To date, these cells have
undergone about 365 doublings in suspension cultures, with
no cells becoming senescent, a property characteristic of
cancer-derived cell lines, e.g. human erythroleukemic
(K562) and small cell lung cancer (H69) cell lines. The
HLE B-3/hGSTA4 anchorage-independent cells had a sig-
nificantly shorter doubling time than wild-type-transfected
and empty-vector-transfected HLE B-3 cells (20 ± 3.4 h
vs. 50 ± 4.3 h).
hGSTA4-4 expression and 4-HNE levels in transfected
cells

The expression of hGSTA4-4 in stably transfected cells was
confirmed by Western blots, which showed no detectable
expression of hGSTA4-4 in the wild-type-transfected or
empty-vector-transfected HLE B-3 cells, but a strong band
in hGSTA4-transfected cells (Fig. 1D). All three clones (C4,
D7 and E1) continued to express high levels of enzymat-
ically active hGSTA4-4 and showed similar effects of
hGSTA4 transfection on their phenotype with a significant
reduction in intracellular 4-HNE levels. Most of the data
presented here were obtained using the representative clone
C4. Although there was detectable constitutive GST activity
towards 4-HNE in WT- HLE B-3 cells, this activity was
about sixfold higher in the transfected cells [1.5 vs. 9.7 nmol
4-HNE consumedÆmin
)1
Æ(mg protein)
)1
], indicating success-
ful expression of enzymatically active hGSTA4-4 in trans-
fected cells. The 4-HNE level in clone C4 used for these
studies was found to be 40 ± 8% of that observed in the
wild-type-transfected or empty-vector-transfected HLE B-3
cells (Fig. 1C). These results further confirm overexpression
of active hGSTA4-4 in the transfected cells.
Anchorage-independent growth
The anchorage-independent growth of phenotypically
transformed cells was confirmed by assay of soft agar
colonies [26]. Clone C4 cells grew into colonies within
3 weeks of plating, while WT-HLE B-3 cells did not form
detectable colonies (data not presented). The colony-

forming ratio of clone C4 (HLE B-3) cells to WT-K562
cells used as positive control in these experiments was
found to be 3 : 1. Taken together, these results confirm
the phenotypic transformation of WT-HLE B-3 cells to
anchorage-independent growth on stable transfection with
hGSTA4.
Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur. J. Biochem. 271) 1693
Effect of transfection with enzymatically inactive
mutant
hGSTA4
To establish whether the observed phenotypic changes were
specifically due to depletion of 4-HNE because of high
activity of hGSTA4-4 towards 4-HNE in the transfected
cells or to some unknown effect of transfection, we prepared
a mutant cDNA of hGSTA4-4 isozyme in which Tyr212
was replaced with phenylalanine. Consistent with the
previous studies [13], recombinant mutant hGSTA4-
4(Y212F) had only  3% of the activity towards 4-HNE
compared with WT-hGSTA4-4 [1.9 vs. 72 lmol 4-HNEÆ
min
)1
Æ(mg protein)
)1
]. There was no noticeable change in
morphology of the cells tarnsfected with mutant hGSTA4
(Y212F) even after six passages (Fig. 1B, m-p). Despite high
expression of mutant protein as indicated by immunolocal-
ization (Fig. 1B, m-f) and Western blot studies (Fig. 1D,
lane 1) using hGSTA4-4 antibodies, there was no significant
change in either their GST activity towards 4-HNE or the

steady-state levels of 4-HNE compared with those of WT-
HLE B-3 cells (Fig. 1C). These results strongly suggest that
overexpression of enzymatically active hGSTA4-4 resulting
in accelerated metabolism of 4-HNE and thereby lowering
of the intracellular concentrations of 4-HNE leads to the
observed phenotypic transformation and immortalization
of WT-HLE B-3 cells.
Microinjection of the active hGSTA4-4 induces
similar phenotypic changes
We also studied the effects of direct microinjection of the
active hGSTA4-4, its inactive mutant, and their expression
vector counterparts into WT-HLE B-3 cells. To monitor
the microinjection of active or inactive hGSTA4-4 recom-
binant protein, the cells were coinjected with OG-dextran, a
fluorescent marker, as detailed in the legend of Fig. 2A. The
Fig. 1. Phenotypic transformation and biochemical characterization of hGSA4-transfected cells. (A) Phenotypic transformation of hGSA4-trans-
fected cells. (a) Control WT-HLE B-3 cells; (b) HLE B-3 cells 2 weeks after hGSTA4 transfection; (c) growth arrest and enlargement of HLE B-3
cells 4 weeks after transfection; (d) budding of rounded cells from giant cells 8 weeks after transfection; (e) anchorage-independent growth of
transformed rounded cells. (B) Transfection of HLE B-3 with WT-hGSTA4 and Y212F mutant hGSTA4 (mut-hGSTA4) with no activity towards
4-HNE: (a-p) a typical phase contrast micrograph of transformed cells after transfection with WT-hGSAT4;(a-f) fluorescence micrograph showing
expression of WT-hGSTA4-4 protein in transformed cells detected immunohistologically using hGSTA4-4 antibodies; (m-p) phase contrast
micrograph of cells 8 weeks after transfection with mut-GSTA4;(m-f) fluorescence micrograph showing expression of mut-hGSTA4-4 protein in
transfected cells. (C) 4-HNE levels in HLE B-3 cells. (D) Expression of hGSTA4-4 protein in transfected cells as detected by Western blots: lane 1,
cells transfected with hGSTA4 Y212F mutant; lane 2, WT-HLE B-3 cells; lane 3, cells transfected with hGSTA4; lane 4, positive control of
hGSTA4-4. Details for transfection, immunofluorescence studies, Western blots and 4-HNE determination are given in Experimental procedures.
1694 R. Sharma et al.(Eur. J. Biochem. 271) Ó FEBS 2004
cells were monitored from 12 to 48 h after injection. After
24 h, cells injected with active protein began to round up
and detach (Fig. 2A, a-p and a-f), whereas those injected
with OG-dextran and inactive protein remained flat and

attached (Fig. 2A, m-p and m-p & f). There was a significant
increase in the percentage (52.5 ± 5%; mean ± SD) of the
round cells in active protein-injected cells over the first 48 h
after injection (data not presented). In contrast, in the cells
injected with the mutant hGSTA4-4 protein, only
8 ± 2.5% of the cells were rounded, which was similar to
the level observed in OG-dextran mock-injected cells (data
not presented).
In the experiments for microinjecting WT-hGSTA4 and
theinactivemut-hGSTA4 (Y212F) cDNA into HLEB-3
cells, the expression vector of eGFP was used as a marker
for successful microinjection. As shown in Fig. 2B, a-p and
a-f, microinjection of WT-hGSTA4 cDNA led to charac-
teristic rounding and anchorage-independent growth within
24 h. Cells microinjected with mut-hGSTA4 cDNA main-
tained their original phenotype and did not undergo any
change (Fig. 2B, m-p and m-f). A small but clearly
noticeable number of cells underwent apoptosis after
microinjection. The apoptotic cells could be identified, as
they showed activation of caspase-3 detected by staining the
cells with antibodies to cleaved caspase-3 (data not shown)
and loss of fluorescence due to extrusion of cytoplasm.
These cells could be easily distinguished from the rounded,
transformed cells. As shown in Fig. 2C, these cells were not
fully rounded and showed only minimal fluorescence of
eGFP which was prominent in rounded, transformed cells.
The percentages of unchanged flat cells, transformed
rounded cells, and apoptotic cells after 24 h and 48 h of
microinjection of WT-hGSTA4 and mut-hGSTA4 cDNA in
a typical experiment are given in Fig. 2D. Together, these

results further indicate that overexpression of active
hGSTA4-4 is required for phenotypic transformation.
Only GST isozymes that have high catalytic efficiency
with 4-HNE have transforming activity
To further establish that high hGSTA4-4 activity was
required for its transforming activity, we microinjected four
different GST isozymes into HLE B-3 cells. For these
experiments, two GST isozymes with high activity and two
Fig. 2. Microinjection of active WT-hGSTA4-4, inactive mutant hGSTA4-4 recombinant protein or the respective expression vector into HLE B-3
cells. (A) (a-p) Phase contrast micrograph of a typical transformed HLE B-3 cell 24 h after cytosolic microinjection with WT-hGSTA4-4 protein;
(a-f) fluorescence micrograph of same cell showing fluorescent marker, OG-dex, coinjected with WT-hGSTA4-4; (m-p) phase contrast micrograph
of a typical HLE B-3 cell after microinjection with inactive mut-hGSTA4-4 protein; (m-p&f) combined phase contrast micrograph and fluorescence
micrograph of same cell indicating delivery of OG-dex marker. Bar denotes 30 lm. (B) (a-p&f) Combined phase contrast micrograph and
fluorescence micrograph of a typical transformed HLE B-3 cell 24 h after nuclear microinjection with WT-hGSTA4 and the marker eGFP cDNAs;
(a-f) fluorescence micrograph of same HLE B-3 cell (fluorescence represents expression of eGFP); (m-p) phase contrast micrograph of typical
HLE B-3 cells 24 h after microinjection with inactive mut-hGSTA4 and eGFP cDNAs; (m-f) fluorescent micrograph of same cells. (C) A small
fraction of microinjected cells undergo apoptosis. (p) phase contrast micrograph of cell undergoing apoptosis; (f) fluorescence micrograph of same
cell. These cells could be distinguished from the transformed cells as they were not fully rounded and expression of fluorescent marker eGFP was
minimal. (D) Quantitation of transformed (unfilled bars), nontransformed (grey bars) and apoptotic cells (black bars) after microinjection with
WT-hGSTA4 or mut-hGSTA4 expression vectors. Details of microinjection and immunofluorescence experiments are given in Experimental
rocedures.
Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur. J. Biochem. 271) 1695
with minimal activity toward 4-HNE were selected. Mouse
enzyme mGSTA4-4 [14] and Drosophila enzyme DmG-
STD1-1 [17] are known to have high activities for 4-HNE
(specific activities: 65 UÆmg
)1
and 32 UÆmg
)1
, respectively).

On the other hand, human enzymes hGSTA1-1 and
hGSTP1-1 have minimal activity towards 4-HNE [27].
These results show that mGSTA4-4 and DmGSTD1-1
(Fig. 3) trigger transformation and hGSTA1-1 and
hGSTP1-1 (Fig. 4) do not. A phase contrast micrograph
and fluorescent micrograph of a typical transformed cell
24 h after microinjection of mGSTA4-4 or DmGSTD1-1
are presented in Fig. 3A and Fig. 3B, respectively. Results
presented in Fig. 3C indicate that most microinjected cells
are transformed within 48 h. In contrast, results presented
in Fig. 4 show that cells microinjected with either hGSTA1-
1 or hGSTP1-1 retain their original phenotype and do not
undergo transformation. These results further support the
idea that the ability of the GST isozymes to induce
transformation is dependent on their ability to conjugate
4-HNE to GSH. Furthermore, these results argue against
the possibility of a nonspecific effect of hGSTA4-4 causing
the transformation.
Effect of hGSTA4-4 overexpression in the CCL-75 cell line
The effect of hGSTA4-4 overexpression was also examined
in a human lung fibroblast cell line, CCL-75, a nonviral
transformed adherent cell line with a finite lifetime of
50 ± 10 population doublings [28]. In these experiments,
when CCL-75 cells were microinjected with active and
mutant hGSTA4-4 proteins in a manner similar to WT-
HLE B-3 cells, comparable results were observed (Fig. 5A–
D). Interestingly, cell rounding was observed in CCL-75
cells 48 h after the microinjection of active protein, a delay
of nearly 24 h compared with WT-HLE B-3 cells. The
reasons for this time lag are not clear. These results also

show that direct injection of active hGSTA4-4 protein or its
cDNA into attached cells causes a characteristic trans-
formed phenotype and further suggest that overexpression
of hGSTA4-4 leading to such transformation may be a
generalized phenomenon.
Effect of hGSTA4-4 on key cell-cycle genes
We also studied the effects of the hGSTA4-4 expression on
some of the key genes involved in cell-cycle regulation and
apoptosis. In the hGSTA4-transfected and phenotypically
transformed, anchorage-independent HLE B-3 cells, we
found upregulation of transforming growth factor, cyclin-
dependent kinase 2, protein kinase C bII, and extracellular
regulatory stress kinase vs. downregulation of p53 (Fig. 6).
These observations are consistent with the idea that
lowering the intracellular concentrations of 4-HNE upreg-
ulated genes involved in promotion of proliferation and
Fig. 3. Cytoplasmic microinjection of recombinant mGSTA4-4 and DmGSTD1-1 in HLE B-3 cells. Cells were microinjected with the respective
protein as described in Experimental procedures and scored 24 h and 48 h after injection. (A) (p) Phase contrast micrograph of a typical
transformed cell 24 h after microinjection with mGSTA4-4; (f) fluorescence micrograph of same cell showing fluorescent marker OG-dex coinjected
with mGSTA4-4. (B) (p) Phase contrast micrograph of a typical transformed cell 24 h after microinjection with DmGSTD1-1; (f) fluorescence
micrograph of same cell showing fluorescent marker OG-dex coinjected with DmGSTD1-1; (C) Bar graph showing percentage of nontransformed
(flat; black bars), transformed (rounded; light grey bars) and apoptotic (dark grey bars) cells 24 h and 48 h after microinjection.
1696 R. Sharma et al.(Eur. J. Biochem. 271) Ó FEBS 2004
downregulated genes (such as p53) that control the cell cycle
and are pro-apoptotic [29].
Discussion
Previous studies strongly suggest that intracellular 4-HNE
can influence signaling mechanisms and, depending on its
concentration, can promote apoptosis [3,6,7,12], differenti-
ation [12], or proliferation [11,12] of cells. GSTs in general,

and hGSTA4-4 and hGST5.8 in particular, are the major
4-HNE-metabolizing enzymes in humans [16]. Dramatic
phenotypic transformation of attached cells on transfection
with hGSTA4-4 into smaller rounded immortalized cells
which grow rapidly in suspension is surprising but it seems
to be consistent with numerous previous studies suggesting
that 4-HNE is involved in cell-cycle signaling mechanisms.
Our results show that transfection or microinjection of cells
with enzymatically active hGSTA4-4 causes the emergence
of the transformed phenotype, whereas hGSTA4-4(Y212F),
a mutant with decreased activity for 4-HNE [13], is unable
to transform cells. This result provides a reasonable basis for
proposing the hypothesis that the observed transformation
of HLE B-3 and CCL-75 cells is a consequence of its
conjugation of 4-HNE, rather than being linked to other
possible effects of hGSTA4-4, such as a hypothetical direct
binding to signaling kinases, as has been described for
Pi-class GSTs [30].
To test further the hypothesis that 4-HNE conjugation
is relevant to cellular transformation, we microinjected
cells with two additional GSTs which are known to
metabolize 4-HNE but are structurally distinct and phylo-
genetically distant from hGSTA4-4. The murine enzyme
mGSTA4-4 has a catalytic efficiency for 4-HNE that is
similar to that of hGSTA4-4 [14]. However, antibodies
against one enzyme do not cross-react with the other [16],
indicating that at least parts of the surface of the two
proteins differ substantially from each other. The second
microinjected protein was DmGSTD1-1 from D. melano-
gaster [31]. This Delta-class insect GST also has a relatively

high catalytic efficiency for 4-HNE conjugation [17]. Insects
and mammals diverged at least 600 million years ago [32],
and hGSTA4-4 is only 22%/40% identical/similar to
DmGSTD1-1. Thus, it is unlikely that DmGSTD1-1 could
replace hGSTA4-4 in any putative regulatory protein–
protein interactions in which hGSTA4-4 may be involved.
Our studies clearly show that microinjection of hGSTA4-4,
mGSTA4-4, and DmGSTD1-1 triggers cell transformation
whereas microinjection of hGSTA4-4(Y212F), hGSTA1-1,
and hGSTP1-1 does not. The three proteins able to
transform cells are structurally dissimilar but are all efficient
at conjugating 4-HNE, whereas those that lack 4-HNE-
conjugating activity also fail to transform cells, even if
they are structurally almost identical with an active
enzyme, as in the case of hGSTA4-4(Y212F). Together,
these results point to the conjugative metabolism of 4-HNE
as the common denominator and the causative principle
in the transformation process, and suggest that the level
of 4-HNE or its glutathione conjugate (GS-HNE) is the
Fig. 4. Cytoplasmic microinjection of recombinant hGSTA1-1 and hGSTP1-1 in HLE B-3 cells. (A) Typical cells 48 h after microinjection of
hGSTA1-1. (p) Phase contrast micrograph showing that cells maintained their original phenotype; (f) fluorescence micrograph of same cells
showing OG-dex fluorescent marker. (B) Typical cells 48 h after microinjection of hGSTP1-1. (p) Phase contrast micrograph and (f) fluorescence
micrograph of same cells. (C) Bar graph showing percentage of nontransformed (flat; black bars) and transformed (rounded; light grey bars) cells
24 h and 48 h after microinjection.
Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur. J. Biochem. 271) 1697
most likely effector of the cell transformation we observed.
This is consistent with a lower 4-HNE level in cells
overexpressing hGSTA4-4 but not in cells overexpressing
the mutant Y212F, which is ineffective in triggering
transformation.

Although a hypothetical substrate other than 4-HNE,
perhaps another Michael acceptor, cannot be ruled out at
present, 4-HNE is the only currently known common
physiological substrate of proteins as different as hGSTA4-
4 and DmGSTD1-1. However, the correlation of 4-HNE-
conjugating activity with the ability to transform cells which
holds for six different proteins [hGSTA4-4, hGSTA4-
4(Y212F), mGSTA4-4, DmGSTD1-1, hGSTA1-1, and
hGSTP1-1] indicates that a causal involvement of 4-HNE
in the mechanism of hGSTA4-4-mediated transformation
of HLE B-3 and CCL-75 cells provides the simplest
explanation of all the available experimental data.
Binding of GSTs, particularly hGSTP1-1 with c-Jun
N-terminal kinase (JNK), modulates stress-mediated signa-
ling for apoptosis. In monomeric form, hGSTP1-1 binds to
JNK and inhibits its activation, but under conditions of
stress such as exposure to UV or H
2
O
2
treatment, it
oligomerizes and dissociates from the JNK complex leading
to abrogation of JNK inhibition [30]. Such interactions of
hGSTA4-4 with JNK or other key kinases may also be
considered as the mechanistic basis for the observed
transformation. However, the inability of mutant
hGSTA4-4(Y212F) to induce transformation argues against
such a possibility because GSTP1-1 with a mutation in its
active site is still able to prevent JNK activation. An effector
domain critical for its binding to JNK (residues 191–201)

has been identified in hGSTP1-1 [33], and it seems unlikely
that mutation of a single active-site residue (Y212F)
would abrogate the binding of hGSTA4-4 to kinases.
Fig. 5. Microinjection of WT-hGSTA4-4, mut-hGSTA4-4 recombinant protein or respective expression vector in CCL-75 cells. (A) (a-p)Phase
contrast micrograph of a typical transformed CCL-75 cell 48 h after cytoplasmic microinjection with WT-hGSTA4-4 recombinant protein and
OG-dex marker; (a-f) fluorescence micrograph of same cell (fluorescence indicates delivery of the marker OG-dex); (b-p) phase contrast micrograph
of a typical transformed CCL-75 cell 48 h after nuclear microinjection of expression vectors of WT-hGSTA4 and eGFP cDNAs; (b-f) fluorescence
micrograph of same cell (fluorescence indicates expression of the marker eGFP); (c-p) phase contrast micrograph of a typical CCL-75 cell 48 h after
cytoplasmic microinjection of mut-hGSTA4-4 protein and the marker OG-dex; (c-f) fluorescence micrograph of same cell (fluorescence indicates
the marker OG-dex); (d-p) phase contrast micrograph of a typical CCL-75 cell 48 h after nuclear microinjection of mut-hGSTA4 and eGFP
cDNAs; (d-f) fluorescence micrograph of same cell (fluorescence indicates expression of the marker eGFP). Details are provided in Experimental
procedures. Bar represents 30 lm. (B–D) Bar graphs showing percentage of transformed (rounded; unfilled bars), unaffected (flat; dark grey bars)
and apoptotic (black bars) cells after cytoplasmic microinjection of recombinant protein of active WT-hGSATA4-4 (B) or inactive mut-hGSTA4-4
(C) and nuclear microinjection of expression vectors (D).
1698 R. Sharma et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Furthermore, GSTP1-1-mediated activation of JNK does
not appear to be applicable to all cell types because, in a
human lung fibroblast cell line, GSTP1-1 does not affect
JNK activation [34]. We show that neither hGSTP1-1 nor
hGSTA1-1 cause transformation but mouse and Drosophila
4-HNE-metabolizing GST isozymes (mGSTA4-4 and
DmGSTD1-1, respectively) show transforming activity
comparable to that of hGSTA4-4. Thus, transformation
does not appear to be due to interaction of hGSTA4-4 with
signaling kinases but seems to be linked to its catalytic
ability to conjugate 4-HNE to GSH.
GSTA4-4-catalyzed conjugation of 4-HNE to GSH
results in the formation of GS-HNE. An increase in the
level of GS-HNE may also be a trigger of transformation.
Previous studies have shown that overexpression of 4-HNE-

metabolizing GST isozymes leads to accelerated formation
of GS-HNE in cells. As confirmed by identification and
quantification of intact GS-HNE in the medium of cells
loaded with radioactive GS-HNE, most GS-HNE thus
formed is transported out of the cells through ATP-
dependent transport processes [6,7]. However, a significant
proportion of intracellular GS-HNE can be metabolized to
the corresponding alcohol, glutathionyl dihydroxynonene
formed through NADPH-dependent reduction of GS-HNE
catalyzed by aldose reductase [35]. In addition, GS-HNE
can also be converted into mercapturic acids, which can
then be x-hydroxylated by CYP-450 [36] to yield more
hydrophilic products. The possibility of GS-HNE or its
metabolites being involved in the mechanisms of the
observed transformation phenomenon is not ruled out by
the present studies. Aldose reductase, which can reduce
GS-HNE, has been shown to mediate mitogenic signaling in
vascular smooth muscle cells [37], and its inhibitors have
been shown to inhibit tumor necrosis factor-a-induced
apoptosis and caspase-3 activation [38]. Channeling of
4-HNE towards accelerated formation of GS-HNE in
hGSTA4-4-overexpressing cells may abruptly change the
overall ÔphysiologicÕ homeostasis of 4-HNE, GS-HNE, and
its metabolites maintained by a co-ordinated action of
4-HNE-metabolizing enzymes including GSTs [16], aldose
reductase [35], aldehyde dehydrogenase [39], and transport-
ers of GS-HNE [6,7]. According to this interpretation, it is
not just the concentration of 4-HNE but also changes in the
homeostasis of 4-HNE and its metabolites that provides the
mechanistic basis for the transformation. This possibility

needs to be explored in future studies.
Our results show that some of the more prominent genes
suggestedtobeinvolvedinpromotingproliferationare
upregulated in hGSTA4-4-overexpressing HLE B-3 cells.
This, along with almost complete suppression of p53, may
account for the observed threefold higher growth rate of the
transformed cells. Our studies are limited to evaluating the
expression of only a few key genes known to be involved in
cell-cycle events. An assessment of the effect of transfection
with hGSTA4-4 and 4-HNE depletion on global gene
expression using cDNA microarrays is planned for the
future. Taken together with the results of previous studies
showing that at higher concentrations, 4-HNE causes
apoptosis [3,6,7,12] and differentiation [12,40], the present
results suggest that the intracellular level of 4-HNE may be
one of the determinants for leading cells towards pathways
for transformation, differentiation, proliferation, or apop-
tosis.
The mechanisms through which 4-HNE affects signaling
processes in a concentration-dependent manner are obscure
and appear to be complex. 4-HNE is a strong electrophile
which reacts with nucleophilic groups of proteins [41,42],
nucleic acids [43,44], and lipids [45]. It interacts with thiols
Fig. 6. Expression of genes known to be involved in cell-cycle regulation. (A,B) HLE B-3 cells (1 · 10
6
) transfected with wild-type, empty vector or
hGSTA4 were lysed in RIPA buffer containing protease inhibitor cocktail, 1 m
M
phenylmethanesulfonyl fluoride and 2 m
M

sodium orthovanadate.
The cell extracts were centrifuged at 15 000 g at 4 °C. Supernatant containing 100 lg protein was loaded in each well and subjected to Western blot
analysis using antibodies against proteins identified in the left hand margins of (A) and (B). Lanes 1, 2, and 3 in both panels represent extracts of
HLE B-3 cells transfected with wild-type, empty vector and hGSTA4, respectively. (C) For comparison of expression of extracellular signal
regulated kinase 1/2, cells (1 · 10
6
) from clone C4 (A4-4) and empty-vector-transfected (VT) HLE B-3 cells were serum starved for 24 h in separate
Petri dishes and then treated with serum-containing medium (10%) for different times. The cells were centrifuged at 654 g (5 min), their extracts
were prepared in RIPA buffer as described in Experimental procedures, and Western blot analyses were performed using antibodies against
extracellular signal regulated kinase 1/2. Lane 1, extract of cells before serum stimulation; lanes 2–6, extracts of the cells after treatment with 10%
serum for 2, 5, 10, 15 and 30 min, respectively.
Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur. J. Biochem. 271) 1699
and also with nucleophilic nitrogen atoms in proteins and
phospholipids, perhaps with varying affinity. It may be
postulated that, at low concentrations, 4-HNE may selec-
tively affect pathways favoring proliferation by interacting
with nucleophilic groups that have high affinity for the
compound. On the other hand, at higher concentrations of
4-HNE, the effects of these interactions may be over-
whelmed by reactions with low affinity groups of cellular
nucleophiles to trigger the pathways favoring apoptosis.
This speculation lacks direct experimental evidence but is
consistent with the previous studies showing that bIandbII
isoforms of phosphoinositide-specific protein kinase C in
several cell types are activated by submicromolar levels but
inhibited by higher levels of 4-HNE [9]. Further studies on
the possible chemical interaction of 4-HNE with cellular
nucleophiles including proteins, nucleic acids and lipids and
possible correlation between these interactions and signaling
cascades may provide clues to the mechanisms by which

4-HNE affects signaling events.
Acknowledgements
Supported in part by NIH grants EY 04396 (to Y. C. A.), CA77495
(to S. A.), ES 07804 (to P. Z.) and CA 76348 (to S. V. S.).
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Ó FEBS 2004 Transformation of cells transfected with hGSTA4-4 (Eur. J. Biochem. 271) 1701