nitrosourea was partially reduced by pyruvate, a H202 scavenger. Our results suggest that overexpression of. MnSOD can cause an imbalance of antioxidant.
Vol. 7, 1 175-1
186, September
1996
Cell Growth
Inhibition
of Cell
Growth and Sensitization Damage by Overexpression of Manganese Dismutase in Rat Glioma Cells1
Weixiong
Larry W. Oberley,2
Zhong,
Terry D. Oberley, Tao Yan, and Daret K. St. Clair Radiation 52242 [W. Middleton University Graduate Kentucky
produced
Frederick
E. Domann,
tive
include
Research Laboratory, The University of Iowa, Z., L W. 0., T. Y., F. E. D.]; Pathology Service, Veterans Memorial Hospital and Department of Wisconsin, Madison, Wisconsin 53705 [T. Center for Toxicology, University of Kentucky, 40536 [D. K. S.]
Iowa City, Iowa William S. of Pathology, D. 0.]; and Lexington,
of overexpression
superoxide
dismutase
of human
the SODs,
dismutation subsequently of HO. MnSOD
(MnSOD)
MnSOD
can cause an imbalance
been
implicated are more
activity
ulated
fense
systems
against
enzymes
oxidative
damage
are the caused
de-
by ROS3
that
MnSOD metastasis
idative
superoxide
dismutase;
can
CAT, catalase; GPX, glutathione
levels
of
have
cancer
been
(1
when
2).
,
anti-
of antioxidant found
a wide
in
to their normal counof decreased MnSOD
damage
and increased led to the eon-
some
sensitized mechanism
SOD
could
increase
injury
cell
if GPX
this
of CuZnSOD,
demonstrate levels
of GPX
stress
in a balanced
in an imbalanced when
SOD
In the
glioma
cells
tential
in tumor
study,
mechanisms
are usually cells cells.
of these
did
resistant
to
increased
relatively
are also
cells
enzymes
ovenexpressed
and
high
low and
tumor
the effects
BCNU,
not ole-
also
Therefore,
and differentiation
cv-
with
in antioxidant
to examine
effects
that
but are often
was
simulta-
ovenexpres-
more
transfeetion.
radiation,
not
provided
in cells
was
MnSOD
cell proliferation ionizing
became
on GPX
by gene
by transfection to
,
of an imbalance
is increased
present
on controlling responses
more
are have
not
of SOD
of H2O2 that
in cells but
in normal
state
even
of GPX
of
between
level
CAT
studies
enzymes
state
elevated
(1 8, 1 9). After
CAT
oxidative
ovenexpression (1 4-1 7). The underbut sensitization
accumulated
when
(1 2, 1 6, 1 9). Antioxidant
may have
against
that
and/or
(1 9). Cells
only
to ox-
in antioxidant
production
hypothesis
H2O2
CuZn-
resistance
of the imbalance
recent
an elevation
oxidative
protection shown
(1 4-1 9). An
Sevenal
cell diffenen-
an increase
in an increased
increased.
of and
of MnSOD,
cellular
to be a result
result
to support
vated
overexpression
can
or CAT
dem-
phenotype
potentiate
that
have
have
by transfection
(9).
cells to oxidative stress is not fully understood,
GPX
cause
and
provides
postulated
and
activity
and
(6-8) reagent
studies
studies
malignant
(1 0-1 3). Although
generally
damage,
the
cells
GPX
recent
of MnSOD
suppress
shown
and
enzymes
sion
(5). Several
of cancer
CAT,
idenee
peroxidase; GSH, reduced glutathione; BCNU, 1 ,3-bis(2-chloroethyl)-1 nitrosourea; BSO, buthionine sulfoximmne; RT-PCR, reverse transcriptionPCR; FBS, fetal bovine serum; DT, doubling time; NBT, nitroblue tetrazohum; MIT, 3,(4,5-dimethylthiazol-2-yI)2,5-diphenyl-tetrazolium; GA, glutathione reductase.
have
form ROS
damage
Low
MnSOD,
by a differentiating
neously
SOD, copper-zinc
including
to oxidative
ovenexpnession
eDNA
tiation
can
Received 5/3/96 revised 6/7/96; accepted 6/29/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to mdicate this fact. 1 This work was supported by NIH grants CA 41267 and DE 10758 to LW. Oberiey, CA49797 to D.K. St. Clair, and a University ofWisconsin Medical School grant to T. D. Oberiey. 2 To whom requests for reprints should be addressed, at Radiation Research Laboratory, 77 Medical Laboratories, The University of Iowa, Iowa City, IA 52242. Phone: (319) 335-8015; Fax: (319) 335-8049. 3 The abbreviations used are: ROS, reactive oxygen species; SOD, superoxide dismutase; MnSOD, manganese superoxide dismutase; CuZn-
diseases
fluid.
ROS-induced cancinogenesis with cell differentiation have
It has been
primary
GPX
is mainly
another
extnaeellular
decreased.
cell proliferation
onstnated
has been antioxidant
the
and
cept that MnSOD may function as a tumor suppressor gene that modulates intracellular nedox state to control ROS-reg-
SOD lying
and
SOD,
of cancer cells when compared cells (3, 4). The correlations with
removing
Antioxidants
are
particularly
MnSOD
SOD,
Introduction
to the
in many
activity
of antioxidant
enzymes.
catalyzes CAT
CuZnSOD
and extraeellular
susceptible
enzymes
enzymes,
terpart
oxidaenzymes
SOD
whereas
in mitochondnia;
is localized
Cells
which we hypothesize results in an elevation of intracellular H202. Overexpression of MnSOD can either inhibit cell proliferation or increase cell death by oxidative agents, depending on the levels of peroxideenzymes,
H2O2,
during
antioxidant
convert H2O2 to water to prevent the pnoducThere are three forms of SOD in mammalian cells.
is located
variety
and
The
and penoxidases.
to 02 and
in the eytosol;
oxidant
on cell proliferation
and response to oxidative stress in rat glioma cells were studied. MnSOD-overexpressing cells had a 2- to 14-fold increase in MnSOD activity, but did not have consistent changes in the activities of CuZnSOD, catalase, or glutathione peroxidase. Cells with more than a 5-fold increase in MnSOD activity became more sensitive to radiation, 1 ,3-bis(2-chloroethyl)-1 nitrosourea, and buthionine sulfoximine and had a lower growth rate than parental and vector control cells. The sensitivity to I ,3-bis(2-chloroethyl)-1 nitrosourea was partially reduced by pyruvate, a H202 scavenger. Our results suggest that overexpression of
metabolism
systems.
CAT,
of O2”
tion
found
manganese
aerobic
in biological
CuZnSOD,
Abstract The effects
to Oxidative Superoxide
by normal
stress
1175
& Differentiation
in rat of MnSOD
and cellular BSO.
The
discussed.
po-
1176
MnSOD Gene Transfection
and Oxidative
Therapy
Results Transfection The
of Human
Al 72
cell
line
was
cDNA obtained
Type
Culture
from
the Al 72 cell line by long-term
originally cell
Collection.
MnSOD originally
human
line
these
cells
soon
after
(data
not
dominated that
and
tually
were
from
then
than
a rat glioma
cell line; this
was
and
MnSOD (data not
that
cells but
by rat glial to our
has
been
-MnSOD
pH3Apr-l
into
reported
cells
that the human
strated genome
Al 72R
of
bands
with
were
from
different
the
genomic
the
plasmids
were
to overexpress
Mn-
+-18S
copies
is no
EcoRI-restniction clone
SOD5
band
cell
same
size
MnSOD
No
from
probably
proximate lines.
The
band cell
of
low
intensity
in the
lines
two
had
of the
expression. equal
RNA intensity
blot
larger
due
of
bands
with
Southern
extra
probably
levels
and
MnSOD
blot correlated
observed
line
to
sample of the
the
intensity
than
band
neo
or nearly
control
MnSOD
Fig. 1. Southern and Northern blot analyses of MnSOD in A172A cells. Lane 1 , parental cell line; Lane 2, neol ; Lane 3, neo2; Lane 4, neol 4; Lane 5, 50D3; Lane 6, SOD4; Lane 7, SOD5; Lane 8, SOD7. A, Southern blotting. Twenty j.tg of genomic DNA were digested by EcoRl, electrophoresed in an 0.8% agarose gel, transferred to a nylon membrane, and probed with digoxigenin-labeled human MnSOD cDNA. Various copies of human MnSOD cDNA were integrated into individual MnSOD-transfected cell lines. B, Northern blotting. Twenty g of total cellular RNA were separated by electrophoresis, transferred to nylon membrane, and probed with 32P-labeled human MnSOD cDNA. C, verification of RNA sample loading by ethidium bromide staining of 285 and 185 RNAs.
the Increased
Northern cell
lines,
transcripts
(Fig.
with
an ap-
transcripts
MnSOD-tnansfected
cell
cell lines of the extra
in the bands
1A). SOD4 and SOD7 cell bands (4 kb and 6 kb) that
induction
-18S
in the
in the
in individual
(Fig.
I
s$4#{248}.
$1
band.
the intensity
mRNA
it
equal intensity, the lower band
same
visualized
in all of the
size
the
(23).
in that
integration of blot of this
eDNA
MnSOD
was
abundant
1 .2-kb
Northern
had
genomic
there
eDNA
clones
greater
extra
I,
of the
because
MnSOD
of nearly fact that
an
4-e$
,
all the bands
sizes
sites
had much that
parental
were
different
the other
two bands cells. The
lower
because
There
from
clone
mRNA
the
bands
the
_wwII_
two
MnSOD eDNA. also represents
human
integrative
coincidentally
the
into the
at least
bands. However, unless occurred, the Southern
suggests
line as
and
C
lines; these bands which has a single
site in the human
in the SOD5
upper
of
was different
clone should show only as seen in the parental the
gene,
of extra
different
did not show any extra exogenous DNA has
observed
were
On the other hand, except SOD5 had extra
eDNA,
indicate
was integrated
it (22).
integration
of MnSOD
cell lines were obblot analysis demon-
in all the cell
and the number
bands
the
(20, 21),
that the Al 72 cell
There
MnSOD within
the
extra
The SOD5
1A).
cell lines
represented
The intensity
eDNA
(Fig. sizes
site
MnSOD-transfected that
MnSOD
cells
two
EcoRl-restriction
were
-.28S
The pBK-CMV-neo plasmid alone was also transfected Al 72R cells as a control. Four MnSOD-overexpressing
into
18).
B
protein
Al 72R is a rat of human glial
and pBK-CMV-neo
cell lines and three neo-vector control tamed under G41 8 selection. Southern
blot
acidic
by others
report
rat Al 72R
these
Al 72R is
by our demonstra-
sequence from Contamination
this is the first contaminated.
cotransfected
SOD.
cells
and even-
Thus,
glial fibrillary
knowledge,
line has been The
eDNA shown).
the
sequence
in
Culture
We concluded cells
cell type.
tion that Al 72R cells expressed
S
on the
proteins
the
confirmed
‘
#{163}5,,,
-
pre-
band.
...-.
-
passaged,
the human
the only or predominant
.
were human
Type
were
A
and the rat band
the only
faster
this
based
American
disappeared,
became
grew
cells
As the cells
shown).
generated
Al 72 cells
CuZnSOD
rodent
arrival
line was that
12345678
Cells.
American
found
rodent
and
band
the rat cells
we
Al 72R from
culture.
but
by
human
CuZnSOD
human
cells,
contaminated of both
Collection
The Al 72R cell
glioma
was
expression
into
of
endogenous
gene
loading
was
equivalent
based
on
1 8S and
28S
RNA
(Fig.
1 C).
bands
gene
MnSOD
were (Fig.
primer
blot analysis
Western
The
parental
protein.
the exogenous using
line
at the
all ofthe
CuZnSOD
in
quantitation
bottom protein
and
amounts
increases
Densitometric
used
protein
cell
equal
However,
significant
tive
from RT-PCR
was
MnSOD
approximately
shown
by
(plasmid)
a human
MnSOD
2).
immunoreactive
3A).
transcripts
confirmed
to measure
the amount
(Fig.
neo
had
control
cell
of immunoreactive
of the Western showed
cell lines had MnSOD MnSOD
individual
blot.
decreased
lines
MnSOD
MnSOD-transfeeted immunoreactive of
of
in the Al 72R cell lines
The
protein. bands
is
immunoreac-
levels
in most
of
123456789
A
14.72
10.43 17.33
1177
kDa 97.4 68
B
-
43
-
29
-
18.4
-
14.3
-
bp
S
-
& Differentiation
12345678
A
-
Cell Growth
Relative Area
100
0.92 0.57
1.14
19.52
18 bp
B
97.4-
Fig. 2. Detection of MnSOD expression by RT-PCA. The lanes contained the following: Lane 1 , 4,x Haelll DNA markers; Lane 2, Al 72A parental cells; Lane 3, ned cells; Lane 4, neo2 cells; Lane 5, neol4 cells; Lane 6, SOD3 cells; Lane 7, SOD4 cells; Lane 8, SOD5 cells; Lane 9, SOD7 cells. A, cDNA from AT reaction was amplified using MnSOD sense 1 and antisense primers. A 450-bp DNA fragment was amplified from both endogenous and exogenous MnSOD genes. B, AT samples were amplified by PCA using MnSOD sense 2 (human-specific) and antisense primers to detect the exogenous (plasmid) MnSOD transcripts. A 21 8-bp DNA fragment was amplified only in the MnSOD-overexpressing cell lines.
lines
but increased
3B). Antioxidant tance
Enzyme
of both
among
levels
mammalian
Activities.
antioxidant
antioxidant antioxidant
glioma
activity
we
enzymes
cell
Because
enzyme
enzymes,
and GPX) in these
in the SOD5
of the imporand
measured
the
the
(MnSOD,
line (Fig.
balance
four
major
CuZnSOD,
cell lines. Table 1 shows
CAT,
that all the
-
43
-
29
-
18.4
-
14.3
-
_
-
Relative
the cell
68
Area
1.00 0.72 0.67
__
_
1.05 0.64
0.78
-.
0.38
1.60
Fig. 3. Westem blot analysis of MnSOD and CuZnSOD in Al 72A cells. Total cellular proteins were subjected to SDS-PAGE for electrophoresis, transferred to nitrocellulose, and detected with antihuman MnSOD or CuZnSOD antiserum. A, MnSOD, 40 g protein (Lanes 1-4) and 15 g protein (Lanes 5-8). B, CuZnSOD, 40 g protein/lane. Lane 1, parental cell line; Lane 2, ned ; Lane 3, neo2; Lane 4, noel 4; Lane 5, SOD3; Lane 6, SOD4; Lane 7, SODS; Lane 8, SOD7. The protein molecular mass protein markers (kDa) are indicated on the left. The relative areas shown at the bottom of the blots were determined by densitometry and have been corrected for the different protein loading.
MnSOD-transfected cell lines had increased (2-14-fold) MnSOD activity, whereas none of the neo cell lines had altered MnSOD
activity
compared cell lines
neo2, and SOD3 fold)
in CuZnSOD
significant
with
changes
the levels
cell line. The neol, decrease (2-2.6-
activity, whereas the SODS cell line had a (1 .4-fold) in CuZnSOD activity. These
increase
observed
to the parental had a significant
in CuZnSOD
activity
of immunoreaetive
agreed
protein
found
blotting
(Fig. 3B). There
activity. in most
GPX activity was generally decreased of the cell lines except for the SOD4
lines,
which
showed
were
no significant
no change.
The
quite
by Western
changes
reason
closely in CAT
(1 .4-2.4-fold) and SOD7 cell for this
is not
populations
within
individual
cell
stained with rabbit antihuman goat antirabbit lgG conjugated fected
cells
displayed
cence
(Fig.
5, C and
control
cells
ogy that
(Fig.
a much
higher
D) than
that
5). After
within
individual
cell
of the cell populations
intensity
of the lines
of
the homo-
to both morpholalso showed mitochondnia
with
rare gold
(Fig.
6). MnSOD-transfeeted
in all the
crease in specific mitochondnial labeling compared to parental and neo control cell lines (Table 2). SOD3 and SODS cells
protein
cell lines, indicating that some or fully active. The results further
demonstrate that the protein parallel to activity. Measuring
levels of MnSOD are not always enzymatic activity is necessary
had
for the studies
of antioxidant
enzymes,
lines,
Increased
SOD activity
fection was
studies. further
MnSOD and
confirmed
activity
CuZnSOD
Immunostaining staining protein
by activity
was
undetectable
activity
was
low
of MnSOD.
was performed to detect in situ and to determine
particularly
in the transfeetants
gel assay in parental
(Fig.
4), in which
and neo cells,
in all of the cell Indirect
for trans-
lines.
immunoreactive the heterogeneity
MnSOD of cell
mitochondria
cells
particularly
SODS
than cells,
aneies
among enzymatic
translational accumulation drial
proliferation.
the levels activity modification of MnSOD Cells
which
MnSOD
per mitoehondnion, for this is unclear.
protein
per mito-
might with
and least The discrepprotein,
to abnormal SODS
cells,
stimulate
normal
cell
mitochon-
immunoreactive
be related and
in-
two transfeeted
had the most
in SOD3 stained
or nucleus
a 12-15-fold
more
the other
of mRNA, might
cytoplasm
showed
per cell and
but less activity
dna per cell, most MnSOD MnSOD activity. The reason and
immunofluoreseent
more
chondnion
in the
neo
clear. The increase in MnSOD protein as measured by Westcnn blotting was higher than the increase in MnSOD activity MnSOD-tnansfected may not be active
observed
and intensity
indicated
with regards
by
of fluores-
parental
and MnSOD levels. Immunogold staining label was almost exclusively localized over beads
being
antibody followed FITC, MnSOD-trans-
(Fig. 5, A and B). The homogeneous
fluorescence
geneity
lines
MnSOD with
rabbit
postand
the
mitochonserum
in
1 178
MnSOD Gene Transfection
Table
1
and Oxidative
en zyme
Antioxidant
activity
A172A noel neo2 neol4 SOD3 SOD4 SOD5 SOD7 Values
b p
6 7 5 4 35 85 13 78
are mean 0.05 compared
