of Cell Growth and Sensitization to Oxidative Damage by ... - CiteSeerX

3 downloads 0 Views 2MB Size Report
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

Suggest Documents