Modulation of transferrin receptor expression by dexrazoxane (ICRF-187) via activation of iron regulatory protein

Biochemical Pharmacology, CopyrIght 0 1997 Elsevier

Vol. 53, pp. 1419-1424, Science Inc.

ISSN

1997.

0006-2952/97/$17.00 + 0.00 PII SOOOS-2952(96)00894-5

ELSEVIER

Modulation of Transferrin Receptor Expression by Dexrazoxane (ICRF- 187) via Activation of Iron Regulatory Protein Gtinter Weiss,* Stefan Kastner,* Jeremy Brock, f Josef Thaler” and Kurt Grtinewald” *DEPARTMENTOF INTERNALMELXCINE,UNIVERSITYHOSPITAL, A-6020 INNSBRUCK,AUSTRIA AND ~DEPARTMENT OF IMMUNOLOGY,WESTERN INFIRMARY,UNIVERSITYOF GLASGOW, GLASGOW Gil 6NT, SCOTLANLQUK

ABSTRACT.

Dexrazoxane

chemotherapy

h as recently

(ICRF-187)

with anthracyclines,

although

been

the reason

demonstrated

to reduce

for this phenomenon

cardiac

toxicity

induced

has remained

obscure

thus far. In

by

order to investigate whether ICRF-187 might exert its effects by modulating iron metabolism, we studied the drug’s potential to influence the maintenance of iron homeostasis in two human cell lines. We demonstrate that ICRF-187

enhanced

the

posttranscriptional THP-1

binding

iron

myelomonocytic

an elevation

of transferrin

ICRF-187

receptor

iron

treatrnent

response

follow

response

to ICR?-187

may contribute

complex

and iron-catalysed

53;10:1419-1424,

(ICRF-187;

cells. Increased

surface

of hydroxyl

activity

receptor;

human

therefore

side-effect

the reason dated

thus far. Besides

by generating

redox

molecule

oxygen

with transfer

catalysing

ferric

electrons

the formation

dose-

induced

suggested

to be a major

cardiotoxicity,

f,ince

* Corresponding

reduce

species

or via formasee 6). The internal oxygen, [7]. An-

cause of anthracyclinelevels

of

radical

author: Dr. Gunter Weiss, Department of Internal Medicine, University Hospital, .4nichstr. 35, A-6020 Innsbruck Austria; Tel. ++43-512-504-3255; FAX ++43-512-504-3293; E-mail: guenter. [email protected] Abbreviatwns: trf-ret, transferrin receptor; IRP, iron regulatory protem; IRE, iron responsive element; e-ALAS, erythrold 5-aminolaevulinate synthase; NO, nitric oxide. Received 5 June 1996; accepted 20 November 1996.

iron

the

from

shown induced

to be present

ICRF-187

could

chelate

product

analogue

as

storage

protective

protein such

iron and due to the

of ICRF-187,

of EDTA.

to find that ICRF-187

chelators

cardiotoxicity

in the heart

toxicity

hydrolysis

is a cycled

iron

iron

to exert

ICRF-187

[6, 81.

open-ring

the

intracellular

appear

that

it was interesting

potential,

that

protein;

anthracycline-mediated

respect,

to molecular

reduced

It was suggested

fully eluci-

of free radicals

RIOCHEM PHARMACOL

tissues

ADR-925,

thracycline-induced formation of radicals causes lipid peroxidation of mitochondrial membranes and endoplasmatic reticulum,

mechanisms to other

namely

further

in

of iron in

the iron-anthracycline

iron regulatory

fact

iron (for review

All these

of the protein

reaction.

anthracy-

directly

after undergoing

by reducing

However,

of free radical

Subsequently,

and sequestration

with

DNA-intercalation either

uptake

[l-5].

has not been

inducers

radicals

can then,

reactions,

thereby

their

are powerful

of complexes

latter

therapy

or daunorubicin

for this phenomenon

anthracyclines tion

of anticancer

iron uptake.

scavenging

which

such as adriamycin

cellular

monocytes;

diomyopathy, limiting

enhanced

expression

by

mechanism

hy IRP.

surface

in

was paralleled

mRNA

of ICRF-187

compared

clines

interaction

for

(IRE),

Inc.

(+)-1,2-bis(3,5-dioxopiperazinyl-

cumulative

factor

elements

to the well-established

via the Haher-Weiss

has been demonstrated to exert protective development of anthracycline-induced carand

regulatory

of trf-ret

cellular

l-yl)propane) effects against

is a frequent

central

responsive

IRE/IRP

increased

Increased

radicals

1997. 0 1997 Elsevier Science

and

and

the

iron

according

expression

mRNA

relationship.

transferrin

(IRP), called

levels which,

to the protective

generation

protein

structures,

due to stahilisation

of trf-ret

a dose-response

KEY WORDS. iron metabolism;

Dexrazoxane

mRNA

trf-ret

stahilisation

to ICRF-187,

loop

erythroleukemic was likely

of cells increased

regulatory

stem

(trf-ret)

regulation,

i.e. IRP activation,

of iron

to RNA

as well as K562

of posttranscriptional events,

affinity

regulation,

ferritin.

In this

can recruit Furthermore,

as desferrioxamine

activity

against

comparable

to that

were

anthracyclineof ICRF-187

[9-111. Therefore, this study was designed to investigate the potential effects of ICRF-187 on the regulation of cellular iron metabolism. Maintenance of cellular

iron homeostasis

erted

by the

posttranscriptionally

cytoplasmatic

proteins,

named

is largely

interaction iron

regulatory

ex-

of specific protein

(IRE’)-1 and IRP-2, with RNA stem loop structures, called iron responsive elements (IRE). IREs are present within the 5’ untranslated protein ferritin erythroid within

regions of the mRNAs for the iron storage and the key enzyme of haem biosynthesis,

Saminolaevulinate the

3’ untranslated

synthase region

(e-ALAS),

of transferrin

and also receptor

(trf-ret) mRNA, the central molecule for iron uptake into cells (for review see 12-14). IRP-1 is a bifunctional protein which can, depending on intracellular iron supply, act

1420

G. Weiss

either

as a cytoplasmic

protein

[15-171.

the case of enhanced radical

nitric

binding IRPs

oxide

function then

ferritin

untranslated it from

at the

ferritin

which

consumption Based

on

chemical of this

enhances

identified

translation

results

overall

to

which intracel-

iron

of IRPs, which and reduced

then

lose

in increased trf-ret

and

concerning

on

erythroleukemic

of IRPs,

and

cellular

cell

line

used for investigating macrophage

human

monocytic

the

American

USA).

Type

dinium

with

10% heat-inactivated

glutamine,

grown

Cells were seeded

50 p,M ferric

nitrate

- 9H,O

Fe(N03)s

and

both

8 hr at 65°C cpm/mL probes

the

human

procedure

as indicated

at 65°C.

10 X Denhardt’s

turated

salmon

quently

with

were exposed

air containing

5%

Hind111 insert) land. Probes England metrically analysis

Germany),

(a gift from

Laevosan,

Aus-

below.

(twice

of dena-

washed

subse-

for 30 min)

for 30 min)

and

at 65°C. x-ray films

with intensifying

screens

at

the 800 bp Accl

cDNA

pBR322;

was a generous

were labelled

scanned

insert

was used.

1.9 kb

HindIII/

gift from Dr. D. W. Cleve-

with [a-32P]dCTP

Boston,

procedure (Vilber

(in

MA,

U.S.A.)

[26]. Autoradiographs using

Lourmat,

the

Bio-Profil

Marne

(DuPont using were

system

La Vallee,

New

the

oli-

densitofor image

France).

Determination Cell Surface Expression anti-human

of Transferrin

Receptor

of human trf-ret was estimated CD 71 monoclonal antibody

Boehringer Mannheim, Germany). mouse IgG2a monoclonal antibody Mountain

View,

CA,

Expression

on

by using a rabbit (obtained from

For isotype control, a anti CD-10 (BectonUSA)

was used.

Both

antibodies were applied as purified immunoglobulin (0.5 p,g in 20 p_L of PBS containing 0.1% azide). Cells were seeded

Assay

and purified

1 mg/mL

were

100

K562 and THP-1 cells were grown in RPM1 plus additives and treated as described above. After harvesting and washing, detergent cell extracts were prepared as described previously [19]. A [32P]-labelled human ferritin H-chain IRE probe was generated by an in vitro transcription procedure

10% dextran

for up to 4 days to XRP-5

Nuclear,

goprimer

[Fe(3+)]

as

3 X

contained

Human transferrin receptor cDNA was kindly by Dr. M.W. Hentze (EMBL, Heidelberg). For

of 0.5 X 106/mL, and (applied

10h

plasmid

(0.2% Ficoll 400, 0.2% and

Blots

RP, Sigma)

B-actin

(FCS),

with

cDNA

SDS (twice

SDS

CA,

for 6 to

-80°C. provided

Dickinson, Gel-retardation

X-OMAT

hybridisation,

0.1 mg/mL

LA Jolla,

solution

BSA),

DNA.

0.1 X SSC/O.5%

on 1%

was blotted

overnight

solution

2 X SSC/O.5%

northern

Munich,

(Stratagene,

hybridised

0.2% sperm

accord-

and prehybridisation

The hybridisation

Chicken

tria) were then added to the culture medium. After incubation for 24 hr, the incubated cells were harvested, washed twice with phosphate-buffered saline and subjected to further

were

RNA

[oy-32P]dCTP-radiolabelled

MD,

(Sigma, Munich, Germany) or 10 (corresponding to 25 FM to 2.5 mM)

hydrochloride-salt

blots

of

from

fetal calf serum

at a density

membranes

(Rockville,

and

gels and

UV-crosslinking

Prepa-

were separated

polyvinylpyrrolidone,

supple-

nonahydrate Sigma,

After

above.

out by acid guaniextraction

RNA

M formaldehyde

obtained

penicillin,

carried

sulphate,

1640 medium

in humidified

from

FM desferrioxamine pg/mL to 1 mg/mL ICRF-187

in RPM1

100 U/mL

at 37°C

THP-1

kg of total

Duralon-UV

(Kodak,

were

was then

thiocyanate-phenol-chloroform

agarose/2.2 onto

for 24 hr as described

RNA

human

of iron metabolism

Collection

were

streptomycin CO,.

Culture

of total

Filters

line

K562

as de-

Blot Analysis

are fre-

respectively.

Cells

mented 2 mM

cell

cell line

autoradiography

SSC, 0.1% SDS, 0.1% sodiutnpyrophosphate,

MATERIALS AND METHODS Cell Culture Techniques

erythroleukemic

and Northern

were stimulated

ration

with

The

After

in the

the

of which

regulation

function,

and

both

bio-

mRNA

uptake

RNA Extraction

USA).

the effects

trf-ret

iron

K562

cell line THP-1,

iron

a putative

we investigated

activation

expression

temperature.

and subsequent

ing to [25]. Ten

mRNA 18-231.

for ICRF-187,

of this of each

[18].

Cells

then

storage

supposition

cpm

gel electrophoresis scribed

at room

12000

20 p.g protein

affinity, the 3’

extract

Approx. with

proteins

RNase, weight

IREs. This

[24].

incubated

probe precip-

the stability

Increased

function

were

following

and ethanol

3

of this RNA by protecting

yet

as described

6 M urea) extraction

of

[12-14,

myelomonocytic quently

mRNA

itation transcript cellular

of

iron

substance

human

these

phenol/chloroform

20 min,

high

for

into cells [12-141.

leads

our

and

with

(20:1),

elution,

mg/mL heparin were added for ten min, and analysis RNA/protein complexes was carried out by non-denaturing

region

as well as low iron uptake

function

stability

Activated

of low molecular towards

and e-ALAS

stability,

mRNA

by a not

affinity

[18-231.

in

IRE-

of IRPs to IREs within

of trf-ret

the IRE-binding

high

stress

5’-untranslated

the expression

iron uptake

decrease

oxidative

of translation

concentrations

and/or

of the labile

binding

digestion

promotes

their

formation

or during

e-ALAS

region

and consequently

lular

intracellular

in repression Conversely,

amide/bisacrylamide

states,

(NO)

and

iron-regulatory

iron deprivation

IREs

mRNA

[IZ-141.

or an

cellular

of IRPs is stimulated

target

resulting

aconitase

During

et d.

by gel electrophoresis

(15%

acryl-

at a density of lo6 cells/mL in 24-well plates and supplemented and stimulated as described above for 48 hr. After harvesting by scraping, cells were washed and resuspended in 50 FL of DMEM containing 2% FCS and 0.1% azide and incubated with the appropriate antibody for 30 min on ice. For reagent control, the first step was carried out only with

ICRF-187 and Transferrin

culture

medium.

Receptor

Incubation

1421

Expression

was stopped

mL of cold DMEM/2%FCS/O.l%

by addition

of 1

azide, and after washing

twice, cells were counterstained

with fluorescein

treatment

c

I

I) ICHF-187 (me/n&) 0.01

isothio-

0.1

1

cyanate-conjugated anti-mouse IgG serum diluted l/100 in DMEM/2%FCS/O.l% azide for 40 min on ice. Stained cells were

analysed

Dickinson,

on

a FACStar

Mountain

flow cytometer

View, CA, USA).

(Becton

Fluorescence

in-

tensities of 5 X 10’ cells were measured for each determination. Data are expressed as mean fluorescence

channel

IRE/IRP

in

relation to isotype and reagent control.

Iron Uptake

Studies

Cells were incubated (RPM1

for six hours in serum-free

1640 with 1 mg/mL HSA)

medium

with appropriate addi-

tives, and then incubated with [59Fe]-labelled transferrin for two hr as described natants

[27]. Radioactivity

was determined,

in cells and super-

and uptake expressed

as ng Fe

taken up per lo6 cells per hr.

Protein

free probe

Determination

Protein concentration ing to Bradford

of cell lysates was estimated accord-

[28] using the protein

BioRad (Richmond,

California,

dye reagent

U.S.A.)

from

and bovine serum

2-ME

albumin as a standard.

Statistical

Analysis

Calculation Student’s

of statistical t-test.

Only

significance

P values <

was carried out by 0.05

were considered

significant.

RESULTS ICRF- 187 Enhances

IRE:-binding

Affinity

of IRP

In order to investigate a pc’ssible effect of ICRF-187 regulation,

on iron

we first performed

gel-retardation

assays to

obtain an estimate for alterations

in IRE-binding

activity of

IRP. In K562 as well as in THP-1

cells, only one IRE/IRP

complex could be detected, corresponding

to human IRP- 1.

As previously described [18, 19, 221, treatment ferric iron nitrate IRP-l-binding cells,

while

(50 FM) for 24 hr caused a decrease in

activity addition

desferrioxamine

of cells with

(100

as compared of the FM)

to untreated

intracellular

iron

control chelator

FIG. 1. Activation of IRP by ICRF-187. KS62 cells were treated with desferrioxamine (D; 100 PM corresponding to 66 &mL), ferric iron nitrate (I; 50 PM), ICRF-187 (10 pg/mL to 1 mg/mL corresponding to 25 PM to 2.5 mM, respectively), or left untreated (control; C). After 24 hr detergent cell extracts were prepared and analysed for the IRE-binding activity of IRP by electromobility shift assay using a [32P]-labelled IRE probe. IRE/IRP complexes and unbound IRE probe are shown in the upper panel, while in the lower panel (extracts plus 2% mercaptoethanol) only IRE/IRP complexes are depicted. One of four similar experiments is shown. Gel-retardation assays with extracts from THP-1 gave results comparable to those shown for KS62 (data not shown).

concentration IRE-binding

2%),

a procedure

known

to fully activate

of IRP in vitro [19], caused maximum

high

affinity binding of IRP to IREs with either treatment

(Fig.

1). This indicates that changes in IRP-1 activity upon iron perturbations

or treatment

with ICRF-187

occur posttrans-

lationally.

resulted in an up to three-fold

increase in IRE/IRP interaction

as compared to the control

(Fig. 1). When cells were exposed to increasing concentrations of ICRF-187, ranging from 10 p_g/mL to 1 mg/mL (corresponding to final ccncentrations between 25 PM to 2.5 mM), a growing stimulation of high IRP to IRE could be observed (Fig. alterations in IRP-binding affinity after or ICRF-187 treatment were evident in

affinity binding of 1). The observed iron perturbations both human eryth-

roleukemic cells (K562, Fig. 1) and human cytic cells (THP-1, results not shown). Addition of 2-mercaptoethanol

myelomono-

to cellular extracts (final

U~regulation

of trf-ret

mRNA

by ICRF-187

We then tested whether the stimulation of the IRE-binding function of IRP-1 by ICRF-187 would result in modulation of trf-ret mRNA concentrations, since the expression of this protein is subjected to posttranscriptional regulation by IRE’s [14, 201. As is evident from Fig. 2, cytoplasmic concentrations of trf-ret mRNA are increased in K562 cells after treatment with desferrioxamine, which is due to stabilisation of trf-ret mRNA following IRE/IRP interaction, as previously demonstrated

[20]. Accordingly,

trf-ret

1422

TABLE 1. Stimulation erythroleukemic and

treatment

of trf-ret expression on K562 human THP-1 human monocytic cells by

Mean channel log fluorescence Treatment

trf-ret mRNA I

1

I

p-a&in mRNA

K562

Control Iron Desferrioxamine ICRF-187 (10 &mL) ICRF-187 (100 kg/mL) ICRF-187 (1 mg/mL)

23.2 19.8 61.2 35.4 58.7 124.0

cells ? + i ? -c -+

THP- 1 cells

4.8 5.91 19.6* 12.6$ 16.2* 21.6*

13.3 10.7 37.2 26.8 49.4 72.6

2 f 2 +t ?

2.4 2.5f 12.9* 8.77 15.4* 12.0”

I FIG. 2. ICRF-187 increases trf-ret mRNA expression. K562 cells were treated as described in the legend to Fig. 1. After 24 hr total RNA was prepared, and 10 pg were subjected to Northern blot analysis. One of three similar experiments is shown. Northern blot analysis of THP- 1 cells provided the same pattern of trf-ret mRNA concentrations depending as pictured for K562 cells for either treatment (data not shown).

mRNA

levels

procedure IRP-1

are

which

reduced

following

also decreases

(Fig. 1 and 2). Densitometric

blots

clearly

demonstrated

ratio relative

“1” corresponding

challenge,

a growing

to the control

increase

where

(N = 3), 1.8 t

(N = 3), and 2.3 2 0.4 for 1 mg/mL

ICRF-187

(N = 3), respectively; <

0.01

*for

P > 0.05

106

pg/ml

in trf-ret

levels were not altered

(Fig. 2, lower panel). The effects observed gated,

were

although

(details

control

for 10 WE/ml 1 mg;;n‘i

are specific, by either

consistent

in both

of

since

treatment

trf-ret

equal

effective

iron

not significant untreated

cell lines

of ICRF-187 viability

cells as checked

(K562

decrease

control

This

appears

expression resulted

in trf-ret

(100

indicates

that

than

ICRF-187.

in a slight,

expression

at

to be more although

as compared

to

cells.

Stimulation

of Cellular Iron Uptake by ICRF-187

Incubation

of K562

in

a significant

mediated

FM

to

in comparison

was relatively

to

0.025)

ICRF-187

with

as compared

small,

kg/mL

in

transferrin-

2 0.05, N = 4

cells per hr for controls)

treated

ICRF-187-treated

in iron uptake

(100

of 250 FM) resulted increase

to 66 kg/mL;

P < 0.025

as compared increase

blue exclusion

<

for cells

corresponding

cells/hr;

with

into K562 cells (0.31

-’ 0.05 ng Fe/lo6

investihere

(P

iron uptake

also observed

and THP-1)

cells

to a final concentration

for 24 hr did not

by trypan

trf-ret

treatment

surface expression

of desferrioxamine

desferrioxamine

in regulating

Conversely,

of trf-ret

to 66 p,g/mL).

concentrations

mRNA

for K562 are shown

cell lines

cellular

on

amounts

administration

FM)

[2.5 mM] of

and

after

corresponding

vs 0.23

dosages

in a decreased

untreated

levels

for ICRF#l87

of these

the indicated

result

mRNA

only the results

(Fig. 2). Treatment with

(250

of

in comparable

as observed

corresponding

p-actin

concentrations

(25 FM)

0.3 for 100 pg/mL

ICRF-187). The changes mRNA

in trf-ret

the ratio is set at

to “lOO%~“] for 10 p,g/mL

resulted PM,

of increasing dosages of the trf-rec/@actin

ICRF-187

P

of

of Northern

ICRF-187 ICRF-187;

a

activity

scanning

mRNA levels upon supplementation of ICRF-187 (1.2 -+ 0.4 [increase mRNA

iron

the IRE-binding

desferrioxamine

0.33

(100

-t 0.06 ng Fe/lo6

to the control; cells).

with desferrioxamine it was consistent

as was

P > 0.05

Although

the

and ICRF-187 and

reproducibly

observed.

not shown). DISCUSSION

ICRF- 187 Increases

trf-ret Surface Expression

As shown in Table 1, ICRF-187 significantly trf-ret expression on the surface of both human

increased erythroleu-

We were able to demonstrate that ICRF-187 influences posttranscriptional regulation of iron metabolism via activation of IRP-1, thus causing a subsequent increase in

kemic (K562) and human myelomonocytic cells (THP-1) after an incubation period of 48 hr, which indicates that the increased concentrations of trf-ret mRNA observed after

trf-ret mRNA concentrations, trf-ret expression and iron uptake. These metabolic changes were observed in two different cell lines, thus pointing to the generality of this

ICRF-187 treatment cause an enhanced sion of trf-rec. As with the activation

mechanism

cell surface expresof IRP-1 and the

increase in cytoplasmic trf-ret mRNA levels, the effects of ICRF-187 on surface expression were dose-dependent. Administration of ICRF-187 at a dosage 0.1 mg/mL [250 FM]

at least in human

cells. The effects of ICRF-187

were comparable to those of desferrioxamine, which indicates that ICRF-187 may act in a similar fashion [29]. Therefore, according to the well-established mechanism of posttranscriptional iron regulation by intracellular iron

ICRF-187

and Transferrin

Receptor

[12-14,

perturbations

1423

Expression

201, the

up-regulation

mRNA levels in response to ICRF- 187 treatment due to stabilisation interaction ICRF-187

of this

mRNA

of trf-ret

tumour potential

should be

mitochondrial

following

IRP/IRE

within its 3’ untranslated region. The effect of on IRP-1 is most likely due to one of two

mechanisms:

direct interaction

phur cluster

of the

conformational

with the central

protein,

change

thus causing

which

enhances

affinity of IRP-1 while its cis-aconitase

iron-sul-

an allosteric

the IRE-binding

function

is reduced

IRP

interaction

mRNA

effect

in a similar

risk-benefit

IRP- 1. However,

for this substance,

hances

trf-ret

expression

vated intracellular

en-

and iron uptake into cells. Ele-

concentrations

of low molecular weight

iron may then cause deactivation

of IRP-1,

above, which promotes ferritin translation

and iron storage

Iron has been shown t3 be of central

importance

for

via its catalytic function for the synthesis

of hydroxyl radical in the so called H&r-Weiss (for review see 30, 31). Most interestingly,

oxidants

[32]. According

thus provia iron-

to the data presented

it remains to be seen to what extent stimulation

transferrin-mediated ICRF-187 catalytic

may

iron

reduce

uptake

toxic

and

radical

sequestration formation

action of iron and iron/anthracycline

thus contributing Nevertheless,

to the protective one

would

uptake of iron after ICRF-187

of by

by the

complexes,

effects of the drug.

assume

that

chelation

administration

and

reduces the

toxic potential of anthracyclines against tumour cells, which should lead to a decreased response rate and reduced disease-free

survival in such patients.

Interestingly,

187 has been proven to exert an anti-tumour itself, which has mainly been attributed is a potent However,

inhibitor ICRF-187

of DNA

ICRF-

potential

by

to the fact that it

topoisomerase

is converted

intracellularly

II [33, 341. to ADR-

925 with a Tliz of 28 hr [35]. While the latter substance has been proven to act as a potent not have an inhibitory

metal chelator

[9], it does

effect towards DNA topoisomerase

II. Therefore, it would also appear reasonable that altemative mechanisms may contribute to the anti-tumour potential of ICRF-187.

Future s:udies will be needed to clarify if

ICRF-187 (in a comparable fashion as described for desferrioxamine) may favourably modulate cell-mediated immune effector function: i.t has been reported that ironloaded macrophages lose their ability to kill intracellular pathogens via interferon-gamma (IFN-y) mediated pathways, while iron chelation by desferrioxamine enhances cytotoxic effector potential of macrophages involving formation interesting ICRF-187,

as previously

described

for

protective

and potential

cytopathic

ef-

as ICRF-187

studies in order to estimate

may require

further

the ultimate clinical

ratio as well as putative new therapeutic

targets

e.g. as a supportive drug for treatment

of

as has already been shown for desferrioxamine

1411. The authors would like to thank Dr. Tracey Howon for support in performing the iron uptake srudies. The supporr of the Austrian research fund “Zur F&erung der Wissenschuftlichen Forschung”, project 10603, is also gratefully acknowledged.

reaction

hydroxyl radicals via the .Yaber-Weiss reaction, tecting local tissue from free radical damage herein,

enzymes such as ribofor an anti-proliferative

iron sequestra-

tion by macrophages has recently been detected as a powerful mechanism to reduce extracellular formation of

catalysed

of its

of ICRF-

as described

[12-141. radical formation

region

fashion

of a drug such

infections

iron-sulphur

of

[40].

The concurrent

the proposed turnover of the central iron-sulphur cluster of

inhibition

expression via IRE/

the 5’-untranslated

reductase may account

desferrioxamine

intensive

[E.C. 4.2.1.3.1

or whether direct interaction

187 with other critical nucleotide

fects

IRP- 1 activation

within

[12-17,391,

[12-171; alternatively, ICRF-187 may reduce the amount of metabolically available iron in the cell, thereby influencing ICRF- 187-induced

of the drug by translational

aconitase

the the

of NO [36-381. Moreover, it will be most to see whether or not activation of IRP-1 by as shown herein, may contribute to the anti-

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