A Cell Culture Assay for the Detection of Cardiotoxicity

LOW-FRIEDRICH, FERDINAND VON BREDOW, AND WILHELM SCHOEPPE

IRIS

An important

step in minimizing

research

is the study

protein”

formation,

assay to monitor

which digestion

systems

as well

proteins.” sible

as to known

Pharmaceuticals

cardiotoxic

subtherapeutic

cardiotoxins relevant

effects:

Cyclosporine

concentrations.

to induce

toxic

drugs.

system

The

“shock

data presented

for cardiotoxicity “Heat

Key Words:

ranges

protein”

the de nova medicine

A evokes

Azathioprine

same effect but at concentration ability

with

“shock

cells

in other

synthesis

of “shock

highly

demonstrate

seems

level.

The

to be restricted

that the proposed

at

exert the

above the therapeutic

obviously

for pos-

formation

and methyl-prednisolone

synthesis

re-

cellular

were tested protein”

as an

were pre-

fetal mice. These

protein ” formation

in transplant

stress

cardiac myocytes

l&day-old

“shock

in medical

the use of “shock

to cell-damaging

and cultured

from

inducing

We propose

response

Isolated method

to typical substances

of animal experiments

systems.

is a cellular

cardiotoxicity.

pared by a trypsin spond

the number

of in vitro model

in vitro

to

model

is animal saving and sensitive.

shock

proteins”;

Cardiac myocytes;

Cardiotoxins;

In Vitro

assay. INTRODUCTION Experiments

in pharmacology

is expensive,

accompanied

are usually dependent

by ethical

problems,

on the use of animals,

and of little public

which

acceptance.

In

addition, the results obtained in in vivo experiments are difficult to interpret due to the fact that they deal with a very complicated system consisting of different organs

and their

interaction.

A probable

solution

to these

problems

is offered

by

the model system cell culture, which allows the monitoring of pharmaceutical effects in an animal-saving and organ-specific manner. Another advantage of the in vitro

test is the ability

wider

to control

range than in the whole

An important letalis media”

and determine

the experimental

field of pharmacological

research

is the measurement

(LD5,,) of new drugs. The development

of experimental

improve the specificity of the assay and save animals can be achieved by determining toxicity in different suring the concentration

dependence

Address ternal

reprint

Medicine,

furt am Main Received

D-6000

Frankfurt

requests Hospital

70, Federal

January

am Main

to: Dr. med.

70, Federal

1, 1990;

revised

of the “dosis strategies

that

is a challenge. More specificity organs separately and by mea-

Medicine, Republic

Iris Low-Friedrich,

of the Johann Wolfgang Republic

in a

of the toxic effect.

From the Department of Nephrology, Center for Internal Goethe-University,

parameters

animal.

Hospital

of the Johann

Wolfgang

of Germany.

Department

Goethe-University,

of Nephrology,

Theodor-Stern-Kai

Center 7, D-6000

for InFrank-

of Germany. and accepted

September

1, 1990.

133 Journalof

Pharmacological Methods

0 1991 Else&r

Science Publishing

25, 133-145 (1991) Co., Inc.. 655 Avenue of the Americas, New York, NY 10010

0160.5402/91/$3.50

134

I. Liiw-Friedrich

et al.

We are engaged ments documented The test system

in the development above,

of a toxicity

assay that fulfills

that is, one that saves a considerable

is based on the phenomenon

of “shock

to stress with the de nova synthesis of certain proteins, The toxic

change

in the environmental

chemical

nature;

the cells respond

such as heavy thesis.

metals

The existence perature effects selective analoguous (Lemeaux

Cells respond

the so-called

shock proteins.

can be of a physical

chemicals,

with

as well

and to toxic

a change

as

agents,

in protein

syn-

of shock proteins was first described in 1962 in a study on temin Drosophila melanogaster (Ritossa, 1962). For almost 15 years the

induction

of proteins

response

to a “heat

was thought shock”

1978). Other

et al., 1978) and plants

to be unique

was detected

to the fly.

In 1978 an

in avian and mammalian

tis-

groups found the same activity in bacteria

(Fink and Zeuther,

1978).

Today,

it is generally

that indeed

shock proteins

all organisms-from bacteria to man-are able to produce (Schlesinger et al., 1982). The detection of a general reaction mech-

anism to toxic agents proves that it is obviously a conserved of the organism, which was very successful in evolution. The mechanism that the presence

of cell protection of these proteins

and after a vital “stress”

duties:

(Schlesinger,

The 90,000-Dalton

strategy for the survival

by shock proteins is still speculative. It seems increases the cellular ability to recreate during 1986). The most frequent

ilies are of 90,000, 70,000, and 30,000 called ubiquitin (8000 D) (Ciechanover ferent

of animals.

proteins.”

to a rise in temperature

or radical-forming

sues (Kelley and Schlesinger, accepted

condition

the require-

amount

shock protein

Dalton mol. wt., a fourth shock et al., 1984). The shock proteins

shock

protein

family

functions in cellular metabolism. It exists as a complex ceptors and as a complex with virus-coded polypeptides

has a variety

fam-

protein is fulfill dif-

of important

with steroid hormone re(Schuh et al., 19851, and

it binds calcium-dependently to actin (Nishida et al., 1986). This shock protein influences cell proliferation by regulating the entry into a new mitosis (lida and Yahara, 1984). The 70,000-Dalton cytoplasm

to nucleus

shock proteins and return

they concentrate “stress,” strengthen the cytoskeleton

move immediately

to cytoplasm

in the (Welch

nucleolus

upon

forming

and Suhan,

after a shock event from

cell regeneration.

1985);

insoluble these

During

aggregates

complexes

the and

are de-

stroyed by an ATP-dependent reaction (Lewis and Pelham, 1985). The low molecularweight shock proteins form large insoluble aggregates in the perinuclear region after a “stress” and dissociate during cell recovery. Ubiquitin, one of these shock proteins, reactions

is coupled to the l -lysine-group of, polypeptides by a cascade of enzymatic (Munro and Pelham, 1985). Polypeptides transformed in that way are pre-

ferred objects of protease digestion. It is assumed that the protease activity is a part of the cellular mechanism that tolerates stress (Schlesinger, 1986); in this way polypeptides are activited, which derepress the shock-protein-coding genes (Figure 1). The data accumulated in the literature clearly indicate that shock proteins are always synthetized when the cell is threatened by a toxic environment. Hence, the detection of shock proteins might be a useful tool in monitoring toxicity. assay based on this phenomenon should fulfill certain requirements:

A toxicity

Cardiotoxicity

1. The shock formation

protein

de nova synthesis

must be demonstrated

by classical

inductors

in the investigated

of shock

protein

cell culture.

2. Well-known toxic agents should be able to provoke the synthesis of shock proteins dependent on the organ source of the cell culture, for example, cardiotoxins should

affect the protein

formation

in myocardial

cell cultures.

3. If potentially toxic agents induce the formation of shock proteins concentrations, they can be regarded as toxins. 4. Non toxic substances reasonable

should

also be unable

to evoke

at reasonable

shock protein

synthesis

at

concentrations.

The data presented assay to detect

in this study might

establish

the test system as a screening

cardiotoxicity.

Stress (heat or toxin) I +

denaturation of cellular proteins

c

“ubiquitin” guided, ATP dependent proteolysis .............................. \ l. . . . . . . . . z : : de-repression of shock protein coding genes >

L- rv] FIGURE 1.

methionine Scheme of “shock protein” formation.

135

136

I. Liiw-Friedrich

et al.

METHODS Preparation of Fetal Mouse Cardiac Myocytes Fetal mouse examine

cardiac

the toxicity

nant mouse with 8-12 to remove

blood,

phate-buffered

myocytes

are prepared

of IO concentrations fetuses.

Briefly,

cut into smaller

to Low et al. (1989).

and placed

To

we need only kill 1 preg-

hearts from 18-day-old

pieces,

saline (PBS) (NaCI,

according

of 1 substance,

fetal mice are washed

in 6 ml of calcium-free

8 g/L; KCI, 0.2 g/L; Na*HPO,,

phos-

1.15 g/L; KH2P04,

0.2 g/L). All preparation steps are performed under sterile conditions with the addition of 25 U/mL of penicillin and 25 kg/mL of streptomycin to the buffers and media. 0.1%).

Next,

4 mL of 0.25%

The pieces

agitation,

which

pernatant

is discarded

in PBS. After

trypsin

are then

when

in PBS is added

digested

stopped

(final concentration

allows

the pieces

the supernatant

under

to settle to the bottom.

and the pieces are again resuspended

15 min of agitation,

of trypsin:

for 15 min at room temperature

in 10 ml of 0.1% trypsin

is again discarded.

The super-

natants of the following two digestion steps are collected. The digestion by dilution of the supernatant containing the cells into equal volumes alpha-medium

(Stanners

et al., 1971) supplemented

slight

The su-

is stopped of ice-cold

with 20% fetal calf serum

(FCS).

The yield of cells can be increased when the heart pieces are flushed cautiously with a stream of buffer. The cells are collected by centrifugation at 4°C in a Sorvall SS 34 rotor at 1000 rpm for 10 min. The cells are washed in 10 mL of alpha-medium supplemented with 20% FCS. They are centrifuged again as described above. Then the pellet

is resuspended

cell number

in 3 mL of alpha-medium

is determined

in a cell-counting

plus 20% FCS. Thereafter,

chamber.

The amount

the

of cells is ad-

justed to 5 x IO5 cells per dish. They are kept for 2 days in the incubator at 37°C in an atmosphere of 5% CO* saturated with water. There the cells attach themselves tightly to the dishes (Nunclon able culture

surface

dishes:

(Dulbecco’s minimum essential mented with 5% FCS, IO mmol/L and 25 kg/mL are exposed

material,

area, 8.8 cm2). After

streptomycin,

polystyrene;

incubation

diameter,

3.5 cm; avail-

they are transferred

to DMEM

medium) (Dulbecco and Freeman, 1959) suppleHepes, 2.5 mmol/L L-glutamine, 25 U/mL penicillin,

pH 7.4. The cells are kept

to the experimental

in this medium

until they

conditions.

Cells Labelling with L-(~ S)Methionine After

incubation

of the cells for a definite

min of heat shock (42°C) exposure, are incubated

for an additional

The labelling is stopped cold PBS, and dissolving Gold,

time

period

with the toxins

0.1 mCi of L-(35S)methionine

2 hr in a water-saturated

by removing the supernatant, the cells in 1 mL of O’Farrell’s

is added.

atmosphere

or after 5 The cells

with 5% CO,.

washing the cells with icesample buffer (O’Farrell and

1973).

SDS-gel Electrophoresis The proteins are precipitated minute (cpm) in each sample cpm in approximately

according to Wessel and Fltigge (1984). Counts per are determined by liquid scintillation, and 100,000

50 j.d O’Farrell’s

sample

buffer are applied

per slot. Gradients

Cardiotoxicity of 5%-15%

of acrylamide

or 10% acrylamide

are used for SDS-gel

electrophoresis

according to Laemmli (1970). The slab gels are run for 8 hr at 160 V. Phosphorylase b (mol. wt. 94,000 Dalton), albumin (mol. wt. 67,000 Dalton), ovalbumin (mol. wt. 43,000 Dalton),

carboanhydrase

wt. 20,100 Dalton) 0.99: The times.

gels shown

Staining

of Proteins

The

proteins

ethanol,

(mol. wt. 30,000 Dalton),

serve as molecular

weight

are representative;

are stained

with

0.1%

markers. each

experiment

Coomassie

80 mL acetic acid, 670 mL water,

and trypsin

inhibitor

(mol.

The value of r* is better was performed

brilliant

blue

than three

R 250 in 250 mL

and 1% glycerol.

Autoradiography The fixed

and stained

slabs are dried

gels are fixed to the film (B-max) in a commercial

on Whatman

for 72-144

3 MM

paper.

Then

hr. The films are developed

the slab and fixed

system.

Materials L-(35S)methionine was supplied by NEN, FCS by Gibco (Eggenstein, FRG), DMEM, alpha-medium, trypsin, and antibiotics by Biochrom KG (Berlin, FRG). Films for autoradiography

(B-max) were

purchased

from Kodak. Poly(oxyethylene)-40-ricin All drugs were taken from commercial highest

purity

available.

The pregnant

from Amersham,

and developer

and fixer

was provided by Sandoz (Nurnberg, packages; the other chemicals were NMRI

mice lived on standard

FRG). of the

diets.

RESULTS To fulfill

the requirements

the myocardial

cells with

of the toxicity

typical

assay formulated

agents well

thesis in other organisms and tissues. Indeed, served: 0.1 mmol/L CdClz induces the formation

known

above,

to induce

and one 30,000-Dalton protein; 0.001% H202 provokes 30,000 Dalton shock protein (Figure 2). The synthesis of the 30,000 Dalton shock protein is evoked 22.5

protein

syn-

the expected effects could be obof two proteins of the 70,000-Dalton

family

0.05 mg Hg/L) HgC12. At concentrations

we incubated

shock

kmol/L

(=

the synthesis

of a

by 0.25 Pmol/L

(=

0.5 mg Hg/L),

additionally

two de nova synthetized proteins of the 70,000-Dalton family can be detected (Figure 3). These findings are in good agreement with practical clinical experience where the first toxic effects of Hg are observed at concentrations of 0.04 mg/L (Beratungsstelle bei Vergiftungen und Giftinformation, Klinikum der Johannes Gutenberg-Universitat, D-6500 Mainz, FRG; personal communication). The same effect is observed with ZnClz: concentrations r5 kmol/L induce the 30,000 Dalton shock protein; ?I00 Fmol/L ZnClz the proteins of the 70,000-Dalton family are expressed, too (data not shown). At a short-term elevation of temperature to 42°C for 5 min, the de nova synthesis of proteins of the 70,000-Dalton family could be documented (data not shown). This pattern of shock protein formation after exposition to dif-

137

138

I. Low-Friedrich et al.

MW

70000

-

30000

-

A

D

FIGURE 2. Shock protein induction by CdCI,, H,Oz, and allylamine. Fetal mouse myocardial cells were incubated with 0.1 mmol/L CdClz (A), 1 x 10m3% HzOz (C), or 0.01 mmolll allylamine (D) for 2 hr (B, control). Afterward, 0.1 mCi of L-(35S)methionine was added; incubation was prolonged for another 2 hr at 37°C. Then the cells were dissolved in O’Farrell’s sample buffer. SDS-gel electrophoresis was run with 100,000 cpm/slot.

ferent culture

“stress”

conditions

leads one to the conclusion

that cardiac

myocytes

in

react exactly like other mammalian cells. The formation of a 71,000-Dalton of rats after an in vivo exercise test has already protein” in the myocardium

“shock been observed

(Hammond

et al., 1982).

The next step of our experimental work consisted of-according to our postulations-the investigation of a substance that has proven cardiotoxicity in clinical and/or morphological examinations. We choose allylamine, an unsaturated aliphatic amine used in the production of plastics and drugs as a stabilizer. Histologically, allylamine causes myocardial fibrosis. As observed electronmicroscopically, the single myocytes undergo necrosis with clumped myofibrils, electron-dense mitochondrial deposits, and cell lysis after application of two gavage doses of 150 mg/ kg allylamine, 24 hr apart (Boor, 1983). Of this substance 0.01 mmol/L (= 0.57 mg/

Cardiotoxicity

MW

70 000

30 000

F FIGURE 3. Shock protein expression induced by HgClz. The cardiac myocytes were treated as described in the legend to Figure 2. (A) 5 pmol/L; (B) 2.5 ~mol/L; (C) 1 pmol/L; (D) 0.5 pmol/L; (E) 0.25 pmol/L HgC&; and (F) control.

L) provokes the de nova synthesis of the 30,OOODalton polypeptide (Figure 2). This result suggests that proven cardiotoxic agents indeed formation

of shock proteins.

The effect to allylamine,

on protein synthesis seems to be restricted to toxins. In comparison we tested other stabilizers-benzoic and sorbic acid-which are defi-

nitely not dangerous to the myocardium. tein synthesis of the cultured myocytes formation

in our assay stimulate the

of “shock

proteins”

might

The chemicals did not influence (Figure 4). These results indicate

be a specific

indicator

the prothat the

of toxic environmental

conditions. As examples

for potentially

cardiotoxic

drugs,

we examined

the whole

group

of

immunosuppressive pharmaceuticals relevant in transplant medicine. The questionable cardiotoxicity of cyclosporine A is discussed but not proven. The drug inhibits the proliferation of immune-competent cells; nephrotoxicity has already been confirmed. Cyclosporine A stimulates the de nova synthesis of a 30,000-Dalton shock protein in cardiac myocytes. This effect can already be detected at concen-

139

140

I. Lbw-Friedrich et al.

MW

30000

-

C

B

F

E

D

FIGURE 4. Incubation of fetal mouse myocardial cells in benzoic acid does not evoke shock protein synthesis. The cells were treated as described in the legend to Figure 2. (A) 1%; (B) 0.1%; (C) 0.05%; (D) 0.01%; (E) 0.001% benzoic acid; and (F) control. trations

of the pharmaceutical

of the amount

of shock

210

protein

ng/mL.

formed

A quantitative

shows

porine A doses (Figure 5). The therapeutic concentration is between 100 and 150 ng/mL. Although a direct correlation doses and human

serum

to the concentrations

levels cannot

be established,

in the cell culture

exert toxic effects already We could demonstrate must be attributed to (poly(oxyethylene)-40-ricin)

densitometrical

an increase

elevated

a comparison

assay indicates

analysis cyclos-

range of cyclosporine A between the cell culture of these values

that cyclosporine

at subtherapeutic levels. that the cyclosporine A effect the

with

on

protein

A might synthesis

pharmaceutical itself as the cyclosporine solvent does not affect the protein synthesis of cultured heart

cells (data not shown). Another standard immunosuppressant, azathioprine, has an acute LDSO in mice in sensitive tissues occurs at much of 650 mg/kg (Elion et al., 1961). Cytotoxicity lower concentrations, that is, in the order of 5- (Boll et al., 1971) IO (Wilson, 1965) t.@mL for bone marrow and lymph node cells in vitro, respectively. Protein synthesis

Cardiotoxicity

20 -

15-

10

0

80

50

20

cyclosporine A

100

1ng 1 ml 1

FIGURE 5. Increasing relative amounts of the 30,000-Dalton shock protein induced by rising concentrations of cyclosporine A. The relative amounts of the 30,000-Dalton shock protein are given as a percentage of the total cellular protein content. Mean f SD obtained from the densitometrical analysis of five representative experiments is shown.

documented thioprine induces 250

by leucine

concentrations

incorporation 260

the de nova synthesis

t.@mL

(Figure

61, which

results of densitometric cyclosporine A.

lymphocytes

and Forbes,

of the 30,000-Dalton is distinctly

measurements

The same effect is obtained

in human

k.g/mL (Smith

above were

is inhibited

shock protein

at concentrations

the pharmacological comparable

with methyl-prednisolone,

at aza-

1967, 1970). Azathioprine

to those

dosages. achieved

a corticosteroid

The with

hormone,

inducing the de nova synthesis of the 30,000-Dalton shock protein at unphysiologically high concentrations (~0.5 mg/mL, Figure 7). The acute LDSO of methylprednisolone is 768 mg/kg in mice and 650 mg/kg both of which are also rather high dosages.

in rats (Hoechst

AC,

1975a,b),

In the treatment of acute rejection crises after organ transplantation, antibody preparations directed against certain immune-competent ceils are employed. These solutions do not influence the protein synthesis of cultured heart cells over a wide concentration

range (data not shown).

The results presented suggest that the study of shock protein synthesis in cultured cells provides a fast survey on the potential toxicity of a complete group of substances.

141

142

I. Liiw-Friedrich

et al.

MW

30000

-

A

FIGURE 6. Shock protein formation induced by azathioprine. scribed in the legend to Figure 2. (A) 100 &ml; (B) 75 &ml; (E) 1 @ml azathioprine; and (F) control.

F

E

B

The cells were treated as de(C) 50 p.g/mL; (D) 25 kg/ml;

DISCUSSION Our

studies

present

a test system for the assessment

of cardiotoxicity

based on

the detection of shock protein synthesis in a myocardial cell culture. Shock protein formation in heart cells is induced by cardiotoxic agents; substances

(sorbic

acid, benzoic

acid, poly(oxyethylene)-40-ricin,

several

two different

an-

tibody solutions) that are suggested to be nontoxic do not evoke shock protein synthesis. Chemicals that exert toxic effects can be divided into damaging and harmless agents by their ability to evoke shock protein formation. Shock protein synthesis is concentration-dependent so that the degree of toxicity can be estimated by the toxin concentration needed for the induction of shock protein formation. It seems that low concentrations of “severe” toxins or high concentrations of “moderate” toxins induce the 30,000-Dalton shock protein, whereas the proteins of the 70,000-Dalton family are expressed at high concentrations of “severe” toxins. This observation needs further investigation and might provide

Cardiotoxicity

MW

30000

-

A

C

D

FIGURE 7. Shock protein induction by methyl-prednisolone. The cultured myocytes were treated as described in the legend to Figure 2. (A) control; (B) 0.25 mg/mL; (C) 0.5 mg/mL; and (D) 0.75 mg/mL methyl-prednisolone.

insight in the different functions of “shock proteins ” in cellular protection. The de nova expression of these proteins is detectable when the cells are morphologically still intact, when they still have vital functions, such as contractility in myocytes, and when metabolism is not roughly disturbed, for example, the cells are still capable of protein synthesis. These results document that the test system reacts even

143

144

I. Liiw-Friedrich

et al.

more

than

sensibly

sensitivity

does

the measurement

of cell death.

of the assay has been demonstrated

In comparison advantages:

to whole-animal

Toxicity

ner. The number

of animals

and the organs

experiments,

is determined

removed

the cell culture

organ-specifically

needed

We

conclude

that the

test provides

several

by these data. in the described

is very low, only one pregnant

is sufficient

to investigate

10 different

sensitive mouse

man-

is killed,

concentrations

of

one substance. We focused more effective were

on the problem of cardiotoxicity. Theoretically, and animal saving when all important organs-liver,

removed

toxicity

to the special hepatocytes period before

and prepared

test to other

simultaneously

organ cell cultures

abilities

for cell culture.

might provide

of the cells investigated.

may change

in production

the assay could be kidneys, heartThe transfer

a different

Cytochrome

levels after

primary

may not be detected

nonsubstrate specific

specificity.

toxins

must

applied to other Whole-animal

to either

culture,

even

lack of correct

investigations

be performed

before

the shock

organ cell cultures. studies are performed

to determine

Several concentration

series requiring many laboratory cannot be avoided completely and their

model can enlighten

the

careful

new pharmaceuticals.

metabolites

due

Therefore,

possible

animals. because

due

P450 isoenzymes

of 24 hr. Some compounds have to be metabolized by cytochrome they become toxic. If the level of isoenzymes is changed, potentially

compounds

of the

performance

protein

letalis

or

of organ-

test system

the “dosis

a

P450 toxic

enzymes

on the effects

of

over

can be

media”

ranges have to be examined,

of

each test

Presumably, the whole-animal experiment it is able to monitor the interactions of

accumulations

the critical concentrations

in different

organs.

The cell culture

and by that way avoid a large number

of in vivo tests performed in search of the toxic dosages. Increasing scientific interests concentrate on the practical

use of shock proteins

in clinical routine: Hyperthermia is discussed as an additional therapeutic strategy for the treatment of cancer, preferably in addition to radiation. After the first heat treatment,

the level of shock proteins

in the tumor

cells increases

and they develop

a temporary resistance to the effects of heat. By measuring the production of shock proteins, clinicians may be able to identify exactly the period of most vulnerability to determine the onset shock proteins in tumor and to determine would

of the next treatment. The development cells has started; test systems to identify

the extent of ischemic

like to add an additional

formation

by proposing

on shock protein This work

are investigated

for the diagnostic

an assay for the detection

(Ember,

1989). We

use of shock

of organ-specific

toxicity

protein based

synthesis.

was supported

to Ms. Ch. Heinecker

damage

indication

of an assay for metabolic insults

by the Scheidel-Stiftung,

for her expert

preparation

Frankfurt

am Main,

FRG. The authors

are indebted

of the manuscript.

REFERENCES Boll I, Klimas

J,Willigerodt

6-Mercaptopurin

und

B (1971) DieWirkungvon Azathioprin

auf mensch-

liches Knochenmark 21:502-504.

in vitro. Arzneimittel-Forsch

Cardiotoxicity Boor

PJ (1983) Allylamine

cardiotoxicity:

lism and mechanism. J.J. Spitzer.

New

metabo-

In Myocardial

York:

Plenum

Injury.

Ed.,

Press, pp. 533-

541.

of shock

Munro,

A, Finley, D, Varshavsky,

ubiquitin

mediated

mechanisms protein

pathway

of energy-dependent

degradation.

Dulbecco,

A (1984) The

proteolytic

by the polyoma

intracellular

/ Cell Biochem

R, Freeman,

and

Nishida,

HRB (1985) What

binding

protein

to actin

SW, Bieber,

S, Hitchings,

RW (1961) A summary

Cancer Chemother

Ember,

Fink K, Zeuther,

PZ, Gold,

Hammond, forms

Useful

E (1978)

Heat

products

shock

CL, Lai, Y-K, Markert of stress

lead

expression

through

metabolic

pathway.

to

or

proteins

in

CL (1982) Diverse

new

patterns

a common

of gene

and

essential

Proc Nat/ Acad Sci USA

79:3485-3488. Hoechst

AC

akute intravenose

Bericht

solubile

am Main,

FRG, 1-4.

AC

akute Main, lida,

forte”

mit Frankfurt

forte”

vergleichende

an Ratten.

mit

Frankfurt

am

FRG, l-4.

H, Yahara,

of the

I (1984) Durable

yeast

synthesis

heat shock proteins

and

other

eucaryotes.

of high

analogues

and

expression

in chicken

embryo

phage

of

on gene

fibroblasts.

Cell

assembly

of structural

of the

head

proteins

of bacterio-

T4. Nature (London) 227:680-685.

hardt,

PG,

Herendeen,

SL, Bloch,

FC (1978) Transient

perature

Schuh,

polypeptides shifts.

M, Tissieres,

A (1982)

Laboratory,

pp.

Heat

proteins:

the

binding

shock

/ Ce// Bio/103(2):321-325.

S, Yonemoto,

W,

Brugge,

J, Bauer,

VJ,

WF, Toft, DO (1985) A 90,000-

protein

common

and the Rous sarcoma

to both steroid virus transform-

pp 60 v-sarc. / Biol Chem 260:14292-

ing protein,

14296. JL, Forbes,

IJ (1967) Use of human

cytes in studies 539. Smith, IL, Forbes,

lympho-

Nature 215:538-

of drug action.

IJ (1970) Inhibition

in human

lymphocytes

of protein

syn-

by thiopurines.

Aust / Exp Biol Med Sci 48:267-276. CP, of

Welch,

Eliceiri,

CL,

ribosome

WJ, Suhan,

of the

mammalian

zation

in

Green,

H (1971)

mouse-hamster

Two

hybrid

of changes

rates of synthesis

in E. co/i following

of

tem-

in the nuclear shock

HRB (1985) Involvement

and nucleolar protein.

functions

of ATP of the 70

Eur MO/ Biol Organ /

4:3137-3143. Low, I, Friedrich,

stress

tranuclear

actin filaments

heat-shock D,

Fltigge,

in the

UI

recovery

presence

study

characteri-

organelles,

and appearance

cyof in-

in rat fibroblasts

after

/ Cell Bio/101:1198-1211.

treatment.

quantitative

response:

in cytoplasmic

and nucleoli,

Wessel, PL, Neid-

JP (1985) Morphological

toskeleton,

(1984)

A method

of protein

for

the

in dilute solution

of detergents

and

lipids.

Anal

Biochem 138:141-143. Wilson,

heat

by

Ex-

Ce// 13:427-434.

Lewis, MJ, Pelham, kd

MJ (1986)

for functions.

Riehl, RM, Sullivan,

types

heat shock

UK (1970) Cleavage the

individual

induced

in Drosophila.

NY: Cold Spring Harbor

search

Stanners,

15:1277-1286.

Lemeaux,

pattern

and DNP

MJ, Ashburner,

Schlesinger,

/ C/in Biol

MJ (1978) The effect

amino-acid

during

shock

cells. Nature New Biol230:52-54.

P and Schlesinger

Laemmli,

Harbor,

in G, cells

99:199-207. Kelley,

T4

440.

thesis

weight

Bacteriophage

Heat Shock: From Bacteria to Man. Cold Spring

Smith, tiber

Vertraglichkeitspriifungen

solubile

molecular

Schlesinger,

receptors

vergleichende

an Msusen.

(197513) Bericht

intravenose

“Urbason

tiber

Vertrlglichkeitsprtifungen

“Urbason

Hoechst

temperature

dalton

(1975a)

LM (1973)

/ Bio/ Chem 248(15):5502-5511.

perientia (Base/) 18:571-573.

MO/ Cell Bio/l2:103-115.

Tetrahymena.

heat

/ Biol Chem

filaments.

Ritossa, F (1962) A new puffing

thiopu-

Biotechnology News 9(14):6-7.

thinking.

90-kDa

GH,

of investigations

Rep 14:93-98.

B (1989) Stress proteins:

wishful

I (1986) Cal-

of the

261:16033-16036. O’Farrell,

Virology 8:396-397.

with 6-[(I’-methyl-4’nitro-5’-imidazolyl)] rine.

myo-

turns on heat

E, Koyasu, S, Sakai, H, Yahara,

modulin-regulated

gene expression. Rundles,

mouse

G (1959) Plaque production

virus.

Elion, CB, Callahan,

fetal

Nature (Land) 317:477-478.

genes?

shock

24:27-53.

in cultured

S, Pelham,

shock

Ciechanover,

proteins

cells. Fxp Cell Res 180:451-459.

cardial

DB (1965) Quantitative

havior

of sensitized

hibitory Imuran, lymphoid

T, Schoeppe,

W (1989) Synthesis

influence

studies

lymphocytes of the

on the destructive cells against

/ Exp Med 122:167-172.

on the be-

in vitro. II. In-

immune

suppressor,

reaction

of sensitized

homologous

target

cells.

145