Cycloheximide increases the thermostability of proteins in Chinese hamster ovary cells

Vol.

177,

May

31, 1991

No.

1, 1991

BIOCHEMICAL

CYCLCHEXIMIDE

INCREASES

Michael

J. John

1

Department of Beaumont Hospital, 2Guelph-Waterloo

Received

April

AND

BIOPHYSICAL

THE THERMOSTABILITY HAMSTER OVARY CELLS

Borrelli', P. Ofenstein',

Yong

J. Lee', and James

Radiation Oncology, 3601 West Thirteen

RESEARCH

OF PROTEINS

Harold E. R. Lepock'

IN

CHINESE

Frey2,

Research Laboratory, William Mile Road, Royal Oak, MI

Program for Graduate Work in Physics, Waterloo, Waterloo, Ontario, Canada N2L 17,

COMMUNICATIONS Pages 575-581

48073

University 361

of

1991

Protein denaturation resulting from temperatures between 42.0°C and 50°C has been observed and implicated as the lethal lesion for hyperthermic cell killing. A logical corollary is that protection against hyperthemic killing requires stabilization of cellular proteins against thermal denaturation. To test this, Chinese hamster ovary cells were treated with the heat protector cycloheximide and then subjected to differential scanning calorimetry to measure protein denaturation. Cycloheximide stabilized proteins that denatured between 42OC and 52OC in control cells by increasing their transition (denaturation) temperature by an average of 1.3OC. In addition, cycloheximide reduced the cytotoxicity of actinomycin D and adriamycin, suggesting that protein stabilization protects cells against stresses other than hyperthermia. 0 1991Academic Press,Inc.

Time-temperature activation KJ/mole

energy (1,2),

analyses for

of hyperthermic

mammalian cell

as calculated to the a wide

using

killing the

of

Eyring

activation energy range of proteins

cell

killing

yield

502 KJ/mole

hypothesis

(3).

this is similar denaturation

of

that protein Differential

denaturation is a lethal result of hyperthermia scanning calorimetry (DSC) has demonstrated that

hyperthermia

does denature

mammalian cell

proteins

with

temperatures (Tm) between 40.0°C and 55.0°C target that represents the lethal hyperthermic

the rate denature

limiting between

applied the

to

the

data,

586 Since

obtained for the thermal (1,3), it has been suggested

transition critical

step, 40.0°C

to

an

(1,2).

phase (4,5). The lesion, i.e.,

would appear to be one or more proteins that and 55.0°C. When critical target analysis was

a prediction

that

the

transition

temperature

of

critical target proteins is near 46.0°C was obtained (4,5). Treatments that sensitize cells to hyperthermia, e.g., short chain alcohols (6,7), decreased Tmc (4) while treatments that protect

Vol.

177,

cells,

No.

1, 1991

e.g.,

to 1.3Oc denaturation

glycerol

(5).

cells

then during against cell enters cells protection.

cell whole

protein

shift. treated

hypothesis lesion.

minutes

(10) that

the

but

Tmc by

that

cycloheximide

reduces

COMMUNICATIONS

protein

(CHM)

1 h before

and

degree of protection and thermotolerance, protection

requires

and

markedly

1 h to

induce

(9).

is

the

lethal

lesion

for

should cause a shift induced by glycerol

CHM

thermal

CHM itself does not protect cells changes that result in protection.

denaturation

l.O°C

but

hyperthermic

in the DSC scan and thermotolerance.

of

rinsing out CHM prior to scanning should reduce this To test this hypothesis, Chinese hamster ovary (CHO) cells were with CHM and then subjected to DSC. The results support this

against When

heating

CHM treatment similar to that

hypothesis. It has

been

several

due to

RESEARCH

increased

provides the same 43.0°C as glycerol

This implies some intracellular

killing cells

Conversely,

to

BIOPHYSICAL

the

10 ug/ml

hyperthermia killing at within

initiates If

supported hyperthermic

with

CHM prior

AND

and thermotolerance,

(8)

These data is a lethal

Treating

removing

BIOCHEMICAL

actinomycin protection

D (AD) against

cellular MATERIALS

then and

protection

and may represent stress

then

or adriamycin cell killing.

provides

hyperthermia

thermotolerance drugs

stabilization 1 h before

stabilization

that

chemotherapeutic

protein

applied

reported

provides

(11,12).

CHM should

If also

the provide

during

treatment

with

(ADM),

CHM did

provide

protection protection

was

protection. either substantial

This finding implies that protein against stresses other than a component

of

some

more

general

response.

AND METHODS

Cell

Culture and Survival Determination Chinese hamster ovary (CHO) cells were grown as either suspension (13) or monolayer (9) cultures in McCoy's 5A medium. The medium contained 10% iron-supplemented calf serum, 26 mM sodium bicarbonate, and 0.07 g/l penicillin G potassium. 0.1 g/l streptomycin sulphate, Spinner flasks were gassed with CO2 in order to adjust the pH to 7.4 (13) before being placed in a 37.0 C warm room. Monolayer cultures were maintained in a 37.0°C humidified incubator containing a mixture of 95% air and 5% CO2. For survival determination, cells were diluted appropriately (foslowing trypsinization for monolayer cells) and flasks containing lethally irradiated plated into 25 cm culture feeder cells (4,000 feeder cells/cm") (14). After l-2 weeks of incubation, colonies were stained and counted. Drus

Treatments Concentrated (ADM) were made DMSO concentration Experiments with

stock solutions of actinomycin D (AD) or adriamycin DMSO and diluted I,OOO-fold in medium. The final of 0.1% has been shown to be noncytotoxic (15). AD and ADM were performed with monolayer CUltUreS.

in

516

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177,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

After incubating cells with the drug for the prescribed time at 37.0°C, the drug was removed with three rinses of medium, after which the cells were trypsinized and subjected to the colony formation assay. CHM (10 ug/ml) was administered for 1 h at 37.0°C prior to and DSC, 43.0°C hyperthrmia, then during one of the following procedures: or chemotherapeutic drug treatment at 37.0°C. When cells were to be treated with CHM and another drug, the second drug was administered to the cells in medium containing CHM. Glvcerol

Treatment and Thennotolerance Induction Cells were incubated for 0.5 h, at 37.0°C, in medium containing 5% glycerol, and subsequently heated in this medium. Thermotolerance was induced by heating the cells for 12 rain at 43.0°C and then incubating them at 37.0°C for 3 h before the second heat treatment. Hvoerthermia Treatment Cells were heated in suspension at a concentration of 3~10~ cells/ml. Five ml of the suspension was added to a 15 ml centrifuge tube. After gassing the tube with a mixture of 5% CO2 and 95% air, its cap was tightened and sealed with paraffin film. The tube was placed (horizontally) into a rack that was submerged in a 43.0°C water bath. A wrist action shaker was used to agitate the cells during hyperthennia. Differential Scanning Calorimetrv Differential scanning calorimetry was performed on live CHO cells accordiny to the proce ure described by Lepock et al. (4,5). Between 3.5 x 10 and 3.8 x 10 it cells were used per scan, the scan rate was l.OOC/min, and each scan was repeated four times. The intrinsic instrumental baseline curvature and increase in Cp of denaturation (Cp) were corrected, as described previously (15). RESULTS

AND DISCUSSION

Treating cycloheximide provided glycerol

Chinese hamster (CHM) 1 h before

the same degree and thermotolerance

prior

to heating, Averaged DSC presented in Fig. exhibited a shift

of

The

protection (Fig. 1).

with 10 ug/ml hyperthermia at

against cell When CHM was

killing removed

43.0°C

as 30 min

protection was reduced markedly (9) (Fig. 1). scans for experimental and control cells are 2. The averaged DSC scan of CHM-treated cells towards higher temperatures with respect to that

control cells. The shift was 42OC and 55OC and indiscernible shown).

ovary (CHO) cells and then during

transitions

above

most at

pronounced temperatures

65OC occurred

in at

of

the region between above 65.0°C (data similar

Tm values

not as

those observed in V79 cells with no significant shifts induced by CHM, as was true in the case of thennotolerant cells (4‘5). At the 46.0°C point on the control curve (Tmc) the shift was 0.8OC + 0.4OC, and the average Tm of peak A (48OC to 52OC on control curve) was increased by 2.0°C. When CHM was removed and the cells allowed to incubate in fresh medium for 30 min prior to DSC (no CHM during DSC) the CHM-induced 577

Vol.

177,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

CHM

\ ii \ f ‘, \ \ ‘\ ‘1 \ eCHM

1IME

AT 43

C

Iyemoved

(h)

Fiqure 1. Survival curves for CHO cells heated at 43.0°. The protective effects of cycloheximide (CHM), glycerol (GLY), and thermotolerance (TT) are illustrated. When CHM was removed 30 min most of its protective effect was eliminated. before heating,

shift of the DSC scan was: eliminated between 48OC and 55OC, and unaffected

between

42OC and 48OC, halved

between

55.0°C

and 65OC (Fig.

2) DSC scans of disorder transitions

isolated cellular of cytoplasmic

organelles have shown that proteins contribute largely

peak A and those of membrane proteins to peak B. However, all components and organelles contain some thermolabile proteins

orderto cellular

1.0 % 18

0.6

ii

0.4 02

5

0.0 0.8 1 36.0

T (“C) Figure 2. Differential scanning calorimetry scans (excess specific heat (Cp) versus temperature) of whole CHO cells. This figure shows the scans from 35.0°C to 65.0°C Cm-treated cells (.-.*...*'.), 30 min before the scan ( ----------). independent scans. The intrinsic increase in cp of denaturation (16) -

1) control cells ( )I 2) 3) cells for which CHM was removed Each curve is the average of four instrumental baseline curvature and were corrected, as described previously for: and

578

Vol.

177,

No.

1, 1991

BIOCHEMICAL

denaturing under the A peak data) and are thus potential The significantly

shift

in the different

critical

target

cells of

the

The glycerol, thermal

increased and

two

similar

possible

thermolabile

(Lepock for the

to

that

mechanisms proteins

for

the 1 h incubation with detectable as an increase The shift in the proteins.

these

two

in

the

thennotolerance,

degree or

protection.

The that

of

For

shift:

newly

for

CHM-treated 1)

a depletion

synthesized

CHM (17), or 2) protein in the Tm of the most Tm of peak A SUppOrtS

CHM, at of

this

thermal

observation

thermostability

65OC suggests

previously

(4,5).

the

mechanisms.

similarity

the

unpublished target.

+ 0.4OC) iS not in Tmc of the

the

(possibly

COMMUNICATIONS

et al., critical

observed

V79 cells

during

thermolabile latter of

of

are

most

proteins) stabilization

and

RESEARCH

at Tmc (0.8OC predicted shift

and glycerol-treated

there

BIOPHYSICAL

(40°C-55OC) candidates

DSC profile from the

(l.OOC)

thermotolerant

AND

is

43.0°C, that

proteins

the

protection suggests all

three

with

provided

by

a common

mode

treatments

Tm values

protective

between

42OC

mechanism.

Glycerol is known to stabilize proteins in a direct manner This does not appear to be the case with CHM. CHM enters cells (5,lS). rapidly, establishes an intracellular steady state concentration within 2 min (lo), and inhibits protein synthesis to 5% of.control levels within 10 min (9). Yet, at least 1 h was required for maximum thermal

resistance

more like immediately. protein

to

develop

thermotolerance, CHM may initiate stabilization,

The

trigger

in i.e,

CHO cells.

and subsequently, for

the

In

thermal protection some action by the

cellular

action

thermal

this

respect,

is cells

CHM is

not manifested that leads to

protection.

may be the

stress

of

protein

synthesis fact that histidinol

inhibition. Support for this hypothesis is provided by the other protein synthesis inhibitors, viz., puromycin (9) and (19) also induce thermoprotection. There are no chemical

StrUCtUral all three

similarities inhibit protein

is unlikely that direct chemical

between CHM, puromycin, synthesis by different

these substances interaction: more

induce likely,

and histidinol, mechanisms.

thermal

resistance

resistance of protein

is

response resulting from their common effect inhibition. This secondary response may be a stabilization thermolabile cellular proteins to prevent their destruction turnover

processes Cycloheximide

or by thermal also protects

chemotherapeutic drugs, 3a and 3b). Hence, the

actinomycin CHM-induced

denaturation. against the

cytotoxicity

D (AD) cellular

and adriamycin changes that

579

and Hence it a

by

a secondary synthesis of by normal of

two

(ADM) (Figs. lead to

Vol.

177,

No.

1, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

a 1

_ -nCHM+AD

1 .D pg/ml

.

2.0

.3CHM+AD

pg/ml ,CHM+ADM

) -1 AAD

1.0

pg/ml

;: 2 2

i~ADh4 n-

‘2

“1 BAD

10-J&

2.0

I

I

2001

I

3.

actinomycin used are treatment

TIME (h)

I

i

2

TREATMENT

TIME (h)

0 TREATMENT

pg/ml

pg/ml 4.0

4.0

/Jg/ml

pg/ml

I

3

Protective effects of cycloheximide (CHM) against: a) D and b) adriamycin cytotoxicity. The drug concentrations indicated in the figures. Figure 3a also indicates that CHM alone had no effect on cell survival.

appear

thennoprotection

Thermotolerance similar

ADM

c

012345 Figure

2.0 _ -flCHM+ADM

-I -1

pg/ml

2.0

to convey

protects

degree

against

as CHM treatment.

resistance

to

other

stresses.

AD and ADM cytotoxicity The observations

(11,12)

that

to

a

CHM and

thermotolerance induce the same magnitude of protection against heating at 43.0°C and against two chemotherapeutic drugs further suggest that protection is induced by a similar mechanism. Clearly, heat shock protein synthesis cannot be the common factor since CHM including that of heat shock proteins inhibits protein synthesis, (20,21). result candidate

Protein of both for

stabilization,

as evidenced

CHM treatment the

by DSC, is

and thermotolerance

and is

a

prime

mechanism that

induces both thermoprotection and The latter may result from the drugs.

resistance to these cytotoxic targets of the drugs being stabilized against with the drugs. In support of this contention, have demonstrated

a common

that

CHM reduced,

chemical initial

by four-fold,

the

interaction experiments amount of

[3HJ-

actinomycin D that associated with the TCA-precipitable cell fraction (M-J. Borrelli, unpublished data). This fraction contained the DNA, the site in the cell where AD binds (22). Protein stabilization protection to a variety general

cellular

proteins already modification of

response present cellular

of

is associated with treatments that impart stresses and may represent one aspect of a to

stress

that

within cells, 3) proteins, 580

may involve:

1) heat

2) post-translational the

synthesis

of

non-

shock

Vol.

177,

No.

1, 1991

proteinacious protective cellular proteins resulting protein

BIOCHEMICAL

AND

BIOPHYSICAL

and/or substances, in stabilization

RESEARCH

4)

COMMUNICATIONS

reassociation

due

to

increased

of protein-

interactions.

ACKNOWLEDGMENTS This research was supported by NC1 grants CA49715, CA4800, and CA40251, William Beaumont Hospital Research Institute grants 89-06 and 89-02, and grants from the Natural Sciences and Engineering Research Council of Canada. REFERENCES A. & Dewey, W.C. (1971) Int. J. Radiat. Biol. 19, 467-477 . 1. Westra, 2. Borrelli, M.J., Thompson, L.L., Cain, C.A. t Dewey, W.C. (1990) Int. J. Radiat. One. Biol. Phys. 19, 389-399. 3. Johnson, F-H., Eyring, H. & Stover, B.J. (1974) The Theory of Rate Processes in Biolosv and Medicine, Wily, New York. 4. Lepock, J-R., Frey, H-E., Rodahl, A.M., & Kruuv, J. (1988) J. Cell. Physiol. 137, 14-24. 5. Lepock, J.R., Frey, H-E., Heynen, M-P., Nishio, J., Waters, B., Ritchie, K.P. & Kruuv, J. (1990) J. Cell. Physiol. 142, 628-634. 6. Massicotte-Nolan, P., Glofcheski, D-J., Kruuv, J. & Lepock, J.R. (1981) Radiat. Res. 87, 300-313. 7. Henle, K.J. (1981) Radiat. Res. 88, 392-402. 8. Mivechi, N.F. & Dewey, W-C. (1984) Radiat. Res. 99, 352-362. 9. Lee, Y-J. 61 Dewey, W.C. (1986) Radiat. Res. 106, 98-110. 10. Shearer, G. & Sypherd, P.S. (1988) Antimicrob. Agents Chemother. 32, 341-345. 11. Donaldson, S-S., Gordon, L.F. & Hahn, G.M. (1978) Cancer Treat. Rep. 62, 1489-1495 (1978). 12. Hahn, G.M. L Strande, D-P. J. (1976) Natl. Cancer Inst. 57, 1063-1067. 13. Borrelli M-J., Wong, R.S.L. & Dewey, W-C-(1986) J. Cell. Physiol. 126, 181-190. 14. Borrelli, M-J., Thompson, L-L., and Dewey, W.C. (1989) Int. J. Hyperthermia 5, 99-103. 15. Borrelli, M-J., Rausch, C-M., Seaner, R., and Iliakis, G. Int. J. Hyperthermia (in press). 16. Lepock, J-R., Rodahl, A.M., Zhang, C., Heynen, M-L., Waters, B., & Cheng, K.H. (1990) Biochemistry. 29, 681-689. 17. Beckman, R-P., Mizzen, L-A., & Welch, W.J. (1990) Science. 248, 850-854. 18. Edington, B-V., Whelan, S-A. & Hightower, L.E. (1989) J. Cell. Physiol. 139, 219-228. 19. Lee, Y-J., Kim, D. & Corry, P.M. (1990) J. Cell Physiol. 144, 401-407. 20. Lee, Y-J., Dewey, W.C. & Li, G-C-(1987) Radiat. Res. 111, 237-253. 21. Lee, Y.J. & Dewey, W.C. J. (1988) J. Cell. Physiol. 135, 397-406. 22. Snyder, J-G., Hartman, N-G., D'Estantoit, B-L., Kennard, O., Remeta, D-P., and Breslauer, K.J. (1989) Proc. Natl. Acad. Sci. USA 86, 3968-3972.

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