Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

Vol. 112, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

May 16, 1983

Pages

DIFFERENTIATED

INHIBITION

965-971

OF DNA, RNA AND PROTEIN

SYNTHESIS IN L1210 CELLS BY 8-METHOXYPSORALEN Peter

Eigil

Nielsen

and William

P. Linnane

III

Department of Biochemistry B, The Panum Institute, University of Copenhagen, Blegdamsvej 3 C, DK-2200 Copenhagen, Denmark I:eceived March 17, 1983 3'he synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (,b95%) at 200 rig/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (~40% inhibition). At 2 ug/ml the RNA synthesis is inhibited %90%. Even at this concentration only a moderate effect is seen on the protein syntheC9lS. These results strongly indicate that the phototoxic action cbf 8-methoxypsoralen is primarily due to inhibition of DNA synt.hesis. I'soralens

are phototoxic

tion

and also

that

the

cells

normal

synthesis

compounds which

cell

of

probably

with

ultraviolet

psoralens

irradiation

due to photoreactions Ou -et.

8-methoxypsoralen

to DNA decreases

and Leick

al.

and Nielsen

(4) have

the --in vitro

thesis

RNA is decreased

i-alen ~ It acid

has also

acceptor

ABBRETIIATIONS:

after

when it

the psoralens shown that template

with

long are

and DNA and

photobinding activity

of

in vi-__

and Champney (6) in protein

phototreatment

tRNA loses

when

These effects

some of

has been photoreacted

8MOP: 8-methoxypsoralen, PBS: phosphate saline, TCA: trichloro-acetic acid.

965

prolifera-

inhibited

in combination

messenger activity

been shown that

capacity

is

(5) and Karathanasis

have shown that of viral

its

cell

has been demonstrated

(1,2,3).

between

RNA respectively.

tro ^-

It

DNA, RNA and protein

have been treated

wave-length

functions.

inhibit

syn-

with

pso-

its

amino-

with

8-meth-

buffered

0006-291 X/83 $1 .50 Copyright (?I 1983 by Academic Press, Inc. All rights of reproduction In any form reserved.

Vol. 112, No. 3, 1983

oxypsoralen fects

BIOCHEMICAL

(7).

However,

in terms of

been established. RNA and protein treatment

the relative action

In the present

study

inhibited

after

The RNA synthesis

the protein

synthesis

showing

is only

these

psoralens

up to six that

a decline is less

slightly

of

ef-

has not

we have measured the

We find

is most strongly treatment.

of the

in L1210 cells

8-methoxypsoralen.

RESEARCH COMMUNICATIONS

significance

the phototoxic

synthesis

with

AND BIOPHYSICAL

hours

DNA,

after

the DNA synthesis within

heavily

two hours affected

while

affected.

MATERIALS AND METHODS 8MOP was purchased from Fluka. L1210 cells were grown in suspension cultures in RPMI-1640 medium (Bibco) containing 5% fetal calf serum and 5% horse serum plus penicillin and streptomycin. Irradiations were performed in glass vessels at room temperature using a Philips TL 2OW/O9 fluorescent light tube (A~365 nm). The light intensity was 2.5 x 1Ol6 quanta x set-l x cmm2. Psoralen

treatment

L1210 cells (exponential phase) were harvested (1500 rpm, 5 min) and resuspended in PBS. 8MOP dissolved in PBS was added to give the desired final concentration and following equilibration for were irradiated for 10 min. 10 min, the cultures DNA,

RNA and

protein

sunthesis

Seven volumes of preheated medium (37OC) were then added and the sample was divided into aliquots of 80 ~1 containing 2 x lo4 1.5 UCi of the 3H-labeled compound ([3Hl-thymidine(20 Ci/ cells. (23 Ci/mmole) and L3H]-phenylalanine(46 Ci/ nunole), [3H]-uridine synthesis respectively) in mmole) (NEN) for DNA, RNA and protein 20 ~1 medium was then added at &he indicated times (Figs. l-3) and the samples incubated at 37 C. DNA synthesis was stopped after 15 min by the addition of 30 ~1 lysis buffer (0.1% sodium dodecyl sulphate 0.4 mM thymidine, 0.7 mg proteinase K, 0.1 mg sodium polyphosphate) and the samples were allowed to stand overnight at room temperature. The samples were then spotted on Whatmann 3 MM filters washed three times in 1N HCl followed by washThe filters were dried and the ings in 96% ethanol and acetone. radioactivity was determined by liquid scintillation counting. The samples for determination of RNA and protein synthesis were spotted on Whatmann 3 MM filters and immediately put in ice-cold 10% TCA. The filters were then washed in 5% TCA, 96% ethanol and acetone and finally dried. Radioactivity was determined by liquid scintillation counting. RESULTS AND DISCUSSION L1210 cells tions

were treated

(2 pg/ml

( 1.5 x 1019 and protein 6 hours.

with

and 200 rig/ml) quanta/cmm2).

synthesis

8-methoxypsoralen and irradiated

After

this

1 the rate

at

treatment

was measured at various

As shown in Fig.

at two concentra365 nm for the times

of DNA synthesis

10 min

DNA, RNA from

0 to

of untrea-

Vol. 112, No. 3, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Hours

Fig. 1. Rate of DNA synthesis following treatment with 8MOPplus long wavelength ultraviolet irradiation. L1210 cells were treated with 8MOPas described and the DNA synthesis was measured for 15 min pulses beginning at the times indicated. e-o-o: control (untreated cells, irradiated cells or cells treated with 8MOPwithout irradiation), A-A-A: 200 rig/ml of 8MOP, o-o-o: 2 ug/ml of 8MOP, ---: background.

ted cells

(or

8MOP without 15

cells

which

of

increases

slightly

during

gradually.

the cells

changes

in the

may also

explain

this

with

200 rig/ml

rate

of DNA synthesis

DNA synthesis observed

2

two hours.

stationary

observed

and the growth

phase.

pool

size

were treated

30% of normal

only

the

DNA synthesis probably

slow

but

At 2 ug/ml

967

is probably

increase

a rapid

is

first

a slight

Thereafter

the normal

of DNA synthesis

washing

When the cells

irradiation

was also

the

'-deoxy-thymidine

observation.

of 8MOP plus

but ~5% of

j.nhibition

cellular

was reached.

within

the

respectively

The rise

from the

may be due to approaching

However,

served

light

decreases

the recovery

Gecline

received

irradiation)

min and thereafter

cue to

have only

decline

same pattern was reached.

due to the

to

in the

~5% was

was obSince

formation

of

BIOCHEMICAL

vol. 112, No. 3, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Hours Rate of RNA synthesis Fig. 2. 30 min pulses beqinninq at the Symbols as in Fig. 1.

psoralen-DNA polymerase

adducts (4)

polymerases

(1,4)

the decline

to reach

after

treatment However,

treatment

was a decrease effect

of

action

to the

of

the DNA for

time

the

in the DNA template. constant

(Fig.

during

2) and the

the amount of

was much less

the DNA synthesis

the

may correspond

was basically

psoralen

the

inhibit

such adducts

The RNA synthesis hours

which

following 8MOP treatment. times indicated were used.

effect

RNA being

dramatic

than

(40% and 90% inhibition

at

the

six

of

the

synthesized.

the one seen on

200 rig/ml

and 2 ug/

ml respectively). The decrease probably

also

DNA. However, practically synthesis

in RNA synthesis

due to the it

is

stopped

introduction

interesting 2 hours

is maintained

after

of psoralen

that after

while

treatment,

(60% at 200 rig/ml). 968

psoralen

treatment adducts

is in the

the DNA synthesis a significant This

may reflect

has RNA

Vol. 112, No. 3, 1983

BIOCHEMICAL

OO

1

AND BIOPHYSICAL

L

I

I

I

2

3 Hours

4

5

3. Rate of protein synthesis ment. Conditions as in Fig. 2.

the DNA-polymerase

ced by 8MOP than

is more sensitive

is the

is

simply

wlhich have not

been damaged by the

At the psoralen tein

synthesis

due to damage of the o!i mRNA (Fig. expect

the

synthesis

the mode of action

2) -Since

effect of

of

of the

protein

synthesis

explanation

thus

with

the old mRNA. in fact,

o:i photobinding

is leads

of psoralens

seen at to the

a pool

time

the pro-

the

This

longer conclusion

synthesis

of mRNA one woulc

due to

the decreased

as a result

a more pronounced

to mRNA (5,6) 969

of genes

3 & 4).

with

synthesis

new mRNA to increase

the

of 8MOP is primarily

contain

protein

that

study

(Figs.

interfering

the cells

on the

turnover

This

affected

DNA thereby

produ-

psoralen. used in this

moderately

if

or more likely genes or parts

concentrations

is only

would be predicted

transcribing

8MOP treat-

to DNA lesions

RNA polymerase,

RNA-polymerase

6

following

Fig.

that

RESEARCH COMMUNICATIONS

times that

of

inhibition

(Figs. the

3 & 4). effects

and tRNA (7,8,9)

seen

BIOCHEMICAL

Vol. 112, No. 3, 1983

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Hours

Fig.

4.

Cumulative treatment. o-o-e: o-o-o : 2 pg/ml of [3HI-Phenylalanine were withdrawn at

--in vitro

are not

significant

of 8-methoxypsoralen A possible the action

of

protein synthesis following 8MOP control, A-A-A: 200 rig/ml of EMOP, 8MOP, A-A-A: 20 ug/ml of 8~0P. was added at time 0 and aliquots the times indicated.

with

and probably

photodynamic singlet

oxygen

also

be regarded

as insignificant.

We conclude

therefore

on Ll210 in inhibition

and we are confident concern

cells

other

psoralens

as well.

membranes (12),

the phototoxic

is primarily

through

has been shown to react

action

thus may

of

due to damage of

of RNA and particularly that

toxicity

of 8-methoxypsoralen

and mitochondrial

that

to the -in vivo psoralens

(10) which

ribosomes

resulting

(11)

other

effect

with

oxypsoralen

regard

this

conclusion

and cell

types

8-meth-

the DNA

DNA synthesis,

can be expanded

to

as well.

REFERENCES

1. 2.

Song, P.-S. and Tapley, 2, 1177-1197 (review). Bordin, F., Baccichetti, Experientia 28, 148.

K.J.Jr. F.

970

(1979)

and Musajo,

Photochem. L.

(1972)

Photobiol.

Vol. 112, No. 3, 1983

3. 4. 5. 6. 7. 8. 9. 13. 11. 12.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Bordin, F., Baccichetti, F., Bevilacqua, R. and Musajo, L. (1973) Experienta 2, 272-273. Ou, C.-N., Tsai, C.-H., Tapley, K.J.Jr. and Song, P.-S. (1978) Biochemistry 17, 1047-1053. Leick, V. and Nielsen, P.E. (1981) Photochem. Photobiophys. 2, 285-290. Karathanasis, S.K. and Champney, W.S. (1981) Biochemistry 20, 3579-3585. (1978) Biochemistry 17, 1054-1059. our C.-H. and Song, P.-S. Bachellerie, J.-P. and Hearst, J.E. (1982) Biochemistry 21, 1357-1363. Nielsen, P.E. and Leick, V. (1982) Biochem. Biophys. Res. comm. E, 179-185. De Mol, N.J. and Beijersbergen van Henegouwen, G.M.J. (1979) Photochem. Photobiol. 2, 331-335. (1978) Photochem. Photobiol. 28, Singh, H. and Vadasz, J.A. 539-545. Salet, C., Moreno, G. and Vizens, F. (1982) Photochem. Photobiol. 36, 291-296.

971