Cordycepin and xylosyladenine: Inhibitors of methylation of nuclear RNA

Vol. 8 1, No. 2, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Pages

Match 30, 1978

INRIBITORS

CORDYCEPIN AND XYLOSYLADENINE: Robert Laboratory

Received

February

I.

Glazer

521-526

OF MEYWfLATION OF NUCLEAR RNA

and Ann L. Peale

of Medicinal Chemistry and Biology National Cancer Institute Bethesda, Maryland 20014

1,1978

SUMMARY

Cordycepin and xylosyladenine inhibited methylation of nuclear RNA to a greater extent than RNA synthesis in L1210 cells --in vitro. Inhibition of base methylation, 2'-0-methylation and 5'cap methylation was equal to, 2 to 5-fold greater and 1.5fold greater, respectively than inhibition of RNA synthesis. Cordycepin was more potent than xylosyladenine in inhibiting 2'-0-methylation of cytidine and adenosine, but not guanosine. These results suggest that impinnent in the 2'-0-methylation of nuclear RNA may be one of the major effects that limits the biological activity of rRNA and mRNA by these drugs. INTRODUCTION The processing believed

of nuclear

rRNA and mRNA via

to be a prerequisite

mRNA.

In particular,

for

the maturation

2'-0-methylation

of endonucleolytic

cleavage

methylation

of the 5'

of nuclear

now believed

to be necessary

translation

species

furanosyladenine) submitted wish bitors these plasmic

precursor

to form

cytoplasmic

to

is

t-RNA and

45s rRNA determines

28s and 18s r-RNA (l-5).

precursors

the formation

processes

mRNA to form

of a stable

initiation

the

Furthermore, the 5' "cap" complex

for

of the synthesis

of

(6-10).

Cordycepin diverse

for

methylation

of functional

of nuclear

specificity

terminus

specific

for

to present

(3'-deoxyadenosine) of nuclear

evidence

of the methylation findings

It's

spectrum

although indicating of total

may be contributory

rRNA and mRNA after

an effective

RNA (11-13).

has a similar publication)

is

treatment

it

inhibitor

congener,

xylosyladenine

of activity is

that nuclear to the with

521

5-fold both

(A.L.

Peale

more potent. nucleosides

RNA than impairment these

of its

(9-S-D-xylo-

and R.I. In this

Glazer,

study,

are more effective synthesis,

of the appearance

we inhi-

and that of cyto-

drugs.

Copyright 0 1978 by Academic Press, Inc. Ail rights of reproduction in any form reserved.

is

Vol. 81, No. 2, 1978

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

MATERIALS AND METHODS Cordycepin and xylosyladenine were kindly provided by Dr. Harry B. Wood, Jr., National Cancer Institute. [3H-methyl]methionine (63 Ci/mmole) and [14C(U)]uridine (462 mCi/mmole) were purchased from New England Nuclear. E. coli alkaline phosphatase and snake venom phosphodiesterase were obtained from Sigma Chemical Co., and 2'-O-methyl nucleosides and m7guanosine were purchased from P-L Biochemicals. Animals, L1210 cells were inoculated into CDFl mice at an inoculum of lo5 cells/O.1 ml Hanks balanced salt solution. Cells were harvested 6 days after inoculation, and were washed once in Dulbecco's medium and further diluted with same medium to 2 x lo7 cells/ml.

the

Incubations. Incubations of L1210 cells were carried out for 60 min at 37" in a shaking water bath at 100 rpm and consisted of: 5 ml of Dulbecco's medium, 5 x 10 7cells, 0.25% glucose, either 50 uCi [3H]methionine (80 mCi/mmole), 5 uCi [14C]uridine (50 mCi/mmole), and either 2.5 x 10e4 M cordycdpin or 5 x 10m5 M xylosyl, adenine as indicated. Cells were preincubated for 30 min with either drug in Dulbecco's medium with 0.25% glucose before the 60 min labeling period. RNA extraction: After incubation, cells were centrifuged at 200 x g for 15 min medium. at 4', and washed once with Dulbecco's Nuclei were prepared according to the procedure of Daskal --et al. (14) using Triton Total nuX-100 as the detergent. clear RNA was extracted by the sodium dodecyl sulfate-phenol extraction method previously described (11). With this procedure, rRNA is extracted with equal parts of 0.1% sodium dodecyl sulfate-O.1 M sodium acetate (pH 5.1)-0.005 M EDTA and phenol 7:2:1 (v/v) containing 0.1% Ghydroxyquinoline) folmixture (phenol:m-cresol:H20, lowed by extraction of heterogeneous RNA from the phenol precipitated interface with an equal volume of 0.1% sodium dodecyl su1fate:O.l M Tris-HCl (pH 9.0):7 M urea Extracts were combined and precipitated at -20" with 2 volumes of 2% potassium (15). RNA precipitates were washed once with 95% ethanol at 4' and t acetate in 95% ethanol. hydrolyzed overnight at 37" with 0.3N KOH. DEAE Sephadex chromatography. Alkaline hydrolysates of nuclear RNA were neuSamples tralized with 0.3 N HCIOI, using 0.1% phenol red as internal indicator. Neutralized were centrifuged at 2,006 x 8 at 4" after remaining at 4' for 20 min. hydrolysates were diluted with 10 ml of 7 M urea-20 mM Tris-HCl (pH 7.6) and adMono-, di- and oligosorbed to DEAE Sephadex equilibrated with the same buffer. nucleotides were eluted with a linear gradient of 0.1 - 0.7 M NaCl in 7 M urea-20 mM Tris-HCl (pH 7.6). Columns were standardized separately with RNase A hydrolysates of 3H-labeled L1210 nuclear RNA. Thin layer chromatography. Mononucleotide, dinucleotide and oligonucleotide respectively, were defractions corresponding to a net charge of -2, -3, and -5.5, salted of urea by adsorption of samples diluted lo-fold with water to columns Each fraction was eluted with 50 ml of 1.0 M triethyl(1.5 x 10) of DEAE cellulose. ammonium carbonate and lyophilized. Lyophilized samples were redissolved in 2 ml of Samples were water and transferred to 13 x 100 mm test tubes and lyophilized again. dissolved in 0.1 ml of 50 mM Tris-HCl (pH 8.5)-0.005 M MgCl2 containing 10 ug of alkaline phosphatase and 20 ug of phosphodiesterase, and incubated for 2 hr at 37' After incubatior+ samples were spotted on cellulose-coated thin layer sheets (17). The solvent systems were: A) ethyl acetate:2-propanol:7.5 M NH4OH: (Eastman Kodak). 1-butanol (3:2:2:1, v/v) and B) 2-propanol:concentrated HCl:H20 (68:17.6:14.3, v/v) (17). RESULTS Alkaline with

hydrolysates

[3H]methionine

of control

and [14C]uridine

and drug-treated showed

522

three

nuclear distinct

RNA double-labeled 3 peaks of H radioactivity

Vol. 81, No. 2, 1978

BIOCHEMICAL

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

A

5if 3 $% n

I-

-2

1

5

VJ

FRWTIONNUMSER

10

i

15

DISTANCE

MIGRATED

(cm)

Figure

1.

DBAE Sephadex chromatography of alkaline hydrolysates of nuclear RNA labeled with [ Hlmethionine and [ Cluridine. Ll210 cells were incubated in the absence (A) or presence of 2.5 x 10e4 M cordycepin (B) or 5 x lo-' M xylosyladenine (C). Total nuclear RNA was extracted, hydrolyzed and chromatographed as described in MATERIALS AND METHODS. A total of 100 fractions were collected but only the first 60 fractions are shown since no radioactivity was eluted in the last 40 fractions. The insets represent the -5.5 oligonucleotide region with the scale on the ordinate decreased lo-fold.

Fi .gure

2.

Thin layer chromatography of DEAE Sephadex fractions of alkaline hydrolysates of nuclear RNA. Nucleotide fractions were separated by DBAB Sephadex chromatography, desalted, freeze-dried and digested with alkaline phosphatase and phosphodiesterase as described in MATERIALS AND METHODS. The abbreviations used for the nucleoside markers are: m%, 5-methylcytidine; m6A, 6-methyladenosine; m%, 5-methyluridine; m7G, 7-methylguanosine; Gm, 2'-O-methylguanosine; Cm, 2'-0-methylcytidine; Am, 2'-0-methyladenosine. A) -2 fraction with solvent system A B) -2 fraction with solvent system B C) -3 fraction with solvent system A D) -5.5 fraction with solvent system A.

523

Vol. 8 1, No. 2, 1978

at -2,

BIOCHEMICAL AND BlOPHYSlCAL RESEARCH COMMUNICATIONS

-3 and -5.5

nucleotide

peak

corporation

net

(Fig.

into

1).

RNA synthesis,

RNA in

the mononucleotide

(-5.5

charge)

tion

but

mononucleotide fraction

charge),

(Figs.

respect

of cordycepin

in contrast

and 71-74%

[14C]uridine

(-3

charge)

to control

of nuclear

and oligonucleotide RNA (Fig.

1A).

dinucleotides

of methylation

inmarkedl:

methylation

of base methylation

inhibition

the mono-

and xylosyladenine

2'-0-methylated

to inhibition

only of

reduced

dinucleotide

containing

with

to inhibition

1B and lC> compared

fraction

fraction

coinciding

even more significantly,

(-2

fractions

by 73-92%

(with

RNA) concentrations

of the dinucleotide

hibited

Methyla! was in-

of 45-582

in the

of the oligonucleotide

(TABLE 1).

Methylated layer

Equipotent

nuclear

inhibited

and 14 C radioactivity

charge

nucleosides

chromatography.

methylcytidine

The mononucleotide

as determined

dinucleotide

fraction

methylguanosine consisted

in each of the

and 2'-0-methylcytidine

as found

for

A (Fig.

(Fig.

inhibited their

separated

by thin

predominantly

of 5-

2A) and B (Fig.

with 2C) while

2'-0-methylguanosine

and xylosyladenine

same extent

systems

were

consisted

2'-0-methyladenosine

of 7-methylguanosine,

Cordycepin

fractions

fraction

in solvent

contained

three

lesser

The

quantities

of 2'-O-

the oligonucleotide

fraction

and 2'-0-methyladenosim

each of the methylated

respective

2B).

fractions

after

(Fig.

nucleosides

DEAE Sephadex

2D1

to the chromatogra-

phy (TABLE 1).

DISCUSSION The present

study

cepin

and xylosyladenine,

nuclear

RNA of L1210

has demonstrated are cells

cytidirrwere

particularly

tion

more sensitive

was far

incorporation. represents though

Inhibition the

5' "cap"

the mononucleotide,

RNA obtained

effective

--in vitro. sensitive than

that

inhibitors

impairment

of the methylation

dinucleotide

by DEAE Sephadex

RNA (16-18)

of adenosine,

agents

and inhibition

of RNA synthesis in

524

cordy-

methylation

in

guanosine

as assessed

was intermediate

represent

drugs,

of sugar

the oligonucleotide

and oligonucleotide

chromatography

anticancer

of base and sugar

2'-0-Methylation to these

of nuclear

the nucleoside

and methyla-

by uridine

fraction

which

in sensitivity. fractions

a mixture

from

Altotal

of RNA species,

it

71.4 + 1.4

Xylosyladenine, 5 x 10-5M

6 42

100

278.8 f 59.4

287.1 -•t- 49.1

670.9 -+ 130.7

14C Total

42

43

100

r,

53.7 + 4.6

186.9 + 18.1

684.8 + 86.6

dpm ( x 10m3)

RJA fraction

-___- _-

27

100

z

RNA hydrolysates

14.0 + 1.2

15.2 f. 1.5

53.1 2 5.0

Incubations and isolation of L1210 nuclear RNA werecarried out as described in MATERIALS AND METHODS. The total radioactivity represents that coinciding with the peaks of -2, -3, and -5.5 charge in Fig. 1. .Each value is the mean + S.E. of 3 separate experiments.

54.4 + 4.1

2.5 x 10-4M Cordycepin,

16.7

130.12

3H

Control

Treatment

-2

of ~1210 nuclear

and xylosyladenine

of methylation

by cordycepin

Inhibition

TABLE1

26

29

100

x

is SE

i

Vol. 81, No. 2, 1978

is

believed

and the

5' "cap"

inhibition plain,

that

BlOCt4EMlCAL

the dinucleotide with

the activity

their

fraction

poly(A)-containing

by cordycepin in part,

AND BlOPHYSlCAl

and xylosyladenine inhibitory

of cordycepin

in impairing

is mainly

associated

heterogeneous

nuclear

of these

effects

RESEARCH COMMUNICATIONS

on nuclear the processing

with

rRNA (16-18)

RNA (18).

methylation RNA synthesis, of precursor

processes

The potent may ex-

and in particular, rRNA (19).

REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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