[CANCER RESEARCH 32, 22—27,January 1972)
Interaction of 1-(2-Chloroethyl)-3-cyclohexyl- 1-nitrosourea
(NSC 79037) with Nucleic Acids and Proteins in Vivo and
in Vitro'
ChunJui Cheng,Shinji Fujimura,DeziderGrunberger,and I. BernardWeinstein2
Institute of CancerResearchand Department ofMedicine, CollegeofPhysicians and Surgeons,Columbia University, New York, New York 10032
SUMMARY
@
@
@
@
The macromolecular
binding of radioactivity
from
1-(2-chiorethyl)-3-cyclohexyl- 1-nitrosourea, labeled with ‘C
in either the cyclohexyl moiety or the ethylene residue, was
studied in the L1210 leukemia-bearing mice, in a suspension of
Ll2lO leukemia cells, and during in vitro incubation with
isolated nucleic acids and proteins. In all three systems,
radioactivity from
C-labeled 1-(2-chloroethyl).
3-cyclohexyl- 1-nitrosourea was extensively bound to proteins,
and there was negligible binding to nucleic acids. Radioactivity
from the ethylene-' 4C-labeled drug was bound to both nucleic
acids and proteins, but the binding was only a fraction of the
observed protein binding of the cyclohexyl label. Of the
various macromolecules
examined for in vitro binding,
poly-L-lysine and albumin were the most active in binding the
cyclohexyl-labeled material; polyguanylic acid, polycytidylic
acid, and tRNA were the most active in binding- the
ethylene-' C-labeled material.
The possibility that the carcinostatic activity of this drug
reflects a dual capacity, namely, modification of cellular
proteins via cyclohexylcarbamoylation
and of nucleic acids via
alkylation, is discussed.
INTRODUCTION
The nitrosoureas offer a relatively new and promising class
of antineoplastic
agents. Of particular interest is the
compound CCNU.3 Because of its high lipid solubiity and
permeability through the blood brain barrier (16), CCNU
might be particularly effective in the treatment of certain
neoplasms of the central nervous system. Indeed, there is
evidence that systemically administered CCNU and BCNU are
effective in the treatment of meningeal leukemia in the mouse
(1 1, 19) and in children who have acute leukemia with central
nervous system involvement (9). In addition, preliminary
@
studies indicate that CCNU and BCNU might exert a beneficial
effect in patients with malignant glial tumors (8, 22, 25).
Despite their clinical interest, the mechanism of action of
CCNU and related mtrosoureas such as BCNU is not known.
The following facts suggest that they do not function as
conventional alkylating agents. (a) In human Hodgkin's
disease, development of resistance to conventional alkylating
agents is not associated with cross-resistance to BCNU (10).
(b) CCNU and BCNU appear to act at phases of the cell cycle
different than those of the known alkylating agents (2, 20,
23). (c) Although the alkylating potency of CCNU is lower
than that of BCNU (24), it is somewhat more effective in the
treatment of leukemia Ll 210 (4). For these reasons, we
undertook a study of the possible binding of 2 different
moieties of CCNU, the
C-labeled cyclohexyl residue and the
‘
4C-labeled
ethylene
residue,
to
cellular
DNA,
RNA,
and
protein, both in vivo and in vitro.
MATERIALS AND METHODS
Radioactive
Chemotherapy
‘
4C
label
CCNU was kindly supplied by the Cancer
National Service Center, Bethesda, Md., with
in 2 separate
positions
of
the
molecule
(Chart
1),
uniformly throughout the carbon atoms of the cyclohexyl ring
(cyclohexyl-' 4C-labeled CCNU; 2.498 mCi/mmole) or in the
carbon atoms of the 2-chloroethyl
moiety (ethylene.
‘
4C-labeled
CCNU;
1 .338
mCi/mmole).
Bovine albumin Fraction V powder (unesterified, fatty
acid-poor form), bovine globulin, 40% @3,30% ‘y(Cohn
Fraction II, III), and horse heart cytochrome c were purchased
from Nutritional Biochemicals Corp., Cleveland, Ohio; calf
thymus histone (HLY, regular) and calf thymus DNA were
from Worthington Biochemicals Corp., Freehold, N. J.;
Escherichia coli B tRNA was from General Biochemicals, Inc.,
Chagrin Falls, Ohio; bovine pancreas RNase A (type 1-A) was
from Sigma Chemical Co., St. Louis, Mo.; RNase T2 was from
Sankyo Co., Tokyo, Japan; Pronase (B grade) was from
Calbiochem,
Los Angeles, Calif.; and poly-L.lysine,
1 This
work
was
supported
by
USPHS
Grant
CA-02332
and
by
poly-L-arginine,
poly U, poly A, poly (G1 U3), poly G, and
Grants Cl-I 7 and 1N-4K from the American Cancer Society.
poly C were from Miles Laboratories, Inc., Elkhart, Ind.
2 Career
Scientist
of the
Health
Research
Council
of the City
of New
York (1-190).
In Vivo Experiments. DBF male mice, weighing around 20
3The abbreviations used are: CCNU, 1-(2.chloroethyl)-3- g, were obtained from Breeding Laboratory, New York, N. Y.
cyclohexyl-1-nitrosourea; BCNU, l,3-bis(2-chloroethyl)-1-nitrosourea;
poly U, polyuridylic acid; poly A, polyadenylic acid; poly G, On the 7th day after i.p. inoculation of 2 X 106 cells of
leukemia Ll2lO, groups of 3 mice received i.p. injections of
polyguanylicacid; poly C, polycytidylic acid.
Received July 9, 1971; accepted August 25, 1971.
0.5 mg of either
C-labeled CCNU (5 @.zCi)
or
22
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Binding ofCCNU to Nucleic Acids and Proteins
@
10 ml of Bray's solution with a Nuclear-Chicago Mark II liquid
scintillation spectrometer.
In Vitro Experiments with Cell Suspensions. Leukemia cells
were harvested as described above and were washed twice with
ice-cold 0.9% NaCl solution. A 10% cell suspension was
prepared
in Krebs-Ringer
phosphate
buffer (pH 7.4)
containing
0.1% glucose. To this was added either
ethylene-' 4C-labeled CCNU (3 j.zCi) in 0.2 ml of dimethyl
sulfoxide. The mice were sacrificed by decapitation 6 hr later,
and brain, liver, and leukemia cells were quickly removed. The
RNA, DNA, and cytoplasmic protein fractions were isolated
by the method of Roberts and Warwick (18), with the
following modifications. The RNA fraction (containing both
tRNA and rRNA) was extracted with phenol, as described
previously (6), and the crude DNA fraction was further
purified by Pronase and RNase T2 digestion. Radioactivity in
the isolated protein and nucleic acid fractions was measured in
4 C-
CI—CH2—cH2 —
*
0U
*
CI—CHZ-—CH2—N—C—NH
NO
Chart 1. Molecularstructure of cyclohexyl-' 4C-labeledCCNU(top)
@
@
and ethylene-1 4C-labeled CCNU (bottom). *, labeled position.
Table 1
C-labeledCCNU to nucleic acids
and proteins in vivo
Binding
or
C-labeled
CCNU,
50
j.zg/ml,
and
the suspension was incubated aerobically at 37°,with gentle
shaking.
After various incubation times, aliquots (50 jil) of cell
suspension were pipetted onto filter paper discs (Whatman No.
3MM). The discs were washed 3 times with 5% cold
trichloroacetic acid, ethanol, ethanol:ether (1 :2, v/v), and
ether. The acid-insoluble radioactivity remaining on the disc
was counted as bound radioactivity, with 10 ml of toluene
liquid scintillation solution.
The DNA, RNA, and protein fractions were isolated 3 hr
after incubation,
and the bound radioactivities
were
determined as described in the in vivo experiments.
In Vitro Studies with Isolated Nucleic Acids and Proteins.
Twenty-five @zgof either
C-labeled CCNU or
Table 2
Binding ofethylene-' 4C-labeledCCNU to nucleic acids
and proteins in vivo
A group of 3 leukemia-bearingmice receivedi.p. injections of 0.5 mg
A group of 3 leukemia-bearingmice received i.p. injections of 0.5 mg
of cyclohexyl-' 4C-labeled CCNU (5 @tCi)per mouse. Six hr after
injection, the mice were sacrificed, and the nucleic acids and proteins
of ethylene-' 4C-labeled CCNU (3 DCi) per mouse. Six hr after receiving
the
injections,
the mice were
sacrificed,
and the nucleic
acids and
were isolated and assayed for radioactivity, as described in “Materials proteins were isolated and assayed for radioactivity, as described in
and Methods.―
“Materialsand Methods.―
Radioactivity pr
Radioactivity
pr
inRNADNAProteinBrain17.410.4Liver18.520.617.5Leukemia
(pmoles/mg)esent
(pmoles/mg)esent
inRNADNAProteinBrain2.288.7Liver0.61.1125.4Leukemia
cells1.19.4431.6
cells26.326.018.5
A
E
I .2
I')
0
0
C-,
E
B
In
0
0
Ui
I
C,
C,
L;J
z
C-,
z
Ui
—I
C,
I
FUi
D
U,
a,
0
z 0.0
E
C
C,
C,
U,
a,
0
@
I
2
3
TIME (HOURS)
Chart 2. Time course of the binding of
4
0
E
C
C-labeled
0
2
3
TIME (HOURS)
CCNU (A) and
4
C-labeled CCNU (B) to the acid-insoluble
fraction of a cell suspensionof leukemia L1210.
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1972
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23
Cheng, Fu/imura, Grunberger, and Weinstein
4 C-labeled
@
@
CCNU
were
incubated
with
250
@ig of
either calf thymus DNA, E. coli tRNA, poly U, poly A, poly
(G, U3), poly G, poly C, bovine albumin, bovine globulin,
cytochrome c, histone, RNase A, or poly-L-lysine in 0.5 ml of
0.05 M cacodylate buffer (pH 7.2) at 37°. After various
incubation times, 50-pl aliquots were placed on filter paper
discs, and the acid-insoluble radioactivity was determined, as
described above.
For the assay of radioactivity bound to poly-L-lysine, 5%
trichloroacetic acid containing 0.25% sodium tungstate (pH
2.0) was used instead of 5% trichloroacetic acid (7).
Chemical Determinations. DNA, RNA, and protein were
determined by the methods previously described by Burton
(3), Dische (5), and Lowry et a!. (12), respectively.
DNA and RNA is actually due to contaminating protein. In
contrast to these results, the radioactivity of the ethylene
moiety was bound to both nucleic acids and proteins of brain,
liver, and leukemia cells to about the same level (Table 2). In
all 3 tissues, the binding of the cyclohexyl moiety of CCNU to
protein fractions was 5- to 20-fold greater than the binding of
the ethylene moiety to either protein or nucleic acids
(compare Tables 1 and 2).
Binding of
C to Nucleic Acids and Proteins in
Leukemia Cell Suspensions. As shown in Chart 2, incubation
of
C- or
C-labeled CCNU with a
suspension of leukemia L1210 cells led to the binding of both
radioactivities to acid-insoluble material, which increased
progressively with time for at least 4 hr. However, the binding
Table 3
Binding
C to nucleic acidsand proteins of
leukemia L1210 cells incubated in vitro
RESULTS
@
@
@
Binding of
C to Nucleic Acid and Proteins in Vivo.
The radioactivity of the cyclohexyl moiety of CCNU was
almost exclusively bound to cellular proteins, rather than to
nucleic acids (Table 1). The specific activity of the protein
fraction from leukemia cells was considerably greater than that
of the protein fractions obtained from liver or brain. In view
of subsequent results obtained with purified nucleic acids and
proteins (see below), it seems likely that the small amount of
radioactivity from the
C-labeled CCNU found in
A 10%suspensionof leukemia Ll210 cellswas incubated aerobically
at 37°for 3 hr with 0.05 mg CCNU/mL Nucleic acids and proteins were
then isolated and assayed for radioactivity, as described in “Materials
and Methods.―
Radioactivity present
inRNADNA
(pmoles/mg)
ProteinCyclohexyl-'
4C-labeledCCNU01.2
2006.7Ethylene-'
4C-labeled CCNU57.524.0
32.0
A
POLY-LYSINE
B
3.0
x____.__.._______x
t RNA
ALBUMIN
01
E
..‘,‘
20.0
U
2.0
z
U
-J
>-
I
/
/
I—
U
z
HISTONE
4.0
CYTOCHROME C
I RIBONUCLEASE
0.75
0
0
U)
a,
0
A
ALBUMIN
DNA
HIS TONE
-
E
C
RIBONUCLEASE
GLOBULIN
CVTOCHROME
0
0
I
2
3
4
5
2
6
24
4
5
6
TIME (HOURS)
TIME (HOURS)
Chart 3. Time course of the binding
ethylene-' 4C-labeled CCNU (B).
3
A
C
of radioactivity
to isolated
nucleic
acids and proteins,
with cyclohexyl-'
4C-labeled
CCNU (A) and
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Binding ofCCNU to Nucleic Acids and Proteins
5.0
P01_v C
@
@
POLY G
@
E
0
L:J
z
U
—I
lower than that obtained with
C-labeled CCNU.
With the
C label, the radioactivity bound to tRNA
was approximately 5-fold higher than the binding to DNA or
albumin. For determination of the effect of base composition,
the binding of
C-labeled CCNU to synthetic
polynucleotides
was also studied (Chart 4). There was
extensive binding to poly C and poly G, lesser binding to a
random copolymer of G and U, in which the ratio of G to U
was 1:3, and a low level of binding to poly A and poly U.
4.0
Dl
@
A, or globulin. There was no detectable binding to purified
calf thymus DNA, to E. coli tRNA (Chart 3A), or to
poly-L-arginine (not shown here).
Chart 3B shows that, as in the in vivo and cell suspension
studies, the
C-labeled CCNU was bound to both
nucleic acids and proteins. However, the level was considerably
3.0
>-
I
I-.
L@J
z
0
DISCUSSION
2.0
0
Alkylation has been suggested as the major mode of action
of CCNU and related nitrosoureas (4, 19, 24), but the precise
mechanism of action is still unknown (see “Introduction―).
U)
0
E
POLY(G,u@) The present study clearly indicates that radioactivity from
C
4 C-labeled
CCNU
is extensively
bound
to
proteins
and is negligibly bound to nucleic acids, both in vivo and in
POLY A
vitro.
On
1 4 C-labeled
the other
CCNU
was
hand,
bound
radioactivity
at
a low
level
from
to
both
ethylene
proteins
and nucleic acids.
POLY U
In the present study, the in vivo labeling of nucleic acids
and proteins in leukemic cells, which was greater than that
2
4
6
with similar components in liver or brain, could simply reflect
0
direct contact with the drug following i.p. injection and/or
TIME (HOURS)
preferential uptake or metabolism by the leukemic cells. With
Chart 4. Time course of the binding of ethylene-' 4C-labeledCCNU respect to the therapy of brain tumors, there was significant
to synthetic polynucleotides.
labeling of brain nucleic acids and proteins. This is consistent
with the previous data of Oliverio et aL (16), indicating that
of radioactivity from the
C-labeled CCNU was radioactivity from cyclohexyl- and ethylene-labeled CCNU
does appear at appreciable concentrations
in both the
20- to 30-fold higher than that of the
C-labeled
cerebrospinal
fluid
and
brain
tissue,
although
these
authors
compound. The specific activities of the isolated nucleic acids
presumably measured both free and bound material.
and proteins are shown in Table 3. As in the in vivo studies,
the radioactivity of the cyclohexyl moiety of CCNU was
The binding of radioactivity from labeled CCNU to cellular
bound almost exclusively to proteins, and that of the ethylene
macromolecules in a form that is trichloroacetic acid-insoluble
group was bound (at a much lower level) to both nucleic acids and not extractable by ethanol or ether suggests covalent
attachment of the cyclohexyl moiety to amino acid residues in
and proteins.
Interaction of
C with Isolated Nucleic Acids and
proteins and of the ethylene moiety to both amino acids and
Proteins. Since CCNU was bound to cellular nucleic acids and base residues in nucleic acids. The precise identity of these
proteins both in vivo and in cell suspension, it was of interest
derivatives remains to be determined. It seems likely that the
to determine whether CCNU would also bind at neutral pH to cyclohexyl ring system remains intact during the protein
isolated nucleic acids or proteins, without the addition of an binding
and
also retains
an amino
group,
since
energy source or enzymatic activation. Chart 3A presents the cyclohexylamine
and N,JY-dicyclohexylurea
have been
time course of the binding of
C-labeled CCNU to identified as major urinary metabolites (1 6). Our studies do
specific proteins and nucleic acids. There was extensive
not indicate whether the carbonyl moiety of CCNU is also part
binding to a variety of proteins, which reached a plateau at of the protein-bound adduct, since carbonyl-labeled material
was not available at the time of these studies. We presume that
about 4 hr, except in the case of poly-L-lysine. The latter
this is the case, since cyclohexylisocyanate,
but not
polypeptide reacted to a greater extent with cyclohexyl
labeled CCNU than with any other material studied, and the cyclohexylamine, mimics certain biological effects of CCNU
(2). By analogy with nitrosoguanidine, which substitutes on
reaction continued for at least 6 hr. There was also extensive
binding to albumin, to a level which was at least 6 times the e- amino group of lysine-forming nitrohomoarginine (13,
15, 21), one might expect CCNU to react with the e- amino
greater than that obtained with histone, cytochrome c, RNase
0
@
@
@
@
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1972
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25
Cheng, Fujimura, Grunberger, and Weinstein
@
group of lysine to yield M -cyclohexylcarbamoyllysine.
Consistent with this is the extensive binding which we
obtained with polylysine and
C-labeled CCNU.
We are currently analyzing the hydrolsis products of this
complex
to definitively
establish
its structure.
The postulated lysine derivative is compatible with previous
evidence
that both
BCNU and CCNU tend to decompose
in
aqueous solution to yield isocyanates which would be
expected to be highly reactive with primary amino groups (14,
16). During the course of the present studies, Bowdon and
Wheeler ( 1) reported the binding of BCNU to lysine residues
of proteins, and therefore it is likely that this mechanism may
apply to a variety of substituted nitrosoureas. The high
reactivity of albumin noted in the present studies suggests that
its lysine residues are more exposed
@
are required
@
than those in several other
proteins which were examined.
The modifications of proteins as well as of nucleic acids
observed with
C-labeled CCNU might be the result
of alkylation of both classes of compounds. Further studies
to determine
whether
the modified
amino
acid
and base residues are similar to those found with more
conventional alkylating agents (1 7). The present results with
synthetic polynucleotides suggest that G and C residues in
nucleic acids are more reactive than are A and U residues. On
the other hand, the unusual reactivity of tRNA with
ethylene-' C-labeled CCNU suggests that factors related to the
secondary and tertiary structure of nucleic acids might also be
important
in exposing specific sites for modification.
7. Gardner, R. S., Wahba, A. J., Basilio, C., Miller, R. S., Lengyel, P.,
and Speyer, J. F. Synthetic Polynucleotides and the Amino Acid
Code, VII. Proc. Nail. Acad. Sci. U. S., 48: 2087—2094, 1962.
8. Hansen, H. H., Selawry, 0. S., Muggia,F. M., and Walker, M. D.
Clinical Studies with 1-(2.Chloroethyl)-3-cyclohexyl-l-nitrosourea
(NSC 79037). Cancer Res., 31: 223—227,1971.
9. Iriarte, P. V., Hananian, J., and Cortner, J. A. Central Nervous
System Leukemia and Solid Tumors of Childhood: Treatment with
1,3-Bis(2-cMoroethyl)-l-nitrosourea
(BCNU).
Cancer,
19:
1187—1194,
1966.
10. Lessner, H. E. BCNU (1,3-Bis(2-chloroethyl)-1-nitrosourea).
Effects on Advanced Hodgkin's Disease and Other Neoplasia.
Cancer,22: 451—456,
1968.
11. Levin, V. A., Shapiro, W. R., Clancy, T. P., and Oliverio,V. T. The
Uptake, Distribution, and Antitumor Activity of 1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea in the Murine Glioma. Cancer Res., 30:
2451—2455, 1970.
12. Lowry, 0. H., Rosebrough, N. J., Fan, A. L., and Randall, R. J.
Protein Measurement with the Folin Phenol Reagent. J. BioL
Chem., 193: 265—275,1951.
13. McCaHa, D. R., and Reuvers, A. Reaction of N-Methyl-iV'-nitro
N-nitrosoguanidine with Protein: Formation of Nitroguanido
Derivatives. Can. J. Biochem. PhysioL, 46: 1411—1415, 1968.
14. Montgomery, J. A., James, R., McCaleb, G. S., and Johnston, T. P.
The
Modes of
1-nitrosourea
In conclusion, the present experimental results suggest that
CCNU
chemically
modifies
proteins
mainly
via
cyclohexylcarbamoylation
of lysine residues and modifies
nucleic acids via alkylation. The dual capacity of this
compound may explain its broad cytotoxicity and its activity
against tumors which are resistant to conventional alkylating
agents (10).
ACKNOWLEDGMENTS
Decomposition
and Related
of
Compounds.
1,3-Bis(2-chloroethyl)J. Med. Chem., 10:
668—674,1967.
15. Nagao, M., Yokoshima, T., Hosoi, H., and Sugimura, T. Interaction
of N-Methyl-N'-nitro-N-nitrosoguanidinewith Ascites Hepatoma
Cellsin Vitro. Biochim.Biophys. Acta, 192: 191—199,1969.
16. Oliverio, V. T., Vietzke, W. M., Williams,M. K., and Adamson, R.
H. The Absorption, Distribution, Excretion, and Biotransformation
of the Carcinostatic 1-(2-Chloroethyl)-3-cyclohexyl-l-nitrosourea
in Animals. Cancer Res., 30: 1330—1337, 1970.
17. Price, C. C., Gaucher, G. M., Koneru, P., Shibakawa, R., Sowa, J.
R., and Yamaguchi, M. Mechanism of Action of Alkylating Agents.
Ann. N. Y. Acad. Sci., 163: 593—600,1969.
We thank Dr. Edgar M. Houspian for valuable support and
suggestions. We are indebted to Dr. Steven K. Carter, Dr. Robert R.
Engle, and the Cancer Chemotherapy National Service Center of the
National Cancer Institute for making the labeled preparations of CCNU
availableto us.
18. Roberts,
J. J., and Warwick, G. P. The Covalent Binding of
Metabolites of Dimethylaminoazobenzene, p-Naphthylamine and
Aniline to Nucleic Acids in Vivo. Intern. J. Cancer, 1: 179—196,
1966.
19. Schabel, F. M., Johnston, T. P., McCaleb, G. S., Montgomery, J.
A., Laster, W. R., and Skipper, H. E. Experimental Evaluation of
Potential Anticancer Agents. VIII. Effect of Certain Nitrosoureas
on Intracerebral L1210 Leukemia. Cancer Res., 23: 725—733,
1963.
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27
Interaction of 1-(2-Chloroethyl)-3-cyclohexyl-1-nitrosourea (NSC
79037) with Nucleic Acids and Proteins in Vivo and in Vitro
Chun Jui Cheng, Shinji Fujimura, Dezider Grunberger, et al.
Cancer Res 1972;32:22-27.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1972 American Association for Cancer Research.