(CANCER RESEARCH 37, 3802-3814, October 1977]
Macromolecular Complexes Produced by Chemical Carcinogens
and Ultraviolet Radiation1
Nancy R. Morin, Paul E. Zeldin, Z. 0. Kubinskl, P. K. Bhattacharya,2
and H. Kublnskl3
Division of Neurosurgery, University of Wisconsin School of Medicine, Madison, Wisconsin 53706
INTRODUCTION
SUMMARY
The effects of several carcinogenic
and noncarcinogenic
chemicals, ultraviolet light, and some presumably noncan
cinogenic analogs of carcinogenic compounds were tested
for their ability to induce in vitro complexes between pun
fied nucleic acids and between nucleic acids and proteins.
Several independent analytical methods were used to mini
mize the possibility of an unrecognized technical artifact.
The results indicate that all of the ultimate tumor-producing
agents tested thus far fall into two distinct groups with me
spect to their ability to form macromolecular complexes.
Class A of these chemicals includes several mono- and
polyfunctional alkylating agents. Both nucleic acid-nucleic
acid and protein-nucleic acid adducts are produced in the
presence of these compounds. Class B is exemplified by
N-acetoxy-2-acetylaminofluorene, salts of beryllium , and ul
traviolet light. Complexes between proteins and nucleic
acids, although not between the nucleic acids themselves,
are produced in the presence of Class B agents. Nonultimate
carcinogenic chemicals such as N-hydnoxy-2-acetylamino
fluonene and 2-acetylaminofluonene do not give rise to any of
these macromolecular complexes. However, they may be
transformed into apparently active forms that then behave
like Class B carcinogens after exposure to mouse or rat liven
extracts (postmitochondnial supennatants). None of the
macmomoleculan complexes were produced by noncancino
genic chemicals. Along with our earlier observations, data
reported in this paper indicate that a significant number and
possibly all of the tumor-producing agents are able to form
macromoleculan complexes. We propose that, if such strong
bonds between various chromosomal macromolecules are
produced inside living cells exposed to carcinogenic agents,
one extreme consequence of their formation may be a non
disjunction of daughter chromosomes during mitosis and/
or a chromosomal
breakdown.
Such effects
were often
observed by other authors in tissues treated with carcino
gens, and chmomosomal aberrations are believed by some
to correlate with malignant behavior.
I This
work
was
supported
in
part
by
USPHS
Grant
CA-16989
and
by
James Picker Foundation Grant R-67-11.
2 Present
address:
Department
of
Physical
from
density
gradient
Chemistry,
Jadavpur
Univer
centnifugation
and gel electrophone
sis experiments suggest (35) the following
sequence of
eventsafter exposure of DNAto BPL. Immediatelyafter the
addition of the carcinogen, DNA changes its appearance
from a smoothly coiled linear molecule into a structure
composed of a small number of rigid loops (‘
‘soap
bub
bles―)kept together by a small number of intramolecular
bridges of unknown chemical nature. The contour length
of this DNA is increased (by as much as 15%) and such
DNA moves faster through sucrose gradients and agarose
polyacrylamide
gels, presumably
because
of its changed
hydmodynamic profile.
After further incubation this apparently strained DNA
molecule starts to break. The accumulation of single
and then double-strand scissions leads to the appearance
of a new fraction
of DNA that sediments
slowly
during
centnifugation and moves more rapidly during gel electro
phomesis than does the bulk of untreated DNA. Under the
electron microscope the breaks are seen to produce “whis
kems―
of single-stranded DNA, which peel off and attach to
another part of the same or to another DNA molecule.
Similarly, molecules with double-strand breaks are often
seen attached to other DNA's end to side. This increased
tendency for intra- and intermolecular binding leads to the
formation of branched, irregular networks of alkylated DNA
molecules. Such “megamolecules―
may be detected by
their increased sedimentation mate in sucrose gradients
and their decreased mobility through gels during electro
phonesis. The largest of the ‘
‘clumps―
are found at the
4 The abbreviations used are: BPL,
tied albumin-kleselguhr;
isty, Calcutta 700032, India.
3 To whom requests for reprints should be addressed, at McArdle Labora
tory for Cancer Research, University of Wisconsin, Madison, Wis. 53706.
Received April 14, 1977; accepted July 14, 1977.
3802
We have been reporting recently on the effects of a
simple monoalkylating agent, BPL,4 which produces in
vitro strong, covalent-like bonds between nucleic acids
and between nucleic acids and various, especially basic,
proteins (3, 5, 46, 59). The formation of intra- and intermo
lecular bridges alters the size of these biologically impor
tant macromolecules, thus changing their sedimentation
rate in sucrose density gradients and their mobility through
agamose-polyacrylamide gels during electrophomesis. Elec
tron microscopic studies of viral DNA together with data
@-propiolactone;MAK, methylesteri
PS, 1 ,3-propanesulone;
MMS.
methyl
methane
sulfonate; AAF, 2-acetylaminofluorane; SDS, sodium dodecyl sulfate; GC,
guanine plus cytosine; EMS, ethyl methanesulfonate; DM5. dimethylsulfate;
NM, nitrogen mustard; N-OH-AAF, N-hydroxy-2-acetylaminofluorena; N-Ac
AAF, N-acetoxy-2-acetylaminofluorene; DM50, dimethyl sulfoxide; MNNG,
1-methyl-3-nitro-1 -nitrosoguanidine.
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Macromolecular Complexes and Carcino genesis
bottom of centrifuge tubes even after a relatively short
centnifugation and are not admitted into the agarose-poly
acrylamide gels. These DNA complexes are stable in form
amide and at high pH, suggesting that covalent-like bonds
are formed between these macromolecules. Thus, some
how paradoxically the appearance of large DNA complexes
in the presence of BPL is the direct consequence of the
breakdown of the polynucleotide, with concomitant forma
tion of some highly reactive centers in the molecule. The
relative proportion of the 2 fractions of alkylated DNA (one
of high molecular weight and the other of low molecular
weight) depends on the concentration of DNA during the
reaction; more of the low-molecular-weight material is seen
at higher dilutions, whereas the formation of the complexes
is facilitated by higher concentrations of DNA during the
exposure to BPL.
The DNA-DNA adducts are not the only ones that could
be produced in the presence of BPL. If RNA or protein are
added in excess to the reaction mixture, DNA-RNA or DNA
protein complexes are formed (46) and may be detected by
the altered sedimentation mateand electrophoretic mobility
of the DNA when compared with the same characteristics
of DNA exposed alone to the carcinogen. Similarly, the
density of DNA is appreciably changed in equilibrium dens
ity gradients.
The artificial
nucleoproteins
formed
under
such conditions are stable in the presence of strong ionic
detergents and, whenever they are tested, are found to be
resistant to the action of high pH, reflecting again on the
nature of the stable bonds formed by the carcinogen. It is
not even necessary to expose nucleic acids to BPL in the
presence of a protein; alkylated DNA and RNA diluted with
a buffer and chromatographed on MAK remain firmly bound
to the protein moiety of the column and cannot be eluted
by high concentrations of NaCI. Both the megamolecules
and the low-molecular-weight fractions of the alkylated
DNA are completely retained. The amount of the nucleic
acid material adsorbed irreversibly to the column is related
to the time of incubation with BPL (before application to
the column) and to the concentration of the chemical.
Nucleic acids and proteins are not the only “targets'
‘
for
the attachment of the alkylated DNA and RNA. We have
noted that, among other materials, centrifuge tubes and
nitrocellulose filters retain such nucleic acids; in fact the
method of filtration serves as a convenient measure of
progress of the reaction between DNA and the alkylating
agent.
Purified RNA and single-stranded DNA react with BPL in
a manner similar to that of native DNA, as suggested by the
same analytical techniques used for the DNA experiments.
The nibonucleoproteins formed in the presence of BPL are
equallystableinionicdetergents.
PS, a carcinogenic and mutagenic chemical, is a small
ring alkylating agent related chemically to BPL. Although
no electron-microscopic studies were carried out on the
morphology of PS-treated DNA, all other techniques men
tioned above revealed no major differences (if any) between
the actions of BPL and PS on DNA and ANA; formation of
both the nucleic acid-nucleic acid and nucleic acid-protein
adducts was observed (59).
Thus, it seemed interesting to establish whether the ability
to attach nucleic acids to other macromolecules is a com
OCTOBER
mon feature of other and, possibly, all carcinogenic agents.
We decided therefore to use a similar battery of techniques
to test whether other alkylating agents and other, unrelated
chemical and physical carcinogens would alter the nucleic
acids in a manner similar to that of BPL and PS. We docu
ment here in somewhat greater detail the effects of alkylat
ing agent MMS on isolated nucleic acids and mixtures of
nucleic acids and proteins. To avoid being repetitious, we
summarized more concisely the effects of some of the other
chemicals. A preliminary report on some of these observa
tions has been published already (36). The chemical and
biological properties of the compounds used in our work
and the carcinogenic and mutagenic activities of UV have
been summarized inseveralreviews(1,8,11,13,18,21,27,
43-45,49,55).
MATERIALS AND METHODS
Most of the techniques used in the present study were
previously described (33, 46, 59). A brief summary follows.
Sources and Preparation of Nucleic Acids. Bacterial
DNA and RNA were obtained from Escherichia co/i, Sarcina
/utea, and Cytophaga
johnsonii
(34). The methods
of 32p@
labeling and nucleic acid extraction have been published
(33). RNA from Ehrlich ascites cells was labeled with radio
active phosphorus and extracted with hot phenol as de
scnibed before (25, 32). [3H]Thymidine-labeled human lym
phocytes were kindly given to us by Dr. M. Bach of the
University of Wisconsin, Madison, Wis. DNA was extracted
from these cells by the modified (34) technique of Manmur
(41).
Sourcesof Carcinogensand Other Chemicals.AAFand
its derivatives were generously given to us by Dr. E. Miller
and Dr. J. Miller from the McArdle Laboratory for Cancer Re
search, University of Wisconsin, Madison, Wis. CG 662 and
chlorpromazine were gifts from Chemie Gmünenthal,
GmbH,
Stolbeng, Rhineland, Germany, and Smith Kline & French
Laboratories, Philadelphia, Pa., respectively. All other
chemicals were of the highest purity available commem
cially. Nevertheless, to avoid any artificial effects of possible
contamination, we usually tested and compared several lots
of such chemicals, purchased from a variety of suppliers.
Although minor quantitative differences were occasionally
noted, the effects of these various lots were comparable or
identical.
Bovine serum albumin, Fraction V, was obtained from
Armour Pharmaceutical Co., Kankakee, Ill. Other proteins
used in this study as well as calf thymus DNA were pun
chased from Worthington Biochemical Corp., Freehold,
N. J.
SucroseDensItyGradientCentrlfugatlon.DNAwascen
tnifuged in either neutral on alkaline 5 to 20% sucrose gra
dients. In addition to sucrose the neutral solutions con
tamed 0.15M NaCI,15 mM sodium citrate,
and 10 mM Tnis
HCI buffer (pH 7.2). In experiments in which DNA was incu
bated with basic proteins such as lysozyme or histone, SDS
was added to the sucrose solutions to a final concentration
of 0.2%. The alkaline sucrose contained 0.1 N NaOH in place
of sodium citrate and Tnis-HCI. The samples were centni
fuged at 42,000 rpm for 90 mm in a Spinco SW 50.1 motor.
The temperature of the rotor was kept at 4°,except for
1977
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3803
N. A. Morin et al.
experiments with SDS-containing sucrose gradients, which
were nun at 20°.Fractions were collected from the bottom.
RNA was fractionated in sucrose containing 0.1 M NaCI and
0.05 M sodium acetate-acetic acid buffer, pH 5.3. The condi
run. The agarose-acrylamide gels were sliced into 1-mm
discs with a set of razor blades positioned 1 mm from each
other. The acrylamide gels used for the electrophoresis of
RNA were carefully transferred onto narrow strips of What
tions of centnifugation and collection were the same as man No. 1 paper, dried, and sliced into 1-mm strips with a
those used for DNA, except that the time of centnifugation
was extended to 150 mm.
MAK Chromatography. The protein-coated kieselguhr
columns were prepared (40) fresh for each experiment. DNA
or RNA was eluted with the NaCI gradient buffered with
sodium-potassium phosphate buffer, under conditions of
constant temperature and flow mate(24, 32). After comple
tion of the experiment, the kieselguhm was extruded from
the glass column by gentle application of compressed air
and then was suspended in water. Several samples of differ
ent volumes were pipetted on planchets and dried, and the
radioactivity was measured. The results were extrapolated
to zero concentration of kieselguhn.
In each series of chromatographic experiments devoted
to testing a single chemical for its ability to alter the elution
profile of a nucleic acid, an experiment was included in
which the radioactively labeled nucleic acid was exposed to
the tested chemical and then chromatographed on columns
built with kieselguhr alone (no protein). The untreated DNA
or RNA did not attach to the column at any time, and less
than 0.1% of the total radioactivity was eventually found
associated with the column material at the completion of
the experiment. None of the chemicals and treatments dis
cussed in this report increased either the initial adsorption
paper cutter. The presence and the distribution of the radio
active label werethen established.The placementof gels on
Whatman filter paper gave an additional advantage, that of
allowing rapid detection of RNA bands by viewing the gels
before slicing under filtered UV (Mmneralight UVS 12, Ultra
violet Products, Inc. , San Mateo, Calif.).
Fiftratlon of Carcinogen-treated DNAthrough Nitrocellu
lose Membranes. The radioactive DNA or RNA was coincu
bated with the tested chemicals at 37°in a buffer containing
0.1 M NaCI and 0.01 M phosphate (or Tris-HCI) buffer, pH
7.2. At various times the aliquots were withdrawn and im
mediately diluted 20-fold into an SDS-contamning NaCI
solution (0.1 M NaCI; 0.5% SDS). The samples were then
incubated for 2 mm at room temperature and applied with
gentle suction onto nitrocellulose filters [25-mm-diameter
BA 85 (old designation, B6); Schleicher & Schuell, Inc.,
Keene, N. H.], presoaked in the same SDS-containing
NaCI solution. After application of the samples, filters were
extensively washed with 40 to 50 ml of the same solution
and dried, and the radioactivity associated with them
was determined in a low-background , gas-flow planchet
counter.
Uv Irradiation. The nucleic acids, alone onwith appropni
ate proteins, were dissolved in 0.14 M NaCIbuffered with 5
or the final retention of nucleic acids on kieselguhr alone.
mM phosphate buffer (pH 7.2). The samples were irradiated
EquilibrIumDensity Gradient Centrifugation in CsCI. on ice, with constant mixing, in 65-mm crystallizing dishes.
DNA and DNA exposed to the tested chemicals both in the
presence and in the absence of proteins were mixed with
CsCl (820 @lof the buffered DNA solution plus 3 ml of
saturated CsCI). In experiments in which DNA-protein asso
ciation was studied, an ionic detergent, Sarkosyl (Geigy
Industrial Chemicals, Ardsley, N. Y.) was added to a final
concentration of 0.5%. The samples were centrifuged for 44
hr at 36,000 rpm in a Spinco SW 50.1 motor and collected
from the bottom. Refractive indices were established for
selected fractions, followed by measurements of radioactiv
Two parallel 15-watt mercury lamps were used, with peak
radiation at 254 nm . The irradiated samples were 2 to 3 mm
thick, and the average incident fluence matethrough the
sample was 0.1 J/sq rn/sec as determined with the aid of an
UV meter (Ultra-violet Products).
ity and,
summarizesits effects on the sedimentation rate in sucrose
in some
experiments,
absombance
at 260 nm.
Analytical centnifugation in a Spinco E ultracentnifuge
was carried out as described before (52). We are grateful to
Dr. W. Szybalski of the University of Wisconsin, Madison,
Wis., for kind permission to use his analytical ultracentnifuge
and facilities.
Gel Electrophoresis. DNA was electrophoresedin agarose
acrylamide gels as described by Peacock and Dingman (47),
and RNA was analyzed in 2.4% acrylamide gels prepared
according to Bishop et al. (7). Both types of gels were
prepared and run with 0.1% SDS in the gel and the buffer.
Our previous observations on carcinogen-treated nucleic
acids and nucleoproteins have indicated that the large com
plexes produced under such circumstances do not enter
the gel and are often lost at the time of extrusion of gels
from Plexiglas tubings (59). For prevention of such losses,
the gels were topped before application of DNA with a thin
layer of Sephadex G-25, which retained the extra-large
clumps of nucleic acids. The Sephadex layer was then
quantitatively recovered at the end of the electrophoretic
3804
RESULTS
Effects of MMS. MMS is a monofunctional alkylating
agent, unrelated chemically to either BPL or PS. Chart 1
plus SOS of E. coli DNA exposed to this compound alone
and in the presence of lysozyme. Under these conditions
untreated DNA sedimented at a rate identical to that ob
served in sucrose gradients without SDS and appeared as a
fairly homogeneous band. Its distribution in the sucrose
SDS gradient was not significantly altered by previous coin
cubation with lysozyme (not shown).
The same DNA exposed to MMS became more heteroge
neous, and a sizable proportion of this nucleic acid sedi
mented more rapidly than did the bulk of control DNA. A
slight but highly reproducible increase in the amounts of an
apparently low-molecular-weight material was observed at
the top of the gradient. Such altered sedimentation patterns
were also seen in sucrose gradients without SDS. The total
amount of radioactive DNA recovered from the gradient was
lower by some 10% than it was in the control. The remainder
of the labeled material was found in the nitrocellulose cen
tnifuge tubes, most of it at the bottom and the nest in the
upperone-third,
forminga ringof radioactivity
atthelevel
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Macromo/ecu/ar Complexes and Carcino genesis
In several other experiments we have found that the de
gree of change in the sedimentation rate during centnifuga
tion and in the mobility of DNA during gel electrophoresis,
both in the presence and the absence of a protein, was
related to the concentration of MMS, the duration of expo
sure to the carcinogen, and the temperature of incubation.
Changes in the sedimentation rate comparable to those
induced at 37°within 20 mm were seen only after several hr
of incubation at 0°.The effects of MMS on DNA were rather
insensitive to the ionic strength and composition of the
“‘Ol
incubation medium. The presence or the absence of diva
lent cations or potassium and the NaCI concentrations be
tween 0.05 and 0.5 M did not appreciably alter the course of
the reaction.
Chart 3 summarizes the results of an isopyknic density
gradient centnifugation experiment, in which the E. co/i
FRACTiONNUMBER
[32P]DNA was incubated with MMS or MMS and lysozyme
Chart 1. Sucrose density gradient centrifugation of 2 @.tgof •
E. coli
in CsCI supple
(“P]DNA
(0) and of DNA incubated with 1 mu MMS for 60 mm at 37@(•) and then centrifuged until equilibrium
and with MMS and lysozyme under the same conditions (x ; 100 @ig
lysozyme
mented with ionic detergent Sarkosyl. The untreated DNA
par sample). The samples ware put on top of 5 to 20% sucrose gradients
containing 0.2% SDS (w/v) and centrifuged in a SW 50.1 rotor for 90 mm at banded in CsCl at the density of 1.71 g/ml, and its incuba
42,000 rpm at 20―.
After the completion of the run, 120-SI fractions were tion with lysozyme did not alter its position in the gradient
collected from the bottom. Close to 99% of the total applied radioactive
(not shown). Incubation with MMS broadened the DNA peak
DNA were recovered in the control gradient; 90% of the MMS-treated DNA;
and increased its average density. This observation was later
and 83% of DNA exposed to MMS in the presence of lysozyme.
confirmed in several experiments by analytical ultracentrif
where the sample was deposited immediately before the ugation used as a more precise tool for detection of density
centnifugation. Such phenomenon was previously observed changes (Chart 4). There were several artifacts commonly
with BPL- or PS-treated DNA (46, 59). Similar changes in the observed during analytical equilibrium density gradient
sedimentation rate of MMS-treated DNA were seen during centnifugation of the alkylated DNA. One of these artifacts
centnifugation in alkaline sucrose gradients.
was the “disappearance―
of the alkylated DNA from the
When the same DNA was exposed to MMS in the presence gradient. In extreme cases almost all of the tested DNA may
of lysozyme, the sedimentation pattern of the [32P]DNAwas become undetectable by this technique (Chart 40), due to
further altered. Only a small portion of the total radioactivity extensive degradation, attachment to the walls of the centni
was recovered at the position of the normal DNA, and most fuge cell, or both. Another, somewhat less often observed
of the material moved much more rapidly through the gra phenomenon (not shown) was the occasional fusion of 2 or
dient, suggesting the formation of large complexes made more DNA bands of different density, presumably due to the
presumably of both the DNA and the protein. The recovery formation of DNA-DNA complexes (see below).
of the radioactive material from the gradient was decreased
The same DNA that showed only minor changes in density
by an additional 6% compared with that of the sample in
which DNA alone was exposed to MMS. Most of the material
found in the centrifuge tubes was at the bottom. A signifi
I
cant portion of the material recovered from the gradient
banded next to the bottom of the tube. With the assumption
that the relationship between the molecular weight and
sedimentation rate for the DNA-protein complexes was sim
ilar to that of native, double-stranded DNA (10), such corn
plexes may have been more than 20 times larger than the
bulk of DNA from which they were derived.
[32P]DNA exposed to MMS alone migrated at a slightly
altered rate through gels during electnophoresis (Chart 2).
The DNA peak was less homogeneous than it was in the
control, and a part of the radioactive material barely entered
the gel. No appreciable changes in DNA migration through
the gel were observed after its incubation with lysozyme
alone. However, coincubation of the DNA with MMS and
lysozyme prevented most of the radioactive material from
entering deepen than 1 mm into the gel. This observation
further indicated that the fast-moving fraction observed
during sucrose centnifugation experiments was composed
of megamoleculesof large molecular weight rather than of
DNA of normal size but with grossly altered hydrodynamic
properties.
OCTOBER
-J
L&@
0
LU
I-
LU
.J
E
0.
U
in
0
MILLIMETERS
OF GEL@
Chart 2. Gel electrophoresis of 2 p.g of E. coli (32P]DNA (0) and DNA
treated with 1 mu MMS for 60 mm at 37°in the presence (x) and in the
absence (•)of 100 g@gof lysozyma. The radioactive material not admitted
into the gel (the Sephadex layer; see “Materials
and Methods―)is shown as
0-mm fraction.
1977
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3805
N. A. Morin et a!.
to about
one-tenth
of its original
molecular
weight,
twice
the concentration of MMS was required to produce compa
mablealterations in DNA's physicochemical characteristics.
We have tested the effects of MMS and MMS plus protein
on DNA's of different GC contents. C. johnsonii (34% GC),
human [3H]DNA (42% GC), and S. /utea (71% GC) DNA's
reacted in a manner very similar to that of E. co/i DNA (50%
GC; see Ref. 34). Therefore, we conclude that the formation
of DNA-DNA and DNA-protein adducts by MMS is basically
independent of the overall base composition of the DNA. It
is also not affected by the kind of radioactive label used for
the experiment.
Microsomal membrane from rat liver (23) exposed to
MMS and DNA and centrifuged until equilibrium in CsCI
(28, 30) attached increased amounts of E. co/i DNA com
I
1.71 .694
DENSITY,g/mI
Chart 3. Isopyknic density gradient centrifugation in CsCI plus 0.5%
Sarkosyl; 6 @L9
of E. coli (32PJDNA(0) and DNA exposed to 2 mu MMS for
60 mm at 37@in the presence (x ) and in the absence (•)of 200 @.tg
of
lysozyrne. The final recoveries of radioactive DNA from the gradient were
10% in the control, 88% in the MM5-treated sample, and 60% in the sample
exposed to both MMS and protein. The balance of the radioactive material
was detected on walls of the centrifuge tubes and at the oil-water interfaces
(ca. 1 ml of oil hadbeenplacedon the top of the samplesat the beginningof
centrifugation to help to balance the rotor and to prevent any water losses
due to evaporation).
after treatment with MMS became much more heteroge
neous when lysozyme was added to the incubation mixture
(Chart 3). In addition to a small amount of material heavier
than 1.71 g/ml, a significant proportion of the radioactivity
was recovered higher in the gradient, some of it at densities
that indicate formation of complexes with the protein. With
the assumption that the density of the protein is close to 1.4
g/mI (see Ref. 22), as much as 30% of the complex may have
been made of this macmomolecule. The bonds between DNA
and protein were apparently strong enough to withstand
prolonged exposure to high concentrations of CsCl and to
the ionic detergent.
From several other experiments we have learned that
lysozyme is not the only protein that can form complexes
with DNA in the presence of MMS. The lysine-rich histone
fraction from calf thymus produced similar changes in the
sedimentation mateand electrophoretic mobility of the alkyl
ated DNA and decreased its density in CsCl. On the other
hand the effects of Fraction V of bovine serum albumin were
less apparent; at a given concentration of the proteins DNA
and MMS, less change in density, sedimentation mate,and
electrophoretic mobility was seen with albumin than with
the 2 basic proteins.
Sonic disruption of DNA (5 to 60 sec at 50 watts in a Heat
Model 185 Sonifier and in an ice-water bath) prior to its
exposure to MMS or MMS and proteins did not qualitatively
change the response to the alkylating action of MMS and
to its ability to produce DNA-DNA and DNA-protein adducts.
However, in addition to the obvious and expected altema
tions in sedimentation rate and electnophonetic mobility,
sonic disruption apparently decreased the sensitivity of
the polymer toward the carcinogen : for DNA reduced in size
3806
Chart 4. Densitometer tracing from photographs of MMS-traated E. coil
DNA at equilibrium. DNA samples (2 pg/sample) were incubated for 60 mm
at 37―
with the indicated concentrations of the carcinogen. At the end of the
incubation time, 4.6 volumes of C5CI solution saturated at room temperature
were added together with C. johnsonii DNA (density marker; density. 1.694
9/mI) and immediately transferred to 12-mm Kel-F cells. The samples were
then centrifuged for 20 hr at 44.770 rpm at 25°in a Spinco Model E
analytical centrifuge. Tracings of the UV photographs were prepared with a
Joyce-Loebel Mark IIIC double-beam microdensitometer equipped with the
cylindrical lens condenser. A, control (no MMS); B and C, 0.8 and 2 mu
concentration of MMS, respectively. Note: The tracings were arbitrarily
aligned in such a mannerthat the peaksof the density marker DNAare
positioned on the same vertical line marked 1.694. No allowance was made
for any possiblechangein densityof c. johnsonii DNAcausedby tracesof
undergraded MMS present in the sample at the time of addition of this DNA.
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Macromolecular
pared to that of the control sample, which was not exposed
Complexes and Carcinogenesis
ical carcinogens. Although this method was used primarily
to MMS. For example, at 8 mM MMS, 200 @g
of membrane in measuring the binding of alkylated nucleic acids to the
immobilized protein, testing for the formation of DNA-DNA,
DNA-RNA, and RNA-RNA adducts was also possible with
this technique.
Chart 6 presents the elution pattern of the purified,
radioactive E. co/i DNA chromatographed on a MAK col
of the membrane band in the control sample without MMS. umn. The same DNA preincubated for 5 mm with 5 mM
The experimental conditions were the same as those used MMS, diluted with the buffer, and then applied showed a
different elution profile; almost one-half of this DNA was
in our previous studies (30).
retained irreversibly on the column. The remaining 50%
The technique of isopyknic density gradient centrifuga
tion in CsCI was also used to demonstrate the formation of was eluted at the same salt concentration as was the
control DNA, and no traces of the low-molecular-wemght
complexes between DNA's derived from various organisms.
In the experiment illustrated in Chart 5, S. !utea [32P]DNA DNA or of DNA megamolecules were detectable in the
(density, 1.731 g/ml) was mixed with unlabeled C. johnsonii
column effluent. An extension of the incubation time to 60
DNA (density, 1.694 g/ml) and centrifuged in CsCI until mm altered the DNA so much that only about 8% of the
equilibrium. The 2 DNA's were easily separated in the applied material was eluted by the salt gradient. Similarly,
gradient. S. lutea DNA incubated alone with MMS had a an increase in the concentration of MMS to 20 mM de
creased the fraction of DNA eluted from the column to less
somewhat higher density and, predictably, was less homo
geneous than that in the control. However, when a more than 30% after 5 mm of incubation (not shown). If the
than 100-fold excess of unlabeled C. johnsonii DNA was incubation mixture of DNA and 10 mM MMS was supple
added to the mixture of MMS and radioactive S. /utea DNA mented at the 3-mm time point with albumin and applied 2
mm later to the column, the retention rate further
shortly before the end of the incubation period, a substan
tial portion of the latter nucleic acid was found banding at increased compared with the sample of DNA alone, which
a density lower than 1.7 g/mI. In a series of related expeni
was incubated for 5 mm with the same concentration of
ments (not shown), we have found that similar changes in MMS. From separate experiments (not shown) we have
the density of the alkylated DNA were seen when a radioac
learned that a similar effect was observed when unlabeled
tive E. co/i DNA was mixed with S. /utea or C. johnsonii calf thymus DNA (600 @g/ml)was added at 3 mm of incuba
unlabeled DNA's. However, the changes were often less tion. Addition of either albumin or the heterologous DNA
obvious due to a smaller density differential between E.
co/i and the other 2 DNA's.
Chromatography on MAK was another technique applied
to studies on macromolecular interactions caused by chem
material (measured as protein) coincubated with 1 @g
of E.
coli [32P]DNA attached 12% of the total nucleic acid. At 20
mM MMS, more than 18% of the total DNA was found in the
membrane band at the density of 1.18 g/mI. In contrast,
less than 5% of the total DNA were recovered at the density
E
E
C,
In
0
0@
DENSITY, g/ml
@
Chart 5. lsopyknic density gradient centrifugation in CsCI of 2 @g
of S.
lutea (UP]DNA together with 300 sg of unlabeled C. johnsonii DNA (0); 5.
lutes (UPIDNA exposed for 60 mm at 37°to I mu MMS (C) followed by the
addition of 300
of unlabeled c. johnsonii DNA at The 50-mm time point
(t@).Arrow, position of c. johnsonii DNA in the 1st and in the 3rd sample, as
established by spectrophotometric measurements at 260 nm after comple
tion of centrifugation.
OCTOBER
0.7
08
M NaCI
Chart 6. MAK column chromatography of 2 @.tg
of E. coli (UPJDNA (0)
and the same DNA exposed to 5 mu MMS for 5 mm (C), 60 mm (x) and for
5 mm in the presence of 200 @g
bovine serum albumin (z@)added at 3 mm.
After the completion of the experiment, the following amounts of the applied
radioactive DNA ware recovered with the column material: control, lass than
1%; 5 mm incubation with MMS, 49%; 5 mm incubation with MMS, plus
albumin, 71%, 60 mm incubation with MMS, 93%.
1977
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3807
N. A. Morin et a!.
did not alter the elution profile of the untreated E. co/i (not
shown).
Chart 7 demonstrates the effects of MMS on DNA as
detected by filtration through nitrocellulose membranes.
We have observed previously (46, 59) that several cancino
genic chemicals, including BPL and PS, cause the retention
of nucleic acids on these filters. In this experiment, radioac
tive E. co/i DNA was incubated with MMS for various lengths
of time, diluted in an SDS-containing buffer to reduce the
“background―
retention, and then filtered through the ni
trocellulose membranes. Although less than 1% of the total
DNA was retained on the filters at zero time and in the
control, more than 80% remained bound after 60 mm of
incubation with 10 mM MMS, in spite of extensive washing
of filters with the buffer and detergent. Thus, the filter
experiment confirmed our earlier observations made with
other agents; i.e., alkylated DNA will attach strongly to
various materials, including nonbiological ones such as
walls of centrifuge tubes and nitrocellulose filters.
Ribosomal RNA of bacterial or mammalian origin ex
posed to MMS and then centrifuged in sucrose gradients or
chromatographed on MAK columns underwent changes
similar to those of DNA. For similar amounts of these 2
nucleic acids, however, about 10 times higher a concentra
tion of the carcinogen was required for RNA to effect
comparable alterations in sedimentation rate or elution pro
file. Presumably, the size of the molecular target might
account for this difference, as suggested by our ultrasonic
experiments on DNA (see above) and confirmed further by
our observation that the larger mRNAwas more sensitive to
MMS than was the smaller nRNA.
Effects of EMS. From a series of several dozen experi
ments similar to those described for MMS, we have learned
that EMS reacts in a qualitatively similar manner to that of
MMS with DNA from various sources; i.e., it changed the
physical characteristics of the molecule in a manner
0
LU
z
I-
LU
>-
>
IU
4
0
0
4
TIME OF INCUBATION, MIN
Chart 7. Retention on nitrocellulose filters of E. coli (@P]DNAincubated
alone at 37°in a buffer ( 0) or In the presence of MMS: 2 mu (C) and 10 mu
(1k). Aliquots were withdrawn at the indicated times diluted in SDS
NaCI solution and filtered through the nitrocellulose filters. The filters ware
then exhaustively washed with the same solution.
3808
suggestive of a limited breakdown of the molecule followed
by the induction of intra- and intermolecular bridges and
the formation of stable complexes with proteins if these
were present in the reaction mixture. However, we have
noted that on the molar basis somewhat more (1.5 to 2 times
more) EMS had to be applied to induce the same degree of
apparent change in the sedimentation mateor in the electro
phonetic mobility than the amounts that were needed for
MMS. Sincethis phenomenonwasobservedwith EMS pur
chased from various manufacturers as well as with different
lots from the same supplier, we presume that this may
reflect a genuine difference between these 2 related chemi
cals.
Other Alkylatlng Agents: DMS, DES, and NM. E. co/i DNA
or rRNA coincubated with DMS and DES changed their
sedimentation rates and electrophoretic mobilities in a
manner suggestive of molecular breakdown and concomi
tant reassembly into larger molecules. The alkylated DNA
and RNA were strongly retained on nitrocellulose filters and
on MAK columns. The changes in the physicochemical
characteristics of these nucleic acids were further accen
tuated by the addition of proteins or unlabeled heterolo
gous nucleic acids to the reaction mixture. Thus, these 2
agents had effects similar to those of MMS and EMS; i.e.,
they produced both the nucleic acid-nucleic acid and nu
cleic acid-protein adducts. However, DMS was significantly
more effective than either MMS or EMS. On the molar basis,
comparable alterations in DNA's sedimentation, electro
phonetic mobility, and retention on MAK columns were ob
served with concentrations of DMS that were 10 to 15 times
lower than those required for MMS. DES was somewhat
less active than DM5 (by a factor of 2 to 3).
E. co/i DNA exposed to NM and analyzed by centnifuga
tion in neutral sucrose or by electrophoresis did not differ
appreciably from the untreated control DNA. In alkaline
sucrose the alkylated DNA moved slightly ahead of the
untreated DNA. This slight difference in mobility could per
haps be interpreted as being due to incomplete separation
of the 2 complementary strands after their cross-linking by
the carcinogenic chemical and thus to a somewhat higher
molecular weight for such pains of complementary DNA
chains (3, 14, 26). No increase in the degree of attachment
of DNAto nitrocellulose membraneswas evident after alkyl
ation with NM. Addition of lysozyme and albumin, however,
to the reaction mixture quite dramatically altered the prop
erties of the alkylated DNA; in both the sucrose centnifuga
tion and gel electrophoresis experiments, ample evidence
was found for the formation of protein-DNA adducts. When
DNA was treated with NM in the presence of lysozyme and
subsequently centrifuged until equilibrium in CsCI plus San
kosyl, a substantial proportion of the radioactive material
banded at densities lower than the untreated DNA or DNA
exposed to NM in the absence of any protein. Part of the
radioactive material was observed at the top of the gradient.
From MAK columns the alkylated DNA was recovered
poorly, and the degree of retention on such columns was
proportional to the time of exposure to NM and to its con
centration. Addition of albumin to the reaction mixture con
taming DNA and NM further enhanced the degree of reten
tion. No such enhancementwas noted when unlabeled loaf
thymus DNA was added instead of albumin.
CANCER RESEARCHVOL. 37
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Macromolecular Complexes and Carcino genesis
C. johnsonii and S. lutea DNA's reacted with NM alone in
a manner quite similar to that of E. co/i DNA;i.e., no major
changes in their physicochemical characteristics were dis
cernible during centnifugation or electrophoresis. Similarly,
addition of a protein to the reaction mixture produced DNA
protein adducts that did not break down in the presence of
ionic detergents. On MAK columns both DNA's were in
creasingly retained after exposure to NM.
Effects of Aromatic Amlnes AAF, N-OH-AAF, and N-Ac
AAF.The2 derivativesof AAFand the parentcompound
itself were tested for their ability to induce macromolecular
complexes in vitro. N-Ac-AAF was studied especially cane
fully since it is believed to be the ultimate (or one of the
ultimate) carcinogenic metabolites of AAF in vivo (27, 4345).
The effects of N-Ac-AAF on the sedimentation of E. co/i
DNA preincubated with this carcinogen in the presence
and the absence of lysozyme may be summarized as fol
lows: no change in the DNA's sedimentation rate was
observed after the DNA was coincubated with either DMSO
(used as solvent for AAF and its derivatives) or N-Ac-AAF.
In several other tests we found no concentration of N-Ac
AAF
and
no
length
of the
incubation
time,
which
E
0.
C,
vi)
0
would
result in alterations of the sedimentation rate, that could
be interpreted as indicating the formation of DNA-DNA
adducts. After incubation of DNA with N-Ac-AAF and lyso
zyme, however, changes in the sedimentation rate clearly
suggested complexes larger than those of untreated DNA.
Such complexes were also observed during electrophoresis
1.0
M NoCl
Chart8. MAKchromatography
of purified23SrRNAfromE.coli.Two @&g
ofrRNAwereincubated
withN-Ac-AAF
at37°
Inasolutioncontaining
0.05u
NaCI and 0.01 u acetate buffer, pH 5.4. IncubatIon time: 10 mm with 100 @su
N-Ac-AAF (0); 20 mm with 40 g@u(0); 10 mm with 200 g@u(@@);
20 mm with
100 @u(x). C, control.
in SDS-containing agarose-acrylamide gels and during
equilibrium density gradient centnifugation in CsCl in the
presence of Sarkosyl (results of all these experiments are
not shown). The N-Ac-AAF-treatedDNA was retained on
MAK columns in proportion to the concentration of the
chemical and to the duration of exposure during incuba
tion. Addition of albumin at the end of the incubation
period (but not of calf thymus DNA) increased the degree
of retention. As was the case with MMS-treated DNA or
DNA exposed to BPL or PS (46, 59), the treated DNA was
found at the end of the experiment either attached “
imre
versibly―to the column or eluted at the “normal―
position
in the gradient. Purified rRNA, however, behaved differently
E
E
0.
U
in this respect. In dozens of experiments we haveobserved
that N-Ac-AAF-treated nibosomal RNA was retained on
MAK columns and that the degree of retention was propor
tional to the time of incubation with N-Ac-AAF and to the
concentration of the chemical. In addition to this effect,
however, an increase was reproducibly observed in the salt
concentration needed to elute the potentially recoverable
fraction of RNA. This increase was again related to the
concentration of N-Ac-AAF and to the time of incubation
and was not observed when RNA was treated with DMSO
alone. The elution patterns of the larger (Chart 8) and the
smaller (Chart 9) rRNA's changed in the same manner. In
the experiments summarized in Chart 9, the 16 S rRNA
material used for testing contained small amounts of DNA,
which eluted as a discrete peak before the RNA, at about
0.7 M NaCI. An increasing concentration of N-Ac-AAF or
prolonged incubation times decreased the size but not the
location in the gradient of this DNA peak. On the other
hand the distance between the DNA and RNA peaks in
M NaCI
Chart 9. MAK chromatography of E. coii 16 5 rRNA Incubated (see the
legend to Chart 8 for details of the incubation) with 200 Mu N-Ac-AAF for 10
mm (0) and 20 mm (0);and for20 mm with500 @.tu
N-Ac-AAF (@7).
C, control.
creased due to the higher salt concentration needed to
elute the alkylated RNA.
The reasons for this behavior of N-Ac-AAF-treated rRNA
on MAK columns are not clear. The slight alteration in
sedimentation
and electrophoretic
mobilities
after treat
ment with N-Ac-AAF(N. R. Monin and H. Kubinski, unpub
lished data) can hardly explain this phenomenon. Conceiv
ably, the base displacement observed by several authors (15,
39), with subsequent alterations in the secondary structure of
nucleic acids, could explain this anomalous elution of NAc-AAF-treated rRNA. We have never observed a simslar
OCTOBER 1977
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3809
N. A. Morin et a!.
change in the elution patterns from MAK columns of either
DNA on RNA exposed to any other carcinogenic or noncar
cinogenic chemical.
We have tested several concentrations of AAF and N-OH
AAF with both DNA and RNA, using all the techniques
that
we have routinely applied for studies on the effects of the
other carcinogenic chemicals. None of these experiments
suggested the formation of any nucleic acid-nucleic acid
or nucleic acid-protein adducts. We attempted to activate
AAF (6, 43), the nonultimate
carcinogen,
with extracts
(postmitochondnial supemnatants) from rat liver prepared as
described by Ames (4). We have observed that the density
of E. co/i DNA is not changed after incubation with either
AAF, DMSO, on the liven extract during equilibrium
density
gradient centnifugation in CsCI plus Sarkosyl. Incubation
of this DNA together with AAF and liver extract, however,
produced radioactive material banding at densities consid
erably lower than that of control DNA, signifying formation
of Sankosyl- and high-salt-stable complexes between DNA
and the extract, which is most likely with the extract's
proteins. Addition of lysozyme to the reaction mixture
further enhanced the effect. The density shift and the
distribution in the gradient of this DNA-protein complex
produced by AAF with the liver extract were comparable to
the ones induced by a corresponding amount of N-Ac-AAF.
Effects of Beryllium. This metal is carcinogenic for a
variety of experimental animals (16). DNA and RNA exposed
to beryllium sulfate (up to 10 mM) did not change its
mobility during electrophomesis and during velocity centnif
ugation in sucrose. Similarly, no change was observed in
both analytical and preparative CsCI gradient. In the pres
ence of proteins, however, complexes were formed, chang
ing the density and the apparent molecular weight of the
radioactive nucleic acids. DNA or RNA exposed alone to
BeSO4were retained on MAK columns (but not on nitrocel
lulose membranes during filtration), and addition of albu
mm (but not unlabeled DNA) at the end of the incubation
period further increased the degree of retention on the
column.
Miscellaneous Chemicals. Several other chemicals,
some known to be carcinogenic and others not generally
considered or thought of as carcinogens, were tested for
their ability to produce macromolecular complexes in vitro.
MNNG, a well-known carcinogen and mutagen (e.g., see
Ref. 19), gave no evidence of its ability to induce nucleic
acid-nucleic acid complexes, and the MNNG-tneated DNA
was not retained on nitrocellulose membranesduring filtra
tion. Addition of basic proteins to the DNA-MNNG reaction
mixture produced DNA-protein adducts that were detecta
ble by a variety of techniques.
Two alkylating antitumor drugs, chlorambucil, an ano
matic derivative of nitrogen mustard, and lucanthone (Mm
acil D), behaved similarly; no change in the physiochemical
characteristics of DNA was observed after exposure to
either of these 2 agents, but the formation of DNA-protein
adducts was observed under appropriate conditions.
Chlompromazine, a drug known for its cytotoxicity (19,
28, 38), belongs to the same category of compounds that
produce nucleic acid-protein complexes. A derivative of
bisureido-diphenylsulfone called CG 662 has been used as
a wide-spectrum antivimal dung (48). According to its manu
3810
facturer (personal communication) the carcinogenic poten
tial of this agent is unknown. In our hands it behaved as if
it were a carcinogen, producing a variety of DNA-protein
adducts. Since a significant number of human subjects has
been exposed to this drug during the 1950's and 1960's (2),
we suggest that a survey of this population for the occur
ence of neoplasia may be justified.
On the other hand several known noncancinogenic chem
icals tested failed to elicit any of the macnomolecular
complexes in vitro. In addition to the 2 nonultimate carcin
ogens AAF and N-OH-AAF (see above), the list includes
antibiotics (penicillin, Actidione), salts of mono- and diva
lent cations (K@,Na@,Mg2@,Mn2@),and a substance that is
known to interact with DNA but that does not produce
tumors in experimental animals (sodium bisulfite, see Refs.
9, 20, 28, 38).
Effectsof UV. Irradiationof DNA for up to 60 mm with
UV did not detectably change its mobility in neutral sucrose
during velocity centnifugation and in gels during electro
phoresis. In alkaline sucrose, the irradiated DNA [not unlike
the NM-treated DNA (see above)] sedimented slightly ahead
of the control DNA, indicating presumably a failure in the
separation of the 2 complementary strands due to cross
linking (e.g., see Ref. 42). DNA irradiated in the presence
of lysozyme formed complexes that moved rapidly in SDS
containing sucrose gradients during centnifugation and
that did not enter gels during electrophoresis. Such lyso
zyme-bound DNA also had a lower density in CsCI-plus
Sarkosyl gradients, forming a series of poorly defined
bands at densities below 1.7 g/ml and a layer on the top of
the gradient. When DNA alone was irradiated and then
filtered through nitnocellulose filters, no increase in the
rate of retention was observed. The same DNA incubated
at 37°with methyl-estenifiedserum albumin (40) formed
strong complexes, which were subsequently retained on
filters, even in the presence of 1% SOS (Chart 10). Incuba
tion at 0°,however, did not promote the formation of
0
LU
z
4
I—
LU
>-
I>
I—
U
4
0
0
4
TIME OF INCUBATION, MIN
Chart 10. Retention on nitrocallulose filters of E. coli (@PJDNAincubated
with MBSA at 0°(x), at 37°(@4,and at 0°with uv irradiation (0). Aliquots
warewithdrawnat times indicated,diluted with the SDS-containingbuffer,
and further treated as described in the legend to Chart 7. 0, U, experiment in
which DNA-MBSA mixture was incubated at 0 and 37°,respectIvely, but the
aliquots were taken into a buffer without SDS.
CANCER RESEARCH VOL. 37
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Macromolecular Complexes and Carcino genesis
complexes. Such [32P]DNA was mixed with methyl-esteni
fied albumin, placed on ice, and irradiated with UV. Aliquots
were withdrawn at various times, diluted immediately in an
SDS-containing buffer, and filtered, and the presence of
radioactive material on the filters was measured. It was
found that under such conditions significant amounts of
DNA attached to the filters (Chart 10).
UV irradiation of RNA (Chart 11) or DNA (not shown)
alone changed the pattern of nucleic acid eluted by a salt
gradient from MAK columns. Both the smaller (Chart 11, A
to C) and the larger (Chart 11, D to F) nibosomal RNA's
were eluted after UV irradiation at the position of the
unimradiated control material. However, the amounts of
radioactive rRNA recovered from the column were inversely
proportional to the time of irradiation and to the molecular
weight of the RNA. That the magnitude of the degree of
retention was indeed related to the size of the target
molecule is further demonstrated by the experiments shown
in Chart 12. No attachment of the irradiated DNA or RNA
was observed in experiments in which the columns were
built with kieselguhm alone, indicating that the protein
moiety is responsible for the retention. We have noticed
that the alterations in the nucleic acid structure responsible
for its increased attachment to albumin are quite stable;
incubation of irradiated DNA for 6.0 mm at either 0°or 37°
did not appreciably change the degree of retention.
DISCUSSION
We have tested a number of unrelated carcinogenic
chemicals to detect the formation of macnomolecular com
plexes in the presence of these compounds. The effects of
UV and its ability to induce DNA-protein adducts were
already studied by several authors (for review see Refs. 51
and 53), and we included this physical carcinogen and
mutagen only for the sake of comparison. Observations
summarized in the present report, together with those
previously published (29, 31, 46, 59), indicate that a much
larger than previously suspected proportion of carcino
genic agents (in fact all such agents tested to date) belong
to one of the 2 classes with respect to their ability to
induce macnomolecular
complexes
(Table 1). Group A,
TIME OF IRRADL@@11O@
MNUTES
Chart 12. Retention on MAX columns of various E. coli RNA's irradiated
with UV. For conditions of irradiation, see Chart 10 and Materials and
Methods.
represented by alkylating compounds such as BPL, PS,
MMS, EMS, DM5, and DES, produces both the nucleic acid
nucleic acid and the nucleic acid-protein adducts; Group
B, which includes NM, N-Ac-AAF (as well as the “activated―
AAF), BeSO4, MNNG, chlorambucil, lucanthone, chlonpro
mazine, CG 662, and UV, is able to form the nucleic acid
protein adducts only. DNA and RNA exposed to any of the
agents from the latter group are retained on MAK columns,
but their mobility changes in sucrose density gradients and
in gels during electrophoresis only if these polymers are
exposed to the action of such carcinogens in the presence
of a protein, especially a basic protein. The protein-DNA
complex induced by these agents has a lower density in
CsCI-plus-Sarkosyl equilibrium density gradients, demon
strating that the artificial nucleoproteins formed by carci
nogenic chemicals are stable in high salts and in the
presence of ionic detergents. Whenever they were tested,
the active carcinogens were found to react with isolated
cellular membranes (28, 29, 31, 59) and with the surface of
intact cells [i.e., plasma membranes (see Ref. 33)] altering
the patterns of interaction of nucleic acids with these
membranes, in most cases by increasing their binding to
membrane proteins and penetration into the intact cells.
Although our laboratory did not study the potential of X
naysand other forms of ionizing radiation for the production
of macromolecular complexes, the limited data available
from the literature (42, 50, 57, 58) suggest that these tumor
inducing physical agents sould also be included in this
group.
Group A compounds score positive in all these tests; in
addition they produce complexes between DNA-DNA, DNA
RNA, and RNA-RNA, as evidenced by the neutral and
alkaline sucrose gradient centnifugation experiments, gel
electrophonesis, equilibrium density gradient centnifuga
tion, and direct electron-microscopic examination of the
alkylated DNA (35). A convenient way for measuring the
“stickiness―
of the alkylated DNA on RNA is filtration of
such nucleic acid through the nitmocellulose membrane
filters. Understandably, chemicals in Group A are in general
more toxic than are those in Group B.
When exposed to carcinogens, all natural nucleic acids
studied show these effects, including DNA's of widely
different
Chart 11. MAK chromatography of Uv-irradiated E. coli rRNA. Five @.tg
of
[UP]rRNA ware placed on ice, irradiated for various times, mixed with 2 mg of
unlabeled E. coli total cellular nucleic acids, and applied to the column. 0,
radioactivity; •,absorbance.
base composition.
Nevertheless,
according
to our
(unpublished) observations, base composition appears to
affect the degree of response of DNA to a given carcinogen,
reflecting presumably the specificity of the chemical. Ex
OCTOBER 1977
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3811
N. A. Morinetal.
Table 1
List of agents producing macromolecu!ar complexes and summary of experimental
evidence.
nucleicacid-protein
Class A compounds are chemicals producing nucleic acid-nucleic acid and
acid-proteincomplexes
complexes.
Class B compounds
only.Chemical
(treat-
encesClass
ment)
ABPL
46PS
59MMS
33EMS
33DMS
JDES
JClass
are agents producing
As, Bb, C, Dd, E, F, Gg, Hh, l,Jj
JMNNG
JChlorpromazine
JLucanthone
JChlorambucil
JCG
JjUV662
As, B, C, Dd, F,Gg, H,
A, B, C, D, E, F, Gg, H,
Aa, B, C, D, Hh,
A,B,C,D,Hh,
A, B, C, Dd, Gg, Hh,
A, B, Dd, E, F, Gg, Hh,
53X-rays
As, B, C, Dd, E, F, Gg, Hh, I, J
33, 51 ,
(?)
a Only
experiments
sucrose
29, 31, 33, 35,
33,
Aa, Bb, C, Dd, E, F, Gg, Hh,
31,33BeSO4
in neutral
Additional refer
Typesof experimentsperformed―
Aa, Bb, C, Dd, E, F, Gg, Hh, Ii, Jj, K
Aa, Bb, C, Dd, E, F, Gg, Hh, I, Jj
Aa, Bb, C, Dd, E, F, Gg, Hh, Ii, Jj
Aa,B,C,Dd,E,F,Gg,H,I,Jj
Aa, B, C,Dd, E, F, Gg, Hh,
Aa, Bb, C, Dd, F, Gg, Hh,
BNM
JN-Ac-AAF
nucleic
42, 50, 57, 58
performed
gradients;
in our
Bb, neutral
laboratory
sucrose
are
listed:
plus
SDS;
As,
velocity
C, alkaline
centrifugation
sucrose;
Dd, gel
electrophoresis; E, CsCIequilibrium density gradient centrifugation; F, density gradient
centrifugation
in CsCI plus Sarkosyl; Gg, MAK chromatography;
Hh, filtration through
nitrocellulose
filters; Ii, interaction with isolated cellular membranes, measured in CsCl
equilibrium
density gradient centrifugation
experiments
(Refs. 29—31); Jj, enhanced
binding to intact cells (33); K, electron microscopic
examination.
Capital letters indicate
experiments in which DNAwas tested; small letters indicate experiments in which RNA
was tested. In all casesthe experimentswere repeatedseveraltimes, with and without
minor changes in the experimental protocol (variations in the concentrations of the
chemicals, time and conditions of exposure, etc.).
peniments aimed at further evaluation of these quantitative
relationships are presently being carried out.
The mechanisms by which the macromolecular corn
plexes reported here are produced may vary from one
carcinogenic agent to another. Smith (51) and his collabo
matonselucidated to a large extent the chemical nature of
the bonds formed between DNA and proteins during UV
irradiation. DNA exposed to various alkylating agents un
dergoes depunination (37) with the formation of a highly
active aldehyde (Ref. 54; we are grateful to Dr. J. Miller for
calling our attention to this possibility). Polyfunctional agents
may directly link the macromolecules (18). In all these
instances strong covalent bonds are produced. Presum
ably, physical proximity of potentially reactive macromole
cules facilitates the formation of such bridges. Thus, basic
proteins that react with nucleic acids through noncovalent
electrostatic interactions (lysozyme, calf thymus histone)
are more likely to produce stable nucleoproteins in the
presence of carcinogenic chemicals than are those proteins
that do not show any electrostatic attraction toward DNA
or RNA (serum albumin). Undoubtedly, further studies are
needed to elucidate all possible and existing mechanisms.
It seems quite remarkable, nevertheless, that such dissimilar
chemicals and physical agents have 2 things in common:
the ability to mutate and to render cells malignant on the one
hand and the ability to produce macromolecular complexes
on the other.
3812
Although all of the experiments described in this report
were carried out in the test tube with purified proteins and
nucleic acids, some observations published by others (17)
and our own unpublished data indicate that at least several
classes, if indeed not all, of the macromoleculam complexes
that we have seen produced in vitro are also induced in
living cells exposed to carcinogenic and mutagenic chemi
cals. Thus, it seems likely that among various chromosomal
lesions caused by carcinogens in vivo, the formation of
intermolecular bridges may be one of the more important
events, although not necessarily the most frequent event.
We speculate that the paucity of observations on these
lesions may be due to the very nature of the commonly
applied extraction procedures, which selectively discrimi
nate against and ultimately exclude the altered macromol
ecules from further analysis (Refs. 17 and 50 and our
unpublished observations).
The idea that intermolecular cross-links play a role in
chemical carcinogenesis is not a new one (for review see
Ref. 18). It is difficult, of course, to be certain beyond any
doubt to what degree such cross-links are involved in the
induction and maintenance of the malignant state since at
present a step-by-step analysis of all the biochemical events
leading to malignant transformation of a cell is clearly
impossible. In our judgement, however, the strong chemi
cal bonds formed between chromosomal macromolecules
are not likely to be easily removed by the cell through the
CANCER RESEARCHVOL. 37
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Macromolecu/ar Complexes and Carcino genesis
known repair mechanisms, primarily due to the great vania
bility and complexity of such lesions. For example, the
number of possible combinations between various chro
mosomal nucleic acids and proteins is truly astronomical.
The DNA-DNi@, DNA-RNA, or DNA-protein adducts may
prevent normal chromosomal transcription , replication,
and separation by “derailing―
the enzymes that use DNA as
a template, by creating permanent “
repressor―molecules
from normally noncovalently attached chromosomal pro
teins and, eventually, by preventing disjunction of the
incompletely divided and cross-linked daughter chromo
somes. In this scenario the final injury to the chromosome
previously exposed to a carcinogen may even come at the
time of the next cellular division, with mitotic apparatus
doing the final damage by pulling and breaking the chro
mosome. In fact, the existence of chromosomal aberrations
in tumor cells and in tissues exposed to carcinogenic
agents has been recognized for a long time (5, 12) and,
since the work of Boveni (see Ref. 56), such aberrations
were repeatedlysuggested as the cause of malignant trans
formation. The chemical reactions described in this report
supply a plausible molecular mechanism through which
this sequence of events may be accomplished.
ACKNOWLEDGMENTS
We thank Donna M. Dregger, James M. Unger, Cynthia T. Komro, Terry
G. Lichtenwald,andMichaelNunleyfor technicalassistance.
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CANCERRESEARCHVOL. 37
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Macromolecular Complexes Produced by Chemical
Carcinogens and Ultraviolet Radiation
Nancy R. Morin, Paul E. Zeldin, Z. O. Kubinski, et al.
Cancer Res 1977;37:3802-3814.
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