Interaction of Carcinogenic Hydrocarbons with Tissues
VIII. Binding of Tritium-labeled Hydrocarbons to the
Soluble Proteins of Mouse Skin*
C. W. ABELLANDCHARLESHEIDELBERGER!
(McArdle Memorial Laboratory, Medical School, University of Wisconsin, Madison, Wisconsin)
SUMMARY
Studies were carried out in which the binding of thirteen carcinogenic and noncarcinogenic hydrocarbons to the soluble proteins of mouse skin was compared. A good
quantitative correlation was found to exist between the binding of the hydrocarbons to
a particular protein fraction (protein fraction 1) obtained from mouse skin, which
migrates toward the cathode with a relative mobility of +0.23, in starch gel electrophoresis, and the carcinogenic activity of the hydrocarbon. The electrophoretic simi
larity, but not identity, between this fraction and "h2" proteins from rat liver was
established.
Studies were also carried out which demonstrated that protein fraction 1 is greatly
reduced in hydrocarbon-induced carcinomas and sarcomas. These observations sup
port the protein deletion hypothesis of carcinogenesis.
The possible relationships between these findings and carcinogenicity are discussed.
Previous investigations in this laboratory have
been concerned with the binding of polycyclic aro
matic hydrocarbons to the soluble proteins of
mouse skin (8,14—16,22,41). A direct quantitative
correlation has been obtained between the binding
of C14-labeled hydrocarbons and carcinogenicity,
* This work was supported in part by a research training
grant, CRTY-5002, and in part by a grant C-1132 from the
National Cancer Institute, National Institutes of Health, Pub
lic Health Service.
A preliminary account of this work has appeared in Proc.
Am. Assoc. Cancer Research, 3:208, 1961.
•f
American Cancer Society Professor of Oncology.
Received for publication March 38, 1962.
1The abbreviations used are as follows:
with the notable exception of the noncarcinogenic
1,2,3,4-DBA,1 which was extensively bound to the
total soluble proteins of mouse skin (16). Pre
application of unlabeled 1,2,3,4-DBA, however,
did not inhibit the binding of 1,2,5,6-DBA-9,10-CU
(16). This observation suggested that these isomers may be bound to different protein receptor
sites. Therefore, it was of considerable interest
to compare the binding of the carcinogenic and
noncarcinogenic hydrocarbons to different protein
fractions as a means of investigating specificity of
binding as related to carcinogenesis.
Separation of mouse skin proteins by cellulose
Compound
Abbreviation
10-Methyl-l,2-benzanthracene
4-Methyl-l,2-benzanthracene
S-Methyl-l,2-benzanthracene
3-Fluoro-10-methyl-l,2-benzanthracene
4-Fluoro-10-methyl-l,2-benzanthracene
1,2,5,6-Dibenzanthracene
1,2,3,4-Dibenzanthracene
9,10-Dimethyl-l,2,5,6-Dibenzanthracene
3,4-Benzpyrene
3-Methylcholanthrene
9,10-Dimethyl-l,2-benzanthracene
Anthanthrene
2-Phenylphenanthrene-3,2'-dialdehyde
10-MBA
4-MB A
3-MBA
3-F-10-MBA
4-F-10-MBA
1,2,5,6-DBA
1,2,3,4-DBA
DMDBA
3,4-BP
3-MC
DMBA
AA
PDA dialdehyde
Chemical
Abstract
Nomenclature
7-Methylbenz[a]anthracene
6-Methylbenz[a]anthracene
5-Methylbenz[a]anthracene
5-Fluoro-7-methylbenz[a]anthracene
6-Fluoro-7-methylbenz[a]anthracene
Dibenz[a,/i]anthracene
Dibenz[a,c]anthracene
7,14-Dimethyldibenz[a,A]anthracene
Benzo[a]pyrene
3-Methylcholanthrene
7,12-Dimethylbenz[a]anthracene
Anthanthrene
2-Phenylphenanthrene-3,2'-dialdehyde
931
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Cancer Research
932
column electrophoresis
(25) and comparison of
the spécifieactivities obtained demonstrated
that
the carcinogenic 1,2,5,6-DBA-9,10-C14 was bound
to a much greater extent than the noncarcinogenic
1,2,3,4-DBA-9,10-C14 principally
in two protein
fractions (8). A comparison of the specific activities
of the protein fraction which migrated toward the
cathode (tubes 20-24) showed that 1,2,5,6-DBA9,10-C14 was bound to an extent approximately
fivefold greater than was 1,2,3,4-DBA-9,10-C14,
whereas comparison of the specific activities of the
protein fraction which migrated toward the anode
(tubes 35-39) demonstrated
that l,2,5,6-DBA-9,
10-C14 was bound approximately
threefold more
than was 1,2,3,4-DBA-9,10-C14.
Since excellent resolution of proteins with small
quantities of material (1-2 mg.) has been obtained
with starch gel electrophoresis, the technic devel
oped and described by Smithies (29, 33) was em
ployed, which has also been used by several inves
tigators (28-30) to study serum proteins. The ca
pacity of starch gel electrophoresis
to separate
complex protein mixtures is illustrated by the fact
that more than 30 protein components were shown
to be present in many human sera (34). More re
cently, mixtures of enzymes, hormones, and pro
teins obtained from tissue extracts have also been
successfully resolved by this technic (34).
Therefore, in the current investigations,
thir
teen tritium-labeled
polycyclic aromatic hydro
carbons were prepared by the procedure described
in the preceding paper (11), and their binding to
the soluble protein fractions of mouse skin, re
solved by starch gel electrophoresis, was studied.
MATERIALS AND METHODS
Preparation and purification of Cu-labeled com
pounds.— 1,2,5,6-DBA-9,10-C14
(specific activity,
42.8 Aic/mg) and DMDBA-9.10-C14
(specific ac
tivity, 4.06 /Lic/mg) were synthesized by Oliverio
and Heidelberger (22). Each compound was chromatographed
on Florisil in 10 per cent benzene in
petroleum ether before use.
Preparation and purification of tritium-labeled
compounds.—All tritium-labeled
polycyclic
aro
matic hydrocarbons
were prepared by the Wilzbach technic (42) in which the compounds were
exposed to high activities of tritium gas. The pro
cedure used for all compounds is fully described in
the preceding paper (11).
Animals.—Female Swiss albino mice obtained
from Taconic Farms, Inc., German town, New
York, were used in all experiments.
They were
maintained in air-conditioned rooms and fed Rockland pellets and water ad libitum. These animals,
weighing approximately
20 gm. each, were chosen
Vol. 22, September
1962
at random from larger groups in all experiments.
Approximately
twenty mice per compound were
used in the experiments in which starch gel electro
phoresis was employed.
Separation of epidermis from dermis in mice.—
The backs of the mice were clipped with hair clip
pers and then shaved with a Sunbeam Shavemaster electric shaver. The animals were killed
with ether, the skins removed, and the adipose
layer and connective tissue scraped from the epidermis-dermis
layer either at room temperature
or after freezing in liquid air (35, 40). According
to a modification of the method of Pinkus (24), the
skins were placed in a 0.5 N NaBr solution at ap
proximately 5°C. for 20 minutes. After removal
from this solution, the epidermis was readily sepa
rated from the dermis with a scalpel. Histological
sections, demonstrating
the separation obtained,
were generously prepared by Dr. Henrik A. Hart
mann, and the photographs of sections of mouse
skin, epidermis, and dermis are shown in Figures
1-3. The tissues were stained with a hematoxylin
and eosin stain.
Procedure for extraction of protein from mouse
skin.—The backs of a given number of mice were
clipped with hair clippers, followed 24 hours later
by the topical application of 0.1 mg. hydrocarbon
(1 mg/ml) in benzene per mouse to the clipped
area, which was approximately 5 sq. cm. The mice
were killed with ether at the appropriate time, the
treated area washed with benzene (except in exper
iments at zero-time), and the skins were removed,
placed on corks, and frozen in liquid air. The adi
pose tissue and blood vessels were removed from
the epidermis-dermis
layer by scraping as de
scribed by Wiest and Heidelberger (40). All sub
sequent manipulations
of the tissue were carried
out in a cold room maintained
at 0-5°C. The
epidermis-dermis
layer was then minced with scis
sors and homogenized with isotonic KC1 in an allglass Potter-Elvehjem
homogenizer (35). A mix
ture of 3 parts of extracting solution to 1 part of
skin by volume was used, and only ten passes of
the pestle were made in each homogenization
in
order to keep zero-time binding at a minimum
(35). The mixture was centrifuged in an Interna
tional Clinical Centrifuge at approximately
1500
X g. An equal volume of isotonic KC1 was added
to the insoluble fraction, and additional protein
was extracted by the use of a reciprocating shaker
over a 24-hour period. This procedure was carried
out 3 additional times, and the soluble fractions
were pooled. The soluble extract was then filtered
through a pad of glass wool to remove suspended
fat and concentrated to a total volume of 1.0 ml.
by reverse dialysis with Carbowax-20M
(17). Ap-
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ABELL AND HEIDELBERGER—Carcinogenic Hydrocarbons
proximately 5 gm. of Carbowax-20M,
which had
previously been dialyzed for 3 days against six
changes of distilled water to remove impurities,
was placed in a dialysis tubing (2X9
cm.) and
introduced into a centrifuge tube containing 40
ml. of protein solution to be concentrated. A large
surface area was provided between the protein so
lution and dialysis tubing containing the Carbowax, and a protein concentration of 12-15 ml/hour
was accomplished, while the salt concentration re
mained constant. A small amount of protein pre
cipitated (8-10 per cent), which was easily re
moved by centrifugation. The unbound radioactive
hydrocarbon and other materials of small molecu
lar weight were removed by passing the protein
solution through a Sephadex G-50 column (1X8
cm.) (26) which had previously been washed thor
oughly with isotonic KC1. The eluant was again
concentrated by Carbowax dialysis, centrifuged at
1500 X g for 10 minutes, and the soluble fraction
containing 2.0 mg/0.05 ml was submitted to electrophoresis.
Fractionation of proteins.—Preliminary experi
ments with column electrophoresis
(25) have af
forded a good separation of the soluble proteins of
mouse skin (8). In the present experiments, since
excellent resolution can be obtained in starch gel
electrophoresis
with small quantities of protein
(1-2 mg.), the technic described by Smithies (29,
33) was used with a few minor modifications. The
following procedure was employed. Approximately
a 13 per cent concentration of hydrolyzed potato
starch (obtained from Connaught Medical Research
Laboratories, Toronto, Canada) in a 0.025 M boric
acid, 0.010 M sodium hydroxide buffer, pH 8.5, was
used. Upon being heated to 95°C. the suspension
gelled, and the trapped gases were removed by
evacuation at 1 mm. of mercury for approximately
1 minute. The starch gel was then added to a tray
(0.6 X 21 X 30 cm.), the cover applied, and the
gel allowed to stand at room temperature for 2-3
hours. The tray containing the gel was then placed
in a cold room maintained at 0-5°C. for 12-14
hours. The tray cover was removed, and the con
centrated protein solution was applied in an indi
vidual slot which held a volume of 0.05 ml. Since
each gel had eight slots, a series of proteins was
run on the same gel. The gel was covered with a
thin film of petrolatum, and vertical electrophore
sis was carried out at 180 v (6 v/cm), 6-7 ma.,
for 30 hours. The buffer employed for the bridge
solution was a 0.30 M boric acid, 0.05 M sodium
hydroxide buffer, pH 8.5. The gel was then re
moved from the tray, placed in a container de
signed for precise slicing, and cut in half horizon
tally.
and Tissues
Protein determination.—Solutions
from Sepha
dex columns were assayed at 280 m/u in a Beckman
Model DU spectrophotometer.
Protein solutions
prepared for electrophoresis were assayed by the
biuret technic (12) at 540 myu in either a Beckman
Model DU or DB spectrophotometer.
Protein concentrations in starch gel were deter
mined in the following way. One-half of the gel
was stained with a concentrated solution of Amido
Black-lOB dye in methanol :water :acetic acid (5:
5:1). The dye that was not bound to protein was
readily removed by washing the gel with metha
nol :water :acetic acid (5:5:1) for 8 hours. The
starch gel was then photographed
with a Panatomic-X film in the Wisconsin Medical School
Photography Laboratory. The relative optical den
sities of the resulting negative were determined in
a Welch densitometer
(23). The values were ob
tained at 3-mm. intervals and corresponded to the
same distance in the gel. Since the relative optical
densities obtained in this way are dependent upon
the conditions of the development of the film, it
was necessary in every experiment to made a sideby-side comparison in the same gel of a series of
protein solutions so that quantitatively
compara
ble results could be obtained. The resolution ob
tained with the proteins of mouse skin is shown in
Figure 4 and Chart 2.
Assay of radioactivity.—Determination
of radio
activity in the starch gel was carried out directly
without elution of the protein. The unstained half
of the gel was washed 3 times in methanol :water :
acetic acid (5:5:1) for 1 hour, 2 times in absolute
methanol for 10 hours, and 4 times in benzene
for 3 hours. Since the starch gel becomes trans
parent in benzene, the section of the gel containing
the separated proteins was cut into 3-mm. sections,
added to 10 ml. of scintillator
solution, which
contained 40 mg. of 2,5-diphenyloxazole
and 0.05
mg. of 1,4 bis-2(5-phenyloxazolyl)-benzene
in tolu
ene, and was counted directly in a Packard TriCarb
liquid scintillation spectrometer.
All tritium-labeled
hydrocarbons were weighed,
dissolved in benzene, and their specific activities
were determined with the liquid scintillation spec
trometer. The efficiency of counting was deter
mined by adding an internal standard, and the
specific activity of each hydrocarbon was expressed
in mc/mg. To determine the radioactivity
in the
total soluble protein, 2 mg. was dissolved in 0.2
ml. of hydroxide of Hyamine 10-X (obtained from
Packard Instrument Co.), added to 9.8 ml. of the
scintillator
solution, and counted in the liquid
scintillation spectrometer.
Specific method for expression of data.—As stated
in the introduction,
the noncarcinogenic
1,2,3,4-
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934
Cancer Research
DBA is bound appreciably to the soluble proteins
of mouse skin (16). Thus, it was of interest to
compare the binding of carcinogenic and noncarcinogenic hydrocarbons in all electrophoretic frac
tions to determine whether or not binding could
be demonstrated of the carcinogenic hydrocarbons
to different fractions from the noncarcinogenic hy
drocarbons. The data obtained are expressed in
the following way. The soluble proteins to which
the noncarcinogenic
and carcinogenic hydrocar
bons were bound were submitted to electrophoresis
in adjacent slots in the same starch gel. The
counts/min, corrected for the differences in specific
activity of the hydrocarbons applied, and relative
2.0'
PROTEIN
CTS/MIN
2000
1.5
1500
1.0
1000
0.5
soo
10
20
TUBE
30
40
NUMBER
CHART1.—The removal of radioactive hydrocarbon from
protein on sephadex columns.
optical densities of each 3-mm. section were de
termined. Since the photograph of the stained gel
was adjusted to the same size as the half of the gel
in which the radioactive determinations were made,
alignment of radioactivity
and relative optical
density were easily obtained. The alignment was
also checked by determination
and comparison of
the mobilities of the protein and radioactive peaks.
The specific activities were then calculated, and
difference curves of mean specific activities over a
three-section
range were graphically represented
between the carcinogenic and noncarcinogenic hy
drocarbon. The protein fractions to which the car
cinogenic hydrocarbon was bound to a greater ex
tent than the noncarcinogenic
hydrocarbon
are
plotted as a positive value, and the protein frac
tions to which the noncarcinogenic
hydrocarbon
was bound to a greater extent are shown as a nega
tive value.
Vol. 22, September 1962
RESULTS
Study of the binding of tritium-labeled hydrocar
bons to the soluble proteins of mouse skin.—At the
appropriate times after application of hydrocarbon
(zero-time, 4 hours, 1 day, 2 days, 3 days), which
will be defined for each compound, the mice were
sacrificed, and the skins were processed as de
scribed in "Materials and Methods."
Porath (26) has demonstrated
that salts and
substances of small molecular weight can be effec
tively separated from protein by the use of dextran
gel (Sephadex) columns. Consequently, this technic was used to remove tritium-labeled
hydrocar
bons which were not covalently bound to the solu
ble proteins of mouse skin. Approximately
2 mg.
of 3-MC-H3 (specific activity, 1.78 mc/mg) was
thoroughly mixed with 20 mg. of mouse skin pro
tein in 5.0 ml. of isotonic KC1. After concentration
to a total volume of 1.0 ml., the protein solution
in which the hydrocarbon was partially dissolved
was passed through a Sephadex G-50 column
which had previously been treated as described
in "Materials and Methods." A demonstration
of
the separation of protein and hydrocarbon is repre
sented in Chart 1. The fraction of protein to the
left of the arrow was used in these studies. A recov
ery of 83 per cent of this protein, free of radioac
tive hydrocarbon, was achieved, and more than 99
per cent of the total radioactivity
added was re
moved. It was demonstrated
that all the radioac
tivity could be removed by a second filtration on
Sephadex. However, in the studies to be described
in the following section, the protein was not sub
mitted to a second Sephadex column. After con
centration with Carbowax 20-M, the proteins were
submitted to starch gel electrophoresis as described
previously. The remainder of the unbound radio
active hydrocarbon was removed from the gel dur
ing the organic wash procedure.
Comparison of the binding of 3-methylcholanthrene-H3 and 1,2,3,4-dibenzanthracene-H3 to the sol
uble proteins of mouse skin.—Four groups of twen
ty mice were treated with 0.1 mg. 3-MC-H3/0.1
ml. benzene/mouse
and four other groups with
1,2,3,4-DBA-H3. The mice were sacrificed imme
diately (zero-time), 1 day, 2 days, and 3 days fol
lowing the application of hydrocarbon. The skins
were removed and treated as previously described.
The zero-time binding of 3-MC-H3 and 1,2,3,4DBA-H3 was negligible in all protein fractions ex
cept albumin, which had approximately 8 counts/
min above background. Heidelberger and Moldenhauer (16) had previously shown that the maxi
mum binding of C14-labeled 3-MC to the proteins
of mouse skin occurred at 2 days. However, the
maximum binding of CH-labeled 1,2,3,4-DBA was
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ABELLANDHEIDELBERGER—Carcinogenic
Hydrocarbons and Tissues
shown to occur at 1 day and decreased slightly at
2 days (16). Comparable results were obtained
with the tritium-labeled compounds and are shown
in Table 1. The relative optical densities of the
proteins resolved in starch gel to which 3-MC-H3
and 1,2,3,4-DBA-H3 were bound were determined
and are shown in Chart 2. Since the optical densi
ties in the fractions were identical for both proteins
studied, only one protein concentration
curve is
given. The counts/min and specific activity curves
obtained for 3-MC-H3 and 1,2,3,4-DBA-H3 at 2
days are shown in Charts 3 and 4. Graphic analysis
of the differences in specific activity of the protein
fractions as shown in Chart 5 demonstrated that
the carcinogenic 3-MC-H3 was bound to a greater
extent than the noncarcinogenic
1,2,3,4-DBA-H3
principally in two protein fractions at 1, 2, and 3
935
Thus the amount of binding of 3-MC-H3 to protein
fraction II was equal at 1, 2, and 3 days. This
experiment was repeated, and similar difference
curves were obtained.
Comparison of the binding of 3,4-benzpyrene-H3
and 1,2,3,4-dibenzanthracene-H3
to the soluble pro
teins of mouse skin.—The procedure described in
the preceding section was used in all the experi
ments concerned with the binding of tritiumlabeled hydrocarbons
to the soluble proteins of
mouse skin.
i.o
0.8
d
o
TABLE 1
SPECIFICACTIVITIESOFTHETOTALSOLUBLE
PROTEINSOFMOUSESKIN
X10"»/MO
ACTIVITT
IN RUÓLES
0.6'
0.4
20
010
40
PBOTEINZerotime0006004hours310391653822531day180255197106202160701006560702021202days2808025511022817035909582851633days160
SECTION30 NUMBER50
COMPOUND3-MC-H33,4-BP-H3DMDBA-H«DMBA-H31,2,5,6-DBA-H31,2,3,4-DBA-H310-MBA-H34-F-10-MBA-H33-F-10-MBA-H33-MBA-H'4-MBA-H3AA-H3PDA
CHART2.—Therelative optical densities of the soluble pro
teins of mouse skin resolved in starch gel. The soluble proteins
of mouse skin were submitted to starch gel electrophoresis in a
NaOH-borate buffer solution, pH 8.5, 180 v, 6-7 Ma., for 30
hours. The same conditions were used in all experiments. The
arrow in Charts 2-14 represents the origin of the electrophore
sis. Protein fractions I and II are indicated.
300
250'
Dialdehyde-H3SPECIFIC
200
days. The extent of binding to a protein fraction
which migrated toward the cathode with a relative
mobility of +0.23 (protein fraction I) was approxi
mately equal to that which was observed in a frac
tion which migrated toward the anode with a rela
tive mobility of —0.65 (protein fraction II) at 1
and 3 days. A comparison of the specific activities
in protein fraction II between the carcinogenic and
noncarcinogenic
hydrocarbon
demonstrated
that
3-MC-H3 was bound approximately
to a fivefold
greater extent than 1,2,3,4-DBA-H3.
However,
comparison of specific activities at 2 days showed
that 3-MC-H3 was bound to a twenty fold greater
extent than 1,2,3,4-DBA-H3 in protein fraction I,
whereas the amount of binding to protein fraction
II was five times greater than that which was ob
served with the noncarcinogenic 1,2,3,4-DBA-H3.
150
3-MC-H0
1,2,3,4-DBA-H0
SO
20
30
50
SECTION NUMBER
CHART8.—The binding of 3-methylcholanthrene-H3 and
1,2,3,4-dibenzanthracene-H3 to the soluble proteins of mouse
skin resolved in starch gel.
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936
Cancer Research
The binding of 3,4-BP-H3 to the soluble pro
teins of mouse skin was observed at 4 hours, 1 day,
and 2 days following the application of hydrocar
bon and was compared with the binding of 1,2,3,4DBA-H3 at 1 and 2 days. Although Heidelberger
and Moldenhauer (16) studied the binding of C14labeled 3,4-BP to the soluble proteins at 1 and 2
days, recent investigations
by Bock (4) utilizing
fluorescence technics have demonstrated
that the
maximum absorption of 3,4-BP in mouse skin oc
curs at approximately
4 hours. Determination
of
400-
Vol. 22, September
1962
question was raised concerning the retention of
the radiochemical purity of 3,4-BP-H3 because of
exposure to light when the compound was applied
to the skins of mice. Consequently,
the following
experiment was carried out. The benzene washing,
obtained from the backs of the group of twenty
mice which had received 3,4-BP-H3 4 hours pre
viously, was concentrated
and applied to paper
for Chromatographie
analysis. Three of the four
Chromatographie systems described in the preced
ing paper (11) were used to determine radiochemi
cal purity. The results indicated that 3,4-BP-H3
remained chemically unchanged, since neither the
presence of compounds other than 3,4-BP which
fluoresce in ultraviolet light nor radioactivity other
than that associated with the 3,4-BP-H3 spot were
found.
o
C
300
3-MC-H3
4250
200
D
B
100
10
20
SECTION
30
NUMBER
40
50
CHART4.—The specific activities obtained from the bind
ing of 3-methylcholanthrene-H3 and 1,2,3,4-dibenzanthraceneH3 to the soluble proteins of mouse skin.
the specific activity of the total soluble proteins
indicated that the maximum binding of 3,4-BP-H3
took place at approximately
4 hours (Table 1).
A comparison of the binding of 3,4-BP-H3 and
1,2,3,4-DBA-H3 to the soluble proteins of mouse
skin by determination
of the differences in mean
specific activities was made, and the graphic anal
ysis obtained is shown in Chart 6. Since the bind
ing of 1,2,3,4-DBA-H3 to all fractions of soluble
protein was extremely low at 4 hours, the com
parison of the binding of 3,4-BP-H3 at 4 hours
was with that of 1,2,3,4-DBA-H3 at 1 day.
At 4 hours, 3,4-BP-H3 was much more exten
sively bound to protein fraction I and, to a smaller
extent, to protein fraction II, than was 1,2,3,4DBA-H3. However, at 1 and 2 days the carcino
genic hydrocarbon
was bound to approximately
the same extent in most of the protein fractions
obtained.
Since observations
by E. C. Miller (18) have
shown that 3,4-BP is readily photo-oxidized,
a
20
SECTION
30
NUMBER
CHART5.—Comparison of the binding of 3-methylcholan
threne-H3 and 1,2,3,4-dibenzanthracene-H3 to the soluble pro
teins of mouse skin.
Comparison of the binding of 9,10-dimethyl-l,2,
5,6-dibenzanthracene-H3
and 1,2,3,4-dibenzanthracene-H3 to the soluble proteins of mouse skin.—Four
groups of twenty mice, two receiving DMDBA-H3
and the other two groups, 1,2,3,4-DBA-H3, were
sacrificed 1 and 2 days following the application
of hydrocarbon.
After extraction and separation
of the soluble proteins of mouse skin, the specific
activity of each section of gel was determined, and
the difference curves obtained are shown in Chart
7. At 1 day, the amount of binding of DMDBA-H3
to all fractions was similar to that obtained with
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ABELLANDHEIDELBERGER—Carcinogenic
Hydrocarbons and Tissues
1,2,3,4-DBA-H3. The amount of binding at 2 days
of the carcinogenic hydrocarbon to protein frac
tion I was, however, approximately 10 times great
er than in the case of the noncarcinogenic hydro
carbon.
It is of interest to note that with the three most
potent carcinogens studied, 3-MC-H3, 3,4-BP-H3,
and DMDBA-H3,
graphic analysis of the differ
ences in mean specific activities over the entire
electrophoretogram
demonstrated
that the car
cinogenic hydrocarbons
were bound primarily in
two protein fractions (protein fraction I and II) at
the time of maximum binding to the total soluble
proteins, to a greater extent than was the noncar
cinogenic 1,2,3,4-DBA-H3. In addition, the amount
937
tion indicated that the time of maximum binding
was 1 day. In addition, the specific activities ob
tained in protein fraction I and II at 1 and 2 days,
as shown by the difference curves in Chart 8, were
considerably less than the values obtained in the
experiments in which 3-MC-H3, 3,4-BP-H3, and
DMDBA-H3 were employed.
Comparison of the binding of 1,2,5,6-dibenzan-
»150vsDBA-H3
lDMDBA-\
1,2,3,4\i1
100-+50-o50
!Ã-
D
1 AY2
DAYSf\f\
£' !
V'1/
l
.1
!/
^&'••
" i'^. .- J
\LJâ
V0
c40
v^
<l
10
30SECTION
20
NUMBERHJ
50
CHART7.—Comparison of the binding of 9,10-dimethyl1,2,5,6-dibenzanthracene-H3 and 1,2,3,4-dibenzanthracene-H3
to the soluble proteins of mouse skin.
-50
20
SECTION
30
NUMBER
40
CHART6.—Comparisonof the binding of 3,4-benzpyrene-H3
and 1,2,3,4-dibenzanthracene-H3 to the soluble proteins of
mouse skin.
DMBA-II3 vs
1,2,3,4-DBA-H3
>. +100-
of hydrocarbon
bound to protein fraction I was
extensive in all three experiments.
Comparison of the binding of 9,10-dimethyl-l,2benzanthracene-H3 and 1,2,3,4-dibenzanthracene-H3
to the soluble proteins of mouse skin.—After the top
ical application of DMBA-H3 and 1,2,3,4-DBA-H3
to four groups of twenty mice per group, the ani
mals were sacrificed at 1 and 2 days, and the skins
were removed and treated as previously described.
The specific activity of each section of gel was
determined, and the difference curves are shown
in Chart 8. Although Heidelberger and Moldenhauer (16) previously had demonstrated
that the
maximum binding of C14-labeled DMBA to the
soluble proteins of mouse skin took place at 2 days,
the results obtained with the tritium-labeled
hy
drocarbon and a different method of protein isola
1 DAY
2 DAYS
O
+50'
10
20
SECTION
30
NUMBER
40
CHART8.—Comparisonof the binding of 9,10-dimethyl-l,2benzanthracene-H3 and 1,2,3,4-dibenzanthracene-H3 to the
soluble proteins of mouse skin.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
938
Cancer Research
thracene-H3 and 1,22,3,4-dibenzanthracene-H3 to the
soluble proteins of mouse skin.—The binding of
1,2,5,6- and 1,2,3,4-DBA-H3 to the soluble pro
teins of mouse skin was studied at 1 and 2 days
following the topical application of hydrocarbon
to four groups of mice. Graphic analysis of the
differences in the specific activities of the protein
fractions obtained (Chart 9) showed that the car
cinogenic 1,2,5,6-DBA-H3 was bound at 2 days to
protein fraction I and II to a greater extent
than was the noncarcinogenic
1,2,3,4-DBA-H3.
Vol. 22, September
1962
compound resulting from substitution on the other
carbon atom (3-position) is not.
In view of these observations it was of interest
to compare the binding of the carcinogenic 10MBA-H3 and 4-F-10-MBA-H3
with that of the
noncarcinogenic
3-F-10-MBA-H3
to the soluble
proteins of mouse skin. Four groups of twenty
mice were given topical applications of 10-MBAH3 and 3-F-10-MBA-H3. The animals were sacri
ficed 1 and 2 days following the application of
hydrocarbon. The binding of 10-MBA-H3 and 3F-10-MBA-H3 to the total isotonic KCl-soluble
proteins at 1 and 2 days was determined as de
scribed in "Materials and Methods," and the re
sults are given in Table 1. Graphic analysis of the
differences in specific activity of the protein frac
tions obtained (Chart 10) demonstrated
that at 2
days the time of maximum binding, the carcino-
1.2, 5,6-DBA-H3 V8
1.2,3,4-DBA-H3
t DAY
3" +Õ50.
10-MBA-H3 va
3-F-10-MBA-H
1 DAY
2 DAYS
+ 100
U
£
0
10
20
SECTION
30
NUMBER
40
50
CHART9.—Comparison of the binding of 1,2,5,6-dibenzanthracene-H1 and 1,2,3,4-dibenzanthracene-H3 to the soluble
proteins of mouse skin.
The amount of binding of the carcinogenic hydro
carbon to protein fraction I was approximately
5
times greater than was that of the noncarcinogenic
hydrocarbon,
whereas the amount of binding to
protein fraction II was 3 times greater.
A comparison of the difference curve obtained
from experiments
on C14-labeled hydrocarbonbound proteins, separated by cellulose column
electrophoresis
(8), with the difference curve ob
tained from experiments utilizing tritium-labeled
hydrocarbon-bound
proteins, resolved in starch
gel, demonstrates that these different technics give
comparable results.
Comparison of the binding of 10-methyl-l ,2-benzanthracene-H3 and 3-fluoro-10-methyl-l,2-benzanthracene-H3 to the soluble proteins of mouse skin.—
In collaboration with Dr. Melvin S. Newman, the
Millers (19, 20) have shown that the compound
resulting from substitution
of a fluorine atom on
one of the carbon atoms (4-position) at the "K"
region in 10-MBA is carcinogenic, whereas the
20
SECTION
30
50
NUMBER
CHART10.—Comparison of the binding of 10-methyl-l,2benzanthracene-H* and S-fluoro-10-methyl-l,2-benzanthracene-H3 to the soluble proteins of mouse skin.
genie 10-MBA-H3 was bound in at least two pro
tein fractions (protein fractions I and II) to a
greater extent than was the noncarcinogenic 3-F10-MBA-H3. Comparison of the specific activities
of protein fraction I showed that the carcinogenic
hydrocarbon
was bound to an extent fourfold
greater than that of the noncarcinogenic
hydro
carbon. The amount of binding of 10-MBA-H3 to
protein fraction II was, however, approximately
3 times greater than that of 3-F-10-MBA-H3. Ex
amination of the difference curve at 1 day showed
that 10-MBA-H3 was bound to an extent only
slightly greater than that of 3-F-10-MBA-H3
in
most of the protein fractions.
Comparison of the binding of 4-fluoro-lO-methyl-
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
ABELLANDHEIDELBERGER—Carcinogenic
Hydrocarbons and Tissues
1,£-benzanthracene-H3and 3-fluoro-10-methyl-1,2benzanthracene-H3 fo the soluble proteins of mouse
skin.—Four groups of twenty mice given 4-F-10MBA-H3 were sacrificed at zero-time, 4 hours, 1
day, and 2 days following application of the hy
drocarbon. Control groups of mice were treated
with 3-F-10-MBA-H3 and sacrificed at comparable
times. After electrophoresis of the proteins the
specific activity of each section of gel was deter
mined, and the difference curves obtained are
shown in Chart 11. The zero-time binding of 4-F-
20
SECTION
939
inactive. The difference in carcinogenic activity
upon skin painting in mice, however, is not so
clearly defined (1, 5). Since 4-MBA-H3 and 3MBA-H3 were bound to approximately the same
extent to the total soluble proteins of mouse skin,
a comparison of the binding of these isomers to
the proteins resolved in starch gel was carried out.
After topical application of 4-MBA-H3 and 3MBA-H3 to the backs of mice, the animals were
sacrificed at 4 hours, 1 day, and 2 days. The
specific activity of each section of gel was deter
mined, and the difference curves between 4-MBAH3 and 3-MBA-H3 are shown in Chart 12. The
time of maximum binding was found to occur at
2 days (Table 1). Graphic analysis of the differ
ences in specific activities of the protein fractions
obtained at 4 hours, 1 day, and 2 days showed
that the weak carcinogen, 4-MBA-H3, was bound
30
NUMBER
10
CHABT11.—Comparisonof the binding of 4-fluoro-10-methyl-l,2-benzanthracene-H3 and 3-fluoro-10-methyl-l,2-benzanthracene-H3 to the soluble proteins of mouse skin.
10-MBA-H* to all fractions of the soluble proteins
of mouse skin was negligible. The time of maxi
mum binding of the carcinogenic hydrocarbon to
the total soluble proteins of mouse skin was at
4 hours (Table 1). Examination of the difference
curves obtained at 4 hours demonstrated that 4-F10-MBA-H3 was bound to several protein frac
tions, and, in particular, to protein fraction I to
a small but significantly greater extent than 3F-10-MBA-H3. Although the binding of 4-F-10MBA-H3 to protein fraction I determined at 1 day
was less than the value obtained at 4 hours, an
increase in specific activity of this fraction was
observed at 2 days.
Comparison of the binding of 4-methyl-l,2-benzanthracene-H3 and 3-methyl-l,e!-benzanthracene-H3
to the soluble proteins of mouse skin.—Dunning (9)
observed that 4-MBA induced sarcomas in rats
upon subcutaneous injection, whereas 3-MBA was
20
SECTION
30
NUMEER
CHART12.—Comparison of the binding of 4-methyl-l,2benzanthracene-H3 and 3-methyl-l,2-benzanthracene-H3 to the
soluble proteins of mouse skin.
in several fractions to a very small but greater
extent than 3-MBA-H3.
Comparison of the binding of anthanthrene-H3
and 1,2,3,4-dibenzanthracene-H3 to the soluble pro
teins of mouse skin.—Daudel et al. (7) have demon
strated that the noncarcinogenic anthanthrene
(AA) was bound to the total soluble proteins of
mouse skin. Their results were confirmed in the
present studies, and a maximum binding of AA-H8
was shown at 1 day (Table 1). Since AA is another
example of a noncarcinogenic hydrocarbon which
is bound to protein, a comparison of the binding
of AA-H3 and 1,2,3,4-DBA-H3 to the soluble pro
teins of mouse skin was studied 1 and 2 days fol
lowing the topical application of hydrocarbon. The
specific activities over the entire electrophoretogram were calculated for both AA-H3 and 1,2,3,4-
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
940
Cancer Research
DBA-H3, and a difference curve is shown in Chart
13. No bound radioactivity
was found in protein
fraction I or II.
Comparison of the binding of 2-phenylphenanthrene-S,£'-dialdehyde-H3 and 1,2,3,4-dibenzanlhracene-H3 to the soluble proteins of mouse skin.—
Somerville and Heidelberger (35) recently demon
strated extensive binding of PDA dialdehyde at
zero-time to the total soluble proteins of mouse
skin. Therefore, it was of interest that a compari
son of the binding of PDA dialdehyde-H3
and
1,2,3,4-DBA-H3 by means of differences in the
AA-II vs
1.2, 3,4-DBA-H3
Vol. 22, September
1962
the other group DMDBA-9.10-C14 (specific activi
ty, 4.75 X IO6 counts/min/mg).
After develop
ment of the fibrosarcomas (16-30 weeks) the mice
were sacrificed, and the tumor, liver, kidney, spleen,
and muscle adjacent to the tumor were removed
and homogenized
in isotonic KC1. The total
amount of radioactivity
in the homogenate was
determined and is shown in Table 3. The results
indicate that 20-25 per cent of the total radioac
tivity injected was bound or carried with the tu
mor, and 0.1-2.0 per cent with the other tissues.
However, precipitation
of the proteins with trichloroacetic acid followed by extensive extraction
with organic solvents as described by Somerville
and Heidelberger (35) demonstrated
that most of
the radioactivity
in the tumor homogenate was
removed. The specific activities of the soluble frac
tion obtained by centrifugation at 1500 X g ranged
from 1 to 17 counts/min/mg
protein. The specific
activities of the insoluble fraction were consider-
»100»
PDA DIALDEHYOE-H3 vs
1,2,3,4-DBA-M3
——1
20
SECTION
30
NUMBER
40
y
ft.
C
ZERO TIME
DAY
.50
I
CHART13.—Comparisonof the binding of anthanthrene-H3
and 1,2,8,4-dibenzanthracene-H3 to the soluble proteins of
mouse skin.
specific activities obtained in each fraction in
starch gel demonstrated that approximately 80 per
cent of the zero-time binding was to the albumin
fraction (Chart 14). In addition, no radioactivity
was found in protein fraction I or II, although the
remaining 20 per cent of bound radioactivity
was
contained in a protein fraction (Section 29-31)
which migrated near protein fraction II.
The results of the binding of these tritiumlabeled hydrocarbons to protein fraction I and II
are summarized in Table 2.
Binding of Cu-labeled hydrocarbons to the tissues
of tumor-bearing mice.—Two groups of four mice
were given subcutaneous injections of 0.5 mg hy
drocarbon/0.1
ml tricaprylin/mouse.
The needle
was passed through the lower left leg muscle, and
the solution was deposited between the skin and
muscle. One group received 1,2,5,6-DBA-9,10-C14
(specific activity, 11.4 X IO6 counts/min/mg)
and
20
30
SECTION NUMBER
CHART14.—Comparison of the binding of 2-phenylphenanthrene-3,2'-dialdehyde-H3 and 1,2,8,4-dibenzanthracene-H3 to
the soluble proteins of mouse skin.
ably higher than those of the soluble fraction and
varied from 1 to 186 counts/min/mg
protein (Ta
ble 3).
Starch gel electrophoresis of the soluble proteins of
mouse skin.—Approximately
2.0 mg. of isotonic
KCl-soluble proteins obtained from twenty mouse
skins as previously described were submitted to
starch gel electrophoresis under the usual condi
tions. The gel was cut and stained for protein,
and the resolution obtained is demonstrated
in
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
ABELLANDHEIDELBERGER—Carcinogenic
Hydrocarbons and Tissues
941
TABLE 2
SPECIFICACTIVITYOFPROTEINFRACTIONS
I ANDII IN STARCHGEL
SPECIFICACTIVITYm CODNTS/MIN/O.D.
COMPOUND3-MC-H33,4-BP-H3DMDBA-H»DMBA-H31,2,5,6-DBA-H310-MBA-H34-F-10-MBA-H34-MBA-H3AA-H'PDA
timeI
II0
Hrs.I
II280
DayI
DaysI
DaysI
II423
II137
17878
7438
42189
3015
1035
6810
00
6215
1612
125
1022
160
Dialdehyde-H31,2,3.4-DBA-H33-F-10-MBA-H33-MBA-H3Zero
00
025
04
206
85
76
71
II143
11549
48278
54120
10180
145160
14775
12280
8620
1024
00
14571
12520
305
55
5Ì
20
83
Protein fraction I: The protein fraction of relative mobility + 0.23 in starch gel.
Protein fraction II: The protein fraction of relative mobility —0.77 in starch gel.
Figure 4. The protein mixture was resolved into
twelve bands. Five of these bands migrated to
ward the cathode and seven toward the anode. The
relative mobilities (Table 4) for mouse skin protein
and mouse serum (Fig. 5) were calculated, based
on the migration of the albumin peak which was
assigned a value of —1.00.
To determine the ionic character of the proteins
which migrated toward the cathode, a neutral in
dicator such as 2-deoxyribose and/or formalde
hyde was introduced into a slot in the starch gel
and subjected to the conditions of electrophoresis
as previously described. Addition of an ammonia
cal solution of silver nitrate resulted in the deposi
tion of silver in the gel. The results demonstrated
that 2-deoxyribose had a relative mobility of
+0.29. Therefore, the movement of the skin pro
teins toward the cathode was affected by electroosmotic back-flow as well as the presence of a
charge on the protein molecules. The isoelectric
point of these proteins has not been determined.
The backs of a group of 50 mice were shaved,
the skins removed, and the epidermis separated
from the dermis as described in "Materials and
Methods." The proteins of the epidermis and der
mis were repeatedly extracted with isotonic KC1,
and the soluble proteins concentrated with Carbowax-20M. Approximately 1.5 mg. of the soluble
proteins from epidermis and 1.7 mg. from dermis
were applied to starch gel. After the electropho
resis had been carried out for 30 hours, the gel
was removed, cut, and stained for protein. The
TABLE3
SPECIFIC
ACTIVITY
OFHYDROCARBON-INDUCED
SARCOMAS
ANDOTHERTISSUES
IN MICE
Tis
sueTumor
counts/
act.
min in homog.
protein
tissue
in per cent of
(counts/
(gm.)6.401.642.712.025.102.740.550.660.651.950.300.430.750.460.37Total
totalinjected18.525.80.20.10.10.10.1010.20.824.40.100.88Spec.
min/mg)S
1*2t3*Liver
of
2I
186S
17I
58S
1I
112111211018127
123Kidney
123Muscle
123Spleen
123Wetweight
S: Soluble fraction obtained by centrifugation at 1500 X g.
I: Insoluble fraction obtained by centrifugation at 1500 X g.
* Sarcoma induced with DMDBA (S.A. = 4.75 X 10«
counts/min/mg).
t Sarcoma induced with DBA (S.A. = 11.4 X 10«
counts/
min/mg).
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
942
Cancer Research
resolution obtained with both epidermal (Slot 2)
and dermal (Slot 1) proteins is shown in Figure 6.
Although the proteins have probably been dena
tured to some extent because of the NaBr treat
ment, the results show that both epidermis and
dermis contained proteins which migrated toward
the cathode in the region where hydrocarbonbound proteins migrate.
Starch gel electrophoresis of rat liver proteins.—
Approximately
1.0 mg. of "h" protein, obtained
through the generosity of Dr. S. Sorof, prepared
by cellulose column electrophoresis
of the super
natant fraction of rat liver, was submitted
to
starch gel electrophoresis (38) under the usual con
ditions. The gel was cut and stained for protein,
and the resolution obtained is shown in Figure 7.
Vol. 22, September
as shown in Figure 8, demonstrated
1962
that "hi" pro
tein can be further resolved into at least three
fractions. The relative mobilities are given in Table
4, and comparison of these values with the value
obtained for protein fraction I shows that the mo
bilities are similar, but not identical.
Starch gel electrophoresis of the soluble proteins
from squamous-cell carcinomas in mice.—One drop
(40 drops/ml) of a 0.3 per cent solution of 3-MC
in benzene was applied topically to the shaved
area of the back of a group of 30 mice. The hydro
carbon was applied 2 times/week for the duration
of the experiment. After development of the squa
mous-cell carcinomas (25-40 weeks) the animals
were sacrificed, the carcinomas removed and ho
mogenized in isotonic KC1. The soluble fraction
TABLE 4
RELATIVEMOBILITIESOFPROTEINFRACTIONS
OBTAINEDIN STARCHGEL
Mouse
m—1.05—
-TM
protein+0.33+0.37+0.40Carcinoma—1.10—1.00—0.65—0.59—0.53—0.48—0.4
protein—0.097+0.13+0.24+0.29+0.37+0.43"hi"
skin—1.10—1.00—0.77—0.68—0.51—0.36—0.21+0.095+0.23+0.32+0.46+0.59Epidermis
and
dermis*—1.08—1.00—0.77—0.65—0.51—0.45—0.30—0.17+0.067+0.10+0.19+0.27+0.35(epid.
1.00—0.66—0.37—0.48—0.43—0.89—0.26—0.21—0.11+0.080Mouse
1.00—0.86—0.74—0.71—0.58—0.52—0.47â€
35Sarcoma—1.06—
only)"h"
* Soluble proteins from both epidermis and dermis were extracted which exhibited similar relative mo
bilities (but dissimilar optical densities) in starch gel.
Of the protein bands resolved, five migrated to
ward the cathode and only one toward the anode.
The relative mobilities were calculated and are
shown in Table 4. Since Sorof (36, 37) has shown
that 80-90 per cent of the azo dye bound to the
soluble proteins of rat liver is contained in the
"h" fraction, comparison of the patterns obtained
in starch gel was made between the "h" proteins
and soluble skin proteins. The fraction with a rela
tive mobility of +0.23 which was present in both
rat liver and mouse skin corresponds to the hydro
carbon-bound
protein fraction I. Sorof et al. (38)
have further shown that the majority of the azo
dye bound to "h" protein is contained in a subfraction, obtained by cellulose column electropho
resis, designated
"slow ha" protein. Approximate
ly 1.0 mg. of "Ii2" protein also obtained from Dr.
Sorof was submitted to starch gel electrophoresis
as previously described. The resolution obtained,
was concentrated
with Carbowax to 1.5-2.0 mg.
protein and separated by starch gel electrophore
sis. The resolution obtained is shown in Figure 9,
and a visual comparison of the pattern obtained
with either the soluble skin proteins or the soluble
proteins of epidermis demonstrated
that the pro
teins of relative mobility +0.23 were greatly re
duced in the tumor. Also, a quantitative
compari
son of the soluble proteins of mouse skin and
squamous-cell
carcinomas was made. Since the
proteins from the two tissues were resolved on dif
ferent gels, the optical densities of the albumin
fraction from skin and carcinoma were equated,
and the optical densities corresponding to all other
fractions were corrected by the same factor. The
protein fraction of relative mobility +0.23 was
reduced approximately
80 per cent in the tumor.
This value represents a minimum reduction be
cause of the high background in the gel.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
ABELLANDHEIDELBERGER—Carcinogenic
Hydrocarbons and Tissues
Starch gel electrophoresis of the soluble proteins of
3-methylcholanthrene-induced
leg sarcomas in mice.
—A group of 30 mice was given injections subcutaneously of 2.0 mg 3-MC/0.2
ml tricaprylin/
mouse. After development of the tumors (30-40
weeks), the animals were sacrificed, the sarcomas
removed and homogenized in isotonic KC1. The
soluble fraction, obtained by centrifugation
at
1500 X g for 30 minutes or at 105,000 X fiffor 2
hours was concentrated with Carbowax to 2.0 mg
protein/0.05 ml solution and applied to starch gel
electrophoresis. The resolution obtained with the
soluble proteins of sarcoma is shown in Figure 10,
and comparison with the resolution obtained with
the soluble skin proteins (Fig. 4) or the soluble
proteins of dermis (Fig. 6) demonstrated
that all
the proteins which migrate toward the cathode
were greatly reduced in the tumor. A quantitative
comparison of the relative optical densities of the
protein fractions obtained, as described in the pre
ceding section, demonstrated at least a 90 per cent
reduction of protein fraction I in the tumor. Com
parison of the soluble proteins from sarcoma and
mouse skin, which migrate toward the anode,
showed that their relative mobilities were dissimi
lar. The relative mobilities for all the protein frac
tions obtained from sarcoma were calculated and
are shown in Table 4. Proteins of relative mobili
ties —0.30, —0.47, and —0.58 were either absent
or present in much lower amounts in mouse skin,
whereas proteins of relative mobilities +0.095,
+0.23, and +0.59 were absent or greatly reduced
in the sarcoma.
Comparison of the resolution of the soluble frac
tion obtained by centrifugation
at 105,000 X g
(Slots 3 and 4) with that obtained by centrifugation
at 1500 X g (Slots 1 and 2) demonstrated
that the
two preparations were similar in protein content.
DISCUSSION
In view of the fact that Berenblum (3) has dem
onstrated that a single application of a carcino
genic hydrocarbon to the skin of mice leads to an
irreversible change, it is assumed that the initial
interactions between hydrocarbon and cell constit
uent are important in carcinogenesis. In the ex
periments described in the preceding section the
time of maximum binding of hydrocarbon to the
soluble proteins of mouse skin was observed to be
from zero-time for PDA dialdehyde to 2 days for
3-MC after application. The results of the experi
ments in which the binding of hydrocarbons
to
skin proteins was observed at time intervals after
maximum binding demonstrated
that the hydro
carbon was bound to a very small extent in all
protein fractions.
943
The importance of establishing the extent of
binding of the hydrocarbon at zero-time has been
discussed extensively by Somerville and Heidel
berger (35). Since the zero-time binding of 3-MCH3, 4-F-10-MBA-H3,
and 1,2,3,4-DBA-H3 to all
fractions of the soluble proteins of mouse skin was
negligible compared with the amount bound in
vivo, the questions raised by Hadler (13) concern
ing the validity of the relationship between hydro
carbon binding and carcinogenicity clearly do not
apply in these studies. In addition, the binding of
PDA dialdehyde to the soluble proteins of mouse
skin at zero-time was principally to the albumin
fractions. Since PDA dialdehyde is not carcino
genic, it is of interest that the extensive binding
to the soluble proteins of mouse skin was observed
in a fraction (albumin) to which the carcinogenic
hydrocarbons studied were not appreciably bound.
The lack of zero-time binding of 3-MC-H3, 4-F10-MBA-H3, and 1,2,3,4-DBA-H3 to the soluble
proteins of mouse skin also demonstrates that all
hydrocarbon not covalently bound to the protein
was removed by the experimental procedure em
ployed. Therefore, the specific activities of each
protein fraction observed at maximum binding
represent hydrocarbon
that is covalently bound.
In all the experiments involving the binding of
carcinogenic and noncarcinogenic hydrocarbons to
the soluble proteins of mouse skin the carcinogenic
hydrocarbons are bound principally in two protein
fractions obtained in starch gel electrophoresis.
The amount of binding to the protein fraction that
migrates toward the cathode (protein fraction I)
with a relative mobility of +0.23 is directly pro
portional to the carcinogenic activity of the hydro
carbon studied. For example, 3-MC-H3, a potent
carcinogen, was bound to protein fraction I to a
twenty fold greater extent than was the noncar
cinogenic 1,2,3,4-DBA-H3.
The weakly carcino
genic 4-MBA-H3 was bound to protein fraction I
to only a slightly greater extent than 3-MBA-H3.
The carcinogens studied were also extensively
bound to a protein fraction which migrated toward
the anode (protein fraction II) with a relative mo
bility of —0.77to a much greater extent than were
the noncarcinogenic hydrocarbons.
However, the
amount of binding of the hydrocarbon to protein
fraction II was approximately
the same with all
the carcinogens studied.
Appreciable binding of the noncarcinogenic hy
drocarbons 1,2,3,4-DBA-H3,
3-MBA-H3, 3-F-10MBA-H3, and AA-H3 to the total soluble proteins
of mouse skin was observed. Upon electrophoresis
of these proteins in starch gel and determination
of the specific activities of each section of gel it
was found that the noncarcinogenic hydrocarbons
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
944
Cancer Research
were bound to all protein fractions except protein
fraction I and II. Therefore, the binding to protein
of the noncarcinogenic hydrocarbons was assumed
to be related to the metabolism and detoxication
of the hydrocarbons rather than to carcinogenesis.
Examination of the difference curves obtained
with 1,2,3,4- and 1,2,5,6-DBA-9,10-C14 in which
cellulose column electrophoresis was employed,
and 1,2,3,4- and 1,2,5,6-DBA-H3 wherein starch
gel electrophoresis was utilized, indicated that the
results observed by the two technics were com
parable. Exchange of tritium from the labeled hy
drocarbons studied with body water did not occur,
since only a negligible number of counts were
found upon the examination of water from the
urine of mice receiving 3-MC-H3 and AA-H3. Con
sequently, these observations, in addition to the
fact that tritium is not exchanged with the tissue
constituents during homogenization, indicates that
possible complications involved in the use of trit
ium-labeled hydrocarbons did not occur.
Further attention was directed toward protein
fraction I, since "slow 112"protein, which has been
shown by Sorof (38) to contain the majority of the
dye that is bound to the soluble proteins of liver
in azo-dye carcinogenesis, has a similar, although
not identical, mobility in starch gel. Therefore,
their involvement in carcinogenesis and electrophoretic similarity suggests that "Ii2" protein of
rat liver and protein fraction I of mouse skin may
have a similar physiological role.
Observations by Sorof et al. (36) have indicated
that the "h" fraction is lacking in azo dye-induced
hepatomas. Additional evidence in favor of the
deletion hypothesis of carcinogenesis is based on
the finding that, in 3-MC-induced carcinomas and
sarcomas in mice, the proteins that migrate toward
the cathode are greatly reduced. These results are
in agreement with the investigations of Barry (2),
who resolved the soluble proteins obtained from
hydrocarbon-induced fibrosarcomas by free-zone
electrophoresis. Of particular interest in the pres
ent studies is the fact that the protein of relative
mobility +0.23 is lacking in the tumors. Addition
al studies with 1,2,5,6-DBA-9,10-C14 and DMBA9,10-CM-induced sarcomas indicated that the hy
drocarbon is not bound to the soluble proteins of
the tumor. These observations are in accord with
the studies of the Millers (21), who demonstrated
that azo dyes were not bound to the soluble pro
teins of hepatomas produced in rats.
The conclusions reached from these studies,
therefore, are as follows. An excellent correlation
between the binding of thirteen polycyclic aro
matic hydrocarbons to a particular protein fraction
(protein fraction I) obtained in starch gel, and
Vol. 22, September
1962
their carcinogenic activities, was demonstrated. It
was clearly shown that the extent of binding ob
served was a result of in vivo processes after a sin
gle application of hydrocarbon. Furthermore, pro
tein fraction I was not present in the induced
carcinoma and sarcoma. Although the studies de
scribed herein do not exclude the possibility that
other constituents concurrently play a primary
role in carcinogenesis, the evidence suggests that
a deletion of this protein(s) may be causal in the
carcinogenic process. These results and conclusions
are in full support of and in agreement with the
deletion hypothesis of carcinogenesis (14, 21, 27,
31).
Since it may be inferred that protein fraction
I is intimately concerned with, and perhaps causal
to, the induction of cancer, somehow, as a conse
quence of its interaction with the carcinogen, this
protein becomes deleted from the ensuing tumor.
If this is so, the question is raised as to how the
deletion of a protein could bring about the new
characteristics of tumor cells that are inherited
by genetic control in subsequent cell divisions.
At present we have no information whether pro
tein fraction I is derived from the nucleus or the
cytoplasm. If the former were the case and this
is a nucleoprotein, it is possible to imagine that it
might combine with deoxyribonucleic acid (DNA)
and exert some distorting influence thereon. Al
ternatively, the protein might act as a carrier of
the hydrocarbon or its carcinogenic derivative to
a site where it could interact directly with DNA.
This concept is supported by the observation of
Heidelberger and Davenport that 1,2,5,6-DBA is
bound to DNA (15), which might lead to some
form of a mutation and which is being further
studied in this laboratory. However, the relevance
of this finding to the process of carcinogenesis has
not yet been established.
If protein fraction I were derived from the cyto
plasm, the problem of information transfer to the
nucleus is more complex. However, it should be
recalled that all present studies (32) indicate that
DNA polymerase is found in the cytoplasm, al
though DNA is present in the nucleus; thus, these
two problems may await similar solutions. Al
though Darlington (6) has indicated that cytoplasmic inheritance may occur, for the most part the
current ideas concerned with the transfer of infor
mation express the belief that the inheritable fac
tors are controlled by DNA, which resides in the
cell nucleus. At least two mechanisms appear to
be possible for the transfer of cytoplasmic changes
to the nucleus. One of these involves the migration
of a protein from cytoplasm to nucleus, where com
bination with DNA might occur. Swanson (39)
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ABELL AND HEIDELBERGER—Carcinogenic Hydrocarbons
has indicated in an abstract that a mechanism of
this type could occur, since he demonstrated that
neutral and basic proteins, isolated from the cy
toplasm of rat tissue, were able to cross the nuclear
membrane and interact with DNA when incubated
with isolated nuclei.
A second mechanism would involve the interac
tion of the hydrocarbon-bound protein with DNA
during mitosis, since it is well known that the
nuclear membrane disappears during mitosis. It
should be recalled that cell division is not uncom
mon in the cells of the Malpighian layer of epider
mis after treatment with carcinogenic hydrocar
bons, and it is from this layer that the squamouscell carcinomas probably arise.
It is obviously of considerable interest to isolate,
characterize, and study the structure of these pro
teins to which the carcinogenic hydrocarbons are
specifically bound. Furthermore, the enzymatic or
metabolic function of these proteins must be deter
mined. Such studies are now under way in this
laboratory and may lead to the critical experi
ments, thus far lacking, that will explain in precise
terms the intimate mechanism of hydrocarbon carcinogenesis.
and Tissues
945
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FIG. 1.—Mouse skin scraped in liquid air. Stained with
H. & E. and magnified X160.
FIG. 2.—Epidermis separated from dermis of mouse skin
after treatment in 0.5 NNaBr. Stained with H. & E. and magni
fied X320.
FIG. 3.—Dermis separated from epidermis of mouse skin
after treatment in 0.5 NNaBr. Stained with H. & E. and magni
fied X320.
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Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
FIG. 4.—Separation of the soluble proteins of mouse skin
in starch gel. The soluble proteins of mouse skin were submitted
to starch gel electrophoresis in a NaOH-borate buffer solution,
pH 8.5, 180 v, 6-7 n:a., for 30 hours. The same conditions were
used in all experiments.
In all gels, the slots containing protein are numbered 1-4
beginning with the slot on the upper side of the photograph.
In the gel represented here, the skin proteins were placed in
Slots 1 and •-'.
FIG. 5.—Separation of the proteins of mouse serum in starch
gel. The serum proteins were placed in Slots 1 and 3.
FIG. 6.—Separation of the soluble proteins of epidermis and
dermis in starch gel. The proteins of epidermis were placed in
Slot a and those of dermis in Slot 1.
FIG. 7.—Separation of "h" proteins in starch gel. The "h"
proteins were placed in Slots 1 and i.
FIG. 8.—Separation of "ho" proteins in starch gel. The "ho"
proteins were placed in Slots 1 and 2.
FIG. 9.—Separation of the soluble proteins of squamous-cell
carcinomas in starch gel. The soluble proteins obtained by
eentrifugation at 1500 X g were placed in Slots 3 and 4 and
those obtained by centrifugation at 105,000 X g in Slots 1
and £.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
4 t
TÕ:t
t t
_••••_
t f t t ttt t t
t t
t
M
11 t 11 t
V*
11 t
t
t
t
t
!
tt tt
tt
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
FIG. 10.—Separation of the soluble proteins of 3-niethylcholanthrene-induced
sarcomas. The soluble proteins obtained
by centrifugation
at 1500 X g were placed in Slots 1 and -Ì,
and those obtained by centrifugation
at 105,000 X «7
in Slots 3
and 4.
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
10
t
t t
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1962 American Association for Cancer Research.
Interaction of Carcinogenic Hydrocarbons with Tissues: VIII.
Binding of Tritium-labeled Hydrocarbons to the Soluble Proteins
of Mouse Skin
C. W. Abell and Charles Heidelberger
Cancer Res 1962;22:931-946.
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