(CANCER RESEARCH 31, 1080-1086,
August 1971]
Modification of the Effect of Vitamin A Acid on the Skin
Tumor Keratoacanthoma by Applications of Actinomycin 1) '
Lawrence Prutkin
Department of Anatomy, New York University Medical Center, New York, New York 10016
SUMMARY
Actinomycin D was topically applied in 1 or 2 doses before
or after varying doses of vitamin A acid were topically applied
to the skin tumor, keratoacanthoma. Vitamin A acid has been
previously shown to cause mucous metaplasia in this tumor.
When actinomycin D was applied prior to vitamin A acid,
mucus was not observed at the macroscopic level. At the fine
structure level, no mucigen droplets were present. Glycogen
was seen in the keratinocytes and macrophages were present in
the dermis. Within the tumor keratinocytes, the Golgi and
rough surfaced endoplasmic reticulum were scanty. When
actinomycin D was applied after vitamin A acid applications,
mucus was macroscopically observed. Mucigen droplets were
identified at the electron microscopy level. In addition,
cross-sections of ribosome crystals appeared in numerous
keratinocytes of the tumor tissue. They were not observed in
normal, untreated epithelium.
INTRODUCTION
Previous
investigations
on
the
experimental
keratoacanthoma have demonstrated that this rapidly growing,
dry, keratotic skin tumor can be markedly altered by the
topical application of vitamin A acid (9, 10, 12). Daily
applications of vitamin A acid for 5 days result in gross and
cytological changes in the epidermal product of the tumor.
The keratoacanthoma secretes a viscous mucus which, at the
ultrastructure level, is seen as mucigen droplets in the
cytoplasm of the keratinocytes. If the drug applications are
stopped, the mucus-secreting tumor reverts back to the dry,
horny state in a few days. The reapplication of the vitamin A
acid again results in mucus production within the
keratoacanthoma. Radioactive vitamin A acid topically applied
to several keratoacanthomas on a time sequence basis has
shown that the drug localizes after 48 hr over the loose
chromatin of the nuclei of the keratinocytes of the
hyperplastic stratum spinosum. The grains are still observed
over the loose chromatin at 72 and 96 hr after the initial
application of the isotope (12).
The purpose of this investigation was to apply the antibiotic
actinomycin D topically to the keratoacanthoma either prior
to or after the application of vitamin A acid to observe if the
'This study was supported in part by American Cancer Society
Institutional Grant IN-14L and in part by Hoffmann-La Roche, Nutley,
N.J.
Received February 8, 1971; accepted March 31, 1971.
1080
antibiotic could inhibit or alter the mucous metaplasia.
Actinomycin D at certain concentrations has been postulated
to affect the site of initiation of RNA transcription and thus
to inhibit synthesis of ribosomal RNA. If vitamin A acid, by
localizing over the loose chromatin, can possibly affect gene
expression, then what modifying effect would the antibiotic
have on the action of vitamin A acid and the subsequent
kinetics of mucous metaplasia?
MATERIALS
AND METHODS
Forty albino male rabbits averaging 1 kg of body weight had
the inner surface of their right ear auricles painted twice
weekly with 1% 7,12-dimethylbenzanthracene in equal parts
of lanolin and mineral oil. By biopsy, it was determined that
the hair follicles were in the anagen phase of growth prior to
their exposure to the carcinogen. After 6 weeks, all the rabbits
had keratoacanthomas on their ears with an average yield of 4
to 7 tumors per ear. These tumors are a result of hyperplasia
of the stratum spinosum of the epidermis and hair follicles (8).
The animals were then divided into several groups which
received various topical doses of actinomycin D either prior to
or after the topical applications of vitamin A acid. Each dose
of actinomycin D consisted of 60 ¿tgin 0.1 ml of acetone
delivered by pipet. Each dose of vitamin A acid consisted of
0.5 g of a 3% concentration (kindly supplied by
Hoffmann-LaRoche, Nutley, N.J.) in a lotion vehicle (lanolin
and mineral oil in equal parts). These procedures are
summarized in Table 1.
The corresponding left ears of all the rabbits served as
controls. In addition, 9 untreated rabbits also served as
controls. The ears of these rabbits received the same dose
amount and schedule of the antibiotics and vitamin A acid as
the ears with tumors on the rabbits (see Table 2). Twelve
tumors produced on 3 additional rabbits were treated only
with the antibiotic with no subsequent applications of vitamin
A acid. Six of these tumors received 2 doses of actinomycin D
2 hr apart, and biopsies were taken 24 and 48 hr later. In
addition, 6 of the remaining tumors received 1 dose of 0.1 ml
of acetone. Three tumors were biopsied 24 hr later, and 3
tumors were biopsied 48 hr later.
All the biopsies performed on all the keratoacanthomas as
well as on the control ears were minced into 1-cu mm pieces
and placed into 5% glutaraldehyde in phosphate buffer for 1.5
hr followed by fixation in 2% osmium tetroxide buffered to
pH 7.4. The tissues were then dehydrated in graded strengths
of ethanol and embedded in Epon 812. Ultrathin and 1-¿/-thick
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Actinomycin
D Applications to the Keratoacanthoma
Table 1
A protocol of the experimental materials and methods used in the study
Experi
ment
No. of
tumors
1
15
2
14
3
32
Time of topical application
of antibiotic or vitamin A acid
1 dose of 60 ng of actinomycin D
in 0.1 ml of acetone
2 doses 2 hr apart of 60 Mgof
actinomycin D in 0.1 ml of acetone
2 doses 2 hr apart of 60 Mgof
actinomycin D in 0.1 ml of acetone
36
1 dose of 0.5 g of 3% vitamin A acid
24
2 doses 24 hr apart of 0.5 g of
3% vitamin A acid
12
In 6 tumors, 2 doses of 60 Mgof
actinomycin D in 0.1 ml of acetone
2 hr apart
In 6 tumors, 1 dose of 0.1
ml of acetone
Time interval between drugs
2 hr later 1 dose of 0.5 g of 3%
vitamin A acid"
2 hr later 2 doses 2 hr apart of 0.5
g of 3% vitamin A acid
2 hr later 2 doses 2 hr apart of 0.5
g of 3% vitamin A acid; 24 hr later
1 dose of 0.5 g of 3% vitamin
A acid
2 hr later, 12/36 tumors received
1 topical dose of the antibiotic**
24 hr later, 11/36 tumors received
1 topical dose of antibiotic
48 hr later, 13/36 tumors received
1 topical dose of antibiotic
2 hr after 2nd vitamin application,
8/24 tumors received 1 topical dose
of antibiotic
24 hr after 2nd vitamin application,
8/24 tumors received 1 dose
of antibiotic
48 hr after 2nd vitamin application,
8/24 tumors received 1 dose
of antibiotic
Time of biopsy
Biopsies taken 24 hr later
7 biopsies taken 24 hr later;
7 other biopsies, 48 hr later
Biopsies taken 24, 48, and 72 hr
after last vitamin A acid
application
All 60 tumors in Experiments 4
and 5 were biopsied 24 hr after
the last application of actino
mycin D
3 tumors biopsied 24 hr later;
3 tumors biopsied 48 hr later
3 tumors biopsied 24 hr later;
3 tumors biopsied 48 hr later
0.5 g of 3% vitamin A acid in a lotion vehicle (lanolin and mineral oil in equal parts).
Actinomycin D, 60 Mgin 0.1 ml of acetone.
Table 2
A protocol of the control materials and methods used in the study
ControlearsNo.
oftreatedearsDose scheduletime
and
topicalapplication
of
of antibiotics
40 corresponding left
ears of the
experimental animals
9 untreated rabbits
Same as Experiment 1 in Table 1
4
6
15
12
18
Same as Experiment 2 in Table
Same as Experiment 3 in Table
Same as Experiment 4 in Table
Same as Experiment 5 in Table
Same as above (1 rabbit, 2 ears
per experiment)
1
1
1
1
sections were cut on a Reichert ultramicrotome, stained with
uranyl acetate followed by lead citrate, and examined in an
RCA Type EMU 2E electron microscope.
visible on the surface of the keratoacanthomas. When only
actinomycin D was applied with no subsequent vitamin A acid
applications, the tumors also remained dry. When actinomycin
D was applied after vitamin A acid applications, the tumors
appeared to be moist 48 hr after the last vitamin application,
although the exúdate was not copious.
When actinomycin D was applied either prior to or after
vitamin A acid, the action of both drugs resulted in tumors
which regressed rapidly (4 to 5 days for almost complete
remission). Untreated tumors usually regress in about 1 to 3
weeks and tumors treated with vitamin A acid only regress in
about 7 to 10 days.
Microscopic Results. Previous studies on the fine structure
of the keratinocytes of the keratoacanthoma subject to 0.5 g
of 3% vitamin A acid reveal contiguous cells with extensive
rough-surfaced endoplasmic reticulum, Golgi reticulum, and
tonofibrils. No glycogen is present. In addition, one can
observe mucigen droplets
in the cytoplasm
of the
keratinocytes
(Fig. 1). These organelles are periodic
acid-Schiff-positive as well as Mayer's mucicarmine and Alcian
RESULTS
blue positive (10).
Examination of tumor tissue subjected to 1 dose of
Gross Results. Previous investigations on the dry, horny actinomycin D followed by 1 dose of vitamin A acid and
keratoacanthoma
have shown that when this skin tumor is biopsies taken 24 hr later reveal the presence of glycogen in
subject to 3 to 5 daily topical applications of 0.5 g of 3% the cytoplasm of many of the keratinocytes (Fig. 2). Also
vitamin A acid, the tumor exudes a copious, viscous, observed are electron-opaque
phagocytic vacuoles which
foul-smelling exúdate (8, 10).
contain glycogen (Fig. 3). When actinomycin D is applied in 2
In this study, when 1 or 2 applications of actinomycin D doses 2 hr apart followed by doses of vitamin A acid 2 hr apart
were applied prior to the vitamin A acid applications, there and biopsies are taken 48 hr later (Table 1, Experiment 2), the
was no visible exúdate nor was there any residue of antibiotics
most striking finding is the altered morphological appearance
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1971
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1081
Lawrence Prut kin
of many of the keratinocytes in the stratum spinosum. These
cells appear to have an increased nucleusrcytoplasm ratio and a
concomitant increase in intercellular space with retention of
the desmosomal attachments.
The chromatin material is
slightly condensed and there is an increase of mitochondria.
Some of the keratinocytes
contain
glycogen. When
actinomycin D is given in 2 doses but vitamin A acid is
increased to 3 applications, biopsies taken 48 hr later reveal a
sharp decrease in the amount of glycogen present in the
keratinocytes. At this time and dose schedule, numerous
autophagic and residual bodies are present in macrophages
within the dermis (Fig. 4). The epidermal cells now have only
a few phagocytic vacuoles and appear to be returning to the
normal nucleusicytoplasm ratio and a negligible intercellular
space. In some of the keratinocytes, if multiple doses of
actinomycin D are used prior to multiple doses of vitamin A
acid, then, within the cisternae of the rough-surfaced
endoplasmic reticulum are located fibrils which have a
periodicity.
When multiple doses of actinomycin D are applied before
the vitamin A acid applications, there is no marked increase in
Golgi or rough-surfaced endoplasmic reticulum. In contrast,
the tumor treated with vitamin A acid only has these 2 cell
organelles in extensive amounts. The mucigen droplets present
in the tumor treated with vitamin A acid only were never
observed when actinomycin D was applied prior to vitamin A
acid.
Applying actinomycin D after vitamin A acid applications
does not prevent the formation of mucigen droplets. Although
the droplets are not as numerous as in the tumor cell treated
only with vitamin A acid, they are nevertheless observed in the
hyperplastic stratum spinosum. They possess the histochemical
properties for acid and neutral mucopolysaccharides as do the
mucigen droplets found in the keratinocyte treated with
vitamin A acid only. The Golgi and rough-surfaced
endoplasmic reticulum are present in increased amounts when
compared to normal, untreated epithelium. In addition,
actinomycin D applied after vitamin A acid results in
numerous macrophages between the epidermal tumor cells.
These macrophages appear to migrate up the epidermal strata
toward the free surface and can be found in all upper strata
(Fig. 5) (see Table 3).
When actinomycin D was applied after 2 doses of vitamin A
acid, ribosome crystals were observed in the cytoplasm of
numerous keratinocytes of the hyperplastic stratum spinosum
(Fig. 6). They were never observed in the stratum basale or in
the strata granulosum or corneum.
When actinomycin
D is applied to normal, untreated
epidermis or to the keratoacanthoma,
either in single or
multiple doses, moderate amounts of glycogen and no mucigen
droplets are present in the keratinocytes. There is some
intercellular edema.
When acetone alone is applied topically either to the
keratoacanthoma or to untreated, normal epidermis, there is a
resultant erythema macroscopically as well as a visible
hyperkeratosis (some scaling). No mucus is observed, nor is
there any apparent effect on the regression of the tumor. At
the fine structure level, there are inflammatory cells in the
dermis. However, no mucigen droplets are observed in any
keratinocyte.
DISCUSSION
Previous studies of topical applications of vitamin A acid to
dry keratoacanthomas
have demonstrated that the tumors
undergo metaplasia with the production of mucus (8—11). The
use of drugs like actinomycin topically applied is difficult to
Table 3
Results, observed at ultrastructure level, of actinomycin D and vitamin A acid applications to the keratoacanthoma
epitheliumNo
or tumor
treated with
onlyNoacetone
treated with
onlySome
vitamin A acid
dropletsSome
mucigen
dropletsNo
mucigen
dropletsNo
mucigen
StratumcorneumStratumgranulosumStratumspinosumStratumbasaleDermisUntreated
D appli
cations after vitamin
applicationScant
A acid
dropletsGlycogen
mucigen
mucigen drop
intercellularmacrophagesScant
lets,
dropletsScant
mucigen
droplets.glycogen
mucigen
incytoplasmNo
present
dropletsNumerous
mucigen
present.no
dropletsGlycogen
mucigen
mucigen drop
intercellularmacrophagesMucigen
lets,
rough-surfaced
Golgi and
endoplasmicreticulum,
nomucigen
dropletsNo
droplets,glycogen
mucigen
incytoplasmNo
present
mucigendroplets,
markedincrease
andrough-surfaced
of Golgi
endo
reticulumNo
plasmic
dropletspresent,
intercellularmacrophages,
markedamounts
andrough-surfaced
of Golgi
endo
plasmic
reticulum,ribosome
crystalsNo
dropletsSparse
mucigen
dropletsMany
mucigen
dropletsSome
mucigen
present,no
droplets,lysosomes
mucigen
containingglycogen,
rough-sur
faced
endoplasmicreticulum
containsfibrils
with periodic
ity,
andrough-surfaced
scant Golgi
endo
plasmic
reticulum.intercellular
macro
phagesNo
dropletsMany
mucigen
inflammatorycellsEpitheliuminflammatorycellsTissue
1082
D applica
tions prior to vitamin
applicationNo
A ucid
dropletsSome
mucigen
inflammatorycells
inflammatorycellsActinomycin
inflammatorycellsActinomycin
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Actinomycin D Applications to the Keratoacanthorna
stated that slight warming will cause progressive fragmentation
interpret precisely because one is unsure of the exact amount
of the isolated ribosome crystals yielding free ribosomes. In
of antibiotic reaching the individual cells of the epidermis.
With the dosage and time schedules described in this paper, this study, actinomycin D applied after vitamin A acid can
actinomycin D, when applied prior to vitamin A acid, can result in ribosome crystals only in the stratum spinosum tumor
prevent the secretion of mucus at the macroscopic level. keratinocyte. In another study (1), in cultures treated with
However, when the antibiotic is applied after vitamin A acid, variable doses of vinblastine, the antileukemic drug, large
then mucus production can still occur. It is conceivable that polyribosome helices formed.
actinomycin, when applied prior to vitamin A acid, has an
In preliminary work being carried out in this laboratory, 1
inhibitory action on transcription of a mRNA coded for dose of 500 jug of puromycin in 0.1 ml of acetone topically
translation of mucigen droplets. The inhibitory action of applied either before or after the topical application of vitamin
actinomycin D has been demonstrated by Reich et al. (13, 14) A acid resulted in no mucus production and no mucigen
as well as by others (4-6). However, when actinomycin is droplets. This complete loss of all mucigen droplets with
applied after vitamin A acid, then mucigen droplet formation
puromycin treatment may be due to the fact that puromycin
is observed. Thus, it appears that the direction of protein
inhibits protein synthesis and inhibits the incorporation of
synthesis (toward glycoprotein formation), once established
sugar and sulfate into mucopolysaccharide (7). Further work
by vitamin A, is not changed by actinomycin D. In addition,
in this area is presently being carried out.
when actinomycin D is topically applied, its effects are local
and do not affect untreated areas or any systemic organs (3).
At the fine structure level, the appearance of glycogen in REFERENCES
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endoplasmic reticulum (9). These 2 organdÃ-es play a role in
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Protein Production. Cancer Res.,28: 1021-1030, 1968.
absence of mucigen droplets seen after antibiotic treatment
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Prutkin, L., and Bogart, B. An Ultrastructural Study of the
followed by vitamin A acid. This is especially noted when
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production. These same observations can be noted in the 12. Prutkin, L., and Bogart, B. The Uptake of Labeled Vitamin A Acid
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Ribosome crystals formed by rosette-like 8-ribosome
13. Reich, E., Franklin, R. M., Shatkin, A. J., and Tatum, E. L. The
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chick embryos (2) and in lizard oocytes (1). Byers (2) has
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form crystals during slow cooling. Barbieri et al. (1) have
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1083
Lawrence Prutkin
.r"
Fig. 1. Mucigen droplets (MD) present in keratinocyte of vitamin A acid-treated keratoacanthoma. Arrows, extensive Golgi apparatus. X 16,800.
Fig. 2. Glycogen (arrows) in a selected keratinocyte subject to 1 dose of actinomycin D followed by 1 dose of vitamin A acid. X 6,475.
1084
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31
Actinomycin
D Applications
to the Keratoacanthoma
•:
•
.'--S
.
f
V
j. '•"
'•'¿v
•
3
A,
«V?
.• ' •
•;•'.
Fig. 3. Photomicrograph depicts glycogen in the cytoplasm (arrow) as well as within phagocytic vacuoles (arrows) of tumor keratinocytes after 1
dose of actinomycin D followed by 1 dose of vitamin A acid. X 15,600.
Fig. 4. Autophagic and residual bodies in the dermis after 2 doses of actinomycin D and 3 doses of vitamin A acid. Biopsies taken 48 hr later.
X 7,400.
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1971
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1085
Lawrence Pnitkin
STRATUM CORNEUM
v
Wi
1;
6
\j
»
Fig. 5. Macrophage in upper strata of tumor when actinomycin D is applied after vitamin A acid. X 10,500.
Fig. 6. Ribosome crystals in cytoplasm of keratinocyte of hyperplastic stratum spinosum subject to 2 doses of vitamin A acid followed by 1
dose of actinomycin D. X 38,500.
1086
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31
Modification of the Effect of Vitamin A Acid on the Skin Tumor
Keratoacanthoma by Applications of Actinomycin D
Lawrence Prutkin
Cancer Res 1971;31:1080-1086.
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