[CANCER RESEARCH 35, 364-369, February 1975]
Mucous Metaplasia and Gap Junctions in the Vitamin A
Acid-treated Skin Tumor,
1
Lawrence Prutkin
Department of Cell Biology, New York University Medical Center, New York, New York 10016
SUMMARY
sia. These observations
metaplasia
Desmosomes
are the usual cell junctions
yield further
information
in the vitamin A acid-treated
on mucous
keratoacanthoma.
found in normal
rabbit epithelium as well as in the untreated keratoacan
thoma. This study reports the finding of a second cell
MATERIALS
AND METHODS
Eighteen albino male rabbits (average weight, 2 kg) had
the inner surface of their left ear auricles painted twice
in equal parts
acid. The gap junction forms early in mucous metaplasia weekly with 1% 7,12-dimethylbenzanthracene
of lanolin and mineral oil. After 7 weeks, all the rabbits had
(after 2 days of application of vitamin A acid) and appears
on their ears, with a yield of 6
before the gross appearanceof mucus. The presenceof the developed keratoacanthomas
After the 7th week (14 applications), the
gap junction occurs when there is an increase in the to 8 tumors/ear.
applications of carcinogen to the ears were stopped. Sixteen
rough-surfaced
endoplasmic reticulum and Golgi cisternae
and vesicles. It is possible that the early appearance of the ofthe 18 rabbits received daily applications of3% vitamin A
acid (kindly supplied by Hoffmann-LaRoche, Inc., Nutley,
gap junction facilitates and mediates the mucous metapla
N. J.) in equal parts of lanolin and mineral oil. Each
sia. This suggestion
is strengthened
by the fact that the gap
junction, once present in the mucus-producing
tumor, is application of vitamin A acid (average, 0.10 g) was applied
to a specific tumor by means of a wooden stick spatula,
sparse when the tumor reverts back to the dry, keratotic
resulting in a thin film of the drug over the surface of the
condition upon cessation of vitamin A acid applications.
tumor. The vitamin A acid applications were continued
daily for 5 days. Two of the vitamin A acid-treated animals
were left alone for 2 weeks. Two of the 1% 7,12-dimethyl
INTRODUCTION
benzanthracene-treated
animals received no vitamin A acid.
junction, the gap junction, when epithelium, normal or
tumorous, is subjected to topical applications of vitamin A
Five additional rabbits served as controls; the inner
The keratoacanthoma is a skin tumor which can be surfaces of their ear auricles were painted only with vitamin
produced in experimental animals (5, 6, 21—24)
and humans A acid in the same amount and schedule as was applied on
(5, 26). It is characterized
by rapid growth, excessive
keratinization, and spontaneous regression. In several previ
the ears with tumors. One separate rabbit was painted only
with lanolin and mineral oil.
ous studies, vitamin A acid was topically applied to the
Biopsieswere taken daily, beginning 24 hr after the initial
tumor in rabbits (22—24). Two significant results were an application of vitamin A acid and continuing 48 hr after the
accelerated regression and the presence of a mucous meta
last application. The biopsies were sliced into 1-cu mm
plasia in the tumor. The dry, keratotic keratoacanthoma
piecesand placed in 4% glutaraldehyde in phosphate buffer
secreted copious amounts of mucus. Cytologically, both the for 1.5 hr, followed by fixation in 2% osmium tetroxide
rough-surfaced
endoplasmic reticulum and the Golgi com
buffered to pH 7.4. The tissues were dehydrated in graded
plex, usually sparse in normal epithelium and in the strengths of ethanol and embedded in Epon 812. Sections 1
untreated keratoacanthoma, weremarkedly increasedin the @imthick and ultrathin sections were cut on a Reichert
vitam A acid-treated tumor. In addition, mucigen droplets
ultramicrotome,
stained with uranyl acetate and then with
were observed in the tumor keratinocytes after vitamin A lead citrate, and examined in a Siemens Elmiskop IA
acid applications. Cessation of the vitamin applications electron microscope.
causes the mucus secreting tumor to revert back to the dry,
keratotic condition.
RESULTS
This study reports the finding of a 2nd cell junction, in
addition to the desmosome, and correlates its appearance
Gross Results. Examination of the keratoacanthomas
with the above-named cell organelles and mucous metapla
after 3 days of daily applications of vitamin A acid revealed
the keratotic tumor to have a moist appearance. There was
no visible change in the tumors prior to Day 3. On the 4th
day, the tumors produceda viscousexudateand, by the 5th
Roche, Inc., Nutley, N. J.
1Thisinvestigation
was
supported
byagrant
fromHoffmann
La
Received August
364
12, 1974; accepted October
17, 1974.
day, the exudate was copious and yellow-white. On the 7th
CANCER RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
Mucous
Metaplasia
day, the exudate was very copious and became crusty.
epithelium
Lifting the crust from the tumor revealed a very soft and a
viscous exudate (mucus). The mucus is scant in normal
very wet tumor.
The 2 vitamin
A acid-treated
animals
that
and to the keratoacanthoma
and Gap Junctions
in rabbits results in a
epithelium and very marked in the tumor. At the ultrastruc
were left alone for 2 weeks now had dry, keratotic tumors
which previously were mucus secreting. Biopsies were taken
of these tumors.
Microscopic Results. The ultrastructural morphology of
the keratoacanthoma
as well as normal rabbit skin epithe
tural level, mucigendroplets are evidentin the keratinocytes
hum
acid-Schiff stain (23, 24).
After 2 days of vitamin A acid treatment, there is an
increase in the rough-surfaced endoplasmic reticulum and
Golgi cisternae and vesicles, although mucigen droplets and
mucus are not evident until the 3rd day. These organelles
has been described
contain
tonofibrils
mic reticulum
(2 1). The normal
epidermal
and a sparse, rough-surfaced
and sparse
Golgi
complex.
cells
endoplas
Desmosomes
are
quite prominent. The keratinocytes of the tumor contain a
sparse, rough-surfaced endoplasmic reticulum, Golgi com
plex, and many tonofibrils. The only evident cell junction is
the desmosome.
Tumors treated with vitamin A acid reveal an increase in
the rough-surfaced
endoplasmic reticulum as well as an
increase
vitamin
in the Golgi complex
(Fig.
A acid applications.
However,
1) after 2 days of
at this time period,
after 3 days of vitamin
A acid application.
The droplets
become more numerous on succeeding days. Previous
investigations
with
have shown that the droplets
Mayer's
increase
mucicarmine
in number
and
as the mucous
positive
stain positive
with
metaplasia
periodic
continues.
DeLuca et a!. (2) haveshown that retinol is required for the
formation of mucus-secreting goblet cells in the rat intes
tine. Levinson and Wolf (13) have studied the effect of
vitamin A acid on glycoprotein synthesisin rabbit keratoa
canthoma. After vitamin A acid treatment, labeled fucose
and glucosamine uptake into glycoprotein was greatly
beside desmosomes,
another cell junction appears. The
membranes at this junction are closely apposed, not fused,
and are distinguished by a pattern ofparticles with periodic
ity of 80 A. The overall thickness of the cell junction is 190
to 200 A. Similar substructure and dimensions have been
since fucose is characteristic of secreted glycoproteins
generally not metabolized to other sugars (17).
described
for the gap junction (7, 8, 14—16).
Examination
of the vitamin A acid-treated
tumor
nocytes on the 3rd day depicts a further increase
presence (after 2 days of vitamin A acid applications) of a
cell junction, in addition to the desmosomê, the substructure
number
addition,
of cisternae
there
of the Golgi complex
is an increase
in the amount
stimulated.
They state that the induction of the synthesis of
a fucose-containingglycopeptide is particularly significant,
A very interesting
kerati
in the
(Fig. 2). In
of rough-sur
and is
finding in the present study is the early
and dimensions of which have been described for the gap
junction.
The gap junction
appears only after the applica
tion of vitamin A acid and is not found in normal rabbit
epithelium
nor in the untreated
keratoacanthoma.
faced endoplasmic reticulum, and mucigen droplets are now
present. The gap junction is found more frequently, al
though not associated with every keratinocyte (Fig. 3).
The structure ofthe gapjunction in this study is similar to
From the 5th to 7th day, mucigen droplets and condensing gap junctions reported in a wide variety of tissues,including
vacuoles are found in most ofthe keratinocytes
(Fig. 4). The
myocardium (16), liver (7, 8), cervical epithelium (16), tissue
extensive
Golgi
complex
contains
smooth-surfaced
mem
culture
cells,
(4, 1 1, 25), basal
cell cancer
(3), and
wool
branes arranged in lamellar arrays of cisternae. There are follicle cells (20). The gapjunction has been implicated as a
many small vesicles and large vacuoles (Fig. 5). “Gaplow-resistance pathway for cell-to-cell coupling. Electrical
junction-bounded
vesicles― (20) are frequently
found (aver
coupling between cells does not imply that currents are
age, 6 of 11 cells) (Fig. 6). In several instances, the gap normally passing from one cell to the other, but shows that
junctions
are associated
with desmosomes
(Fig. 7).
areas exist for the preferential exchange of small ions
Those tumors that secreted mucus and reverted back to between cells. Lowenstein (14) has suggested that the
the dry, keratotic condition have keratinocytes that show a junction may be instrumental in exchangingsubstancesthat
sparse number of mucigen droplets and a sparse number control cellular growth and differentiation. This hypothesis
(average, I of 19 cells) of gap junctions.
has been reported by other investigators (1 1, 16, 25).
Normal epidermal cells subjected to vitamin A acid
The present study has demonstrated
that gap junctions
produce a slight increase in the Golgi complex and rough
form early (before the gross appearance of mucus) and
surfaced endoplasmic reticulum and occasional gap junc
continue
to increase
in number
along with the Golgi
tions and mucigen droplets by the 7th day.
complex and rough-surfaced
endoplasmic
reticulum in
mucous metaplasia. It is possiblethat the early appearance
of this cell junction facilitates and mediates the mucous
DISCUSSION
metaplasia and is not a by-product of mucous metaplasia.
The biosynthesis of glycoproteins has received considera
This suggestion is strengthened by the fact that the gap
ble investigative
attention.
Present evidence indicates that
junction, once present in the mucus-producing
tumor, is
the Golgi saccule is the site where carbohydrate is synthe
sparse when the tumor reverts back to the dry, keratotic
sized and is added to protein, the latter synthesized
on the
condition upon cessation of vitamin A acid applications.
ribosomes of the rough-surfaced endoplasmic reticulum (9,
We are now studying the gap junction with tracer
10, 12). Neutra and Leblond (18, 19) reported that the Golgi
materials (lanthanum and horseradish peroxidase) and the
saccule then becomes the mucigen droplet.
freeze-fracture technique to confirm the findings obtained
The topical application of vitamin A acid to normal with thin-section electron microscopy.
FEBRUARY
1975
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
365
L.
Prutkin
REFERENCES
Glycoprotein
Synthesis
in Skin Tumors (Keratoacanthomas).
Cancer
Res., 32: 2248—2252,1972.
1. Bennett, M. V. L. Function of Electronic Junctions in Embryonic and
Adult Tissues. Federation Proc., 32: 65—75,1973.
2. DeLuca, L., Scumacher, M., and Nelson, D. P. Localization of the
Retinol-dependent
Fucose-Glycopeptide
in the Goblet Cell of the Rat
Small Intestine. J. Biol. Chem., 246: 5762—5765, 1971.
3. Flaxman, A. B. Growth in Vitro and Induction of Differentiation in
Cells of Basal Cell Cancer. Cancer Res., 32: 462—469, 1972.
4. Furshpan, E. J., and Potter, D. D. Low ResistanceJunctions between
Cells in Embryos and Tissue Culture. Current Topics Developmental
Biol., 3: 95—127, 1968.
5. Ghadially, F. N. Comparative
Morphological Study of the Keratoa
canthoma of Man and Similar Experimentally
Produced Lesions in
the Rabbit. J. Pathol. Bacteriol., 75: 441-453, 1958.
6. Ghadially, F. N. The Experimental Production of Keratoacanthomas
in the Hamster and Mouse. J. Pathol. Bacteriol., 77: 277—282, 1959.
7. Goodenough, D. A., and Revel, J. P. A. Fine Structural Analysis of
Intercellular Junctions in the Mouse Liver. J. Cell Biol., 45: 272—290,
1970.
8. Goodenough,
D. A., and Stoeckenius.
The isolation of Mouse
Hepatocyte Gap Junctions. Preliminary Chemical Characterization
and X-ray Diffraction. J. Cell Biol., 54: 646—656, 1972.
9. Jamieson,
J. D., and Palade, G. E. Intracellular
Transport
of
Secretory Proteins in the Pancreatic Exocrine Cell. I. Role of the
Peripheral Elements of the Golgi Complex. J. Cell Biol., 34: 577—596,
1967a.
14. Lowenstein,
W. R. Cellular Communication
through Membrane
Junctions. Arch. Intern. Med., 129: 299—305, 1972.
15. McNutt,
N. S., Hershberg,
R., and Weinstein,
R. S. Further
Observations on the Occurrence of Nexuses in Benign and Malignant
Human Cervical Epithelium. J. Cell Biol., 51: 805—825,1971.
16. McNutt,
N. S., and Weinstein,
R. S. The Ultrastructure
ofthe Nexus.
A Correlated Thin-Section and Freeze-Cleave Study. J. Cell Biol., 47:
666—687, 1970.
17. Melchers, F. Biosynthesis of the Carbohydrate
Portion of immuno
globin. Biochem. J., 125: 241—247, 1971.
18. Neutra, M., and Leblond, C. P. Synthesis of the Carbohydrate
of
Mucus in the Golgi Complex as Shown by Electron Microscope
Radioautoradiography
of the Goblet Cells from Rats Injected with
Gucose-H3. J. Cell Biol., 30: 119—136,1966.
19. Neutra,
M., and Leblond, C. P. Radioautographic
Comparison
of the
Uptake of Galactose-H3 in the Golgi Region of Various Cells
Secreting Glycoproteins
or Mucopolysaccharides.
J. Cell Biol., 30:
137—150,
1966.
20. Orwin, D. F. G., Thomson,
R. W., and Flower, N. E. Plasma
Membrane Differentiations of Keratinizing Cells ofthe Wool Follicle.
I. Gap Junctions. J. Ultrastruct. Res., 45: 14—15,
1973.
21. Prutkin, L. An Ultrastructure
Study ofthe Experimental Keratoacan
thoma. J. invest. Dermatol., 48: 326—336, 1967.
22. Prutkin, L. The Effect of Vitamin A Acid on Hyperkeratinization
and
the Keratoacanthoma.
J. Invest. Dermatol., 49: 165—172, 1967.
23. Prutkin, L. The Effect of Vitamin A Acid on Tumorigenesis
and
Protein Production. Cancer Res., 28: 1021—1030, 1968.
10.Jamieson,
J. D.,andPalade,
G. E.Condensing
Vacuole
Conversion
24. Prutkin, L. Antitumor Activity of Vitamin A Acid and Fluorouracil
and Zymogen Granule Discharge in Pancreatic
Exocrine Cells:
Used in Combination on the Skin Tumor, Keratoacanthoma.
Cancer
Metabolic Studies. J. Cell Biol., 48: 503—522, 1971.
Res., 33: 128—133,1973.
11. Johnson, R. G., and Sheridan, J. D. Junctions between Cancer Cells in
Culture: Ultrastructure and Permeability. Science, 174: 717—719, 25. Revel, J. P., Yee, A. G., and Hudspeth, A. J. Gap Junctions between
1971.
Electronically Coupled Cells in Tissue Culture and Brown Fat. Proc.
NatI. Acad. Sci. U.S., 68: 2924—2927, l971.
12. Kawasaki, T., and Yamashina,
I. Metabolic Studies of Rat Liver
Plasma Membranes Using D-l-14C Glucosamine. Biochim. Biophys. 26. Takaki, Y., Masutani, M., and Kawada, A. Electron Microscopic
Acta,225: 234—238, 1971.
Study of Keratoacanthoma.
Acta Dermato-Venereol.,
51: 21—26,
13. Levinson, S. S., and Wolf, G. The Effect of Vitamin A Acid on
1971.
366
CANCER
RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
Mucous Metaplasia and Gap Junctions
,
•¿ *i
r^~+
f
L .*> •¿
Fig. 1. Micrograph depicts Golgi cisternae and vesicles (arrows) in the keratoacanthoma after 2 days of vitamin A acid applica
tions. Tissues in this and succeeding micro
graphs were stained with uranyl acetate and
lead citrate, x 40,000.
Fig. 2. Arrows point to the increase in the
number ofcisternaeof the Golgi complex after
3 days of vitamin A acid applications, x
40,000.
FEBRUARY
1975
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
367
@
@
@
@
@
,,f@
-
@S. .. .
L.
-
.:
‘
@-‘@
. S.
S
,@@S@-••@@
S
:@
-.
,
,-
S@•
-@ t'@:@
‘: @!
S
S
.
Prutkin
@
-@
‘
Fig. 3. Gap junction (arrows) in the
vitamin A acid-treated keratoacanthoma.
x 84,000.
@
Fig. 4. Mucigen droplet and condens
ing vacuole in the keratinocyte subjected to
vitamin A acid. x 32,000.
Fig. 5. The extensive Golgi complex in
mucous metaplasia
containing
smooth
surfaced cisternae,
small vesicles, and
large vacuoles. x 36,000.
W@c1'5@J?@@
@‘.._@.;--
.@-
;. .‘S .4:'
@
@
,
S.
k@,@:.@:@srZd
..a
@
,@-‘
,
;‘_ k.@.
-
@
@_:-
•-
•@
..5.
,@....
:@--@t―@
@‘r .@.
S
.
@,
-.f..@
..
S
o;@
.@,
,4@ .
.‘@‘-“
368
,
..
_@
@,
‘;
-%
@@;@@::-
1
,
.,:.@,.._..
@
---
@@@;;:
,$‘
@
@
•
1I@
S
@‘r
CANCER RESEARCH
VOL. 35
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
Al UC()USAletap/aria arid (lap J tine1io@is
Fig.
mucous
Fig.
with a
mucous
6. Gap junction-bounded
vesicle in
metaplasia. x 129.000.
7. Gap junction (GJ) is associated
desmosome (D) in several instances in
metaplasia. x 136,500.
FEBRUARY
1975
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.
3n5)
Mucous Metaplasia and Gap Junctions in the Vitamin A
Acid-treated Skin Tumor, Keratoacanthoma
Lawrence Prutkin
Cancer Res 1975;35:364-369.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/35/2/364
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1975 American Association for Cancer Research.