Download Newt Squamous Carcinoma Proves

[CANCER RESEARCH 52, 4858-4865, September 15, 1992]
Newt Squamous Carcinoma Proves Phylogenetic Conservation of Tumors as
Caricatures of Tissue Renewall
Nikolaos P. Zilakos, Panagiotis A. Tsonis, Katia Del Rio-Tsonis, and Ralph E. Parchment2
Division of Pharmacology and Toxicology, Hippie Cancer Research Center, 4100 South Kettering Blvd., Dayton, OH 45439-2092 [N. P. Z., R. E. P.], and
Laboratory of Molecular Biology, Department of Biology, University of Dayton, Dayton, OH 45469-2320 [P. A. T., K. D. R-TJ
ABSTRACT
Carcinogenesis (i.e., the malignant transformation of normal
stem cells or, in the case of clonal malignancy, perhaps a single
Although regeneration-competent newts like Notophthalmus virìdestem cell) in renewing tissues such as epithelia results in an
scens have been reported to be resistant to carcinogenesis, we have been
exaggeration or "caricature" of tissue renewal (5). It is a cari
able to induce transplantable epidermal squamous cell carcinomas with
10-20% incidence by implanting 20-methylcholanthrene s.c. into the
scapular region, a tissue that cannot regenerate. As soon as 1 week after
exposure to this carcinogen, malignant cells were present in the treated
skin, and after 4 weeks, macroscopic tumors of infiltrating squamous
carcinoma cells positive for Type IV collagenase and/or ras"" p21 had
dissolved areas of the epidermal basement membrane and colonized the
dermis. Analysis of Ki-67 expression revealed that these tumors grow
via a high growth fraction rather than a short cell cycle time. Morpho
logical and immunohistochemical analyses showed that these tumors
caricature the biology of the renewing epidermis: the presence of basallike cells; differentiating cells; apoptotic cells; and keratinized horn
pearls with an exaggerated or overabundant stem cell compartment as
compared to the differentiated cell compartment. Immunochemical
analyses indicated that the squamous carcinomas arose from the epi
dermis rather than the mucous glands. Thus, the principle that malig
nant tumors caricature the process of tissue renewal originally estab
lished in rodent tumors appears to be valid down the phylogenetic tree
at least to regeneration-competent amphibia. Such a broad conservation
indicates that the caricature principle also holds in human tumors.
INTRODUCTION
Carcinogenesis was originally thought to transform differen
tiated cells into immature, embryonic ones, whose rapid prolif
eration formed the tumor ( 1). The conversion of a cell of mature
phenotype to embryonic phenotype was termed dedifferentiation (2). This model did explain the relative lack of differenti
ated tissue in the tumor. However, recent data from rodent
models indicate that a stem cell model should replace the dedifferentiation model (1). Neoplasms occur only in cell popu
lations with proliferative ability ("renewable" tissues); postmitotic, terminally differentiated cell populations do not become
malignant. If any differentiated characteristics are evident in a
tumor, they will always resemble those of the tumor's tissue of
origin, since this is the basis of pathological diagnosis. Thus,
the target cell in carcinogenesis is a cell capable of tissue re
newal and determined for a particular differentiation, and the
only cell that has these characteristics is the stem cell (3). This
stem cell model was confirmed by Pierce et al. (l, 4), who
showed that differentiated cells in a tumor are postmitotic even
though they are produced by malignant stem cells; areas con
taining only differentiated tumor cells did not transplant the
tumor, whereas areas containing undifferentiated cells did, and
these tumors included differentiated cells.
Received 12/5/91; accepted 7/7/92.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Supported in part by Grant CA51325 from the National Cancer Institute; by
benefactors of the Hippie Cancer Research Center such as the Dayton Exchange
Club, the Phi Beta Psi Sorority, the Moraine Lyceum Society, and Kettering Fair
mont High School; and by the Research Institute of the University of Dayton. The
Developmental Studies Hybridoma Bank is maintained by a contract from the
NICHO (NO1-HD-2-3144).
2 To whom requests for reprints should be addressed, at Mod- l,Rm2023,ORR,
CDER, FDA, 8301 Muirkirk Road, Laurel, MD 20708.
cature because of the exaggerated production of stem and pro
genitor cells in relationship to differentiated cells, and the im
mature cells accumulate, even under conditions when normal
stem cells would be quiescent. Malignant stem cells respond
abnormally to steady-state mechanism(s) in their tissues of or
igin, and thus tissue renewal is no longer balanced to cell loss.
However, other regulatory mechanisms continue to operate.
For example, both physiological cell death by apoptosis (6) and
terminal differentiation occur in malignant epidermal tissue, as
evidenced by the presence of apoptotic cells and keratinized
horn pearls. In addition, some tissue organization is also main
tained after malignant transformation, as evidenced by the compartmentalization of proliferating and terminally differentiated
cells.
The stem cell basis for malignancy (i.e., the "caricature"
model) is an important principle of tumor biology with ramifi
cations for developing curative, nontoxic cancer therapy in hu
mans (5), but only if this principle is conserved phylogenetically. To challenge the phylogenetic conservation of this
principle, we have chosen a primitive amphibian, the newt
Notophthalmus viridescens, as a model. The results show that
malignant tumors in N. viridescens obey the caricature principle
just like rodent tumors.
MATERIALS
AND METHODS
Animals. Adult newts (amphibia Urodela), N. viridescens, of both
sexes were used in this study under protocols approved by the Institu
tional Animal Care and Use Committee at the Hippie Cancer Research
Center. The animals were purchased from Charles D. Sullivan Com
pany, Inc. (Nashville, TN) and were maintained in plastic aquaria at a
room temperature of 22-25°C. The animals were fed live Newberry
shrimp or frozen beef liver given every other day. After each feeding the
water of the aquarium was changed.
Administration of 20-Methylcholanthrene. In preparation for sur
gery, the newts were anesthetized with MS222 (Tricaine methanesulfonate). Then a 10-15-Mg crystal of 20-MCA3 was implanted under
neath the epidermis just above the scapula. All recipients of the
carcinogen were examined every week for the appearance of tumor.
When tumors reached approximately 3-5 mm in diameter, the animals
were sacrificed and the tumors were excised.
Tumor Transplantation. Animals with tumors of 3-5 mm diameter
were anesthetized by immersion in a 1 mg/ml solution of MS222 for 5
min. The surgical area was irrigated with a gentamicin-penicillin-streptomycin-fungizone solution, and then the tumor was resected with a
scalpel, placed in a sterile Petri dish containing McCoy's 5A medium
(Gibco), and minced with scalpels into the appropriately sized pieces
(usually 1-2-mm cubes) for transplantation. Tumor was transferred
with forceps into a second newt after antibiotic irrigation as described
above. After recovering from anesthesia, the tumor recipients were
placed into a 10-liter tank with water to a 1-inch depth. This water was
changed every hour for 6 h, and the newts were not fed during these 6
h to control infection rates.
3 The abbreviation used is: 20-MCA, 20-methylcholanthrene.
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NEWT SQUAMOUS
CARCINOMA
CARICATURES
Histology. MCA-affected tissues were fixed in Bouin's solution or
with 10% neutral buffered formalin and then embedded in paraffin.
Sections of 5-7 /im were cut using a rotary microtome. Paraffin sections
were stained with hematoxylin-eosin or a modified trichrome Malloryazan method (7) that accentuates the basement membrane below the
epidermis of the skin.
Immunohistochemistry. In preparation for indirect immunofluorescent studies, the tissue was embedded in optimal cutting temperature
(OCT) medium, and frozen sections were cut at 6-10 Mmin a cryostat
at -18°C. Primary antibodies were applied at 37°Cfor 60 min. The
slides were washed 3x10 min in phosphate-buffered saline and then
reacted with fluorescein isothiocyanate-conjugated goat anti-rabbit or
anti-mouse antibody. The primary antibodies used were (a) Ki-67
(AMAC, Inc., Westbrook, Maine), a mouse IgG, monoclonal antibody
that reacts with a nuclear antigen expressed in the nucleus of cycling
cells predominantly during the S phase of the cell cycle (8); (b) anti-type
I keratin polyclonal rabbit anti-serum that reacts with a peptide se
quence from the carboxyl terminus of the Xenopus K81A type I keratin
(no. 1920; a gift from Dr. T. Sergant and Dr. I. David); (e) WE3
(purchased from the Developmental Studies Hybridoma Bank, depos
ited by R. A. Tassava), a mouse IgG monoclonal antibody that rea >
with secretory epithelial cells in mucous glands of the skin (9); (d)
anti-ras" monoclonal antibody produced against amino acids 96-118
of the v-ras" sequence that reacts with both c-ras" and v-ras11proteins
(Quality Biotech, Trenton, NJ). The results of the negative controls
consisting of secondary antibody alone, which uniformly gave no immunostaining, are not shown.
In addition, S-^m sections from formalin-fixed, paraffin-embedded
specimens were incubated overnight at 4°Cwith 10 Mg/ml murine IgG2a
monoclonal antibody against Type IV collagenase (Zymed Labs, Inc.)
in 1% normal goat serum in phosphate-buffered saline, washed three
times in phosphate-buffered saline, incubated for 60 min at room tem
perature with goat anti-mouse IgG secondary antibody labeled with
horseradish peroxidase (Vector Labs), washed again three times in
buffer, and then developed with the HistoBlack detection system
(Vector Labs) and counterstained lightly with 0.1% Metanil yellow.
RESULTS
Similar to other vertebrates, the normal skin in the scapular
region of N. viridescens is composed of an epidermal layer of
keratinocytes approximately 4-6 cell layers thick, separated
from the underlying dermis by a basement membrane (Fig. I, A
and li). The epidermis contains a superficial layer called the
corneum striatum, which contains the keratinized remains of
terminally differentiated keratinocytes. Basal cells and keratinocyte stem cells are located at the bottom of the epidermis,
near the basement membrane, and cells move through the spi
nal layer into the corneum striatum as they differentiate. Mitotic figures were very infrequent in the epidermis. Fig. IB
shows a higher magnification of normal skin stained with Mallory-azan to accentuate the collagen fibrils and basement mem
brane. Note that the cells with the largest nuclearcytoplasmic
ratio are localized near the basement membrane. Also note the
presence of apoptotic cells (small arrow) in the intermediate
layers as well as in the corneum striatum. Thus the general
features of epidermal cell biology in rodents, such as basally
situated stem cells, apoptotic bodies, and production of cornified, terminally differentiated cells, are all present in newt epi
dermis.
Melanocytes with easily visible melanotic granules were con
centrated at the basement membrane, but, in contrast to mam
mals, they are found exclusively on the dermal side (Fig. 1, A
and B, large arrows). The dermis is composed of a thin layer of
sparse collagen fibrils containing infrequent leukocytes and mu
cous glands lined with secretory epithelium. As the dermis ap
proached the subcutaneous muscle layer, the collagen fibrils
TISSUE RENEWAL
become denser. Antibody against Type I keratins did not stain
any of the cell types in the dermis or underlying tissues (data
not shown).
The limbs of newts show strong regenerative capability, and
previous work has shown that chemical carcinogens or ionizing
radiation do not produce tumors in regeneration-competent
tissue, even though they produce stable mutations in the ge
nome (10-13). Spontaneous tumors do not occur in these re
gions either (13). To bypass this obstacle to tumor formation,
we implanted 20-methylcholanthrene in the subcutaneous tis
sue in the scapular region. This area shows no capacity for
regeneration (14), so we reasoned that tumor formation would
be maximized there.
Fig. 1C shows a section through scapular tissues after 2
weeks of 20-MCA exposure. The epidermis appeared normal,
with retention of the corneum striatum, a middle spinal layer,
and basal layer. However, the dermis was now almost com
pletely devoid of mucous glands, and the deeper layers had lost
the dense collagen matrix and musculature. Inflammatory cells
were common throughout the deep tissue sites and the dermis.
Fig. 1C shows one suspicious area (*), where basal cells and
dermal melanocytes appeared to be in abnormally close contact.
Fig. \D shows a higher magnification of this area. There were
a few cells in the dermis that are morphologically identical to
basal keratinocytes. These keratinocyte nests in the dermis were
found as early as 1 week after 20-MCA implantation.
To determine if these early appearing keratinocyte nests in
the dermis were composed of malignant cells, we determined
whether transplanted skin exposed to 20-MCA for only 1 week
would form tumors in the flank of untreated animals. Three of
10 of the transplants (30%) formed squamous carcinomas after
transplantation (Table 1). In contrast, untreated scapular re
gions did not form tumors after transplantation (Table 1).
These data suggested that the keratinocyte-like cells appearing
in the dermis after 1 week of exposure to 20-MCA were likely
the first manifestation of malignant transformation.
Three weeks after implanting 20-MCA, small nodules of 1-2
mm diameter were observed under the epidermis in the scapular
regions of some of the treated animals. By the end of 4 weeks,
macroscopic tumors of 3-5 mm were visible in the scapular
region (Fig. 2, A and B). The tumors appeared s.c. and caused
some mild focal ulcérationconsistent with deep invasion. No
superficial tumors or papillomas were observed. After a total of
8 weeks, no additional tumors were observed in animals that
were negative for tumor at the end of 4 weeks, and no animals
were found to have multiple tumor nodules. Autopsy of tumorbearing animals revealed no obvious métastasesto liver, lungs,
or pleural cavity. Tumor incidence scored after 4 weeks in four
sequential experiments was 18%, 10%, 18%, and 10%, respec
tively (Table 2). The animals with tumor exhibited no decrease
in activity or appetite.
These tumors were excised, and portions were examined histologically (Fig. 2, C and D). Examination revealed tumors
containing tightly associated, atypical/irregular epithelial cells
with pleiomorphic nuclear and cytoplasmic shape and active
cytoplasm, which had penetrated the basement membrane and
proliferated downward into the dermis (Fig. 4A). Horn pearl
formation by spindle cells was particularly noticeable in cen
tralized areas of tumor nests (Fig. 2, C and D). Apoptotic cells
were found throughout the tumor (Fig. 1C). Surrounding the
keratinized pearls were large cells with nuclei, showing pleio
morphic size, shape, and chromatin condensation, that resem
bled cells in the overlying epidermis, although the nuclei of the
tumor cells contained less heterochromatin, and nucleoli were
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NEWT SQUAMOUS
CARCINOMA
CARICATURES
TISSUE RENEWAL
B
Fig. 1. Histology of normal skin (I and //)
and skin after I week of exposure to 20-methylcholanthrene (C and D). A, low-power mi
croscopic appearance of normal skin in N. viridescens after staining with hematoxylin and
eosin. C, corneum strlatum; BC, basal cells;
SC, spinal cells; MG, mucous glands; CT, con
nective tissue of the dermis. Melanocytes are
also evident (arrow) on the dermal side. B,
higher-power view of normal skin after stain
ing with Mallory-azan to accentuate the collagenous matrix. CF, collagen fibers of the der
mis; BM, basement membrane; MG, secretory
mucous glands; M, muscle. Apoptotic cells
(small arrow) arc evident in the spinal layer of
the epidermis. Melanocytes (large arrow) are
evident at the dermal face of the basement
membrane. C, appearance of scapular tissues
after 2 weeks of 20-MCA exposure. The epi
dermis appears normal, but inflammatory cells
have completely replaced the mucous glands,
dense collagen matrix, and some musculature
evident in A. *, one area where basal cells and
dermal melanocytes are in abnormally close
contact. /-'. a higher magnification of the * area
in C, showing cells with basal keratinocyte
morphology abnormally located on the dermal
side of the basement membrane. Note the ab
sence of hyperplasia in the epidermis.
s**"!:
t /
* *">/•*•.
w* •¿
•¿
f
Table 1 Formation of tumors by transplanted tissues at various stages
of carcinogenesis
The tissues were surgically excised and transplanted into the dermal tissue of
a secondary recipient. After the indicated time of incubation, the transplantation
site was examined histologically to determine the tumor incidence.
Transplanted
tissueNormal
scapular skin
Skin after 1-2 weeks
exposureSquamous
of MCA
carcinomasTime
of
incubation
(weeks)g
44Tumor
>"f
(%)0/7
incidence
(0)
3/10 (30)
(squamous
carcinoma)
3/10 (30)
4/10 (40)
more apparent (Fig. 3/4). Loose, fibrous-connective tissue
served to envelop these nests of tumor cells, and occasional
spindle-shaped fibroblasts were found at the connective tissuetumor tissue boundary (Fig. 2D). The tumors contained very
•¿
little stromal component. Blood vessels were present but rare
inside the tumor. These observations showed that all of the
differentiated components of normal epidermis were found in
the tumor: basal cells; keratinizing cells (differentiation); and
apoptotic cells. The features are characteristic of squamous
carcinoma of the skin (15).
If these malignancies caricatured tissue renewal in the epi
dermis, then cellular and biochemical elements of epidermis
should be present but exaggerated. Fig. ID shows a Malloryazan stained section that dramatically illustrates the keratinized
layers of differentiated tumor cells and the keratin filaments.
This stain also accentuates the apoptotic bodies, the underlying
spindle fibroblast, and the narrow strip of loose connective
tissue surrounding the squamous tumor. In Fig. 3/4, note the
presence of multiple layers of undifferentiated cells between the
basal and corneal layers in the pearls of the tumor, in contrast
to a single basal layer in normal epidermis. So within the tumor
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NEWT SQUAMOUS
CARCINOMA
CARICATURES
TISSUE RENEWAL
Fig. 2. Squamous carcinomas in the scapu
lar regions of N. viridescens 4 weeks after im
plantation of 20-methylcholanthrene crystals
into the underlying dermis. No superficial tu
mors or papillomas were observed. I. dorsal
macroscopic view of a tumor; B, lateral mac
roscopic view of a tumor showing ulcérationat
the tumor's surface; C and D, histological sec
tions of squamous carcinomas stained with
hematoxylin and eosin or Mallory-azan. All
tumors exhibit identical histological charac
teristics which identify them as squamous car
cinomas (IS): tightly associated, atypical/
irregular epithelial cells with pleiomorphic
nuclear and cytoplasmic shape and active cy
toplasm, producing keratinized horn pearls
(HP), which are evidence of terminal differen
tiation. Melanocytes (arrow) often accompany
the infiltrating tumor. Apoptotic cells AC, are
found throughout the tumor. The tumors con
tain very little stromal component, and blood
vessels are rare. Note the exaggeration of nor
mal tissue renewal by the tumor, which indi
cates the caricature principle.
.
Table 2 W-Methylcholanîhrenecarcinogenesis in the scapular region
of Nolophlhalmus viridescens
"Location" refers to the anatomical site where the majority of tumor resided
and not to the site of origin of the tumor. 20-MCA crystals (10-15 Mg) were
implanted into the scapular region of newts (both males and females), and tumor
incidence was scored after 4 weeks. No additional tumors appeared after 4 weeks.
However, evidence of differentiation was retained, such as cells
morphologically similar to the normal cells in the corneum
striatum layer of normal newt skin.
Biochemical characteristics of the tumor reflected its carica
ture of epidermal renewal. The malignant cells, but not the
Animals
No. of
surrounding normal dermal components, stained strongly pos
Experiment
treated
tumors ("<>>
Histology
Location
itive with antibody against Xenopus Type I keratin (Fig. 3Ä).
carcinomaSquamous
1234303040685(18)3(10)7(18)7(10)Squamous
This same keratin antibody strongly stained epidermal keraticarcinomaSquamous
carcinomaSquamous
nocytes of the normal skin and the secretory epithelium of the
carcinomaSubcutaneousSubcutaneousSubcutaneousSubcutaneous
mucous glands (Fig. 3C). Furthermore, WE3 antibody specific
for secretory epithelia of the mucous glands (Fig. 3D) did not
the number of undifferentiated cells and differentiated cells was stain the squamous carcinomas (Fig. 3£).This result suggested
unbalanced. In normal skin, there was usually one basal cell per the conclusion that the squamous carcinomas arose from the
epidermis rather than the mucous glands.
corneal cell, but in the tumor usually there were six undiffer
Accumulation of malignant tissue could be due to either in
entiated cells per keratinizing cell. This overproduction of im
mature (stem?) cells is a common feature of tumors which creased growth fraction (an increase in the number of cells in
caricature the process of tissue renewal in their tissues of origin.
the cell cycle) or decreased cell cycle time (no change in the
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NEWT SQUAMOUS CARCINOMA CARICATURES TISSUE RENEWAL
Fig. 3. Squamous carcinoma of the skin car
icatures the cellular and biochemical aspects of
normal epidermis. . I. a detailed appearance of
horn pearls in tumor tissue underlying normal
epidermis, showing the increased proportion of
basal cells per corneal cell in the tumor com
pared to normal epidermis, even though features
of keratinocyte differentiation are retained. B,
antibody to Type 1 keratin from X. laevis
strongly stains the tumor cells infiltrating the
dermis, indicating that the tumors originated
from malignant transformation of epithelial
cells. The normal cells interspersed between the
tumor cells show negligible staining. AT, ab
sence of tissue. C, anti-keratin antibody, show
ing strong staining of normal keratinocytes in
the epidermis. In /'. WE3 antibody specifically
reacts only with secretory epithelial cells of the
mucous
glands
but notdoes
epidermal
keratinocytes.
In /:'. WE3
antibody
not stain
the tumor
cells, suggesting the conclusion that the squamous carcinoma derives from the transforma
tion of epidermal keratinocytes. The reactive tis
sue is a normal mucous gland surrounded by
tumor. /. antibody against Ki-67, a nuclear
marker expressed only in cycling cells, showing
strong reactivity with >90% of the tumor cells.
NT, no tissue. When combined with the low in
cidence of mitotic figures, these data prove that
these tumors caricature tissue renewal and form
tumors by increased growth fraction rather than
shortened cell cycle time. G, hematoxylin-eosin
stained section of tumor similar to that used for
Ki-67 immunochemistry in F.
number of cells in cycle but rather an increase in the number of
cells produced per unit time) or some combination of the two.
The caricature model predicts retention of normal cell cycle
time but an exaggerated proportion of cells in cycle. In confir
mation of this prediction, the mitotic index was <0.01% in both
malignant tissue and normal epidermis. Antibody against the
Ki-67 nuclear antigen of cycling cells (8), whose expression is
independent of mitotic index (16), stained nearly 90% of the
tumor cells, with the exception of the flattening cells near the
horn pearls (Fig. 3, F and G). Taken together, these data sug
gested that tumor formation was due to an exaggerated produc
tion and uncontrolled expansion of stem cells, rather than to an
increased rate of cell proliferation.
A characteristic of squamous carcinoma of the mammalian
skin is invasiveness into adjacent tissues, a property exhibited
by the newt tumors perhaps as early as 1 week after 20-MCA
exposure (Fig. ID). In fact, the majority of the tumor mass
resided in the dermal spaces and even invaded the striated mus
cle layer (Fig. 4/4). This observation suggested that the malig
nant cells had penetrated or destroyed the basement membrane,
colonized the subcutaneous space, and proliferated. In some
specimens, it was possible to find regions of discontinuity in the
epidermal basement membrane where malignant cells were in
filtrating into the dermis (Fig. 4B, arrow). Areas such as this
likely represented the initial site of invasion, and subcutaneous
tumor growth was subsequent to basement membrane dissolu
tion and local invasion. Malignant cells expressing Type IV
collagenase were found in these newt squamous carcinomas
(Fig. 4C), although only in a small subpopulation (<5%). How
ever, these few positive cells may provide a basement membrane
lesion that allows other, collagenase-negative transformed cells
to invade. Also note the apparent degradation of interstitial
college fibers as well (Fig. 4B). Immunofluorescence staining
for the rasH p21 protein showed strong reactivity in all of the
malignant cells invading the dermis (Fig. 4D).
These tumors were transplantable into dermal sites in the
flanks of secondary hosts with a 30-40% success rate (Table 1).
In contrast, normal epidermis and normal scapular tissue did
not form tumors after transplantation (Table 1). The trans
planted tumors invaded surrounding dermis en bloc and caused
compression of the normal dermis, producing a superficially
detectable lesion (Fig. 5, A and A); there was no evidence of
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NEWT SQUAMOUS CARCINOMA CARICATURES TISSUE RENEWAL
Fig. 4. Mechanism of invasion of 20-methylcholanthrene-induced squamous carcinomas
into the dermis and subdermal tissue. A, deep
tissue invasion is a commonly occurring char
acteristic of squamous carcinoma (IS). In JV.
viridescens, some tumors have invaded the
deep muscle layers under the dermis by 4-5
weeks after administration of 20-MCA. B,
Mallory-azan stained section showing a dis
continuity in the basement membrane {arrow)
and dissolution of interstitial collagen associ
ated with tumor infiltration into the dermis. C,
antibody to mammalian Type IV collagenase
stains some of the tumor cells in the dermal
space. The very dark cells are melanocytes. D,
tumor cells invading the dermis express high
levels of ras" p21 protein, which in mamma
lian tumors is associated with increased cell
motility and increased metastatic behavior
(28-37).
infiltrating single cells. These transplanted tumors continued to
express the characteristics of squamous carcinoma, including
horn pearl formation (Fig. 5Q.
DISCUSSION
Many previous studies have shown that limbs from Urodela
amphibia including N. viridescens are resistant to chemical carcinogenesis (10-13, 17, 18) and spontaneous tumorigenesis
(13), but these studies were attempting to challenge the hypoth
esis that a regeneration-competent
tissue is resistant to chemi
cal carcinogenesis, not that chemical carcinogenesis does not
occur in newts. Because we previously identified a newt with a
spontaneous melanoma in the scapular region (19), an area
without regenerative capability, we suspected that the key to
experimentally producing cancers in this species might be to
implant carcinogens in regeneration-incompetent tissue like the
scapular region (14). Our results confirmed this idea. These
squamous malignancies are distinct from spontaneously re
gressing skin tumors (20, 21). These data focus the hypothesis
of amphibian resistance to carcinogenesis upon mechanisms
specific to regeneration-competent
tissues. This conclusion is
consistent with the higher incidence of tumors found in am
phibia like Animi that undergo metamorphosis and loose their
regenerative ability (13, 14).
This is the first report of chemically induced, transplantable
squamous carcinoma of the skin in Urodela. These malignan
cies occur via rapid transformation of epidermal keratinocytes
(and not secretory epithelial cells of the mucous glands), in
contrast to the prolonged exposure time required to induce
melanomas with 20-MCA in Triturus cristatus (22). It is inter
esting that 100% of the induced tumors were squamous carci
noma, especially when a promoting agent, such as phorbol es
ter, was not coadministered. Perhaps oxygen free radicals
produced by inflammatory cells at the site of carcinogen
implantation
(Fig. 1C) were functioning as endogenous
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NEWT SQUAMOUS CARCINOMA CARICATURES TISSUE RENEWAL
IV collagenase, which degrades basement membrane collagen
(27). In addition, high levels of ras expression increase motility
in other model systems (28) and induce Type IV collagenase
(29) and possibly thereby increase the probability of migration
of squamous carcinoma cells through the basement membrane
(30-32). These changes may also explain why ras is important
in MCA carcinogenesis (33). In human systems, mutated ras,
rather than overexpressed ras, is associated with an increased
propensity to metastasize (34-37). Either the malignant cells or
the inflammatory reaction may be responsible for the destruc
tion of interstitial collagen fibrils. There are reports of degra
dation of interstitial collagens and/or induction of the required
collagenases as a result of spontaneous tumorigenesis or expo
sure to 20-MCA (20, 38, 39).
Carcinomas contain areas of differentiated tissue that indi
cate their tissues of origin, a basis of pathologic diagnosis.
Pierce et al.(l) showed that the differentiated areas are derived
from the undifferentiated cancer cells, although differentiation
is accompanied by abrogation of the malignant phenotype (1).
Undifferentiated areas could reform differentiated areas, and in
every case the malignant cells could only produce differentiated
tissue consistent with their tissue of origin (1, 3-5, 40). Tumor
formation appeared to be due to a high growth fraction, i.e.,
abnormally high or "exaggerated" expansion of a stem cell
compartment, which was not balanced with the demand for
differentiated cells. These results in tumor models led Pierce et
al. (S) to propose that malignancies caricature tissue renewal,
exaggerating stem cell expansion. Thus, carcinogenesis is the
acquisition of a malignant phenotype by a normal stem cell,
which "remembers'" its differentiation program but continues to
,m
^
-i .w _ •¿* '
Fig. S. Characterization of epidermal squamous carcinomas after transplanta
tion into secondary hosts. The tumors were transplantable into dermal sites in the
flanks of secondary hosts with a 30-40% success rate (Table 1) and grew to 4-6
mm after 3-4 weeks. A, ventral view of the macroscopic appearance of a tumor
that formed after transplantation. In B, the transplanted tumors invade surround
ing dermis en bloc and cause compression of the normal dermis, producing the
superficially detectable lesion; there was no evidence for infiltrating single cells. In
C, these transplanted tumors continue to express the characteristics of squamous
carcinoma, including apoptosis and horn pearl formation.
promoters of malignant transformation, as has been proposed
for murine models (23-26).
The squamous carcinoma cells invade deeply into the dermis,
often to the level of cartilage/bone in association with dissolu
tion of basement membrane and interstitial collagen fibers
(Fig. 4B). A subpopulation of malignant cells expresses Type
divide even when there is no need to replace differentiated
tissue.
20-MCA-induced squamous carcinomas of the skin in
N. viridescens also appear to caricature normal tissue renewal.
There is significant and rapid tissue production without fre
quent mitosis, similar to normal epidermis. In fact, the ratio of
basal cells to keratinized cells in the tumor increases dramati
cally over the nearly 1:1 ratio of normal epidermis. Further
more, >90% of the malignant cells express Ki-67, indicating
tumor growth from exaggerated tissue renewal by transformed
keratinocytes. The malignant cells continue to express an epi
dermal keratin and reform squamous pearls after transplanta
tion into secondary recipients. These data indicate that squa
mous carcinoma in newt caricatures the process of tissue
renewal. Thus, the caricature principle proposed by Pierce and
Spears (5) is phylogenetically conserved down to amphibia.
This phylogenetic conservation implies that human malignan
cies also obey this principle.
The development of nontoxic therapies for human cancers
depends upon conservation of critical biological principles
across phylogenetic lines, especially cancer as a caricature
of tissue renewal and the embryonic control of cancer. This
present study shows that newt malignancies caricature tissue
renewal and indicates that the caricature principle is conserved
phylogenetically. The next critical step is to test whether em
bryonic regulation of malignancy is also phylogenetically con
served, using the transplantable tumors described in this report.
If so, the newt may provide a valuable model system for eluci
dating the mechanisms of such embryonic regulation. In fact,
similar mechanisms may be responsible for the resistance of the
newt limb to chemical carcinogenesis, because it is not resistant
to the mutagenic activity of these carcinogens (17, 18). The
retention of embryonic control mechanisms in the limbs of
adult Urodela but not Anura would explain why the former
4864
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NEWT SQUAMOUS
CARCINOMA
CARICATURES
show increased resistance to chemical carcinogenesis and spon
taneous tumorigenesis (10, 13).
ACKNOWLEDGMENTS
The authors wish to acknowledge the expert assistance in manuscript
preparation by Ann Barhorst and technical assistance for histology by
Stephanie Redwine.
REFERENCES
1. Pierce, G. B., Shikes, R., and Fink, L. M. Cancer: A Problem of Develop
mental Biology, pp. 68-87. Englewood Cliffs, NJ: Prentice-Hall, 1978.
2. Hay, E. D., and riseli man. D. A. Origin of the blastema in regenerating limbs
of the newt Trilurus viridescens, Dev. Biol., 3: 26-59, 1961.
3. Pierce, G. B., and Johnson, L. D. Differentiation and cancer. In Vitro (Rockville), 7: 140-145, 1971.
4. Pierce, G. B., Dixon, F. J., and Verney, E. L. Teratocarcinogenic and tissue
forming potentials of the cell types comprising neoplastic embryoid bodies.
Lab. Invest., 9: 583-602, 1960.
5. Pierce, G. B., and Speers, W. C. Tumors as caricatures of the process of tissue
renewal: prospects for therapy by directing differentiation. Cancer Res., 48:
1996-2004, 1988.
6. Kerr, J. F. R., Wyllie, A. H., and Currie, A. R. Apoptosis: a basic biological
phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer,
20:239-257, 1972.
7. Delides, G. S. Practical Histológica! Techniques. Athens, GA: University of
Athens Press, 1978.
8. Bruno, S., Crissman, H. A., Bauer, K. D., and Darzynkiewicz, Z. Changes in
cell nuclei during S phase: progressive chromatin condensation and altered
expression of the proliferation-associated nuclear proteins ki 67, cyclin
(PCNA), plOS, and p34. Exp. Cell Res., 196: 99-106, 1991.
9. Tassava, R. A., Johnson-Wint, B., and Gross, J. Regenerate epithelium and
skin glands of the adult newt react to the same antibody. J. Exp. Zool., 239:
229-240, 1986.
10. Tsonis, P. A. Effects of carcinogens on regenerating and non-regenerating
limbs in amphibia. Anticancer Res., 3: 195-202, 1983.
11. Tsonis, P. A. Studies of the effects of cancer inducing and promoting chem
icals on newt regenerating and non-regenerating limbs. Arch. Biol., 96: 331336, 1985.
12. Zavanella, T., and Losa, M. Skin damage in adult amphibians after chronic
exposure to ultraviolet radiation. Photochem. Photobiol., 34:487-492,1981.
13. Tsonis, P. A., and Del Rio-Tsonis, K. Spontaneous neoplasms in amphibia.
Tumor Biol., 9: 221-224, 1988.
14. Wallace, H. Vertebrate limb regeneration. Chichester, England: John Wiley
and Sons, 1981.
15. Kwan, T. H., and Mihm, M. C., Jr. The skin. In: S. L. Robbins and R. S.
Cotran (eds.), Pathologic Basis of Disease, pp. 1426-1427. Philadelphia: W.
B. Saunders Company, 1979.
16. Ostmeier, H., and Suter, L. The Ki-67 antigen in primary human
melanomas—its relationship to mitotic rate and tumor thickness and its
stability. Arch. Dermatol. Res., 281: 173-177, 1989.
17. Tsonis, P. A., and Eguchi, G. Abnormal limb regeneration without tumor
formation in adult newts directed by carcinogens 20-methylcholanthrene and
benzo(a)pyrene. Dev. Growth Differ., 24: 183-190, 1982.
18. Zilakos, N. P. The regeneration of Trituras limbs deformed by a carcinogen.
In Vivo (Athens), 5: 381-384, 1991.
19. Zilakos, N. P., and Tsonis, P. A. A spontaneous melanoma-like tumor in the
adult newt Triturus cristatus. Tumor Biol., 12: 120-124, 1991.
20. Wirl, G. Collagenolytic activity and cancerogenesis in the skin of the newt
Triturus cristatus. Arch. Geschwulstforsch., 40: 111-115, 1972.
21. Pfeiffer, C. J., Nagai, T., Fujimura, M., and Tobe, T. Spontaneous regressive
epitheliomas in the Japanese newt, Cynops pyrrhogaster. Cancer Res., 39:
1904-1910, 1979.
TISSUE RENEWAL
22. Leone, V. G., and Zavanella, T. Some morphological and biological charac
teristics of a tumor of the newt Triturus cristatus laur. In: M. Mizell (ed.),
Biology of Amphibian Tumors, pp. 184-194. New York: Springer-Verlag,
1969.
23. Bhisey, R. A., and Sirsat, S. M. Selective promoting activity of phorbol
myristate acetate in experimental skin carcinogenesis. Br. J. Cancer, 34:
661-665, 1976.
24. Birnboim, H. C. Superoxide aniónmay trigger DNA strand breaks in human
granulocytes by acting at a membrane target. Ann. N.Y. Acad. Sci., 551:
83-94, 1988.
25. Swauger, J. E., Dolan, P. M., and Kensler, T. W. Role of free radicals in
tumor promotion and progression by benzoyl peroxide. Prog. Clin. Biol.
Res., 340D: 143-52, 1990.
26. Kensler, T. W., Egner, P. A., Taffe, B. G., and Trush, M. A. Role of free
radicals in tumor promotion and progression. Prog. Clin. Biol. Res., 298:
233-48, 1989.
27. Thorgeirsson, U. P., Turpeenniemi-Hujanen, T., and Liotta L. A. Cancer
cells, components of basement membranes, and proteolytic enzymes. Int.
Rev. Exp. Pathol., 27: 203-34, 1985.
28. Partin, A. W., Isaacs, J. T., Treiger, B., and Coffey, D. S. Early cell motility
changes associated with an increase in metastatic ability in rat prostate cancer
cells transfected with the v-Harvey-ros oncogene. Cancer Res., 48: 60506053, 1988.
29. Garbisa, S., Negro, A., Kalebic, T., Pozzatti, R., Muschel. R., Saffiotti, U.,
and Liotta, L. A. Type IV collagenolytic activity linkage with the metastatic
phenotype induced by ras transfection. Adv. Exp. Med. Biol., 233: 179-186,
1988.
30. Treiger, B., and Isaacs, J. T. Expression of a transfected v-Harvey-ros onco
gene in a Dunning rat prostate adenocarcinoma and the development of high
metastatic ability. J Urol., 140: 1580-1586, 1988.
31. Schwarz, L. C., Gingras, M. C., Goldberg, G., Greenberg, A. H., and Wright,
J. A. Loss of growth factor dependence and conversion of transforming
growth factor >'!,inhibition to stimulation in metastatic H-ras-transformed
murine fibroblasts. Cancer Res., 48: 6999-7003, 1988.
32. Waghorne, C„Kerbel, R. S., and Breitman, M. L. Metastatic potential of
SP1 mouse mammary adenocarcinoma cells is differentially induced by ac
tivated and normal forms of c-H-ros. Oncogene, /: 149-155, 1987.
33. Brown, K., Buchmann, A., and Balmain, A. Carcinogen-induced mutations in
the mouse c-Ha-ros gene provide evidence of multiple pathways for tumor
progression. Proc. Nati. Acad. Sci. USA, 87: 538-542, 1990.
34. Yoshida, K., Hamatani, K., Koide, H., Abe, Y., Ikeda, H., Tsuchiyama, H.,
Nakayama, E., and Shiku, H. Analysis of ras gene expression in stomach
cancer by anti-ras p21 monoclonal antibodies. Cancer Detect. Prev., 12:
369-376, 1988.
35. Sheng, Z. M., Barrois, M., Klijanienko, J., Micheau, C., Richard, J. M., and
Riou, G. Analysis of the c-Ha-ros-l gene for deletion, mutation, amplification
and expression in lymph node métastasesof human head and neck carcino
mas. Br. J. Cancer, 62: 398-404, 1990.
36. Deng, G. R., Liu, X. H., and Wang, J. R. Correlation of mutations of
oncogene r I lu KMat codon 12 with metastasis and survival of gastric cancer
patients. Oncogene Res., 6: 33-38, 1991.
37. Slebos, R. J. C., Kibbelaar, R. E., Dalesio, O., Kooistra, A., Stam, J., Meijer,
C. J. L. M., Wagenaar, S. S., Vanderschueren, R. G. J. R. A., van Zandwijk,
N., Mooi, W. J., Bos, J. L., and Rodenhuis, S. K-RAS oncogene activation as
a prognostic marker in adenocarcinoma of the lung. N. Engl. J. Med., 323:
561-565, 1990.
38. Seilern-Aspang, F., Mazzucco, K., and Christian, I. Der Einfluss von Methylcholanthren auf Synthese, Vernetzung und Abbau des Kollagens der
Mausedermis und die mögliche Bedeutung dieses Abbaues fürmalignes
Wachstum. Z. Naturforsch., 24b: 894-901, 1969.
39. Rohrbach, R., Thomas, C., Rumpier, R., and Lau, M. Die Kollagenasenak
tivitätund der Kollagengehalt der Haut wahrend der Carcinogenese. Verh.
Dlsch. Ges. Pathol., 54: 442-50, 1970.
40. Pierce, G. B., and Wallace, C. Differentiation of malignant to benign cells.
Cancer Res., 31: 127-134, 1971.
4865
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Newt Squamous Carcinoma Proves Phylogenetic Conservation
of Tumors as Caricatures of Tissue Renewal
Nikolaos P. Zilakos, Panagiotis A. Tsonis, Katia Del Rio-Tsonis, et al.
Cancer Res 1992;52:4858-4865.
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