(CANCER RESEARCH 50. 6062-6067. September 15. 1990|
Production of Hydrogen Peroxide by Murine Epidermal Keratinocytes following
Treatment with the Tumor Promoter 12-O-Tetradecanoylphorbol-13-acetate'
Fredika M. Robertson,2 Andrew J. Beavis, Tatiana M. Oberyszyn, Sean M. O'Connell, Anthea Dokidos,
Debra L. Luskin. Jeffrey D. Laskin, and John J. Reiners, Jr.
Departments of Surgery [F. M. R., A. J. B., S. M. O., T. M. O.] and Environmental anil Community Medicine ¡A.D., J. D. L.J, University of Medicine and Dentistry of
A>M'Jersey-Robert H'ood Johnson Medical School, New Brunswick, ¡\ewJersey, 08903; Joint Graduate Program in Toxicology and Pharmacology, School of Pharmacy,
Rutgers University, Piscataway, i\'J 08X55 ¡D.L. L.J; and The University ofTexas-M. D. Anderson Cancer System, Science Park- Research Division, Smithrille, Tetas
78957 1J. J. R.I '
tetradecanoylphorbol-13-acetate (TPA) treatment of mice (10 MB;24 h),
two cytokeratin-positive populations of cells were identified that were
heterogeneous with respect to size and density. These two TPA-derived
cell populations oxidized levels of DCFH that were time and dose
dependent and were between 2- and 10-fold higher than levels of DCFH
oxidized by cells isolated from acetone-treated mice. The ability of
catalase, the enzyme that detoxifies hydrogen peroxide, to suppress
DCFH oxidation to control levels suggested that intracellular hydrogen
peroxide was responsible for the enhanced rate of DCFH oxidation in
epidermal cells isolated from TPA-treated mice. The ability of mouse
epidermal keratinocytes to oxidize DCFH in response to TPA treatment
was confirmed using a cloned keratinocyte cell line. These results suggest
that specific subpopulations of keratinocytes produce elevated levels of
intracellular peroxides following treatment with TPA either in vivo or in
culture.
of carcinogens (26) and tumor promotion (6, 7, 10-13, 23).
Recent studies suggest that keratinocytes also have the ca
pacity to produce ROI. Fischer et al. (27, 28) reported that
phorbol ester tumor promoters induced lumino! oxidation in
isolated epidermal cells from newborn SENCAR mice, as meas
ured by chemiluminescence. Because the production of chemiluminescence could be inhibited by the inclusion of Superoxide
dismutase, they concluded that the epidermal-derived oxidant
was Superoxide anión.
Since ROI have been circumstantially implicated in the proc
ess of TPA-dependent tumor promotion in mouse skin (22, 27,
28), the present studies examined the cellular sources of these
active oxygen species. Using flow cytometric techniques, spe
cific subpopulations of cells present in the epidermis following
TPA exposure that oxidized the hydroperoxide-sensitive dye,
DCFH, were identified. These studies provide evidence that
keratinocytes as well as leukocytes produce hydroperoxides
following stimulation with TPA. These observations indicate
that both cell types may be significant sources of ROI during
phorbol ester-mediated tumor promotion.
INTRODUCTION
MATERIALS
ABSTRACT
The ability of murine epidermal cells to produce intracellular hydrogen
peroxide was analyzed by flow cytometry and the measurement of 2',7'dichlorofluorescin (DCFII) oxidation. Epidermal cells isolated from ace
tone-treated CD-I mice for 24 h were relatively homogeneous in cell size
and density and oxidized low levels of IX l'I I. However, following 12-0-
ROI,' including Superoxide anión, hydrogen peroxide, and
hydroxyl radicals, are known to induce biological damage by
reacting with nucleic acids and proteins as well as various
membrane components (1-11). Several different types of DNA
damage associated with ROI production have been reported,
including chromosomal
aberrations, sister chromatid ex
changes, and mutations (12-18). These observations have lead
a number of investigators to suggest that ROI are involved in
the process of mouse skin tumor promotion and carcinogenesis
(19-22).
Currently, little information is available on the specific cell
ular origin of ROI. Several studies suggest that free radical
species generated in the skin may be derived from Langerhans
cells, i.e., resident skin macrophages, as well as inflammatory
leukocytes that have infiltrated into the dermis and epidermis
following tumor promoter treatment (23-25). These cells are
known to produce ROI in response to TPA in vitro and have
been postulated to play a role in both the metabolic activation
Received 7/12/89; accepted 6/19/90.
The cosis of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' This project was supported in part by New Jersey Commission on Cancer
Grant 88593 CCR (F. M. R.) and National Cancer Institute Grants CA-34469
(J. R. R.) and CA 51443 (F. M. R.).
2To whom requests for reprints should be addressed, al the Department of
Surgery, UMDNJ-Robert Wood Johnson Medical School. New Brunswick, NJ
08903."
3The abbreviations used are: ROI, réactive
oxygen intermediates; TPA. 12-0tetradecanoylphorbol-13-acetate;
NCS, new-born calf serum; PBS, phosphate
buffered saline; DCFH-DA, 2',7'-dichlorofluorcscin
diacetate; DCFH, 2',7'dichlorofluorescin; DCF, 2',7'-dichlorofluorescein;
FITC, fluorescein isothiocyanate; PMN, polymorphonuclear cells.
AND METHODS
Chemicals. Propidium iodide, trypsin (bovine pancreatic; type XI)
and catalase were purchased from Sigma Chemical Co., St. Louis. MO.
TPA was purchased from LC Services. (Woburn, MA). DCFH-DA was
obtained from Molecular Probes (Eugene, OR). Eagle's minimal essen
tial medium. Joklik's medium, fetal calf serum, and NCS were pur
chased from Grand Island Biologicals (GIBCO), Grand Island, NY.
Rabbit anti-keratin antiserum (K2) was generously provided by Dr.
Stuart Yuspa (NIH. Bethesda, MD). K2 antiserum was generated from
mouse M, 60.000 keratins and recognizes both acidic and basic keratins
present in basal and suprabasal keratinocytes (29, 30). FITC-conjugated
goat anti-rabbit IgG secondary antibody was purchased from Cooper
Laboratories (Malvern, PA).
Isolation of Mouse Epidermal Cells. Twenty-four h prior to TPA
treatment, the dorsal skin of female CD-I mice (25-30 g; Charles River
Breeding Laboratories, Wilmington, MA) was shaved. TPA (10 ^g),
dissolved in 0.2 ml of acetone, was applied directly to the shaved area.
Control mice received 0.2 ml of acetone alone. After 24 h, the mice
were sacrificed and skin samples from the treated areas were excised,
cut into small pieces, and floated, dermal side down, in a culture dish
containing 0.25% trypsin in PBS (pH 7.3). After 1 h at 37°Cfollowed
by 1 h incubation at 25'C, the epidermal layer was scraped from the
dermal layer using fine edged forceps and a scalpel blade. Ten ml of
Eagle's minimal essential medium supplemented with 2% FCS were
then added to the cultures to inhibit trypsin activity. A single cell
suspension of isolated epidermal cells was obtained by forcefully pipeting the suspension 4-6 times. Cells were then filtered through 150-^m
mesh to remove squames and debris. Viability of the cells was assessed
by propidium iodide exclusion (31) and was found to be greater than
95%.
Cell Cultures. The mouse epidermal keratinocyte cell line was pre
pared from primary cultures of newborn mouse keratinocytes (CD-I)
as described previously (32). Cells were grown continuously in monolayer cultures under low calcium (<0.05 mM) conditions using Joklik's
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KERATINOCYTE
PRODUCTION
OF H¡O¡
medium supplemented with 10% Chelexed NCS.
Flow Cytometric Analysis of Hydrogen Peroxide Production. Hydro
gen peroxide production by epidermal cells was measured using DCFHDA. DCFH-DA is a nonpolar compound that readily diffuses into cells
where it is hydrolyzed to the nonfluorescent polar derivative. DCFH.
and is trapped within the cells (33). In the presence of intracellular
hydroperoxides and peroxidases. DCFH is oxidized to the highly flu
orescent compound DCF (33, 34). Thus, cellular fluorescence intensity
is directly proportional to levels of intracellular hydroperoxides and
can be monitored by flow cytometry (33). Cells (1 x IO6)were incubated
at 37"C with 5 n\\ DCFH-DA with and without the addition of 1000
units of catatase. Catalase was used to determine the amount of DCFH
oxidation that was specifically related to the presence of intracellular
hydrogen peroxide rather than to other hydroperoxide substrates.
DCFH oxidation was measured 30 min later using an EPICS Coulter
Profile analytical flow cytometer (Coulter Cytometry). Experiments
were repeated 6 times, with similar results, with the variability of DCF
oxidation ±10channels on a log scale. Green fluorescence intensity
data were collected (510-550 nm) using 550 dichroic and 525-nm
bandpass filters. For each analysis. 10,000-20,000 events were accu
mulated. Optimal alignment of the instrument was performed daily and
typical coefficients of variation for forward angle light scatter and
integrated green fluorescence were less than 2. Quantitation of fluores
cence is represented as the mean fluorescence channel number. Each
histogram has 256 channels that arc displayed on a 3-decade log scale.
Immunofluorescence. Disaggregated epidermal cells (I x 10") were
B
fixed with 2% paraformaldehyde, washed twice, and permeabilized with
0.05% Triton X-100 for 15 min at 4'C. Cells were then washed twice
with PBS and incubated with a 1:500 dilution of K2 antibody in PBS
containing O.tcl gelatin. After 30 min at 4°C,cells were washed three
times in PBS and incubated for an additional 30 min with a 1:500
dilution of FITC-conjugated goat anti-rabbit whole molecule IgG sec
ondary antibody. The cells were subsequently washed three times in
PBS and analyzed on the flow cytometer. To determine the amount of
nonspecific antibody binding, control cultures were incubated with
FITC-conjugated goat anti-rabbit IgG secondary antibody. Experiments
were repeated at least 6 times with similar results. The data are
expressed as one representative histogram.
RESULTS
Effect of TPA on Mouse Epidermis. In the present studies,
light microscopy was used to characterize the effects of TPA
on mouse epidermis. Female CD-I mice were treated with
acetone or TPA (2 or 10 ßg)for 4 or 24 h. Dorsal skin was
then removed and disaggregated, and cytospin preparations of
epidermal cells were prepared, stained with Harris' modified
acid hematoxylin stain, and examined microscopically. Cell
numbers were determined using a hemocytometer. On the
average, 10* cells were isolated per epidermal preparation iso
lated from individual mice treated with acetone alone or with 2
or 10 MgTPA for 4 h, while 2-3 x 10" cells were isolated from
the epidermis of mice treated with 10 ng TPA for 24 h. We
found that cell populations isolated from acetone-treated mice
consisted of approximately 95% keratinocytes and 5% leuko
cytes. The keratinocytes appeared relatively homogeneous with
respect to size. The numbers of infiltrating PMN in the epider
mis were dependent upon the time of exposure to TPA (Fig. 1)
as well as the dose of TPA. Application of 10 pg TPA to mouse
skin for 24 h resulted in a 2-3-fold increase in the total number
of cells recovered from the epidermis, including increases in
both keratinocytes and leukocytes. The keratinocytes appeared
to be heterogeneous in size following TPA treatment. Vacuolization was apparent within the cytoplasm of the larger, more
granular keratinocytes. These cells also had an increased cytoplasmic:nuclear ratio.
Characterization of Mouse Epidermal Cell Subpopulations by
Flow Cytometry. To further characterize the effects of TPA on
Fig. 1. Light micrograph of Giemsa-staincd epidermal cells isolated from CD1 mice that had been treated with arcione (.-t). 10 /jg TPA for 4 h (Ä).and 10 ng
TPA for 24 h(C). (xlOO).
mouse epidermis, single cell suspensions of epidermal cells were
analyzed using laser light scatter properties. Analysis of cell
size (forward angle light scatter) and density/granularity (log
90-degree light scatter) revealed one relatively homogeneous
population of epidermal cells isolated from acetone-treated
mice (Fig. 2, left). This population consisted of cells that were
intermediate in size and density. These results correlated with
the morphological observations described above. Cells isolated
from mice treated for 4 h with 2 or 10 ^g of TPA did not differ
significantly with respect to size and density compared to cells
isolated from acetone-treated mice. In contrast, there were two
distinct subpopulations in cells isolated from mice treated for
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KERATINOCYTE PRODUCTION OF H2O¡
24 h with both 2 and 10 ^g TPA. Cells within population 1
that were isolated from mice treated with either 2 or 10 ng TPA
were more heterogeneous with respect to both size and density
than observed in cells within population 1 isolated from ace
tone-treated mice (Fig. 2. right). Cells isolated 24 h following
treatment of mice with either concentration of TPA contained
a second population of cells that were similar in size to those
cells within population 1 but had increased density (Fig. 2,
right).
To identify the keratinocytes within the epidermal subpopu
lations, we used K2, a polyclonal antibody that binds mouse
cytokeratins in both basal and suprabasal layers of the epidermis
(29, 30). Fig. 3, left, represents a comparison of the cell size
versus cytokeratin-specific antibody binding, represented as log
green fluorescence on the Y-axis. Epidermal cells isolated from
acetone-treated mice that bound K.2 antibody were relatively
homogeneous with respect to size; however, there was hetero
geneity with respect to the amount of antibody bound by cell
population 1. Cytokeratin-positive cells were identified in both
subpopulations 1 and 2 of cell samples isolated from epidermis
treated with 10 ^g TPA for 24 h (Fig. 3, right). The greatest
amount of antibody was bound by the smallest cells, which were
contained within population 2, a population of cells not present
in acetone-treated epidermis.
Effect of TPA on DCFH Oxidation by Murine Epidermal
Cells. The next series of studies examined the dose and time
dependence of TPA-stimulated DCFH oxidation by epidermal
cell subpopulations (Table 1). Epidermal cells isolated 4 h
following treatment with 2 or 10 ^g TPA oxidized 1.9- and 3.2fold greater amounts of DCFH, respectively, compared with
cells isolated from mice treated with acetone alone.
Since cells isolated 24 h following treatment of murine epi
dermis with 2 or 10 jug TPA resulted in a dramatic alteration
Control
TPA
0>
U
Cell Size
Cell Size
Fig. 2. Flow cytometric scattergram for analysis of cell size (X-axis) and
granularity/density (Y-axis) of epidermal cells isolated from acetone-treated CD1 mice (left) and mice treated with 10 MgTPA for 24 h (right).
Table 1 Quantitation of dose- and lime-dependent DCFH oxidation by isolated
epidermal celts following exposure of mice to 2 and 10 ng TPA for 4 and 24 h or
by cloned epidermal keratinocytes following treatment with 500 ng TPA for 6 It
Data are expressed as mean channel number of log and linear intensity of
green fluorescence as well as fmol DCFH oxidized/cell.
Mean channel
no.TreatmentEpidermisAcetoneTPA.
oxidized
increase1.93.23.44.45.810.82.6DCFH
(fmol/cell)13418826928334743671
h2 4
M10
MgTPA.
hSubpopulation
24
12
Mg10
MgSubpopulation
34.8145.6166.74075Linear8.916.527.929.938.951.590.63.07.8Relative
22
Mg10
MgCloned
keratinocytesAcetoneTPA.
500 ng. 6 hLog80.5103122.7124.91
in size and granularity, cells within each subpopulation were
analyzed for DCFH oxidation. Cells within subpopulation 1
isolated from mice treated for 24 h with 2 /ug TPA oxidized
3.4-fold higher amounts of DCFH than cells isolated from
control animals, while cells within subpopulation 1 isolated
from mice treated with 10 MgTPA for 24 h oxidized 4.4-fold
higher levels of DCFH. Cells within subpopulation 2 isolated
from mice treated with 2 /¿gTPA for 24 h oxidized 5.8-fold
higher amounts of DCFH and cells within subpopulation 2
isolated from mice treated with 10 ^g TPA for 24 h oxidized
10.8-fold higher levels of DCFH.
Since DCFH oxidation can be catalyzed by any hydroperoxy
compound that can serve as a substrate for peroxidases, includ
ing hydrogen peroxide, the specificity of the reaction for intracellular hydrogen peroxide production was determined by meas
uring the ability of catalase, an enzyme that detoxifies hydrogen
peroxide, to inhibit DCFH oxidation. Following isolation of
the epidermal cells from acetone-treated and TPA-treated mice,
catalase (1000 units) was added to the cells prior to incubation
with DCFH-DA. Following preincubation with catalase (1000
units/30 min). DCFH oxidation within both subpopulations 1
and 2 from cells isolated from TPA-treated mice was inhibited
to levels below that found in non-catalase-treated population 1
of epidermal cells prepared from acetone control mice.4 This
result suggested that DCFH oxidation in epidermal cells de
rived from TPA-treated mice represents intracellular hydrogen
peroxide levels.
Effects of TPA on Hydrogen Peroxide Production in Cloned
Epidermal Keratinocytes. To confirm that epidermal keratino
cytes could produce hydrogen peroxide, we used a cloned CD1 mouse keratinocyte cell line. Exposure of cloned mouse
keratinocytes to TPA produced a dose-dependent stimulation
of DCFH oxidation over a concentration range of 50-500 ng
TPA. A maximum increase of 2.6-fold stimulation was detected
following exposure of cloned keratinocytes to 500 ng TPA for
6 h (Table 1; Fig. 4), which was suppressed by preincubation of
cultured keratinocytes with catalase.4
DISCUSSION
Cell Size
Cell Size
Fig. 3. Two parameter flow cytometric analysis of cytokeratin-specific K2
antibody binding (Y-axis) compared with epidermal cell size (X-axis) of cells
isolated from acetone-treated mice (left) or cells isolated 24 h following treatment
with lOjigTPA (right).
Previous investigators have reported that topical application
of TPA to mouse skin stimulates keratinocytes to both prolif4 F. M. Robertson, unpublished data.
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KERATINOCYTE
LJ
ÛQ
PRODUCTION
Ol H3O2
present studies we use flow cytometric detection of DCFH
oxidation that allows the measurement of hydrogen peroxide
production at the 10~g M concentration range (33). This level
of sensitivity exceeds the 10~5 M-10~6 M levels measured using
300
200
" ß*10
100
1000
RELATIVE LOG GREEN FLUORESCENCE
Fig. 4. Flow cylomelric histogram of DCFH oxidation by cultured mouse
epidermal keratinocytes following treatment with acetone (A) or 500 ng TPA (B)
for 6 h.
erate (25, 35) as well as have dramatically altered keratinization
patterns (32, 36). In the present studies, we observed a 2-3-fold
increase in the number of cells following treatment of murine
epidermis with 2 or 10 ng TPA for 24 h, which confirms reports
of TPA-induced epidermal hyperplasia. There are several ex
planations for expanded cell populations in the epidermis after
TPA treatment. The increase in numbers of epidermal cells is
partially due to a decrease in the cell cycle time of basal
keratinocytes following TPA treatment of mouse skin (35).
Furthermore, leukocytes, including polymorphonuclear cells
(PMN) and monocytes/macrophages,
are known to actively
migrate through the dermis into the epidermis following TPA
treatment (25). Macrophage migration is directed by the release
of mediators such as interleukin 1 that act as chemoattractants
during inflammation and tissue injury (37). TPA, by itself, is
known to be a potent chemoattractant for macrophages but not
for PMN (38). These observations suggest that leukocytes may
migrate to the local site of TPA application due to direct effects
of TPA as well as the release of soluble mediators released from
keratinocytes and activated Langerhans cells (39).
Morphological examination of cells isolated from TPAtreated animals revealed dramatic increases in cytoplasmic vacuolization and cytoplasmicinuclear ratio in keratinocytes and
leukocytes, as was also reported by Lewis and Adams (25). Our
flow cytometric analysis confirms morphological observations
that there are dramatic alterations in size and density of kera
tinocytes following TPA treatment.
Based on laser light scatter properties and reactivity with K2,
a cytokeratin-specific antibody that binds to both basal and
suprabasal cells, we identified a subpopulation of keratinocytes
that was present in both acetone-treated and TPA-treated epi
dermis. Furthermore, flow cytometric analysis of TPA-treated
skin revealed the presence of a unique cell population, desig
nated as subpopulation 2. Cells contained within this subpopulation bound similar amounts of cytokeratin-specific antibody
as did cells within subpopulation 1. Cells within subpopulation
2 were smaller and more dense than keratinocytes in population
1. Further cytometric and histologie analysis of this subpopu
lation with antibodies that bind basal or suprabasal cells differ
entially (29, 30) as well as DNA cell cycle probes (40, 41) are
required to determine the proliferarne capacity of these cells.
These studies may also clarify the contribution that this unique
subpopulation makes to the process of carcinogenesis.
Several studies circumstantially implicate ROI in the process
of TPA-dependent tumor promotion (27, 28, 42-46). In the
conventional biochemical methods for ROI detection such as
scopoletin or phenol red oxidation (47, 48). In addition to
providing information about low levels of hydroperoxides pro
duced by epidermal cells, cytometric analysis provides quanti
tative information about cellular subpopulations that can not
be obtained using microscopy or spectrophotometric methods.
Thus, the assay system used in the present studies has a sensi
tivity range that allows detection of levels of keratinocyteassociated hydroperoxides and may therefore be useful in ex
amining the effects of TPA on specific epidermal subpopula
tions.
The oxidation of DCFH-DA has been used by a number of
investigators to measure the intracellular hydrogen peroxide
levels in both monocytes and neutrophils (33. 49. 50). The basis
for the oxidation of DCFH-DA was initially reported by Keston
and Brandt (34). Although DCFH oxidation has been used as
an indirect measure of intracellular hydrogen peroxide levels, a
limitation of this assay is that hydroperoxides other than hy
drogen peroxide may also be able to catalyze DCFH oxidation.
Several investigators have circumvented this limitation by meas
uring the hydrogen peroxide-specific DCFH oxidation as the
difference between DCFH oxidation by cells in the presence
and absence of catatase (33, 34). This is the approach that we
have taken to establish the specificity of epidermal cell produc
tion of hydrogen peroxide for the studies described. Because
the addition of catalase was effective in inhibiting DCFH oxi
dation, the DCFH oxidation measured in epidermal cells, as
well as cultured keratinocytes. was primarily dependent upon
hydrogen peroxide.
Although studies have indicated that ROI may be important
in the tumor promotion process, the cellular sources of ROI in
mouse epidermis following TPA treatment remain unclear.
Inflammatory leukocytes are known to release ROI as part of
their normal phagocytic and bactericidal activities (51-53).
Since TPA has been shown to cause an influx of inflammatory
leukocytes into the dermis and epidermis (25) and is a potent
stimulator of the respiratory burst in vitro (54, 55), it has been
suggested that these cells may be the major contributors of ROI
in the epidermis. Our current study suggests that subpopula
tions of epidermal cells that contain keratin positive cells oxi
dize significant amounts of DCFH in a dose- and time-depend
ent manner in response to TPA treatment. Although the iso
lated epidermal cell subpopulations that we analyzed contained
both keratinocytes and leukocytes, it is unlikely that elevated
levels of DCFH oxidation observed in TPA-treated epidermis
could completely be accounted for by enhanced oxidative me
tabolism by leukocytes. Epidermal cell preparations isolated
from acetone- and TPA-treated mouse skin contained approx
imately 5-10% leukocytes and 90-95% keratinocytes. Analysis
of the histograms suggest that the population of cells that
produced elevated levels of hydrogen peroxide was over 92%
keratin positive. Furthermore, the studies using cultures of
cloned murine keratinocytes confirmed the ability of keratino
cytes to oxidize DCFH that was catalase inhibitable.
The ability of keratinocytes to produce ROI may be a part of
their normal function as the principal cell involved in barrier
functions of the host. Since the skin is continually challenged
with infectious agents, the production of ROI by keratinocytes
may play a role in host defense mechanisms. Keratinocytes have
been shown to be phagocytic (56). and the observed oxidative
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KERATINOCYTE
PRODUCTION
metabolism may be related to this functional activity, as is
observed with leukocytes (51-53). We have found that keratinocytes produce elevated levels of ROI after exposure to cytokines derived from lymphocytes and macrophages, such as 7interferon and tumor necrosis factor (57, 58). The in vitro levels
of DCFH oxidation by keratinocytes were substantially lower
than those detected in vivo. This difference may be due to the
ability of TPA to induce in vivo release of mediators produced
by keratinocytes, leukocytes, and lymphocytes that act in con
cert to enhance cellular ROI production and release. Therefore,
the production of ROI by keratinocytes exposed in vivo to TPA
may be due to direct effects of phorbol ester as well as be a
result of cytokines released by infiltrating leukocytes and lym
phocytes (59).
Epidermal cells, including keratinocytes as well as leukocytes,
contain enzymes for both the generation as well as the detoxi
fication of ROI. A number of investigators have reported that
the normal balance between the production and detoxification
of ROI is dramatically altered in epidermal cells following TPA
treatment (60-67). Reiners et al. (65) reported that the specific
activity of catalase is decreased in epidermal cells isolated from
SENCAR mice following TPA treatment. Conversely, the spe
cific activity of xanthine oxidase, an enzyme that produces
Superoxide anión and hydrogen peroxide, was found to be
increased in keratinocytes following TPA treatment (66, 67).
These results suggest that keratinocytes have the capability to
produce ROI and, further, that TPA modulates ROI-generating
enzymatic activity within these cells.
In summary, our observations demonstrate that epidermal
cell subpopulations produce ROI and that the level of produc
tion of ROI is enhanced following TPA exposure. Since these
subpopulations consisted of both keratinocytes and leukocytes,
these observations therefore suggest that keratinocytes may
function as effector cells that produce ROI as well as act as
target cells for ROI-induced damage during the process of
tumor promotion.
12.
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28.
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Production of Hydrogen Peroxide by Murine Epidermal
Keratinocytes following Treatment with the Tumor Promoter 12O-Tetradecanoylphorbol-13-acetate
Fredika M. Robertson, Andrew J. Beavis, Tatiana M. Oberyszyn, et al.
Cancer Res 1990;50:6062-6067.
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