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Carcinogenesis |
in the Regulation of Ornithine Decarboxylase Expression by Oxidative Stress1
Department of Environmental Health Sciences, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, Maryland 21205
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mutant. PKC- activity
was completely inhibited in response to H2O2 in
cells overexpressing mutant PKC- compared with cells transfected
with a blank plasmid. Induction of ODC mRNA, protein, and activity was
also completely inhibited in cells expressing the PKC- mutant after
H2O2 treatment. Activation of an ODC
promoter-luciferase reporter construct by H2O2
was attenuated in mutant cells compared with control cells, further
confirming that ODC is regulated transcriptionally by PKC-. However,
fold-increases in ODC mRNA and protein were much less than the increase
in activity, suggesting that ODC may also undergo posttranscriptional
regulation in the presence of oxidants. Taken together, these studies
provide new insight into the regulation of ODC by oxidants and suggest
that PKC- may play a critical role in this regulation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of ODC in murine keratinocytes treated with the tumor
promoter TPA has been associated with PKC activation, and there is data
that support transcriptional regulation of ODC by PKC-
(13)
. The current studies investigate the role of PKC-
in the regulation of ODC by the oxidative agent
H2O2. Oxidants derived from
reactive oxygen species and organic hydroperoxides can contribute to
tumor promotion, a process of selection and clonal expansion in which
the expression of genes, such as ODC, that regulate cell
growth are modulated in initiated cells (14)
. The pathways
controlling the expression of such genes by oxidants in mammalian
systems are not well defined. Oxidant tumor promoters also activate
PKCs (15)
. PKC is a multienzyme family of serine/threonine
kinases (16)
that is classified into three groups:
(a) classical PKCs (, ßI, ßII, and 
), which are
Ca2+, phosphatidylserine, and diacylglycerol/TPA
dependent; (b) novel PKCs (, 
, 
, 
, and µ),
which are phosphatidylserine and diacylglycerol/TPA dependent; and
(c) atypical PKCs (
, 
, and 
), which are
Ca2+ and diacylglycerol/TPA independent.
Regulation of ODC and tumor promotion is commonly studied in skin or
cultured keratinocytes because skin is a good model for studying
two-stage carcinogenesis. The present studies, performed in mouse
papilloma cells, focused on the role of PKC- in the induction of ODC
by H2O2, because although
various PKCs are expressed in mouse epidermis, mainly the novel types,
PKC- and PKC-, are associated with keratinocyte differentiation
(17
, 18)
, a course that if deregulated could progress into
a neoplastic phenotype in skin. Our choice to study PKC- was further
influenced by the observations that expression of PKC- protein in
cultured keratinocytes is dependent on high Ca2+
concentrations, whereas PKC- expression is independent of
Ca2+ levels (17)
. The mouse PE cells
used in these studies were sustained in low Ca2+
levels to maintain them in a proliferative state. We find that
H2O2 induces ODC activity
severalfold and that this induction appears to be mediated by PKC-.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generation of Stably Transfected Cell Lines.
Cells expressing dominant-negative PKC-
(PEPKC-DN) were
generated by transfection of the plasmid SRD-DK376A (20)
with lipofectamine (Life Technologies, Inc., Gaithersburg, MD)
according to the supplier’s directions. Control cells were transfected
with a blank plasmid (PEcontrol). G418-resistant
clones were isolated by clonal dilution, expanded, and characterized.
ODC Assay.
After treatment of confluent PE cultures, the cells were rinsed with
pyridoxal phosphate (50 µg/ml)-containing PBS and harvested into
Eppendorf tubes. The cells were subjected to three cycles of freezing
on dry ice and thawing at 37°C. The cleared lysates were used to
determine ODC activity as described previously (21)
.
PKC Assay.
Treated cells were harvested and lysed in 20 mM Tris-Cl (pH
7.5), 1 mM MgCl2, 1 mM
EDTA, 1 mM EGTA, 4 mM
diisopropylfluorophosphate, 1 mM sodium vanadate, 25
µg/ml leupeptin, 25 µg/ml pepstatin, 1% Triton X-100, and 50
mM mercaptoethanol for 30 min on ice. The cleared lysates
were used to determine PKC activity by adding 2–4 µg of protein in
an assay mixture containing 50 mM Tris-Cl (pH 7.5), 1
mM CaCl2, 15 mM
MgCl2, 10 µM TPA, 0.25 mg/ml of
phosphatidylserine, 50 µM ATP, 1 µCi of
[-32P]ATP, 2.5 mM DTT, and 50
µM of a PKC- pseudosubstrate region-derived peptide
(22)
. The reactions were incubated at room temperature for
15 min and spotted onto phosphocellulose discs (Life Technologies,
Inc.). The discs were washed twice with 1% phosphoric acid and twice
with distilled water and analyzed by liquid scintillation. Nonspecific
PKC activity was determined as described but in the absence of TPA or
phosphatidylserine. The specific PKC activity was obtained by
subtracting the nonspecific activity from the total activity.
Immunoblot Analysis.
Cells were lysed in PBS by subjecting the extracts to three cycles of
freezing in dry ice and thawing at 37°C. Cleared cell lysates (10
µg protein) were analyzed by Western blot analysis as described
previously (23)
with polyclonal antibodies specific to
PKC- (Santa Cruz Biotechnologies, Inc., Santa Cruz, CA). ODC protein
expression was determined with a polyclonal ODC antibody.
RNA Isolation and Northern Analysis.
Total RNA was isolated with RNA Stat-60 (Tel-Test, Inc., Friendswood,
TX). RNA (20 µg/lane) was fractionated in formaldehyde-agarose gels
and transferred onto Nytran membranes (Schleischer-Shuell, Keene, NH)
according to the manufacturer’s directions. A mouse ODC cDNA probe was
labeled with [-32P]dCTP as illustrated in a
random primer labeling kit (Boehringer Manneheim, Indianapolis, IN),
and hybridization and washes were done as described previously
(24)
. Hybridization to a 24-bp oligonucleotide
complementary to 18S RNA (5'-ACGGTATCTGATCGTCTCGAACC-3') that was
end-labeled with [32P]ATP by T4 kinase (Life
Technologies, Inc.) was used to control for differences in loading and
transfer efficiency.
Transfections and Luciferase Assay.
PE cells were transiently transfected with 3 µg/ml of -4362/+131
ODC-luciferase (25)
, 0.2 µg/ml of pRLTK (Promega Corp.,
Madison, WI), and 12 µl/ml of lipofectamine (Life Technologies, Inc.)
according to the manufacturer’s directions. Cells were treated with
200 µM H2O2,
and luciferase activity was determined 6 h after treatment with
the dual-luciferase reporter assay system (Promega). Luciferase
activities were determined with a Berthold LB9505 luminometer.
Adenoviral Vectors and Infection.
Constitutively active PKC- (20)
was subcloned into the
modified adenoviral shuttle vector pAdEG1 (26)
, and virus
was purified essentially as described (26, 27, 28)
.
PEPKC-DN cells were
infected with virus (100 plaque-forming units/cell) for 2–4 h in
serum-free media. Infection media was then replaced with normal growth
medium, and cells were allowed to grow for 24 h, after which they
were treated with H2O2 and
analyzed for ODC activity. The fluorescence of green fluorescent
protein was monitored with a confocal fluorescence microscope (Carl
Zeiss, Jena, Germany) at 488 nm argon excitation, fitted with a 515-nm
filter.
Statistical Analyses.
Statistical significance of the differences in the means was assessed
by one-way ANOVA, followed by Bonferroni’s multiple comparison test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, treatment of PE cells with graded concentrations of
H2O2 for 5 h resulted
in a dose-dependent induction of ODC activity with maximal activity
observed at 200 µM
H2O2. Cells treated with
200 µM of
H2O2 for 5 h typically
exhibited a 10–12-fold increase in ODC activity compared with a
25-fold increase with the tumor promoter TPA (100 ng/ml), a
well-established inducer of ODC activity (data not shown).
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). Calphostin alone had no effect on constitutive levels
of ODC activity.
To further probe the role of PKC types in the regulation of ODC by
oxidants, cells were stably transfected with a dominant-negative
PKC- construct and characterized by immunoblot analysis and PKC
activity measurements. PKC- appeared as a doublet of phosphorylated
and unphosphorylated proteins as determined by immunoblot analysis of
control and transfected cells (Fig. 2A
). As demonstrated in Fig. 2A
, Lanes 2
and 3, there was a 3-fold increase in mutant protein in
PEPKC-DN cells compared
with native protein in PEcontrol cells. The
dominant-negative PKC- is mutated in its ATP-binding site and
therefore lacks the ability to autophosphorylate and activate itself.
However, a slower migrating phosphorylated protein was still obtained
in PEPKC-DN cells. We
speculate that this outcome is likely attributable to the fact that
wild-type PKC- and its kinase-inactive mutant are both
phosphorylated on tyrosine residues by tyrosine kinases
(30)
. PKC activity, as determined with a peptide designed
against the PKC- pseudosubstrate region, was increased 3-fold in
cell extracts isolated from
H2O2-treated
PEcontrol cells. However, this activity was
completely inhibited in extracts from
H2O2-treated
PEPKC-DN cells (Fig. 2B
), consistent with a functional loss of PKC-. PKC
activity in extracts isolated from TPA-treated
PEcontrol cells was increased 22-fold, but this
activity was only decreased by 50% in
PEPKC-DN cells after
treatment with TPA. Presumably, PKC activity was only partially
inhibited in extracts from TPA-treated
PEPKC-DN cells because,
although the PKC- pseudosubstrate peptide has high specificity for
PKC-, it is not absolute and can be phosphorylated by other PKC
types. Also, in the presence of TPA, the concentration of the
dominant-negative PKC- in
PEPKC-DN cells may not
be high enough to overcome the particularly robust activation of
multiple types of PKCs.
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DN cells after
treatment with 200 µM
H2O2. A transient induction
in ODC activity was observed in PEcontrol cells,
with maximal ODC activity observed between 5 and 7 h of treatment
(Fig. 3A
), followed by a rapid decline in enzymatic activity. This
transient induction of ODC by various stimuli is attributed to a rapid
degradation of ODC through an antizyme-dependent mechanism
(31)
. ODC activity was not increased at any time point in
PEPKC-DN cells upon
treatment with H2O2,
suggesting that PKC- may be required for induction of ODC expression
by H2O2 (Fig. 3A
). By contrast, a partial inhibition of ODC induction in
response to TPA was seen in
PEPKC-DN cells compared
with PEcontrol cells at a low dose (20 ng/ml;
Fig. 3B
). No inhibition was seen at a higher dose (100
ng/ml) of the phorbol ester (data not shown). This result suggests that
for TPA, unlike H2O2,
inhibition of PKC- in
PEPKC-DN cells has
limited impact upon ODC induction and that elevation of other PKC
enzymes is important in signaling for enzyme induction.
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is a mediator in the induction of ODC
by H2O2,
PEPKC-DN cells were
infected with virus expressing a constitutively active PKC- attached
to green fluorescent protein. This approach was taken to achieve a high
level of constitutively active PKC- expression that could overcome
the dominant-negative effects observed in
PEPKC-DN cells. About
80–90% of the cells expressed constitutively active PKC-, as
determined by the presence of green fluorescent protein (Fig. 4B
). The distribution of PKC- was concentrated in the
perinuclear region, although some protein was also observed in the
cytoplasm. In cells infected with virus containing green fluorescent
protein only (positive control), the protein was expressed diffusely
throughout the cells (Fig. 4C
). Overexpression of
constitutively active PKC- in
PEPKC-DN cells increased
basal ODC activity 4-fold in untreated cells. Moreover, this maneuver
restored ODC inducibility as activity was further enhanced 8-fold after
H2O2 treatment (Fig. 4D
).
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DN Cells after
H2O2 Exposure. is important in the regulation of ODC
enzyme activity by H2O2,
the next step was to determine whether the effect on induction was
transcriptional and/or posttranscriptional. The kinetics of appearance
of ODC message and protein levels in cells treated with
H2O2 was analyzed by
Northern and Western analyses. Significant increases in ODC mRNA levels
in PEcontrol cells were initially observed after
4 h of treatment, and peak message levels (3-fold increase) were
maintained up to 7 h of treatment (Fig. 5
). No induction of ODC mRNA occurred in
H2O2-treated
PEPKC-DN cells (Fig. 5
).
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). A similar time-dependent, albeit smaller increase in
maximal ODC protein (7-fold at 6 h) was observed in
PEcontrol cells after
H2O2 treatment (Fig. 6B
). However, no increase in ODC protein was observed in
PEPKC-DN cells (Fig. 6C
), except for trace elevation of ODC protein after 7 h of H2O2 treatment, which
did not correspond to any increase in ODC activity at the same time
point.
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is
involved in the transcriptional regulation of ODC, activation of an ODC
promoter-luciferase reporter construct by
H2O2 was investigated in
PEcontrol and
PEPKC-DN cells.
Normalized luciferase activity was increased 2.7-fold in
PEcontrol cells after
H2O2 treatment; however,
similar treatment of
PEPKC-DN cells did not
elicit any increase in luciferase activity (Fig. 7
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A direct role for ODC in tumor promotion is evident in studies where targeted expression of high levels of ODC to mouse skin produced tumors after initiation with a carcinogen, in the absence of administration of a tumor promoter (5) . The present studies examined the regulation of ODC by H2O2, primarily because oxidants are known tumor promoters in vivo, and secondly, the precise mechanisms by which oxidants cause promotion are not clear. The current studies demonstrate that ODC activity is induced in murine keratinocytes by H2O2, an action common to most, if not all, tumor promoters in skin. There appear to be several mechanisms by which oxidants can trigger signal transduction pathways leading to enhanced gene expression. For instance, butylated hydroperoxide and H2O2 activate the mitogen-activated protein kinase cascade (33 , 34) , a critical signaling pathway for cellular proliferation. This well-characterized pathway is stimulated by growth factors, mitogens, and stress responses and is initiated by tyrosine kinases that sequentially activate ras, c-raf, and other downstream kinases. Deregulation of this pathway, as might occur in ras-transformed cells, is associated with ODC overexpression (35) .
Another important signaling pathway in the cell, the multienzyme PKC
family, which mediates many of the actions of the potent tumor promoter
TPA, can also be activated by
H2O2. However,
H2O2 activates PKC- by a
mechanism that differs from TPA activation of this kinase. Unlike TPA,
stimulation of cells with
H2O2 does not require
PKC- translocation to the membrane for activation, and the activity
of PKC- isolated from
H2O2-treated cells can be
measured independently of lipid cofactors (36)
.
Investigations into the mechanisms of activation of PKC indicate that
oxidants may modify cysteine residues on several PKCs, leading to their
activation (37)
. Other studies show tyrosine
phosphorylation of major PKC isoforms, including PKC-, in the
presence of H2O2, and
suggest that this may be a mechanism of activation by this oxidant
(30)
.
Cellular expression of the dominant-negative PKC- completely
prevented the inducibility of ODC by
H2O2, suggesting that this
PKC isoform specifically regulates ODC in response to oxidants and
perhaps other actions of oxidants in mouse skin. However, there are
contrary views as to the role of PKC- in tumor promotion and cell
transformation. For instance, whereas a dominant-negative PKC-
mutant inhibited sis-induced transformation and
platelet-derived growth factor-BB-mediated anchorage-independent colony
formation in NIH3T3 cells (38)
, expression of this mutant
in 3Y1 fibroblasts induced c-src-mediated transformation of
these cells and TPA-induced anchorage-independent colony formation
(39)
. These data reflect how little is known on the
physiological role of PKC-. The complexity of PKC- regulation and
function is further demonstrated by recent studies that find PKC- to
be a substrate for c-src, which phosphorylates this kinase at tyrosine
residues and promotes its degradation (40)
.
Overexpression of PKC- in vivo and in vitro
has also been shown to inhibit tumor promotion and induce apoptosis,
respectively (41
, 42)
, supporting a role for PKC- as a
tumor suppressor. However, the physiological relevance of
overexpressing this protein remains to be determined because
overexpression may overwhelm endogenous protein levels, leading to
nonspecific PKC- localization and substrate specificity. This
consideration is especially important in light of very recent data
showing differential localization of PKC- to cellular, nuclear, or
Golgi membranes in response to different agonists (43)
.
Such a differential localization may influence substrate specificity of
PKC-, and hence, its biological response, especially if there is
colocalization with a substrate. Unfortunately, knowledge of the
physiological substrates of PKC- that may aid in understanding its
diverse physiological response is scant.
The fact that we demonstrate an increase in ODC mRNA (3-fold) in
response to H2O2 that does
not correspond to an increase in ODC protein (7-fold) or enzyme
activity (12-fold) suggests the additional involvement of
posttranscriptional regulation of ODC by oxidants. It is well
documented that ODC undergoes rapid degradation upon stimulation; it
has also been shown that ODC can be phosphorylated in situ
and in vitro and that phosphorylated ODC is more stable and
has a higher catalytic activity than the unphosporylated protein
(12)
. Although we report that PKC- is required for the
induction of ODC activity by oxidants and although our data support a
role for transcriptional regulation, we cannot rule out the possibility
of posttranscriptional effects, such as phosphorylation of ODC by
PKC-, to explain the full increase in ODC activity.
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ACKNOWLEDGMENTS |
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constructs, Dr. David Johns for the viral vectors, Dr. Ajit Verma for
the ODC promoter-luciferase vector, and Dr. Thomas O’Brien for the ODC
antibody. |
FOOTNOTES |
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1 This work was supported by NIH Grants CA 44530
and Center Grant ES 06052. 
2 To whom requests for reprints should be
addressed, at Johns Hopkins University, School of Hygiene and Public
Health, 615 North Wolfe Street, Room 7032, Baltimore, MD 21205. Phone:
(410) 955-4712; Fax: (410) 955-0116; E-mail: tkensler{at}jhsph.edu
3 The abbreviations used are: ODC, ornithine
decarboxylase; PKC, protein kinase C; TPA,
12-O-tetradecanoylphorbol-13-acetate.
Received 1/28/00. Accepted 6/13/00.
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) cells were treated with 200 µM
H2O2, and ODC activity determined at the
indicated times. Data are represented as means of three independent
experiments; bars, SE. B,
PEcontrol (
) and PEPKC-
) cells were treated with 20 ng/ml TPA for 5 h, and ODC
activity was determined. Data are represented as means of triplicate
plates assayed in triplicate; bars, SD. *, significant
difference (P < 0.05) from TPA-treated
PEcontrol cells.
