From the
Mammalian DNA polymerase
DNA polymerase
The promoters of the human, bovine, and rodent
The
The present investigation was undertaken to examine
the idea that the response of a mammalian DNA repair gene (
Recombinant rat
Large-scale digestion of
Beginning about
day 10, fluids from actively growing cells were tested by ELISA for
anti-
The epitopes for mAbs 14S and 30S were in the 31-kDa
domain(87-335), as they gave cross-reactivity with all domains
tested except the 8-kDa
domain(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80) .
The epitopes for mAbs 17S and 4S were in the region between residues 75
and 154, as these mAbs reacted with
CHO-K1 cells were purchased from the American Type Culture
Collection (Rockville, MD). These cells were grown per ATCC
recommendations. GC-1 cells (gift from Dr. S. Mitra, Sealy Center for
Molecular Science, Galveston) were maintained as described (Dunn et
al., 1991). Actively dividing CHO cells were exposed to different
effectors, e.g. MNNG (10 and 30 µM) for 4 h or
the indicated time periods; TPA (500 nM), dibutyryl cAMP (100
µM), and forskolin (1 µM) for 4 h. Different
concentrations of TPA (10, 100, 200, 500, and 1,000 nM),
dibutyryl cAMP (1, 10, 100, and 500 µM), and forskolin (1,
5, 50, and 100 µM) were used to study the optimum
concentration of these effectors. The effect of TPA (500 nM)
was also studied from 1 to 12 h.
Actively growing cells in the presence or absence of
effectors were used for
Different amounts of pure recombinant rat
Regulation of
The effect of MNNG-induced DNA damage on the
up-regulation by TPA was also examined (). TPA treatment
up-regulated
We explored
Mammalian cells respond
to treatment with DNA-damaging agents via several intracellular signal
transduction pathways that alter gene expression (Fornace et
al., 1989; Deng and Nickoloff, 1994; Nelson and Kastan, 1994;
Xanthoudakis et al., 1994). In one such pathway protein kinase
C appears to be activated, resulting in phosphorylation of proteins
through a cascade, eventually leading to induction of the so-called DNA
damage-inducible genes (Buscher et al., 1988; Fornace et
al., 1989; Wilson, 1990). In earlier studies, the protein kinase A
pathway was implicated in DNA-damaging agent induction of
In the current study, we selected protein kinase A
signaling agents such as dibutyryl cAMP, forskolin, and the DNA
damaging agent MNNG to study their effect on expression of
After
observing
The precise mechanism of the
The phorbol ester TPA is known in some cases to influence
gene transcription by binding to and activating protein kinase C, which
in turn regulates synthesis and post-transcriptional modification of
transcriptional activator proteins, such as AP-1, among others. CHO
cells, when treated with as little as 10 nM TPA for 4 h,
showed a strong increase in
It was interesting to observe
that the up-regulation of
In summary, we report new immunological
probes for mammalian
CHO-K1 cells were exposed
to different concentrations of MNNG for 2 h either with or without
simultaneous exposure to TPA. The results shown are averages
representative of two independent experiments. The
CHO-K1 cells were exposed to different concentrations
of TPA for 4 h either with or without simultaneous exposure to MNNG.
The results shown are averages representative of two independent
experiments. The
We thank Drs. Amalendra Kumar, Rajendra Prasad, Julie
K. Horton, William A. Beard, and David A. Konkel for helpful
discussions and critical reading of the manuscript, and also Kay Miller
for typing the manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
-pol), a DNA repair
polymerase, is known to be constitutively expressed in cultured cells,
but treatment of cells with the DNA-alkylating agents MNNG or methyl
methanesulfonate has been shown to up-regulate
-pol mRNA level. To
further characterize this response, we prepared a panel of monoclonal
antibodies and used one of them to quantify
-pol in whole cell
extracts by immunoblotting. We found that treatment of Chinese hamster
ovary cells with either DNA-alkylating agent up-regulated the
-pol
protein level 5-10-fold. This induction appeared to be secondary
to DNA alkylation, as induction was not observed with a genetically
altered cell line overexpressing the DNA repair enzyme O
-methylguanine-methyltransferase. We also found
that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of
wild type Chinese hamster ovary cells increased expression of
-pol
protein (
10-fold). Any interrelationship between this TPA response
and the DNA-alkylation response was studied by treatment with
combinations of MNNG and TPA. The
-pol up-regulation observed with
MNNG treatment was abrogated by TPA, and conversely the up-regulation
observed with TPA treatment was abrogated by MNNG.
(
-pol)
(
)is
involved in DNA repair in mammalian cells, based upon inhibitor studies
with cultured cells and permeabilized cell systems (Miller and
Chinault, 1982; Smith and Okumoto, 1984; Dresler and Kimbro, 1987;
DiGiuseppe and Dresler, 1989). The catalytic specificity of
-pol in vitro is consistent with a role in gap-filling synthesis in vivo, to restore double-stranded DNA at short-gapped
intermediates produced during excision repair (Wang and Korn, 1980;
Mosbaugh and Linn, 1983; Randahl et al., 1988; Singhal and
Wilson, 1993).
-Pol has been shown to be responsible for the
single-base gap-filling synthesis step in base-excision repair in
several mammalian crude nuclear extract in vitro systems
(Wiebauer and Jiricny, 1990; Dianov et al., 1992; Singhal et al., 1995).
-Pol is considered a constitutively
expressed enzyme in vertebrates, and in most cells and tissues (Chang
and Bollum, 1972).
-Pol enzymatic activity and mRNA are expressed
at a relatively low level and are independent of cell growth and cell
cycle stage (Chang and Bollum, 1972; Zmudzka et al., 1988;
Nowak et al., 1989). However,
-pol mRNA and enzymatic
activity are regulated in a tissue-specific fashion in rodents (Hirose et al., 1989; Nowak et al.,1989),
and
-pol mRNA is induced by treatment of cells with some
DNA-damaging agents, but not by others. For example, in Chinese hamster
ovary (CHO) cells, treatment with the DNA-alkylating agents MMS or MNNG
causes an induction in
-pol mRNA level (Fornace et al.,
1989).
-pol
genes have been cloned and characterized. The promoters are G +
C-rich and have binding cis-elements for initiation site-binding
protein(s) and two well known transcriptional activators, Sp1 and
ATF/CREB, within a core promoter of
100 base pairs 5` of the mRNA
start site (Widen et al., 1988; Widen and Wilson, 1991; Chen et al., 1995). These promoters lack typical TATA or CCAAT
elements. In the human promoter, the palindromic sequence GTGACGTCAC at
-40 to -49 upstream of the transcription start site is a
typical activating transcription factor/cAMP-response element
(ATF/CRE). This element, which was found to be essential for human
-pol promoter activity in transient expression experiments (Widen
and Wilson, 1991), mediates protein kinase A-dependent stimulation of
the cloned promoter in CHO cells (Englander and Wilson, 1992a). The
element also mediates stimulation of the cloned promoter after
treatment of CHO cells with MNNG (Kedar et al., 1991), and
this up-regulation was not seen in genetically altered CHO cells
deficient in protein kinase A activity, indicating that this particular
signal transduction pathway is required for the response to MNNG
(Englander and Wilson, 1992a, 1992b).
-pol core promoter is
up-regulated by expression of the activated Harvey p21
protein (Kedar et al., 1990), consistent with the
idea that activation of the protein kinase C signal transduction
pathway may also be important in
-pol gene expression. However,
the cloned
-pol promoter does not have functional binding sites
for the transcriptional activator AP-1, a principal protein kinase C
pathway target for transcriptional regulation (Widen et al.,
1988). This is in contrast to the proposed mechanism of up-regulation
of the so-called DNA damage-inducible and oxidative stress-responsive
genes by protein kinase C stimulation with TPA (Macfarlane and Manzel,
1994; Stevenson et al., 1994) and subsequent
activation of AP-1. In this case, treatment of cells with TPA
stimulates the DNA binding activity of AP-1 by dephosphorylation of the
c-Jun subunit; thus, protein kinase C activation appears to lead to
elevation of a protein phosphatase activity and dephosphorylation of
c-Jun's phosphorylation sites at residues 227 and 252 (Boyle et al., 1991). It also has been found that TPA treatment can
directly stimulate members of the ATF/CREB superfamily of
transcriptional activator; for example, ATF-2 (Zu et al.,
1993) is stimulated due to phosphorylation by protein kinase C at
Ser-340 and Ser-367, within the DNA-binding domain (Sakurai et
al., 1991).
-pol)
to DNA alkylation damage may be regulated by the growth factor/protein
kinase C/mitogen-activated protein kinase cascade. Several growth
factors, including platelet-derived growth factor BB (Graves et
al., 1993) and epidermal growth factor (Arteaga et al.,
1994), are known to activate protein kinase C and the mitogen-activated
protein kinase cascade in their signal transduction process. The growth
factor/protein kinase C/mitogen-activated protein kinase cascade has
recently been found to be antagonistic against the protein kinase A
signal transduction system in CHO cells, rat adipocytes (Sevetson et al., 1993), and in human aortic smooth muscle cells (Graves et al., 1993). Thus, protein kinase C involvement in
regulating
-pol transcription could have implications for the DNA
damage-induced response in
-pol level. We found an induction of
-pol level in CHO cells after treatment with either MNNG or TPA
alone. These two agents, however, when used in combination were
mutually antagonistic. These interesting results are discussed in the
context of DNA damage-induced regulation of a DNA repair gene and the
potential for modulation of this regulation by the protein kinase C
signal transduction system.
Isolation and Purification of
-Pol
-pol was purified from Escherichia
coli RR1 (pRK 248clts, pRC-R
1) as described by Kumar et
al.(1990). The molecular mass of the
-pol protein was
39
kDa, as expected from the open reading frame in the cDNA from mammalian
sources (SenGupta et al., 1986; Zmudzka et al.,
1986). The concentrations of stock solutions of
-pol were
calculated from ultraviolet absorbance values using the molar
absorption coefficient
= 2.12
10
M
cm
.
Controlled Proteolysis with Trypsin
-pol with trypsin was carried
out in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride by the
procedure of Kumar et al.(1990). This digestion generated 31-,
27-, and 8-kDa fragments of
-pol. By changing the concentration of
trypsin to a substrate:enzyme ratio of 10:1 (w/w), two more fragments
of 10- and 12-kDa were produced from the 27-kDa domain.
(
)These fragments, along with overexpressed 16/18-kDa
peptides of
-pol, were used to determine the epitope for several
monoclonal antibodies (mAbs) raised to the whole protein.
Generation of Anti-
-pol Monoclonals
Immunization, Hybridization, Selection, and
Cloning
Eight-week old BALB/c mice were inoculated
intraperitoneally with purified bacterial recombinant -pol (75
µg) in a 0.5-ml emulsion with complete Freund's adjuvant. Two
weeks later, the animals were boosted with a 0.5-ml subcutaneous
inoculation of purified
-pol (75 µg) with incomplete
Freund's adjuvant. The following week, serum from the mice was
tested by ELISA against a highly purified antigen preparation to
determine the level of specific anti-
-pol activity. Two weeks
following the second injection, the most reactive mice were given an
intravenous injection of 0.1 ml containing 100 µg of highly
purified
-pol, and their spleens were harvested for fusion 4 days
later. The general procedures used for cell fusion, selection, cloning,
and propagation have been previously described (Köhler et
al., 1976; Showalter et al., 1981).
-pol activity. By controlled washing of antigen-antibody
complexes on the plate, this test was designed to select for antibodies
that would also give strong immunoblot signals. Cells from positive
wells were expanded into 24-well plates and cloned by limiting dilution
in 96-well plates on a feeder layer of compatible thymocytes. Cloned
cells were passaged in ascites tumors by intraperitoneal injection of 3
10
cells into adult BALB/c mice primed 2 weeks
previously with 0.5 ml of pistrane (2,6,10,14-tetramethylpentadecane;
Aldrich) injected intraperitoneally. The ascitic fluids resulting
7-10 days later were harvested, clarified, and the immunoglobulin
purified to homogeneity for further use. Cell lines were designated as
4S, 6S, 9S, 10S, 14S, 15S, 16S, 17S, 18S, 22S, 23S, 30S, 35S, 38S, 40S,
and 41S, respectively.
Analysis and Characterization
Antibody subclasses
of all monoclonals obtained were determined using a commercially
available isotyping kit (Amersham). The kit is essentially a sandwich
immunoblot using specific anti-subtype reagents. Supernatant fluid from
cultures of each line as tested for immunoglobulin class by ELISA and
immunoblot assays using secondary antibodies specific for IgG
(-chain) and IgM (µ-chain). Six cell lines (10S, 14S, 18S,
22S, 35S, and 38S) were found to produce IgG antibodies with the
subclass of IgG
, IgG
, IgG
,
IgG
, IgG
, and IgG
, respectively.
The remaining 10 cell lines (4S, 6S, 9S, 15S, 16S, 17S, 23S, 30S, 40S,
and 41S) produced IgM. Large-scale isolation of some of the mAbs (10S,
17S, 18S, 22S, 35S, and 38S) was accomplished; these were tested by
ELISA and immunoblot assays against various domain peptides of rat
-pol. In a subsequent experiment, we compared the specificity of
each mAb for rat and human
-pols. mAbs 6S, 18S, 22S, 35S, 40S, and
41S gave stronger reaction against rat
-pol than human
-pol,
while mAbs 10S, 17S, 23S, and 38S were stronger against human
-pol
than rat
-pol. The mAbs 4S, 14S, and 30S shared almost equal
avidity for human and rat
-pol; mAb 9S, 15S, and 16S show
relatively less reactivity with
-pol, as compared to the other
mAbs.
-pol, 31-, 27-, 18-, and
10-kDa fragments. Epitopes for the other mAbs were found between amino
acids 140 and 335. In the cases of 23S, 40S, and 41S, we detected no
reaction with the 8-kDa domain, 16- or 18-kDa fragments. The epitopes
of these mAbs may be contributed by different regions of
-pol,
resulting in the cross-reactivity to the 31-, 27-, 12-, and 10-kDa
domains. The mAbs 6S and 38S have epitopes in the 12-kDa domain
(230-335).
Cell Culture
Immunoblots
-pol detection. For rapid cell lysis,
exponentially growing tissue culture cells were washed twice with
ice-cold phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride, 2.7 µg/ml aprotinin, and 0.5
µg/ml each of leupeptin, pepstatin A, and chymostatin. The cells
were lysed in a buffer containing 10 mM Tris-HCl, pH 7.5, 1%
sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and
the protease inhibitors mixture as described above. The lysate was
centrifuged at 8,000
g for 5 min, and the supernatant
fraction (20 µg of protein unless otherwise specified) was
electrophoresed in a 12.5% SDS-polyacrylamide gel. Proteins in the gel
were transferred electrophoretically to nitrocellulose membrane. Equal
loading of samples in different lanes and transfer to nitrocellulose
membrane was verified in all cases by staining the membrane with
Ponceau S.
-Pol was measured by incubating the membrane with mouse
anti-
-pol monoclonal antibody 18S, and then with antibody to mouse
immunoglobulin G (IgG) conjugated to horseradish peroxidase.
Immobilized horseradish peroxidase activity was detected by enhanced
chemiluminescence.
-pol (2 to 20 ng) were also electrophoresed along with various
unknown samples on a 12.5% SDS-polyacrylamide gel and blotted to a
nitrocellulose membrane;
-pol was detected as described. The
amount of
-pol was determined from the linear portion of a
dose-response curve (Fig. 1) obtained by plotting concentration versus integrated optical density. The integrated densitometry
signals of
-pol bands from autoradiograms (proportional to
chemiluminescence) were measured using Millipore BioImage Visage
Software on a Sun Sparc Station II Workstation (BioImage Products, Ann
Arbor, MI). We found that densitometric signals of recombinant
-pol were
7-fold higher when added to whole cell extract than
for the same amount of
-pol run alone (without extract). The
magnitude of this effect was reminiscent of that observed with crude
cell extract in the activity gel assay for
-pol enzymatic activity
(Swack et al., 1985). Hence, the values for
-pol level
reported for different cell lines and tissues were calculated on the
basis of standard curves derived after addition of purified recombinant
-pol to each whole cell extract.
Figure 1:
Standard curve for
-pol quantification. Varying amounts of purified rat
-pol,
from 5 to 20 ng, were mixed with 20 µg E. coli whole cell
extract and electrophoresed on 12.5% SDS-PAGE, electrotransferred to a
nitrocellulose membrane, and probed with mAb 18S. Integrated OD was
measured as described and then plotted against
-pol.
Monoclonal Antibody Characterization
Purified
rat -pol was used as antigen to produce a panel of mouse
monoclonal antibodies (mAbs). Sixteen hybridoma cell lines producing
antibody were isolated, and the antibodies were purified; hybridomas
were selected for their ability to produce probes for immunoblotting.
For epitope mapping of the mAbs, controlled proteolysis of
-pol
was used to prepare 5 domain peptides, as summarized in Fig. 2.
We also overexpressed and purified an 18-kDa N-terminal segment of
-pol corresponding to residues 1-154. The final preparation
of the 18-kDa peptide also contained a smaller peptide, designated as
16 kDa, sharing the same N-terminal sequence. These 7 peptides were
transferred to membrane, which then was probed separately with each mAb
for epitope localization (Fig. 2). Epitopes for 12 of the mAbs
were not localized to a smaller region, whereas epitopes for 10S, 16S,
18S, or 22S were localized to a respective 10-20 amino acid
segment (Fig. 2). The epitope for 35S is in the 140-220
segment of
-pol (Fig. 2), and the epitope for 18S is between
residues 140 and 154 (Fig. 3A). The immunoblotting
signal produced by 18S could be verified by competition with a
synthetic peptide corresponding to the region
KYFEDFEKRIPREEM
, as this peptide blocks 18S
reactivity with intact
-pol (Fig. 3B).
Figure 2:
Epitope mapping of monoclonal antibodies.
Epitope mapping was performed using an immunoblotting assay.
Recombinant rat -pol and its various fragments generated by
controlled proteolysis with trypsin were subjected to 15% SDS-PAGE. The
proteins were electrotransferred to nitrocellulose membranes. Each blot
was probed separately with a mAb. Epitopes were identified by comparing
the reactivity of each mAb with rat
-pol and its various
fragments. The placing of mAb or mAbs shows their epitope position on
-pol (A and B). 8- (1-80) and 31-kDa
(87-335) fragments were produced by 1:1,000, trypsin:
-pol
digestion, whereas 27- (140-335), 10- (140-220), and 12-kDa
(230-335) fragments were produced with trypsin at a
trypsin:
-pol ratio of 1:10 (w/w) at 25°C. 18 kDa (1-154)
and a smaller 16-kDa fragment with the same N terminus were
overexpressed and purified from E. coli (C). These
peptides and
-pol fragments obtained from selective digestion with
trypsin were used to identify epitopes of monoclonal
antibodies.
Figure 3:
Epitope mapping and specificity of mAb 18S
to various DNA polymerases. PanelA, immunoblot of
overexpressed human or rat -pol along with its various domains (8,
10, 12, 27, and 31 kDa) produced by selective tryptic digestion, plus
overexpressed fragments (16 and 18 kDa), DNA polymerase
, the
large fragment of E. coli DNA polymerase I, and human
immunodeficiency virus type-1 reverse transcriptase. The immunoblot was
probed with anti-
-pol 18S mAb. The migration positions of
-pol and its various peptides are indicated on right-hand side.
The positions of protein markers are shown on the left-hand side of the
photograph. PanelB, competition of the 18S mAbs
epitope with a synthetic peptide of
-pol (corresponding to
residues 140-154). All lanes (lanes 1-6) have an
equal amount of pure
-pol (100 ng). Lanes 1-6 were
probed with mAb 18S which was preincubated for 2 h at 25°C with 0,
0.1, 1, 2, 5, and 10 µg of synthetic peptide,
respectively.
Monoclonal antibody 18S did not cross-react with reference enzymes
human DNA polymerase , human immunodeficiency virus type-1 reverse
transcriptase, or with Klenow fragment (Fig. 3A), but it
did react strongly with human
-pol, as shown in Fig. 3A and summarized under ``Materials and Methods.'' Finally,
none of the mAbs exhibited neutralizing activity against purified
-pol on poly(dA)
oligo(dT) as template-primer (data not
shown). Once this panel of high-titer
-pol specific mAbs was
available, we characterized one of them (18S) as a probe for
quantitative immunoblotting. Whole cell extracts from a variety of
sources, including bovine testis (known to contain a high level of
-pol; Hirose et al.(1989) and Nowak et
al.(1989)), were surveyed by quantitative immunoblotting. Among
the sources tested, the
-pol level was higher in bovine testis
than in the other sources.
Regulation of
Transient expression activity of the cloned human -Pol Expression in
Vivo
-pol
promoter in transfected CHO cell can be altered through the protein
kinase A signal transduction pathway (Englander and Wilson, 1992b),
which is activated by MNNG treatment of cells (Kedar et al.,
1991; Englander and Wilson et al., 1992a). With the
availability of mAb 18S, we could examine the effect on
-pol
protein levels of altering protein kinase A by treating CHO cells with
dibutyryl cAMP or forskolin. The
-pol level in our wild type CHO
cell line was found to be
7 ng/mg protein in the whole cell
lysate. Treatment for 4 h with forskolin (1 µM), or with
dibutyryl cAMP (100 µM), resulted in an increase to 18 and
15 ng/mg, respectively. Dexamethasone (500 nM) treatment
caused only a modest increase in
-pol level, to 10 ng/mg of whole
cell protein. It is known that MMS or MNNG treatment of CHO cells
increases
-pol mRNA level (Fornace et al., 1989) and that
MNNG treatment activates the cloned
-pol promoter in transient
expression experiments through an effect on the ATF/CREB
transcriptional activator (Kedar et al., 1991; Englander and
Wilson, 1992a). We examined
-pol protein levels in CHO cells
treated with either MMS or MNNG. CHO cells treated with either MMS or
MNNG showed an increase in
-pol protein level (Fig. 4, A and B); with MNNG, the highest level was observed
2.5-5 h after treatment (Fig. 4B).
Figure 4:
Effect of MMS and MNNG treatment on
-pol expression in CHO-K1 cells. PanelA, CHO-K1
cells were exposed for 4 h at varying concentrations of MMS as
indicated at the top of each lane (lanes 2-5). Lane
1, CHO-K1 cells were grown in presence of 1% Me
SO
(vehicle); lane6, recombinant rat
-pol. PanelB, CHO-K1 cells were grown in the absence (lane 1) or presence of 30 µM MNNG (lanes
2-6) for the time (h) shown at the top of each lane. Lane7, recombinant rat
-pol; and lane8, CHO-K1 cells grown in presence of 30 µM MNNG for 5 h and probed with nonimmune IgG. PanelC, GC-1 cells (lanes1-3) and
CHO-K1 (lanes4-6) cells were grown in the
absence (lanes1 and 4) or presence of 10
µM (lanes2 and 5) or 30
µM MNNG (lanes3 and 6) for 4
h. Purified recombinant rat
-pol (lane7) was
used as a reference. Whole cell lysates of CHO-K1 (20 µg) and GC-1
(12 µg) were subjected to 12.5% SDS-PAGE, electrotransferred to a
nitrocellulose membrane, and immunoblotted with 18S. Levels of
-pol in GC-1 cells were 7, 6, and 6 ng/mg whole cell protein in lanes 1-3, respectively, whereas in CHO-K1 cells levels
were 7, 28, and 50 ng/mg whole cell protein in lanes
4-6, respectively.
Next, to
determine whether this up-regulation in -pol level was linked to
DNA alkylation, we studied the effect of MNNG treatment using a
genetically altered CHO cell line termed GC-1. GC-1 cells, which were
derived from our wild type CHO cell line, strongly overexpress the
alkylation damage DNA repair enzyme O
-methylguanine DNA methyltransferase and,
therefore, are resistant to cell killing by MNNG (Dunn et al.,
1991). These cells are expected to have much less MNNG-induced
methylation damage to DNA, by virtue of removal of methyl groups from
DNA by the overexpressed DNA repair methyltransferase. Our results with
GC-1 cells are shown in Fig. 4C. In contrast to wild
type CHO cells, GC-1 cells did not show any increase in
-pol
expression after MNNG treatment. These results suggest that the
-pol response to MNNG treatment is, indeed, a DNA alkylation
damage response.
-pol by phorbol ester (TPA)
treatment in CHO cells was examined as a function of time and TPA
concentration. Maximum expression occurred 4 h after treatment with 500
nM TPA (Fig. 5); the effect of different TPA
concentrations also depended upon the time of exposure. When cells were
exposed to TPA in the presence of actinomycin D (5 µg/ml)
expression of
-pol did not increase, suggesting that the TPA
induction of
-pol required transcription (Fig. 5A).
These results on up-regulation of the endogenous
-pol gene by
phorbol ester, and presumably the protein kinase C pathway, may be
consistent with our earlier results with mouse 3T3 cells;
overexpression of activated Harvey p21
strongly
up-regulated the cloned
-pol promoter (Kedar et al.,
1990). Although the
-pol promoter does not have a known protein
kinase C responsive DNA element, it is possible that stimulation could
occur through the ATF/CREB protein which is known to be a key
transcriptional regulator of
-pol gene expression (see
``Discussion'').
Figure 5:
Effect of phorbol ester (TPA) on -pol
expression in Chinese hamster ovary (CHO-K1) cells. PanelA, CHO-K1 cells were treated with 500 nM TPA (lanes2-11) for different time intervals from
1 to 24 h as indicated at the top of each lane. The level of
-pol measured was: 7, 14, 20, 53, 42, 46, and 31 ng/mg whole cell
protein in lanes 1-7, respectively. CHO-K1 cells were
exposed first to actinomycin D for 15 min (5 µg/ml) with subsequent
addition of 500 nM TPA for 8 h (lane9), for
4 h (lane 10), and for 1 h (lane11), and
the levels of
-pol in each lane was
7 ng/mg whole cell
protein. Lane1 is without TPA. PanelB, CHO-K1 cells were grown in the absence (lane1) or presence of 10 nM (lanes2 and 6), 100 nM (lanes3 and 7), 500 nM (lanes 4 and 8), and 1
µM TPA (lanes 5 and 9) for 2 (lanes2-5) or 4 h (lanes6-9).
Whole cell lysate (20 µg) was loaded in each lane, electrophoresed,
and immunoblotted as described under ``Materials and
Methods.'' The position of
-pol is indicated by an arrow.
Effect of Simultaneous Treatment with TPA and
MNNG
The results described above and findings recently reported
by others on antagonism between the protein kinase A and protein kinase
C signal transduction systems (Graves et al., 1993; Sevetson et al., 1993), prompted us to study the effect of treatment
with combinations of TPA and MNNG on -pol protein level. In these
experiments, CHO cells were treated with increasing concentrations of
either TPA or MNNG, in the presence of different concentrations of the
other agent. First, TPA had an abrogating effect on up-regulation of
-pol by MNNG (): the up-regulation by MNNG in these
experiments was as much as 8.5-fold and was seen at concentrations as
low as 0.1 µM; TPA at each concentration tested blocked
the up-regulation.
-pol by as much as 14-fold in these experiments, and
this induction was either blocked or partially blocked at each
concentration of MNNG (). These results, in a general
sense, indicate that the mechanism of activation by these two agents is
not identical, and the results are consistent with the possibility that
the mediator of the MNNG effect and the mediator of the phorbol ester
effect interact with one another to form a dead-end complex, or have an
antagonistic effect on the same target protein in
-pol gene
expression (e.g. ATF/CREB).
-pol expression in vivo using new
mAbs as probes for immunoblotting. Of the 16 mAbs isolated, we
localized the epitope for four (10S, 16S, 18S, and 22S) to 10-20
amino acid regions of
-pol (Fig. 2). None had an epitope in
the N-terminal domain (1-75), and none inhibited the
-pol
DNA polymerase activity (data not shown). 35S and 18S were found to be
good probes for immunofluorescence staining and immunoblot analysis of
whole cell extracts, respectively. 18S did not show cross-reactivity
with the other polymerases tested here (Fig. 3A), in
contrast to earlier mAbs reported by Recupero et al.(1992).
18S could be used for quantitative measurements of
-pol and could
also be used in combination with a synthetic epitope peptide to verify
identity of the
39-kDa immunoblot signal by competition. In this
immunoblot assay, the efficiency of 18S for detection of
-pol was
found to be enhanced by the presence of other proteins in the extract;
hence, when pure
-pol was added to a whole cell extract, more
signal was detected than with assays of pure
-pol alone. This
difference in detection efficiency was taken into account in our
quantification of absolute
-pol levels in the whole cell lysate
(see ``Materials and Methods'').
-pol
gene expression. We observed up-regulation of
-pol mRNA in CHO
cells after exposure to the DNA-alkylating agents MMS or MNNG (Fornace et al., 1989), and this up-regulation required transcription.
Furthermore, induction by MNNG was observed with a cloned
-pol
promoter in transient expression experiments with CHO cells; this
induction is mediated by the ATF/CREB transcriptional activator (Kedar et al., 1991; Narayan et al., 1995), and required the
cellular protein kinase A signal transduction system (Englander and
Wilson, 1992b).
-pol
protein in CHO cells. An initial finding of interest was that treatment
of cells with either MMS or MNNG led to an increase in
-pol
protein level. We had found earlier that the increase in
-pol mRNA
level was not accompanied by a significant increase in
-pol
activity level in CHO cells. This earlier work involved activity gel
analysis to quantify
-pol; although the analysis was conducted in
a range of proportionality between activity gel signal and crude
extract applied to the gel (Fornace et al., 1989), and the
analysis showed only a very modest increase (about 1.5-fold) in
-pol enzymatic activity level. The present results represent a
more complete assessment of
-pol protein level in the crude
extract, and measurement by immunoblotting with mAb 18S is considered
to be more quantitative than by activity gel analysis.
-pol protein up-regulation, we examined the interesting
question of whether the induction is secondary to DNA alkylation. To
approach this issue, we studied the effect of intracellular
overexpression of a DNA methyl group DNA repair enzyme, O
-methylguanine DNA-methyltransferase, on the MNNG
induction of
-pol (Fig. 4B). Our results showed
that the induction did not occur in an otherwise isogeneic cell line
(GC-1) carrying the overexpressed DNA methyltransferase repair enzyme.
The mere presence of overexpressed methyltransferase itself did not
produce a change in steady-state level of
-pol, in the absence of
MNNG. Furthermore, there have been no reports that the
methyltransferase itself alters transcriptional regulation. The
results, taken together, indicate that this transcription-based
stimulation of
-pol expression is through a DNA damage response
pathway, rather than through a direct effect of MNNG, on protein kinase
A or on the transcriptional machinery, as has been proposed for the
oxidative stress response involving AP-1 (Devary et al., 1991;
Xanthoudakis et al., 1994).
-pol promoter DNA damage response remains to be revealed, but
clearly the response is mediated by the ATF/CREB transcriptional
activator (Kedar et al., 1990; Englander and Wilson, 1992;
Narayan et al., 1995). Evidence for this includes the finding
that ATF/CREB protein purified from MNNG-treated cells is more active
in stimulating in vitro transcription initiation than the
corresponding activator protein from normal cells (Narayan et
al., 1995). The increases in
-pol protein level observed here
when CHO cells are grown in the presence of dibutyryl cAMP or forskolin
confirmed that the endogenous
-pol promoter is capable of
responding to activation of the protein kinase A signal transduction
pathway.
-pol level, and this up-regulation
required transcription because cells pretreated with actinomycin D did
not show any TPA-mediated increase in
-pol (Fig. 5A). Hence, these experiments, in a general sense,
suggest that both this pathway and the protein kinase A pathway, which
mediates the influence of DNA alkylation damage by MNNG, regulate the
-pol gene. We note that involvement of the growth factor/protein
kinase C/mitogen-activated protein kinase pathway had been suggested by
earlier
-pol promoter transient expression experiments with
overexpression of activated Harvey p21
in 3T3
cells (Kedar et al., 1990).
-pol expression afforded by MNNG or TPA
was not additive. Instead, these agents were antagonistic to each other
at all concentrations tested. This observation suggests that mediators
of these stimulatory effects interact at some point with the same
component in the signal transduction or transcriptional apparatus.
Protein kinase C pathway transcriptional activators, such as c-Jun, can
dimerize with ATF/CREB and influence transcriptional activation by
proteins binding at the ATF/CRE site (Benbrook and Jones, 1990),
whereas in the case of some ATF/CREB family members, protein kinase C
can stimulate phosphorylation of the ATF/CREB protein (Sakurai et
al., 1991). AP-1 also has been reported to recognize and activate
transcription through binding at both AP-1 and ATF elements (Buckbinder et al., 1989). Further studies will be required to understand
how TPA treatment can induce or abrogate
-pol promoter expression.
However, taken together, these results point to the interesting
possibility that the DNA damage response for
-pol in mammalian
cells can be regulated by agents that alter the activity of the protein
kinase C signal transduction pathway. Similar observations on possible
antagonism between the protein kinase A and C pathways have been
reported for others genes (Graves et al., 1993; Sevetson et al., 1993).
-pol, including a mAb (18S) that can be used
to measure the
-pol protein level in a crude cell extract. We
demonstrated that DNA alkylation is required for the induction of
-pol gene expression, secondary to MNNG treatment of CHO cells.
Induction of the
-pol level in CHO cells also was observed after
phorbol ester treatment. When simultaneously applied to cells, MNNG and
phorbol ester were antagonistic toward the up-regulation seen with
either agent alone. The results may be interpreted as an example of
antagonistic effects of stimulation of the protein kinase A and protein
kinase C signal transduction pathways.
Table: Effect of TPA on the MNNG-induced up-regulation
of -pol expression in CHO-K1 cells
-pol level in
untreated CHO-K1 cells was 7 ng/mg of whole cell protein. Value in
parentheses is the relative
-pol level, ratio of
-pol in
treated cells/untreated cells.
Table: Effect of
MNNG on the TPA-induced up-regulation of -pol expression in
CHO-K1 cells
-pol level in untreated CHO-K1 cells was 7 ng/mg
of whole cell protein. Value in parentheses is the relative
-pol
level, ratio of
-pol in treated cells/untreated cells.
-pol, DNA polymerase
; CHO, Chinese hamster ovary; MGMT, O
-methylguanine DNA methyltransferase; MMS, methyl
methanesulfonate; MNNG, N-methyl-N`-nitro-N-nitrosoguanidine; mAb,
monoclonal antibody; protein kinase A, cAMP-dependent protein kinase;
ELISA, enzyme-linked immunosorbent assay; ATF/CREB, activating
transcription factor/cAMP-response element binding; PAGE,
polyacrylamide gel electrophoresis; TPA,
12-O-tetradecanoylphorbol-13-acetate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.