(Received for publication, March 14, 1995; and in revised form, September 22, 1995)
From the
We studied the effect of ionizing radiation on the activation of
the AP-1 transcription factors and the regulation of basic fibroblast
growth factor (bFGF) gene expression in drug-sensitive human
breast carcinoma (MCF-7) cells and its drug-resistant variant
(MCF-7/ADR) cells. Northern blot and gel mobility shift assays showed
that 135 cGy of ionizing radiation induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression in MCF-7/ADR cells. In MCF-7 cells, however, we
observed little/no induction of bFGF gene expression and AP-1
binding activity after the stress. Nevertheless, MCF-7 cells
transfected with plasmids containing c-jun gene contain high
levels of bFGF protein. H-7 (60 µg/ml), a potent protein kinase C
(PKC) inhibitor, inhibited the stress-induced AP-1 binding activity and bFGF gene expression in MCF-7/ADR cells. Corroborating this
observation, overexpression of PKC induced bFGF gene
expression in MCF-7 cells. Taken together, these results suggest that
stress-induced bFGF gene expression is mediated through the
activation of PKC and AP-1 transcription factors. Differences in the
levels of PKC activity and AP-1 binding factors may be responsible for
differential expression of bFGF among breast cancer cell lines.
Although there are large differences in response to ionizing radiation
between MCF-7 and MCF-7/ADR cell lines, we observed no significant
differences in radiocytotoxicity between them.
It has been known for many years that alteration of the micro-
or macroenvironment of a cell can trigger the highly complex cellular
stress management system. Recent studies have shown that, in addition
to the well known stress proteins, environmental stresses such as
ischemia/hypoxia (Plate et al., 1993; Millauer et
al., 1994), tumor promoters (Winkles et al., 1992), and
radiation (Witte et al., 1989; Haimovitz-Friedman et
al., 1991) are all stimuli for triggering the synthesis of
angiogenic factor(s). The observation of the induction of these
angiogenic proteins under these circumstances could have significant
implications on the process of tumorogenesis. Upon these stresses,
tumor cells may induce neovascularization by synthesizing and releasing
diffusible tumor-derived angiogenic factors such as vascular
endothelial growth factor and bFGF (Shing et al.,
1985; Klagsbrun et al., 1986; Plate et al., 1993;
Soutter et al., 1993).
A fundamental question which remains unanswered is how the stresses stimulate the synthesis of these angiogenic factors. DNA sequencing studies suggest that angiogenic factor-related genes such as bFGF contain AP-1 cis-acting regulatory elements (TPA response element: TRE) (Kim et al., 1989a, 1989b; Shibata et al., 1991). In vitro DNA binding and in vivo footprinting experiments demonstrated that these regulatory elements are recognized by AP-1 transcription factors (Jun and Fos family proteins) (Angel et al., 1988; Deng and Karin, 1993). The activity of AP-1 transcription factor is regulated by the induction of jun and fos gene transcription and post-translational modification of their products (Binetruy et al., 1991; Boyle et al., 1991). Several researchers have reported that c-jun and c-fos genes are expressed in response to a wide range of stresses including heat shock exposure (Andrews et al., 1987; Bukh et al., 1990), UV irradiation (Angel et al., 1985; Hollander and Fornace, 1989; Stein et al., 1992), ionizing radiation exposure (Sherman et al., 1990; Hallahan et al., 1991a), and treatment with chemical agents (Andrews et al., 1987; Hollander and Fornace, 1989; Shibanuma et al., 1990) in mammalian cells. Moreover, these stresses also increase AP-1 binding activity (Piette et al., 1988; Hallahan et al., 1993). Thus, we hypothesized that the stress-induced activation of AP-1 transcription factors is responsible for the expression of the bFGF gene as a result of exposure to ionizing radiation. In this study, we also investigated differences between drug-resistant and -sensitive human breast carcinoma MCF-7 cells in response to ionizing radiation. We observed that the radiation-induced expression of jun and fos genes, AP-1 binding activity, as well as bFGF gene expression occurred more prominently in drug-resistant MCF-7/ADR cells compared to drug-sensitive MCF-7 cells. Nonetheless, there was no significant differences in the cytotoxic effects of ionizing radiation between these two cell lines.
Figure 1:
Accumulation of c-jun mRNA
after x-irradiation in MCF-7 or MCF-7/ADR cells. Cells were irradiated
with 135 cGy and incubated at 37 °C for the intervals (0.25, 0.5,
1.25, 3, 6, and 8 h) indicated at the bottom of each lane. Cells were
harvested and RNA was isolated. An equal amount of RNA (30 µg) was
loaded onto each lane of an agarose-formaldehyde gel for separation.
After separation, RNA was blotted onto a nitrocellulose membrane,
hybridized with P-labeled probes for c-jun and
GAPDH mRNA, and autoradiographed (panel A). The
autoradiography of c-jun mRNA was analyzed with a densitometer (panel B). C, RNA from untreated control cells. GAPDH, GAPDH probe was used to verify the equivalent amounts
and integrity of RNAs loaded in each lane. Autoradiograms were all from
the same blot, which was stripped and rehybridized with different
probes.
Figure 2: Northern blots of c-jun and c-fos mRNA after x-irradiation in MCF-7 or MCF-7/ADR cells (panel A) and quantitative analysis of c-jun mRNA (panel B). Cells were irradiated with 75 cGy and incubated at 37 °C for the intervals (0.25, 0.5, 1.25, 3, 6, and 8 h) indicated at the bottom of each lane. Northern blot analysis was performed as described in Fig. 1. C, RNA from untreated control cells. GAPDH, GAPDH probe hybridized to the same blot to verify RNA uniformity.
Figure 3:
Detection of AP-1 binding activity in
nuclear extracts from x-irradiated MCF-7 or MCF-7/ADR cells. Cells were
irradiated with 135 cGy and incubated at 37 °C for the intervals
indicated at the top of each lane (0.25-3 h). The gel
mobility-shift assay was performed with a P-labeled AP-1
oligonucleotide and nuclear extracts (5 µg) prepared from
irradiated cells. Competition assays were performed by adding a
200-fold molar excess of an unlabeled AP-1 or an unlabeled SP1
oligonucleotide. C, nuclear extracts from untreated control
cells. TPA, nuclear extracts from cells treated with 1
µg/ml TPA for 1 h. Closed arrow indicates AP-1 binding
activity. Open arrow indicates free
P-labeled
oligonucleotide fragment (FREE).
To confirm the presence of c-Jun, which binds to the TRE in the bFGF promoter, anti-c-Jun antibody was used to precipitate AP-1 complexes from nuclear extracts. Protein-antibody complexes were removed by centrifugation. The AP-1 binding activity of nuclear extracts from x-irradiated cells was decreased by adding anti-c-Jun antibody followed by a secondary antibody (Fig. 4). These results confirmed that c-Jun protein is involved in the observed AP-1 binding activity.
Figure 4:
Immunospecific inactivation of the AP-1
binding activity in nuclear extracts of MCF-7/ADR cells by anti-c-Jun
antibody. Nuclear extracts (5 µg) from control (odd
numbers) and x-irradiated (135 cGy) cells (even numbers)
were reacted with 0.5 µg of polyclonal antibody developed against
c-Jun (lanes 5, 6, 9, and 10) and
precipitated with 2.5 µg of goat anti-rabbit 2nd antibody (lanes 9 and 10). Nuclear extracts were used in the
assay without 4 °C incubation (lanes 1 and 2),
with 4 °C overnight incubation (lanes 3-10), or with
the addition of 2nd antibody only (lanes 7 and 8).
Gel mobility-shift assay was performed as described in Fig. 3. Closed arrow indicates AP-1 binding activity. Open arrow indicates free P-labeled oligonucleotide fragment (FREE).
Figure 5: Northern blot analysis of bFGF mRNA from x-irradiated MCF-7 and MCF-7/ADR cells. Cells were irradiated with 135 cGy and incubated at 37 °C for the intervals (0.25, 0.5, 1.25, 3, 6, and 8 h) indicated at the bottom of each lane. Northern blot analysis was performed as described in Fig. 1. Arrows indicate the location and the size of bFGF mRNAs (kb) on the right side of the panel. C, RNA from untreated control cells. GAPDH, the housekeeping gene, GAPDH mRNA. See legend of Fig. 1for further details.
Figure 6:
Western blots with an anti-c-Jun or
anti-bFGF antibody. MCF-7 cells were transfected with pRSV-c-Jun or
pCMV-c-Jun containing c-jun cDNA gene. Transient transfectants
were lysed with sample buffer. Equal amounts of protein (30 µg)
from cell lysates were separated by SDS-PAGE, transferred onto a
nitrocellulose membrane, and processed for immunoblotting with c-Jun
polyclonal antibody or bFGF monoclonal antibody. C, protein
from nontransfected control MCF-7 or MCF-7/ADR cells. Molecular weights
( 10
) are shown on the right.
Figure 7:
The level of PKC protein (A) and
PKC activity (B) in MCF-7 or MCF-7/ADR cells. Panel
A, Western blot with an anti-PKC polyclonal antibody. Lysates
from equal amounts of protein (30 µg) were separated by SDS-PAGE,
transferred onto a nitrocellulose membrane and processed for
immunoblotting with PKC
antibody. Molecular weight (
10
) is shown on the right. Panel B, PKC
activity was measured by using an Amersham PKC assay kit. Data from two
separate experiments are plotted.
Figure 8: Effect of H-7 on the expression of c-jun gene in MCF-7/ADR cells. No DRUG, cells were irradiated with 135 cGy and incubated at 37 °C for the intervals (0.25, 0.5, 1.25, 3, 6, and 8 h) indicated at the bottom of each lane. H-7, cells were treated with 60 µg/ml H-7 for 1 h before, during, and after x-irradiation. Northern blot analysis was performed as described in Fig. 1. C, RNA from untreated control cells. GAPDH, internal standard GAPDH mRNA. See legend of Fig. 1for further details.
Figure 9: Effect of H-7 or HA1004 on the radiation-induced AP-1 binding activity in MCF-7/ADR cells. A, untreated control cells. B, cells were irradiated with 135 cGy and nucleoproteins were extracted immediately after irradiation. C, cells were irradiated and nucleoproteins were extracted 0.5 h after irradiation. D, cells were treated with 60 µg/ml H-7 for 1 h before, during, and after irradiation. Nucleoproteins were extracted 0.5 h after irradiation. E, cells were treated with 60 µg/ml HA1004 for 1 h before, during, and after irradiation. Nucleoproteins were extracted 0.5 h after irradiation. The gel mobility-shift assay was performed as described in Fig. 3. Closed arrow indicates AP-1 binding activity.
Figure 10: Effect of H-7 on bFGF gene expression. NO DRUG, cells were irradiated with 135 cGy and incubated at 37 °C for the intervals (0.25, 0.5, 1.25, 3, 6, and 8 h) indicated at the bottom of each lane. H-7, cells were treated with 60 µg/ml H-7 for 1 h before, during, and after irradiation. Incubation intervals after irradiation were shown at the bottom of each lane. Northern blot analysis (panel A) and quantitative analysis of 7-kb mRNA (panel B) were performed as described in Fig. 1. Arrows indicate the location and size of bFGF mRNAs (7, 3.7, 1.4 kb) on the left side of the panel. C, RNA from untreated control cells. GAPDH, GAPDH probe was used to confirm the amount and integrity of RNAs loaded in each lane. See legend of Fig. 1for further details.
Figure 11:
Expression of bFGF gene in MCF-7
cells which were transfected with the plasmid containing the bovine PKC cDNA. Panel A, Western blot analysis was
performed as described in Fig. 7. MCF-7, parental MCF-7
cells; MCF-7-vector, MCF-7 cells were transfected with
pSV
M(2)6 vector without the insert of PKC
gene; MCF-7-PKC
, MCF-7 cells were transfected with
pSV
M(2)6 vector containing PKC
encoding cDNA; MCF-7/ADR, multidrug-resistant MCF-7/ADR cells. Molecular
weight (10
) is shown on the right. Panel B, Northern blot analysis of mRNA from x-irradiated
MCF-7-vector and MCF-7-PKC
cells. Cells were irradiated with 135
cGy and then incubated for various times (0.25-8 h) indicated at
the bottom of each lane. Northern blot analysis was performed as
described in Fig. 1. The apparent reduction of the bFGF mRNA level in MCF-7-PKC
3 h after irradiation was the result
of underloading of the sample. Arrows indicate the location
and the size of bFGF mRNAs (kb) or rRNA on the right or left side of the panel, respectively. C, RNA
from unirradiated control cells. GAPDH, internal standard
GAPDH mRNA.
The effect of H-7 on
c-jun gene expression, AP-1 binding activity, and bFGF gene expression was investigated in MCF-7/ADR cells (Fig. 8Fig. 9Fig. 10). H-7 (60 µg/ml) markedly
suppressed the levels of c-jun, whereas the drug did not
affect the GAPDH mRNA level (H-7 in Fig. 8). Data from
gel mobility-shift assay demonstrated that treatment with H-7 (60
µg/ml) significantly suppressed the radiation-induced AP-1 binding
activity (lane D in Fig. 9). In contrast, HA1004 (60
µg/ml), an H-7 analogue that is a potent inhibitor of cAMP and
cGMP-dependent protein kinases and a weak inhibitor of PKC, did not
suppress the radiation-induced increase in the AP-1 binding activity (lane E in Fig. 9). Northern blot and quantitative
analysis in Fig. 10demonstrated a 2-fold or more reduction of
the 7 kb bFGF mRNA level in the presence of 60 µg/ml H-7.
To confirm the role of PKC in the regulation of bFGF gene
expression, MCF-7 cells were transfected with plasmid
pSV
M(2)6 containing bovine PKC
cDNA (Fig. 11). As compared to either parental MCF-7 or MCF-7-vector
cells, PKC
was overexpressed approximately 5-fold in
MCF-7-PKC
cells as determined by densitometric analysis (Fig. 11A). Interestingly, MCF-7-PKC
cells
displayed bFGF gene expression as detected by Northern blot
analysis (lane C of MCF-7-PKC
in Fig. 11B). Elevated levels of c-Jun and c-Fos proteins
were also observed in MCF-7-PKC
cells (data not shown). Moreover,
the level of bFGF mRNA increased after irradiation with 135 cGy in
MCF-7-PKC
cells.
Figure 12: Effect of H-7 on the radiation dose-survival curves of MCF-7 and MCF-7/ADR cells. Panel A, x-ray survival curves for untreated MCF-7 and MCF-7/ADR cells. Panel B, MCF-7 cells were treated with H-7 (60 µg/ml) for 1 h before and during irradiation. Panel C, MCF-7/ADR cells were treated with H-7 (60 µg/ml) for 1 h before and during irradiation. NO DRUG, untreated control cells. Point, mean of four separate experiments; Error bars, one standard deviation of the data for each point.
Our data from Fig. 1Fig. 2Fig. 3demonstrated that ionizing
radiation activated the expression of jun and fos genes and increased AP-1 binding activity in human breast cancer
cells, particularly multidrug-resistant MCF-7/ADR cells. Our
observations are consistent with results obtained in human epithelial
cells (Hallahan et al., 1991a) and human sarcoma cell line
RIT-3 (Hallahan et al., 1993). Several researchers have also
reported that UV radiation or HO
treatment
stimulates the expression of both c-jun and c-fos, as
well as AP-1 binding activity in HeLa S3 cells (Devary et al.,
1991). Transcriptional activation of the c-jun gene is exerted
through the distal and proximal AP-1 binding sites (TRE) in its
promoter (Angel et al., 1988). In vitro DNA binding
and in vivo footprinting experiments demonstrated that TRE is
recognized by either c-Jun homodimers (Angel et al., 1988:
Deng and Karin, 1993) or c-Jun/activating transcription factor-2
heterodimer (van Dam et al., 1993, Herr et al.,
1994). The transcriptional activity of these binding factors is
stimulated by the phosphorylation of c-Jun (Binetruy et al.,
1991; Devary et al., 1992; Pulverer et al., 1991;
Smeal et al., 1991, 1992) and possibly activating
transcription factor-2 (Abdel-Hafiz et al., 1992). Studies
from Datta et al.(1992) suggest that x-ray-induced
transcriptional activation of the c-jun gene is mediated
through the formation of reactive oxygen intermediates and PKC is
involved in the signal pathway. Our data from Fig. 8and
Hallahan et al. (1991a) demonstrated that treatment with
isoquinolinesulfonamide derivative H-7, a potent PKC inhibitor, prior
to radiation attenuated the increase in c-jun transcripts. The
transcriptional regulation of c-fos is not identical to that
of c-jun. The induction of c-fos transcription is mediated by
two major cis-elements: the serum response element (SRE) and
the v-sis conditioned medium induction element. The SRE is
recognized and stably bound by a homodimer of serum response factor
(Treisman, 1986). The binary SRE-serum response factor complex
interacts with another factor, the ternary complex factor (TCF)
(Treisman, 1992). The TCF, which belongs to the family of E26
transformation-specific (Ets) proteins (Dalton and Treisman, 1992), is
homologous to Elk-1 (Hipskind et al., 1991). The activity of
TCF is rapidly increased in response to cell stimulation with various
agents, such as growth factors, which lead to mitogen-activated protein
kinase activation. Mitogen-activated protein kinase, also known as the
extracellular signal-regulated protein kinase (ERK) can be activated by
UV and x-irradiation (Radler-Pohl et al., 1993; Stevenson et al., 1994). It appears to be responsible for the
phosphorylation of TCF (Gille et al., 1992; Marais et
al., 1993) and subsequently its transcriptional activity (Zinck et al., 1993). The activity of v--sis-inducible
factor, which binds to serum induction element is also enhanced by
phosphorylation. However, it is regulated by a different signaling
pathway: a cytoplasmic protein tyrosine kinase (Nordheim et
al., 1994).
Stress-induced AP-1 binding activity is due to the
posttranslational modification of both newly synthesized and
preexisting Jun and Fos proteins (Boyle et al., 1991; Smeal et al., 1991). c-Jun contains three domains: a DNA binding
domain, a transcription activation domain, and a regulatory domain. In
the unstimulated state, c-Jun is constitutively phosphorylated by
glycogen synthase kinase-3 or casein kinase II at serines (Ser-243 and
Ser-249) and threonines (Thr-231 and Thr-239) close to its C-terminal
DNA binding domain. Phosphorylation in this region markedly reduces the
DNA binding and transcription ability of c-Jun (Boyle et al.,
1991). Dephosphorylation in this region occurs upon stresses such as
phorbol ester tumor promoter TPA treatment (Boyle et al.,
1991) and UV irradiation (Devary et al., 1992) and results in
enhanced AP-1 binding activity. Boyle et al.(1991) reported
that TPA-activated PKC is responsible for site-specific
dephosphorylation of c-Jun. It has been known that PKC can be activated
by stress such as heat shock (Wooten, 1991) and ionizing radiation
(Hallahan et al., 1991b). Our data (Fig. 7) and Lee et al.(1992) showed that MCF-7/ADR cells contain markedly
elevated amounts of PKC. These observations suggest that
differences in the level of PKC
and the level of AP-1
transcription factors (c-Jun and c-Fos) may be responsible for the
differential effects of stress on various cell types.
Several researchers have suggested that PKC mediates stress-induced bFGF gene expression. Tumor promoters such as phorbol 12-myristate 13-acetate, mezerein, and phorbol 12,13-didecanoate activate PKC. These promoters induce the accumulation of bFGF mRNA and its protein in human dermal fibroblasts (Winkles et al., 1992). The enhancement of bFGF gene expression by these tumor promoters is reduced by treatment with H-7. These results are consistent with our observations, which demonstrate the reduction of x-ray-induced bFGF gene expression by H-7 (Fig. 10). Although the dose of H-7 (60 µg/ml) we use is relatively specific for PKC, we cannot rule out H-7 as a general kinase inhibitor. Nonetheless, these results and data from Fig. 11strongly indicate that the x-ray-induced bFGF gene expression is likely mediated through activation of PKC. Data from Fig. 3, Fig. 5, Fig. 9, and Fig. 10also show a good correlation between the AP-1 binding activity and an increase in bFGF gene expression. Moreover, Fig. 6shows that bFGF level was elevated in cells which were transfected with plasmids containing human c-jun cDNA. Taken together, our data have indicated that the enhancement of bFGF gene expression is related to an increase in PKC and AP-1 binding activity after ionizing radiation exposure.
We believe that many critical questions still remain to be answered to understand the mechanisms of regulation of bFGF gene expression after x-irradiation. However, our proposed model will provide important information to understand how environmental stresses induce bFGF gene expression and subsequently lead to tumor angiogenesis. This model will also provide a framework to study the critical steps in tumor development and metastasis.